Dining Out for Life

by Heidi Fellner

Join Sven Sundgaard in Raising Money for The Aliveness Project

This time of year, it’s very easy to feel poor: months of high energy bills, taxes, and home improvement projects all pile on at once. This season in particular, our budgets are stretched even thinner, as more and more of us face pay cuts and layoffs, plus multiple international crises have required our aid as a nation and as a people. It’s all too easy to forget that we are comparatively very wealthy. The truth is that getting takeout coffee, renting a movie, or eating out at a restaurant may seem like basic living expenses to us, but in most parts of the world, they’re luxuries.

It’s pure genius, really, that someone had the idea of turning what Americans do best—spending money on a luxury—into an international fundraising event called Dining Out for Life. It works like this: Most of us have room in our budget for eating a meal out now and then, so if we just do so on April 29 at one of many participating restaurants throughout the Twin Cities, we will raise around $127,000 for The Aliveness Project.

Last year, Dining Out for Life accounted for 14 percent of the organization’s total budget—truly a staggering percentage, especially in light of what it is able to do for Minnesotans living with HIV/AIDS. Under one roof, it provides meals; a food shelf; integrative therapies like massage and acupuncture; case management; health and wellness seminars; and holiday baskets.

The Aliveness Project can do so much for so little thanks to a veritable army of volunteers who donate nearly 550 hours of their time each week. Their valuable skills are put to use in assisting more than 1,600 individuals each year, which amounts to about one in four people diagnosed with HIV/AIDS in Minnesota.

Perhaps the most significant service the organization provides clients is the most difficult to quantify: It offers a supportive, safe place of community.

Tim Marburger, Director of Fundraising and Special Events for The Aliveness Project, says, “It’s definitely a freeing place where you can just be, and not have to worry about what people might think if they find out you’re HIV-positive. Often, when you think of AIDS, you think, ‘It’s going to be dark, and everybody’s going to be sad,’ but hearing the laughter and the conversation here—it’s really neat to know that people can just be themselves.”

HIV/AIDS doesn’t always grab headlines these days, but the rates of new infection in some demographics, including African-Americans and women of every race, are on the rise. What’s more, the Minnesota AIDS Project (MAP) reports that the rate of new infections among gay and bisexual men under 24 has doubled between 2001 and 2008.

Marburger laments, “It’s just a terrible increase. I came to know a young man who came to us when he was 18. And I can’t even fathom to think of being HIV-positive at 18. What does life look like? Some people say that the meds are kind of like taking chemo every day, and they can just wreak havoc on a person’s body. As a youth minister, I’m worried about seeing the influx and the growth. They haven’t seen the people die like we did.”

While rates of infection are discouraging, the other side of the statistics is that people who have been diagnosed with HIV are living much longer lives. It is a wonderful thing, but it results in a broader client demographic for HIV/AIDS organizations and activists.

Marburger, who has been troubled in recent years that The Aliveness Project’s current facility is not easily accessible to an aging population, explains, “Our building now is on two different levels. Thank God people are living longer with the meds, but that doesn’t make stairs any easier.”

Responding to the call, The Aliveness Project has purchased a new building in the Kingfield neighborhood of Minneapolis that will be able to accommodate not only the growing HIV/AIDS population in Minnesota, but also more people with special needs.

Marburger points out, “The new place will have an elevator that will take you to your massage therapy, or to the first floor for your meal or the food shelf.”

Right now, however, the new 12,000-square-foot space needs major renovations. It’s no small task, but Marburger hopes it all will be ready by the fall of 2010. It would be especially nice to see completion then, as The Aliveness Project is celebrating its 25th year of service, and the new space would be a lovely anniversary gift to both patrons and staff who have done so much for Minnesota.

In order for that to happen, we Minnesotans must go out to eat on April 29. Participating in Dining Out for Life never has been easier, thanks in no small part to Marburger, who has re-upped almost all of the Twin Cities restaurants that have participated in past years, plus added a few new ones, for a total of more than 120 this year. He is especially pleased that diners in Afton, Duluth, Rochester, and Stillwater have local restaurant options this year.

Marburger declares, “You can have pizza or lobster, or anything in between. Dining Out for Life is an easy way to get involved.”

The fundraiser is in need of outgoing volunteers who can function as restaurant “ambassadors.” It only requires inviting friends and families to a particular restaurant, socializing, collecting additional donations, and working with the establishment’s staff to make sure the event runs smoothly. A sign-up sheet and a list of participating restaurants are available at The Aliveness Project website.

“Dine out. Do good. What more can one say?” Marburger asks.

What more, indeed.

Dining Out for Life
Apr. 29
(612) 822-7946
www.aliveness.org/dol.htm

Next, we asked whether low concentrations of r-roscovitine and alsterpaullone could potentially inhibit virus replication in primary cells. We have previously shown that r-roscovitine (cyc202) is able to inhibit virus replication both in primary cells as well as cells lines [48]. The IC50 in latent infected cells was from 0.36 μM to 1.8 μM depending on the cell type. Here we utilized a combination of a low 0.01 μM concentration of each r-roscovitine and alsterpaullone, which normally would not inhibit viral replication when used in monotherapy. Results in Figure 5C indicate that the addition of low concentrations of both drugs effectively inhibited a field isolate of HIV-1 in PBMC infections. The combination of these two drugs at such low concentrations had no apparent toxic effects in active PBMCs (data not shown). Collectively, these results imply that cdk2 inhibitors that target the G1/S (cyc202) and early S (alsterpaullone) phases may effectively block viral replication in primary cells when infected with HIV-1 field isolates.

