Stimulation of cis-diamminedichloroplatinum(II) accumulation by modulation of passive permeability with genistein: an altered response in accumulation-defective resistant cells

The effect of the tyrosine kinase inhibitor genistein on the accumulation of cisplatin (DDP) was investigated in DDP-sensitive and -resistant human 2008 ovarian carcinoma cell lines. DDP accumulation after a 1-h exposure was maximally increased by concurrent 40 micrometer genistein. The maximal stimulation of accumulation was observed after 2 h of total genistein exposure and was 83 +/- 13% (n = 5) higher than controls. With resistant C13(*) cells, however, the stimulation of accumulation was delayed until 4 h and was increased only 46 +/- 18% compared to controls. Revertant RH4 cells that retained the accumulation defect behaved like the C13(*) cells. Genistein stimulated [3H]mannitol accumulation (a marker of passive permeability) by 43 +/- 9% (n = 3) in 2008 cells, and the effect was maximal after 2 h of total genistein exposure. Changes in [3H]mannitol accumulation in 2008 parent cells were highly correlated with DDP accumulation (r = 0.9010). These experiments also revealed that [3H]mannitol accumulation after 2 h in C13(*) cells was reduced 38% compared to 2008 cells, a decrease that reflected the DDP accumulation defect. Fluid-phase pinocytosis determined with lucifer yellow CH as a marker showed no difference between 2008 and C13(*) cells and no effect of genistein. Genistein was demonstrated to clearly inhibit protein-tyrosine phosphorylation initiated by the epidermal growth factor receptor kinase. Differences were noted in the phosphotyrosine pattern between the 2008 and C13(*) cells. Under the conditions that had the maximal effect on DDP accumulation in 2008 cells, genistein decreased the IC50 of DDP 8.2-fold in 2008 cells and 4.7-fold in C13(*) cells. We conclude that: (a) genistein stimulates DDP accumulation by modulating the passive permeability of the plasma membrane; (b) C13(*) cells are less permeable to passively diffusing small molecules, which offers a mechanism for the DDP accumulation defect without invoking carrier proteins; (c) the effect of tyrosine kinase inhibition on passive permeability is altered in C13(*) cells; and (d) pinocytosis contributes insignificantly to DDP accumulation. Genistein, a dietary isoflavone, thus seems to be a promising clinical candidate for combination with DDP.     

Ribonuclease A variants with potent cytotoxic activity

Select members of the bovine pancreatic ribonuclease A (RNase A) superfamily are potent cytotoxins. These cytotoxic ribonucleases enter the cytosol, where they degrade cellular RNA and cause cell death. Ribonuclease inhibitor (RI), a cytosolic protein, binds to members of the RNase A superfamily with inhibition constants that span 10 orders of magnitude. Here, we show that the affinity of a ribonuclease for RI plays an integral role in defining the potency of a cytotoxic ribonuclease. RNase A is not cytotoxic and binds RI with high affinity. Onconase, a cytotoxic RNase A homolog, binds RI with low affinity. To disrupt the RI-RNase A interaction, three RNase A residues (Asp-38, Gly-88, and Ala-109) that form multiple contacts with RI were replaced with arginine. Replacing Asp-38 and Ala-109 with an arginine residue has no effect on the RI-RNase interaction. In addition, these variants are not cytotoxic. In contrast, replacing Gly-88 with an arginine residue yields a ribonuclease (G88R RNase A) that retains catalytic activity in the presence of RI and is cytotoxic to a transformed cell line. Replacing Gly-88 with aspartate also yields a ribonuclease (G88D RNase A) with a decreased affinity for RI and cytotoxic activity. The cytotoxic potency of onconase, G88R RNase A, and G88D RNase A correlate with RI evasion. We conclude that ribonucleases that retain catalytic activity in the presence of RI are cytotoxins. This finding portends the development of a class of chemotherapeutic agents based on pancreatic ribonucleases.     

Absence or reduction of Fhit expression in most clear cell renal carcinomas

The FHIT gene at human chromosome region 3p14.2 straddles the common fragile site, FRA3B, and numerous homozygous deletions in cancer cell lines and primary tumors. Also, the 3p14.2 chromosome breakpoint of the familial clear cell kidney carcinoma-associated translocation, t(3;8)(p14.2;q24), disrupts one FHIT allele between exons 3 and 4, fulfilling one criterion for a familial tumor suppressor gene: that one allele is constitutionally inactivated. Because the FHIT gene sustains biallelic intragenic deletions rather than mutations, there has not been evidence that the FHIT gene frequently plays a role in kidney cancer, although replacement of Fhit expression in a Fhit-negative renal carcinoma cell line suppressed tumor growth in nude mice. We have now assessed 41 clear cell renal carcinomas for expression of Fhit by immunohistochemistry. Normal renal tubule epithelial cells express Fhit uniformly and strongly, whereas 51% of the tumors are completely negative, 34% of tumors show a mixture of positive and negative cells, and 14% are uniformly positive, although usually less strongly positive than the normal epithelial cells. Most interestingly, there was a correlation between complete absence of Fhit and the G1 morphological grade and early clinical stage. Morphological grades G2 and G3 exhibited a mixture of positive and negative cells with a tendency for a higher fraction of negative cells in G3. Fhit inactivation is likely to be an early event in G1 tumors and may be associated with progression in G2 and G3 tumors.     

