Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (K_i) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.     

Separation and quantitation ofcis- andtrans-thiothixene in human plasma by high-performance liquid chromatography

A high-performance liquid chromatographic procedure is described for the separation ofcis andtrans isomers of thiothixene, a thioxanthene derivative used as an antipsychotic agent. A radial compression module (RCM-100) was used with both silica and cyanopropyl cartridges. A fixed-wavelength UV detector (254 nm) was used in these studies for quantitation. Mesoridazine is used as an internal standard because of its separation characteristics and reproducible quantitation. C₁₈ Sep-Pak cartridges are used for biological sample cleanup. Plasma samples from patients treated with thiothixene (Navane) were assayed forcis andtrans-thiothixene. Notrans-thiothixene was detectable andcis-thiothixene concentrations ranged from 0 to 22.5 ng/ml.     

The ‘peace of the market’ in the midst of violence: A symbolic analysis of markets and exchange in western Guatemala*

The peasant markets of the western highlands of Guatemala present a field of public solidarity and constitute sources of sociocultural information. The information is expressed in the specific physical arrangements and in the types of interactions carried out in each context. The open, quasi festive plaza presents one model of and for behavior that corresponds to the traditional Indian conceptual framework, the public reunion strengthens the group solidarity and serves as a barrier against external aggression. The compartmentalized enclosed market, instead, follows and serves as a model which corresponds to Ladino conceptual guidelines, one which emphasizes the power of the product over the ethnic category as an organizational parameter. The traditional open plaza constitutes a communitas‐type framework where multiple interactions are peacefully conducted and the ‘Other’ is identified through the practice of bargaining.     

Surgical Stress Delays Prostate Involution in Mice

Androgens control growth of prostate epithelial cells and androgen deprivation induces apoptosis, leading to prostate involution. We investigated the effects of surgical stress on prostate involution induced by androgen ablation and determined the underlying mechanisms. Androgen ablation in mice was induced by surgical castration and administration of the anti-androgenic drugs bicalutamide and MDV3100. Surgical stress was induced by sham castration under isoflurane anesthesia. Surgical stress delayed apoptosis and prostate involution induced by anti-androgenic drugs. These effects of stress were prevented by administering the selective beta2-adrenoreceptor antagonist ICI118,551 and were also blocked in BAD^3SA/WT mice expressing phosphorylation-deficient mutant BAD3SA. These results indicate that apoptosis and prostate involution in response to androgen ablation therapy could be delayed by surgical stress via the beta2-adrenoreceptor/BAD signaling pathway. Thus, surgery could interfere with androgen ablation therapy, whereas administration of beta2-adrenoreceptor antagonists may enhance its efficacy.     

DNA synthesis during larval development and compensatory growth in the mesonephros of Xenopus laevis.

Autoradiography following tritiated thymidine administration to Xenopus laevis tadpoles of stages 45–48 of larval development has revealed that, as the cells of the mesonephric kidney differentiate during organogenesis, there is a marked decrease in the percentage of cells synthesizing DNA (from 100% at stage 45 to less than 9% at stage 48). In the adult this figure is of the order of 0.1%. This reduced DNA synthetic activity was found to take place in the cells of both the proximal and distal tubules of the nephrons. Special mucous cells which serve as markers of distal tubules were not observed to synthesize DNA after the onset of their differentiation at stage 48 of larval development.Through the partial extirpation of tissue in one kidney of adult Xenopus laevis males, DNA synthesis was reactivated in differentiated cells. The increased DNA synthetic activity following partial unilateral nephrectomy was found to be maximal after 6 days of recovery when 19% of cells synthesize DNA. This increased DNA synthetic activity was found to occur only in the cells of the distal tubules, both mucous and nonmucous cells, while the cells of the proximal tubules did not respond to this reactivation.The apparent inability of proximal tubule cells to synthesize DNA following partial unilateral nephrectomy is discussed.     

The regulation of growth in the mesonephric kidney of adult Xenopus laevis by an endogenous inhibitor of proliferation.