Discussion

In contrast with the latest progress in the understanding of HIV-1 infection, its pathogenesis and mechanism of action-especially in relation to therapies, are still at its infancy. However few well established pathways including cell signaling involving kinases and markers of cell cycle progression have been shown to be tightly regulated in HIV-1 infected cells and therefore provide viable targets for treatment. Cdks are attractive targets for drug development since their activity, required for the correct timing and ordering of the cell cycle, is frequently deregulated in cancer. Numerous small molecule inhibitors of cdks have been identified and proven effective in treating tumors. This is mainly due to the increased sensitivity of the transformed cells to inhibitors and to the changes that are associated with cdk activity and levels in a cell. However the consequences of cdk inactivation are complex and can result in disparate outcomes depending on the tumor type and the genetic context that drives their expression.

We investigated whether targeting the cdk/cyclin axis could inhibit the growth of HIV-1 infected cells and assessed this hypothesis using multiple cdk inhibitors. Along these lines, we searched for various inhibitors targeting multiple cdk/cyclin pathways using published literature and our own search by means of small libraries of compounds. We selected first generation inhibitors with low-high IC50 in various cell types and identified their cell growth inhibition efficiencies in HIV-1 infected and uninfected cells. Results in Table 1 clearly show that there are various compounds that specifically target HIV-1 infected cells. In the high selectivity group, alsterpaullone demonstrated the best selectivity to block viability of all HIV-1 infected cells tested and little blockage to control cells at the concentrations tested. Indirubin-3'-monoxime, indirubin-3'-monoxime-5-indo, purvalanol A and r-roscovitine also inhibited growth of infected cells to varying degree. Consequently, we decided to focus and study the mechanism of alsterpaullone in the current manuscript.

Our results with titration of alsterpaullone showed that HIV-1 infected cells were more vulnerable to apoptosis in a concentration dependent manner. Many of these so called latent infected cells harbor various forms of virus and have a certain level of leakiness and expression of singly and doubly spliced messages in the absence of any inducers. Therefore, there is viral transcription in many of these cells especially when they are treated and fed with 10% fetal bovine serum, which provides enough cytokine and growth factor signaling to produce leaky viral transcription in these cells.

We then focused on the cdk2/cyclin A complex since it has been shown to be involved in early S phase transition of cell cycle, is important for cellular DNA synthesis, and is a target of alsterpaullone. Interestingly, when we used immunoprecipitation to detect the kinase activity of endogenous cdk2/cyclin A, we found great inhibition with alsterpaullone in infected cells. However, upon western blot analysis of cdk2 and other cyclins in drug treated cells, we found lower levels of cdk2 and cyclins in infected cells and not in uninfected cells. Downregulation of cdk2, cyclin A, cyclin T, and cyclin E in infected cells is interesting and may indicate that cdk/cyclin complexes in HIV-1 infected cells are inherently different in their behavior, partner binding or post-translational modifications, among other factors, which may contribute to its high sensitivity to alsterpaullone. Consistent with the cleaved caspase-3 and PARP levels, FACS analysis also showed a dramatic difference in infected versus uninfected cells. Results in Figure 4 clearly show that, in infected cells (OM10.1 and ACH2), the G1 phase population has decreased and the S phase population has increased, as well as an increase of almost ten-fold in the apoptotic population. This implies that the G1/S checkpoint in infected cells is either non-existent or severely defective which may be the ultimate mechanism of how these cdk inhibitors kill HIV-1 infected cells. Importantly, there was no viral release after treatment of the infected cells with alsterpaullone (data not shown) even though the cells were apoptosing.

When using primary cells, we found similar IC50 of inhibition in infected PBMCs as well as an additive effect of r-roscovitine (cyc202) with low concentrations of alsterpaullone. Both of these drugs, which target G1/S and the early S phase at low concentrations, do not kill infected or uninfected cells. However, the addition of low concentrations of both drugs to the infected cells selectively inhibits viral replication in primary cells. We therefore concluded that to inhibit HIV-1 activated transcription, one may need to use multiple cdk inhibitors that inhibit critical cdk/cyclin complexes that are needed for HIV-1 transcription, and low concentrations of these drugs may have a synergistic effect in infected cells.

Finally, alsterpaullone is also a potent GSK-3α/GSK-3β inhibitor [49]. GSK-3α/GSK-3β are implicated in the regulation of glycogen synthesis, the Wnt signaling pathway, cell cycle control, transcriptional regulation, and apoptosis [50]. The ability GSK-3α/GSK-3β to regulate this vast array of cellular processes may be related to its numerous substrates including, glycogen synthase, axin, β-catenin, APC, cyclin D1, c-Jun, c-myc, C/EBPα/β, NFATc, RelA and CREB to name a few [50,51]. Interestingly, Tat induces GSK-3β activity, which can be reversed by the addition of the GSK-3β inhibitor lithium [52]. Furthermore, the GSK-3β inhibitors lithium and VPA can protect against Tat and gp120 mediated neurotoxicity [53-55]. Sui et al. investigated the role of GSK-3β in NF-kB regulated neuronal apoptosis [56]. They found that neurons exposed to HIVADA-macrophage conditioned medium (MCM) displayed decreased NF-kB activity in a Tat dependent manner. GSK-3β inhibition through the lithium or indirubin treatment blocked NF-kB inhibition, the suppressive binding of RelA to HDAC3, and neuronal apoptosis [56]. Lithium treatment also inhibits HIV-1 replication of both T- and M-tropic viruses in PBMCs as well as TNF stimulated J1.1 cells [57]. Therefore, the inhibition of GSK-3β may have implications for the treatment of neuroAIDS as well as in the inhibition of HIV-1 replication in PBMCs. Future experiments will shed light on the mechanism of inhibition in various viral strains and its possible tropism in infected cells.