PCR analysis of microsatellite DNA markers: possibility of erroneous determination of genotypes

A comparative study of two techniques for the PCR genotyping of highly polymorphic tandem repeats was carried out by the example of a triplet repeat in the myotonin protein kinase gene. Sequencing denaturing gels were shown to yield more precise results in the analysis of amplification products.     

Transcutaneous lower blepharoplasty

Transcutaneous lower blepharoplasty as a standard procedure for cosmetic lower blepharoplasty is being replaced by transconjunctival blepharoplasty and laser resurfacing techniques. Nevertheless, there remain specific indications for the transcutaneous approach and its modifications. The accomplished facial plastic surgeon will be able to choose the appropriate technique based on the patient's concerns, the eyelid pathology, and his or her clinical experience.     

Survival of cylindrical implants in composite grafted maxillary sinuses

PURPOSE: This retrospective study investigated the survival of dental implants placed in the maxilla after composite grafting of the sinus and an average of 55 months of loading. PATIENTS AND METHODS: Maxillary sinuses of 88 patients were grafted with autogenous cancellous bone combined with dense hydroxyapatite particles. After an average healing period of 3.4 months, hydroxyapatite-coated titanium endosseous implants were placed. A total of 388 implants were placed in grafted sinus floors, and 82 were placed in onlay grafted nonsinus position in the canine region. The implants were loaded with overdentures and fixed bridges 4 months (mean) after implantation, with a follow-up for a mean of 55 months. RESULTS: The cumulative implant survival was calculated according to the Kaplan-Meier method. Implant survival from the time of loading was 89% in full reconstructed cases and 90% in partially edentulous cases. The overall cumulative implant survival rate, including the loss in the surgical stage, was 82%. CONCLUSION: Implant loss in composite grafted maxillae after 70 months of follow-up was similar to loss in nongrafted maxillae.     

Methods for successful follow-up of elusive urban populations: an ethnographic approach with homeless men

Public health is paying increasing attention to elusive urban populations such as the homeless, street drug users, and illegal immigrants. Yet, valid data on the health of these populations remain scarce; longitudinal research, in particular, has been hampered by poor follow-up rates. This paper reports on the follow-up methods used in two randomized clinical trials among one such population, namely, homeless men with mental illness. Each of the two trials achieved virtually complete follow-up over 18 months. The authors describe the ethnographic approach to follow-up used in these trials and elaborate its application to four components of the follow-up: training interviewers, tracking participants, administering the research office, and conducting assessments. The ethnographic follow-up method is adaptable to other studies and other settings, and may provide a replicable model for achieving high follow-up rates in urban epidemiologic studies.     

Laser temperature jump study of the helix<==>coil kinetics of an alanine peptide interpreted with a 'kinetic zipper' model

The kinetics of the helix<==>coil transition of an alanine-based peptide following a laser-induced temperature jump were monitored by the fluorescence of an N-terminal probe, 4-(methylamino)benzoic acid (MABA). This probe forms a peptide hydrogen bond to the helix backbone, which changes its fluorescence quantum yield. The MABA fluorescence intensity decreases in a single exponential relaxation, with relaxation times that are weakly temperature dependent, exhibiting a maximum value of approximately 20 ns near the midpoint of the melting transition. We have developed a new model, the kinetic version of the equilibrium 'zipper' model for helix<==>coil transitions to explain these results. In this 'kinetic zipper' model, an enormous reduction in the number of possible species results from the assumption that each molecule contains either no helical residues or a single contiguous region of helix (the single-sequence approximation). The decay of the fraction of N-terminal residues that are helical, calculated from numerical solutions of the kinetic equations which describe the model, can be approximately described by two exponential relaxations having comparable amplitudes. The shorter relaxation time results from rapid unzipping (and zipping) of the helix ends in response to the temperature jump, while the longer relaxation time results from equilibration of helix-containing and non-helix-containing structures by passage over the nucleation free energy barrier. The decay of the average helix content is dominated by the slower process. The model therefore explains the experimental observation that relaxation for the N-terminal fluorescent probe is approximately 8-fold faster than that for the infrared probe of Williams et al. [(1996) Biochemistry 35, 691-697], which measures the average helix content, but does not account for the absence of observable amplitude for the slow relaxation in the fluorescence experiments (<10% slow phase). If we assume that the activation barrier for the coil-->helix rate is purely entropic, the model can also explain the maximum in the temperature dependence of the relaxation time for the fluorescent probe. Parameters that best reproduce the melting curves and the ratio of relaxation times predict a value of the cooperativity parameter sigma which is approximately 3-fold larger than previously reported values obtained from fitting equilibrium data only. The helix growth rate of approximately 10(8) s-1 that reproduces the experimental relaxation times is approximately 100-fold slower than those observed in molecular dynamics simulations. These parameters can be used to simulate the kinetically cooperative formation of a helix from the all-coil state.