The effect of dorsal lymph sac implanted tissue fragments, of a 100,000g kidney supernatant, and of various kidney-derived ultrafiltrate fractions on the percentage of DNA synthesizing cells in the mesonephric kidney of Xenopus laevis following partial unilateral nephrectomy was investigated autoradiographically. Using Amicon filters with cut-off values of MW 50,000 and 10,000, three ultrafiltrate fractions were obtained: a fraction containing molecules of MW 50,000 and less, a fraction containing molecules of MW 10,000 and less, and one containing molecules in the range of MW 10,000 to 50,000. The ultrafiltrates containing molecules of less than 10,000 MW were found to depress DNA synthetic activity on the sixth postoperative day by 30 to 40%, while the fraction containing molecules between MW 10,000 and 50,000 showed no significant effect. It has been concluded that an endogenous inhibitor of proliferation, with the attributes of a chalone, is present in the fraction of less than 10,000 MW. The loss of inhibitor action following Pronase treatment of the ultrafiltrate suggests that the inhibitor substance may be a protein or polypeptide, or that such constituent may be the carrier for the active agent. Since a depression in DNA synthetic activity of 60% was obtained in normal adult mesonephric kidneys following the injection of the ultrafiltrate, it is concluded that both compensatory growth and reparative growth in the kidney of Xenopus laevis are regulated by a G₁ kidney chalone of less than 10,000 MW.     

Assessment of the Resistance to Uranium (VI) Exposure by Arthrobacter sp. Isolated from Hanford Site Soil

Production of nuclear fuel has resulted in hazardous waste streams that have contaminated the soil and groundwater. Arthrobacter strains, G975, G968, and G954 were used in the prescreening tests to evaluate their tolerance to UO₂ ²⁺ and investigate bacteria-U(VI) interactions under oxidizing pH-neutral conditions. Experiments have shown G975 is the fastest growing and the most uranium tolerant strain that removed about 90% of uranium from growth media. Atomic Force Microscopy images exhibited an irregular surface structure, which perhaps provided a larger surface area for uranium precipitation. The data indicate that aerobic heterotrophic bacteria may offer a solution to sequestering uranium in oxic conditions, which prevail in the vadose zone.     

Polyanionic Candidate Microbicides Accelerate the Formation of Semen-Derived Amyloid Fibrils to Enhance HIV-1 Infection

Polyanionic candidate microbicides, including cellulose sulfate, carrageenan, PRO 2000, were proven ineffective in preventing HIV-1 transmission and even cellulose sulfate showed increased risk of HIV acquisition in the Phase III efficacy trials. Semen plays critical roles in HIV-1 sexual transmission. Specifically, amyloid fibrils formed by fragments of prostatic acidic phosphatase (PAP) in semen termed semen-derived enhancer of virus infection (SEVI) could drastically enhance HIV-1 infection. Here we investigated the interaction between polyanions and PAP248-286, a prototype peptide of SEVI, to understand the possible cause of polyanionic candidate microbicides to fail in clinical trials. We found anionic polymers could efficiently promote SEVI fibril formation, most likely mediated by the natural electrostatic interaction between polyanions and PAP248-286, as revealed by acid native PAGE and Western blot. The overall anti-HIV-1 activity of polyanions in the presence or absence of PAP248-286 or semen was evaluated. In the viral infection assay, the supernatants of polyanions/PAP248-286 or polyanions/semen mixtures containing the free, unbound polyanionic molecules showed a general reduction in antiviral efficacy, while the pellets containing amyloid fibrils formed by the polyanion-bound PAP248-286 showed aggravated enhancement of viral infection. Collectively, from the point of drug-host protein interaction, our study revealed that polyanions facilitate SEVI fibril formation to promote HIV-1 infection, thus highlighting a molecular mechanism underlying the failure of polyanions in clinical trials and the importance of drug-semen interaction in evaluating the anti-HIV-1 efficacy of candidate microbicides.     

Lipidomic Analysis of α-Synuclein Neurotoxicity Identifies Stearoyl CoA Desaturase as a Target for Parkinson Treatment

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