Conclusion

PCIs may be ideal candidates for HIV-1 transcription inhibition, since they target non-essential cellular proteins and avoid emergence of mutant resistant viruses. We previously reported that r-roscovitine (a first generation PCI) is a potential inhibitor of HIV-1 replication. PCIs are among the most promising novel antiviral agents to emerge over the past few years. In the current work, we evaluated twenty four cdk inhibitors for their effect on HIV-1 replication in vitro and found that alsterpaullone is a potent inhibitor of HIV-1 transcription. FACS analysis showed a more dramatic difference in apoptosis of infected versus uninfected cells, where the G1 phase population has decreased and the S phase population has increased. This implies that the G1/S checkpoint in infected latent cells is either non-existent or severely defective which may be the ultimate mechanism of how these cdk inhibitors kill HIV-1 infected cells.

Methods

Cell lines and reagents

The latently HIV-1-infected promyelocytic OM10.1 cell line, the latently infected promonocytic U1 cell line and the uninfected corresponding HL-60 and U937 cell lineages, as well as infected J1-1, ACH2 and their uninfected counterparts Jurkat and CEM (12D7) cells were cultured at 37°C up to 1 × 105 cells per ml (early log phase of growth) in RPMI-1640 medium supplemented with heat-inactivated fetal bovine serum (10%), streptomycin, penicillin antibiotics (1%) and L-glutamine (1%) (Gibco/BRL, Gaithersburg, MD, USA). OM10.1, ACH2, J1-1 contain a single integrated copy of HIV-1 genome, whereas U1 cells harbor two copies (one wild type and one mutant) of the viral genome in parental U973 cells.

Cdk inhibitors

The cdk inhibitors used in this study were: aloisine A (270-385-M001), alsterpaullone (270-275-M001), bohemine (270-390-M001), CGP74514A (270-391-M001), compound 52 (270-248-M001), 9-cyanopaullone (270-282-M001), 6-dimethylaminopurine (480-050-M100), indirubin-3'-monoxime (270-271-M001), 5-iodo-indirubin-3'-monoxime (270-424-M001), N-6-(Δ2-Isopentenyl)-adenine (350-034-M100), kenpaullone (270-274-M001), olomoucine (350-013-M005), N9-isopropylolomoucine (270-397-M001), purvalanol A (270-246-M001), (r)-roscovitine (350-251-M001), (s)-roscovitine (350-293-M001) were purchased from Alexis Co. (San Diego, CA, USA). 6-benzyloxypurine (387606), 2,6-diaminopurine (247847), 2,6-dichloropurine (D73103), flavone (F2003) were purchase from Sigma-Aldrich (St. Louis, MO, USA). Indirubin-3'-monoxime-5-sulfonic acid (402088), iso-olomoucine (495622), WHI-P180 (681500) were purchased from Calbiochem (La Jolla, CA, USA). The cdk inhibitor, flavopiridol was a gift from Dr. Ajit Kumar at The George Washington University Medical Center. All inhibitors were prepared in 10 mM stock solution. 2,6-dichloropurine and diethylmaleate were dissolved in ethanol, flavone was dissolved in acetone, flavopiridol and pyrrolidinedithiocarbamic acid were dissolved in water and 5-aminosalicylic acid was dissolved in hydrochloric acid. All other inhibitors were all dissolved in DMSO.

Drug screening and cell counting

The initial screening assays included use of HIV-1 infected and uninfected cells that were treated with 24 inhibitors at four concentrations including 0.01, 0.1, 0.5, 1, 5, and 10 μM. Two to six days after treatment (experiment-dependent), cell viability was primarily determined by trypan blue exclusion as well as change of color in media from both infected and uninfected cells. Cells (~100 cells that did not clump) were counted for the number of non-viable cells every 24-48 hours. Subsequent focusing experiments used MTT and flow data to check for viability and apoptosis.

Protein extracts and immunoblotting

Nuclear and cytoplasmic extracts from uninfected and infected cells were prepared. Cells were collected, washed once with PBS and pelleted. Cells were lysed in a buffer containing containing Tris-HCl pH 7.5, 120 mM NaCl, 5 mM EDTA, 0.5% NP-40, 50 mM NaF, 0.2 mM Na3VO4, 1 mM DTT and one tablet complete protease inhibitor cocktail per 50 ml. Lysis was performed under ice-cold conditions, incubated on ice for 30 minutes and spun at 4°C for 5 minutes at 14,000 rpm. The protein concentration for each preparation was determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Cell extracts were resolved by SDS PAGE on a 4-20% tris-glycine gel (Invitrogen, Carlsbad, CA, USA). Proteins were transferred to polyvinylidene difluoride microporous membranes using the iBlot dry blotting system as described by the manufacturer (Invitrogen). Membranes were blocked with Dulbecco's phosphate-buffered saline (PBS) 0.1% Tween-20 + 3% BSA. Primary antibody against specified proteins was incubated with the membrane in blocking solution overnight at 4°C. Antibodies against cdk2 (M-2), cyclin E (M-20), cyclin A (H-432), poly (ADP-ribose) polymerase PARP 1/2 (H-250), caspase-3 (H-277), and actin (C-11) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cyclin T1 (ab27963), GSK3-α (9338), and GSK3-β (9332) antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Membranes were washed twice with PBS + 0.1% Tween-20 and incubated with HRP-conjugated secondary antibody for one hour in blocking solution. Presence of secondary antibody was detected by SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA). Luminescence was visualized on a Kodak 1D image station (Carestream Helath, Rochester, NY, USA).

Immunoprecipitation and in vitro kinase assay

For immunoprecipitation (IP) 2 mg of extract from alsterpaullone-treated (0-5.0 μM) CEM, ACH2, OM10.1 and Jurkat cells were immunoprecipitated at 4°C overnight with cyclin A antibody. The next day complexes were precipitated with A/G beads (Calbiochem) for two hours at 4°C. IPs were washed twice with appropriate TNE buffer and kinase buffer. Reaction mixtures (20 μl) contained final concentrations: 40 mM β-glycerophosphate pH 7.4, 7.5 mM MgCl2, 7.5 mM EGTA, 5% glycerol, [γ-32P]ATP (0.2 mM, 1 μCi), 50 mM NaF, 1 mM orthovanadate, and 0.1% (v/v) β-mercaptoethanol. Phosphorylation reactions were performed with IP material and 200 ng of histone H1 in TTK kinase buffer containing 50 mM HEPES (pH 7.9), 10 mM MgCl2, 6 mM EGTA, and 2.5 mM dithiothreitol. Reactions were incubated at 37°C for 1 hour and stopped by the addition of 1 volume of Laemmli sample buffer containing 5% β-mercaptoethanol and ran on a 4-20% SDS-PAGE. Gels were subjected to autoradiography and quantitation using a Molecular Dynamics PhosphorImager software (Amersham Biosciences, Piscataway, NJ, USA).

MTT Viability Assay

Five thousand cells were plated per well in a 96-well plate and the next day cells were treated with 0.25 μM alsterpaullone or DMSO. Forty-eight hours later, 10 μl MTT reagent (50 mg/ml) was added to each well and plates incubated at 37°C for 2 hours. Next, 100 μl of DMSO was added to each well and the plate was shaken for 15 minutes at room temperature. The assay was read at 570 nM using a SpectraMax 340 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Flow Cytometry

For cell cycle analysis, cells treated with or without drugs and subsequently collected by low speed centrifugation washed with PBS without Ca2+ and Mg2+ and then fixed with 70% ethanol. For fluorescence-activated cell sorting (FACS) analysis, cells were stained with a mixture of propidium iodide buffer (PBS with Ca2+ and Mg2+, 10 μg/ml RNase A, 0.1% Nonidet P-40, and 50 μg/ml propidium iodide) followed by FACS analysis. Cells were washed twice with cold PBS without Ca2+ and Mg2+, resuspended in 1 × binding buffer (10 mM HEPES-NaOH pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and 5 μl of propidium iodide/105 cells, and incubated at room temperature for 15 minutes. Cell histograms were acquired using CELLQuest software (BD Biosciences, Bedford, MA, USA) and analyzed by ModFit LT software (Verity Software House, Topsham, ME, USA). Detection of apoptosis through annexin V and PI staining was done according to the manufacturer's protocol (BD Pharmingen, San Jose, CA, USA). In brief, cells were washed three times in PBS and re-suspended in binding buffer at 1 × 106 cells/ml. An aliquot of 1 × 105 ells was stained with annexin V-FITC and PI for 15 minutes at room temperature. Analysis was performed on a BD FacsCalibur flow cytometer. Cells were considered to be early apoptotic if they exhibited staining for annexin V, but not PI. The double positive population was considered to be in the late stage of apoptosis.

PBMC Infection

Phytohemagglutinin-activated PBMCs were kept in culture with IL-2 for 2 days prior to each infection. Isolation and treatment of PBMCs were performed by following the guidelines of the Centers for Disease Control. Approximately 5 × 106 PBMCs were infected with either pNL4-3 (MOI:1) or primary HIV-1 strain (THA/92/00NSI; 5 ng of p24 gag antigen). Other HIV-1 mutant viruses (AZT, 3TC, TIBO and protease) were also used for PBMC infections (data not shown). All viral isolates were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program. After 8 hours of infection, cells were washed and fresh medium was added. Drug treatment was performed (only once) immediately after the addition of fresh medium, supernatants from the infected PBMCs were collected and used directly for reverse transcriptase (RT) assays or p24 assays.

Luciferase Assay

TZM-bl cells were transfected with pc-Tat (1 μg) using the Lipofectamine reagent (Invitrogen) according to the manufacturer's instructions. TZM-bl cells contain an integrated copy of the firefly luciferase gene under the control of the HIV-1 promoter (obtained through the NIH AIDS Research and Reference Reagent Program). The next day, cells were treated with DMSO or the indicated compound at increasing concentrations. Forty-eight hours post drug treatment, luciferase activity of the firefly luciferase was measured with the BrightGlo Luciferase Assay (Promega, Madison, WI, USA) and luminescence was read from a 96 well plate on an EG&G Berthold luminometer (Berthold Technologies, Oak Ridge, TN, USA).

RT and p24 assays

For RT assays, viral supernatants (10 μl) were incubated in a 96-well plate with RT reaction mixture containing 1× RT buffer (50 mM Tris-HCl, 1 mM DTT, 5 mM MgCl2, 20 mM KCl), 0.1% Triton, poly(A) (10-2 U), poly(dT) (10-2 U) and [3H]TTP. The mixture was incubated overnight at 37°C and 5 μl of the reaction mix was spotted on a DEAE Filter mat paper (PerkinElmer, Shelton, CT, USA) washed four times with 5% Na2HPO4 and three times with water, and then dried completely. RT activity was measured in a Betaplate counter (Wallac, Gaithersburg, MD). For p24 assays, supernatants from infected cells were centrifuged for 8 minutes at 1200 rpm to remove contaminating cells. p24 levels in the supernatants were then assayed by enzyme-linked immunosorbent assay (AIDS Vaccine Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD, USA) by following the manufacturer's instructions.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

†Both IG and EA share first authorship of the current manuscript. IG performed western blot analysis, transfections, MTT assays and luciferase assays. EA performed drug treatment studies, western blot analysis, cell cycle analysis, kinase assays and virus infections. KK aided in the preparation of the manuscript and in the experimental design. FK coordinated the research, experimental design and drafting of the manuscript. All authors have read and approved the final manuscript.

Acknowledgements

We would like to thank the members of the Kashanchi lab for experiments and assistance with the manuscript. This work was partly funded by AI043894, AI074410, and AI078859.

References

1. Greene WC, Peterlin BM: Charting HIV's remarkable voyage through the cell: Basic science as a passport to future therapy.

   Nat Med 2002, 8:673-680. PubMed Abstract | Publisher Full Text

2. Karn J: Tackling Tat.

   J Mol Biol 1999, 293:235-254. PubMed Abstract | Publisher Full Text

3. Price DH: P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II.

   Mol Cell Biol 2000, 20:2629-2634. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

4. Adams M, Sharmeen L, Kimpton J, Romeo JM, Garcia JV, Peterlin BM, Groudine M, Emerman M: Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts.

   Proc Natl Acad Sci USA 1994, 91:3862-3866. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

5. Antoni BA, Stein SB, Rabson AB: Regulation of human immunodeficiency virus infection: implications for pathogenesis.

   Adv Virus Res 1994, 43:53-145. PubMed Abstract | Publisher Full Text

6. Dingwall C, Ernberg I, Gait MJ, Green SM, Heaphy S, Karn J, Lowe AD, Singh M, Skinner MA, Valerio R: Human immunodeficiency virus 1 tat protein binds trans-activation-responsive region (TAR) RNA in vitro.

   Proc Natl Acad Sci USA 1989, 86:6925-6929. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

7. Feng S, Holland EC: HIV-1 tat trans-activation requires the loop sequence within tar.

   Nature 1988, 334:165-167. PubMed Abstract | Publisher Full Text

8. Weeks KM, Crothers DM: RNA recognition by Tat-derived peptides: interaction in the major groove?

   Cell 1991, 66:577-588. PubMed Abstract | Publisher Full Text

9. Weeks KM, Crothers DM: RNA binding assays for Tat-derived peptides: implications for specificity.

   Biochemistry 1992, 31:10281-10287. PubMed Abstract | Publisher Full Text

10. Herrmann CH, Rice AP: Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor.

    J Virol 1995, 69:1612-1620. PubMed Abstract | PubMed Central Full Text

11. Baskaran R, Dahmus ME, Wang JY: Tyrosine phosphorylation of mammalian RNA polymerase II carboxyl-terminal domain.

    Proc Natl Acad Sci USA 1993, 90:11167-11171. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

12. Zhang J, Corden JL: Phosphorylation causes a conformational change in the carboxyl-terminal domain of the mouse RNA polymerase II largest subunit.

    J Biol Chem 1991, 266:2297-2302. PubMed Abstract | Publisher Full Text

13. Boeing S, Rigault C, Heidemann M, Eick D, Meisterernst M: RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion.

    J Biol Chem 2010, 285(1):188-196. PubMed Abstract | Publisher Full Text

14. Meinhart A, Cramer P: Recognition of RNA polymerase II carboxy-terminal domain by 3'-RNA-processing factors.

    Nature 2004, 430(6996):223-226. PubMed Abstract | Publisher Full Text

15. Wei P, Garber ME, Fang SM, Fischer WH, Jones KA: A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.

    Cell 1998, 92:451-462. PubMed Abstract | Publisher Full Text

16. Yang X, Gold MO, Tang DN, Lewis DE, Aguilar-Cordova E, Rice AP, Herrmann CH: TAK, an HIV Tat-associated kinase, is a member of the cyclin-dependent family of protein kinases and is induced by activation of peripheral blood lymphocytes and differentiation of promonocytic cell lines.

    Proc Natl Acad Sci USA 1997, 94:12331-12336. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

17. Zhu Y, Pe'ery T, Peng J, Ramanathan Y, Marshall N, Marshall T, Amendt B, Mathews MB, Price DH: Transcription elongation factor P-TEFb is required for HIV-1 tat transactivation in vitro.

    Genes Dev 1997, 11:2622-2632. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

18. Bieniasz PD, Grdina TA, Bogerd HP, Cullen BR: Recruitment of a protein complex containing Tat and cyclin T1 to TAR governs the species specificity of HIV-1 Tat.

    EMBO J 1998, 17:7056-7065. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

19. Garber ME, Wei P, KewalRamani VN, Mayall TP, Herrmann CH, Rice AP, Littman DR, Jones KA: The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein.

    Genes Dev 1998, 12:3512-3527. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

20. Isel C, Karn J: Direct evidence that HIV-1 Tat stimulates RNA polymerase II carboxyl-terminal domain hyperphosphorylation during transcriptional elongation.

    J Mol Biol 1999, 290:929-941. PubMed Abstract | Publisher Full Text

21. Garber ME, Mayall TP, Suess EM, Meisenhelder J, Thompson NE, Jones KA: CDK9 autophosphorylation regulates high-affinity binding of the human immunodeficiency virus type 1 tat-P-TEFb complex to TAR RNA.

    Mol Cell Biol 2000, 20:6958-6969. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

22. Akoulitchev S, Makela TP, Weinberg RA, Reinberg D: Requirement for TFIIH kinase activity in transcription by RNA polymerase II.

    Nature 1995, 377:557-560. PubMed Abstract | Publisher Full Text

23. Lu H, Zawel L, Fisher L, Egly JM, Reinberg D: Human general transcription factor IIH phosphorylates the C-terminal domain of RNA polymerase II.

    Nature 1992, 358:641-645. PubMed Abstract | Publisher Full Text

24. Zhou M, Halanski MA, Radonovich MF, Kashanchi F, Peng J, Price DH, Brady JN: Tat modifies the activity of CDK9 to phosphorylate serine 5 of the RNA polymerase II carboxyl-terminal domain during human immunodeficiency virus type 1 transcription.

    Mol Cell Biol 2000, 20:5077-5086. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

25. Obeyesekere MN, Herbert JR, Zimmerman SO: A model of the G1 phase of the cell cycle incorporating cyclin E/cdk2 complex and retinoblastoma protein.

    Oncogene 1995, 11:1199-1205. PubMed Abstract

26. Clark E, Santiago F, Deng L, Chong S, de La Fuente C, Wang L, Fu P, Stein D, Denny T, Lanka V, et al.: Loss of G(1)/S checkpoint in human immunodeficiency virus type 1-infected cells is associated with a lack of cyclin-dependent kinase inhibitor p21/Waf1.

    J Virol 2000, 74:5040-5052. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

27. Duan L, Ozaki I, Oakes JW, Taylor JP, Khalili K, Pomerantz RJ: The tumor suppressor protein p53 strongly alters human immunodeficiency virus type 1 replication.

    J Virol 1994, 68:4302-4313. PubMed Abstract | PubMed Central Full Text

28. de la Fuente C, Maddukuri A, Kehn K, Baylor SY, Deng L, Pumfery A, Kashanchi F: Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics.

    Curr HIV Res 2003, 1:131-152. PubMed Abstract | Publisher Full Text

29. Diwan P, Lacasse JJ, Schang LM: Roscovitine inhibits activation of promoters in herpes simplex virus type 1 genomes independently of promoter-specific factors.

    J Virol 2004, 78:9352-9365. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

30. Knockaert M, Greengard P, Meijer L: Pharmacological inhibitors of cyclin-dependent kinases.

    Trends Pharmacol Sci 2002, 23:417-425. PubMed Abstract | Publisher Full Text

31. Schang LM: Effects of pharmacological cyclin-dependent kinase inhibitors on viral transcription and replication.

    Biochim Biophys Acta 2004, 1697:197-209. PubMed Abstract | Publisher Full Text

32. Schang LM, Coccaro E, Lacasse JJ: Cdk inhibitory nucleoside analogs prevent transcription from viral genomes.

    Nucleosides Nucleotides Nucleic Acids 2005, 24:829-837. PubMed Abstract | Publisher Full Text

33. Schang LM: Cyclin-dependent kinases as cellular targets for antiviral drugs.

    J Antimicrob Chemother 2002, 50:779-792. PubMed Abstract | Publisher Full Text

34. Whitlock JA, Krailo M, Reid JM, Ruben SL, Ames MM, Owen W, Reaman G: Phase I clinical and pharmacokinetic study of flavopiridol in children with refractory solid tumors: a Children's Oncology Group Study.

    J Clin Oncol 2005, 23:9179-9186. PubMed Abstract | Publisher Full Text

35. Schang LM: Advances on cyclin-dependent kinases (CDKs) as novel targets for antiviral drugs.

    Curr Drug Targets Infect Disord 2005, 5:29-37. PubMed Abstract | Publisher Full Text

36. Schang LM, Bantly A, Knockaert M, Shaheen F, Meijer L, Malim MH, Gray NS, Schaffer PA: Pharmacological cyclin-dependent kinase inhibitors inhibit replication of wild-type and drug-resistant strains of herpes simplex virus and human immunodeficiency virus type 1 by targeting cellular, not viral, proteins.

    J Virol 2002, 76:7874-7882. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

37. Milovanceva-Popovska M, Kunter U, Ostendorf T, Petermann A, Rong S, Eitner F, Kerjaschki D, Barnett A, Floege J: R-roscovitine (CYC202) alleviates renal cell proliferation in nephritis without aggravating podocyte injury.

    Kidney Int 2005, 67:1362-1370. PubMed Abstract | Publisher Full Text

38. Pumfery A, de la Fuente C, Berro R, Nekhai S, Kashanchi F, Chao SH: Potential use of pharmacological cyclin-dependent kinase inhibitors as anti-HIV therapeutics.

    Curr Pharm Des 2006, 12:1949-1961. PubMed Abstract | Publisher Full Text

39. Fischer PM, Gianella-Borradori A: Recent progress in the discovery and development of cyclin-dependent kinase inhibitors.

    Expert Opin Investig Drugs 2005, 14:457-477. PubMed Abstract | Publisher Full Text

40. Schultz C, Link A, Leost M, Zaharevitz DW, Gussio R, Sausville EA, Meijer L, Kunick C: Paullones, a series of cyclin-dependent kinase inhibitors: synthesis, evaluation of CDK1/cyclin B inhibition, and in vitro antitumor activity.

    J Med Chem 1999, 42:2909-2919. PubMed Abstract | Publisher Full Text

41. Leost M, Schultz C, Link A, Wu YZ, Biernat J, Mandelkow EM, Bibb JA, Snyder GL, Greengard P, Zaharevitz DW, et al.: Paullones are potent inhibitors of glycogen synthase kinase-3beta and cyclin-dependent kinase 5/p25.

    Eur J Biochem 2000, 267:5983-5994. PubMed Abstract | Publisher Full Text

42. Martinez A, Castro A, Dorronsoro I, Alonso M: Glycogen synthase kinase 3 (GSK-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation.

    Med Res Rev 2002, 22:373-384. PubMed Abstract | Publisher Full Text

43. Zaharevitz DW, Gussio R, Leost M, Senderowicz AM, Lahusen T, Kunick C, Meijer L, Sausville EA: Discovery and initial characterization of the paullones, a novel class of small-molecule inhibitors of cyclin-dependent kinases.

    Cancer Res 1999, 59:2566-2569. PubMed Abstract | Publisher Full Text

44. Dominguez I, Green JB: Missing links in GSK3 regulation.

    Dev Biol 2001, 235:303-313. PubMed Abstract | Publisher Full Text

45. Guendel I, Carpio L, Easley R, Van Duyne R, Coley W, Agbottah E, Dowd C, Kashanchi F, Kehn-Hall K: 9-Aminoacridine inhibition of HIV-1 Tat dependent transcription.

    Virol J 2009, 6:114. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text

46. Wu W, Kehn-Hall K, Pedati C, Zweier L, Castro I, Klase Z, Dowd CS, Dubrovsky L, Bukrinsky M, Kashanchi F: Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation.

    Virol J 2008, 5:41. PubMed Abstract | BioMed Central Full Text | PubMed Central Full Text

47. Chugh P, Fan S, Planelles V, Maggirwar SB, Dewhurst S, Kim B: Infection of human immunodeficiency virus and intracellular viral Tat protein exert a pro-survival effect in a human microglial cell line.

    J Mol Biol 2007, 366:67-81. PubMed Abstract | Publisher Full Text

48. Agbottah E, de La Fuente C, Nekhai S, Barnett A, Gianella-Borradori A, Pumfery A, Kashanchi F: Antiviral activity of CYC202 in HIV-1-infected cells.

    J Biol Chem 2005, 280:3029-3042. PubMed Abstract | Publisher Full Text

49. Meijer L, Skaltsounis AL, Magiatis P, Polychronopoulos P, Knockaert M, Leost M, Ryan XP, Vonica CA, Brivanlou A, Dajani R, et al.: GSK-3-selective inhibitors derived from Tyrian purple indirubins.

    Chem Biol 2003, 10:1255-1266. PubMed Abstract | Publisher Full Text

50. Frame S, Cohen P: GSK3 takes centre stage more than 20 years after its discovery.

    Biochem J 2001, 359:1-16. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

51. Buss H, Dorrie A, Schmitz ML, Frank R, Livingstone M, Resch K, Kracht M: Phosphorylation of serine 468 by GSK-3beta negatively regulates basal p65 NF-kappaB activity.

    J Biol Chem 2004, 279:49571-49574. PubMed Abstract | Publisher Full Text

52. Maggirwar SB, Tong N, Ramirez S, Gelbard HA, Dewhurst S: HIV-1 Tat-mediated activation of glycogen synthase kinase-3beta contributes to Tat-mediated neurotoxicity.

    J Neurochem 1999, 73:578-586. PubMed Abstract | Publisher Full Text

53. Dou H, Birusingh K, Faraci J, Gorantla S, Poluektova LY, Maggirwar SB, Dewhurst S, Gelbard HA, Gendelman HE: Neuroprotective activities of sodium valproate in a murine model of human immunodeficiency virus-1 encephalitis.

    J Neurosci 2003, 23:9162-9170. PubMed Abstract | Publisher Full Text

54. Dou H, Ellison B, Bradley J, Kasiyanov A, Poluektova LY, Xiong H, Maggirwar S, Dewhurst S, Gelbard HA, Gendelman HE: Neuroprotective mechanisms of lithium in murine human immunodeficiency virus-1 encephalitis.

    J Neurosci 2005, 25:8375-8385. PubMed Abstract | Publisher Full Text

55. Everall IP, Bell C, Mallory M, Langford D, Adame A, Rockestein E, Masliah E: Lithium ameliorates HIV-gp120-mediated neurotoxicity.

    Mol Cell Neurosci 2002, 21:493-501. PubMed Abstract | Publisher Full Text

56. Sui Z, Sniderhan LF, Fan S, Kazmierczak K, Reisinger E, Kovacs AD, Potash MJ, Dewhurst S, Gelbard HA, Maggirwar SB: Human immunodeficiency virus-encoded Tat activates glycogen synthase kinase-3beta to antagonize nuclear factor-kappaB survival pathway in neurons.

    Eur J Neurosci 2006, 23:2623-2634. PubMed Abstract | Publisher Full Text

57. Kumar A, Zloza A, Moon RT, Watts J, Tenorio AR, Al-Harthi L: Active beta-catenin signaling is an inhibitory pathway for human immunodeficiency virus replication in peripheral blood mononuclear cells.

    J Virol 2008, 82:2813-2820. PubMed Abstract | Publisher Full Text | PubMed Central Full Text

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Three Minnesota businesses — including one based in Albert Lea — have earned the Governor’s Award for Innovative Measures in Workers’ Compensation from the Minnesota Department of Labor and Industry (DLI) and the Workers’ Compensation Advisory Council.

This first-ever award recognizes the employers — Innovance Inc. of Albert Lea, Mayo Clinic of Rochester and Starkey Laboratories, Inc. of Eden Prairie — for implementation of programs and strategies that make their workers’ compensation system, management and approach outstanding.

“I want to congratulate these three organizations for their workers’ compensation efforts, innovation and performance,” said Steve Sviggum, DLI Commissioner. “Our system needs to continually focus on the two most important stakeholders:  the injured worker and the business that pays the premium.”

The awards will be presented during the second annual Workers’ Compensation Policy Summit June 14. At the summit, these three employers will be given the opportunity to speak about their outstanding practices in workers’ compensation. Following is a brief description of the companies and their innovative measures; more information is available online at www.dli.mn.gov/Summit.

Innovance Inc.

Innovance Inc., with 337 employees, is a multi-location manufacturing operation with processes including sheet metal fabrication, metal stamping, grinding, sanding, painting, welding, aluminum extrusion, assembly, rolling, bending and installation. Through effective safety and health programs supported by management and employees, the number of workers’ compensation injuries at Innovance has dropped from 74 in 2005 to 18 in 2009; its total cost incurred per claim has also dropped from $7,578 in 2005 to $934 in 2009.

“We have an active safety committee and the total support of middle and upper management,” said LaVerne Schroeder, Innovance safety/environmental coordinator, “without this teamwork approach, none of this would be possible. Innovance also would not have been able to accomplish this great achievement without the assistance and participation of our partners and — most of all — our employees.”

Mayo Clinic

Mayo Clinic, with 31,955 employees, is a multi-specialty medical clinic and hospital providing medical care, education and research. Among its many safety and health programs and strategies, it has focused on eliminating situations that could lead to musculoskeletal injuries in direct patient care, material handling and computer/office-related tasks, the major cause of workers’ compensation injuries at Mayo Clinic. It also provides an extensive return-to-work program that has been expanded to include any employee affected by personal injury or illness, regardless of the nature or cause. In 2009, the Mayo Clinic Return to Work section realized a $1.4 million annual cost savings while serving 1,270 employees with work and nonwork-related injuries resulting in vocational restrictions.

“Mayo Clinic is thrilled to receive this award recognizing its workers’ compensation program,” said Laura A. Mundt, claims section head, recovery and claims services, Mayo Clinic. “We are proud of the integrated approach from many areas within Mayo Clinic that make this program a success. Thank you for this special honor.”

Starkey Laboratories Inc.

Starkey Laboratories Inc., with 22 facilities in more than 18 countries and 1,760 U.S. employees, is a world leader in the design, development and distribution of comprehensive hearing solutions. To combat the rising number of repetitive stress injuries that were common in the light electronic assembly and administrative work of the company, Starkey incorporated conventional approaches for eliminating musculoskeletal injuries, such as encouraging stretching exercises, increasing job rotation and providing adjustable, ergonomic workstations. It also went beyond such approaches by securing the services of a certified Rolfer, a form of massage therapy. Since introducing the measures 15 years ago, carpal tunnel syndrome cases have been virtually eliminated from the workplace — there have been six carpal tunnel claims since 1999, with an estimated savings of up to $20 million.

“On behalf of all the employees of Starkey, we are honored to accept this award,” said Larry Miller, vice president, human resources, Starkey Laboratories Inc. “We take great pride in protecting our employees from workplace injuries. It is exciting to get recognition for our long-standing efforts to make sure we can do everything possible to provide a safe workplace. Aside from experiencing greatly reduced workers’ compensation costs, our efforts help make Starkey a great place to work for 1,500 Minnesotans.”

The policy and education conference in June will feature multiple breakout sessions led by experts and stakeholders in workers’ compensation and occupational safety and health. The conference will examine current issues that affect employers, employees, insurers, medical providers, legislators, attorneys and others who comprise Minnesota’s workers’ compensation system. No taxpayer dollars will be used to fund this conference.

The Workers’ Compensation Advisory Council advises the Department of Labor and Industry commissioner about matters of workers’ compensation and submits its recommendations for proposed changes to the workers’ compensation statutes to the proper legislative committees.

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