Cytokine-mediated activation of cultured CNS microvessels: a system for examining antigenic modulation of CNS endothelial cells, and evidence for long-term expression of the adhesion protein E-selectin

Much of what is known of endothelial responses to cytokines has been derived from in vitro studies using cultured human umbilical vein endothelial cells (EC). Less is known of CNS EC responses and whether intact endothelium responds similarly to cultured cells. We have used techniques by which rat CNS microvessels can be isolated, then cultured in vitro, to study the response of intact endothelium to activation with cytokines. These microvessels are composed of viable EC and perivascular cells, predominantly pericytes. Expression of EC activation antigens in multicellular systems such as cultured microvessels can be assessed quantitatively using immunofluorescence laser cytometry. Interferon gamma increased immunologically reactive major histocompatibility complex class II antigens (< 300 to 2,398 +/- 225 average fluorescence intensity), while tumor necrosis factor alpha induced an increase in vascular cell adhesion molecule-1 (2,167 +/- 171) and E-selectin (1,628 +/- 315). CNS EC appeared to respond similarly to cultured EC with the exception that E-selectin expression was not transiently expressed but was maintained by microvessel EC for 24 and 48 h. Cultured CNS microvessels provide a good system for studying EC activation.     

Dosing-induced stress causes hepatocyte apoptosis in rats primed by the rodent nongenotoxic hepatocarcinogen cyproterone acetate

It has been proposed that several nongenotoxic compounds act as hepatocarcinogens by suppressing the apoptosis that would normally act to remove damaged or potentially initiated cells from the liver. During our investigations of this hypothesis using a widely applied protocol, we have found that the stress induced by the process of gavage dosing can induce massive apoptosis in livers uniquely primed by withdrawal of the hepatomitogen cyproterone acetate from the hyperplastic rat liver. This effect of gavage dosing was not seen in livers of naive animals. Apoptosis was measured by both in situ end labeling (ISEL) of the DNA damage associated with programmed cell death and conventional hematoxylin and eosin (H&E) staining of apoptotic morphology. Apoptotic rates measured by H&E increased significantly from 0.005 +/- 0.010% on Day 11 to 0.657 +/- 0.315% of hepatocytes on Day 15, 4 days after cessation of 10 days dosing with CPA (120 mg/kg). The readministration of CPA suppressed > 89% of this Day 15 apoptosis. However, the readministration of vehicle alone (corn oil) caused a 390% increase in apoptosis to 2.56 +/- 1.31% of hepatocytes. Similar results were obtained using ISEL. Measurements of liver to body weight ratios and total DNA per liver reflected these changes in cell loss by apoptosis. In a second experiment, CPA was administered for 10 days as before then animals were subjected to readministration of CPA in corn oil, CPA in saline, corn oil, saline, or sham dosed. Again, apoptosis was dramatically suppressed by the readministration of CPA in either vehicle but was dramatically increased to around 2% of hepatocytes in all other groups, including the sham dosed group. Data on food consumption provided no evidence for a reduction in food intake as a causative agent but rather pointed to a less efficient usage of food in the stressed animals. The ability of stress to induce liver apoptosis should be borne in mind in the design and interpretation of future toxicological studies aimed at understanding the putative suppression of apoptosis by liver nongenotoxic carcinogens and other toxicants.     

Molecular analysis of the F plasmid traVR region: traV encodes a lipoprotein

The nucleotide sequences of the conjugative F plasmid transfer region genes, traV and traR, have been determined. The deduced amino acid sequence of TraV indicated that it may be a lipoprotein; this was confirmed by examining the effect of globomycin on traV-encoded polypeptides synthesized in minicells. An open reading frame that may represent a previously undetected transfer gene, now designated trbG, was identified immediately upstream of traV. The deduced product of traR was found to share amino acid similarity with proteins from the bacteriophages 186 and P2 and with the dosage-dependent dnaK suppressor DksA.     

Overt behavior problems and serotonergic function in middle childhood among male and female offspring of alcoholic fathers

A large body of literature indicates that the serotonergic system is involved in behavioral regulation, as evidenced by the inverse relationship between impulsive aggression and serotonergic function found in adult alcoholics and nonalcoholics. However, studies of this relationship among child and adolescent offspring of alcoholics (COAs) have not previously been done. This study examines the potentially parallel relationship between behavioral dysregulation and low serotonergic function in young COAs. The relationship is of potential interest as a phenotypic marker of biological vulnerability to aggressiveness, which itself has been hypothesized to be a risk factor for later antisocial alcoholism. The present work is part of an ongoing prospective study of the development of risk for alcohol abuse/dependence and other problematic outcomes in a sample of families subtyped by the fathers' alcoholism classification. We examined the relationship between overt behavior problems in middle childhood (mean age = 10.5 +/- 1.7 years) and whole blood serotonin (5-HT) in a subsample of the offspring (N = 32 boys and 12 girls). Using a Child Behavior Checklist (CBCL) index of behavioral undercontrol, we obtained results indicating that high total behavior problem (TBP) children had lower levels of whole blood 5-HT than did low-TBP children (p < 0.01). These results support the hypothesis that there is an inverse relationship between whole blood serotonin levels and behavior problems in young male and female COAs. A father's alcoholism status was not significantly related to his child's 5-HT level, i.e., the child's phenotypic expression of behavioral dysregulation was more reliably connected to serotonergic function than was paternal alcoholism.     

Re-expression of p16INK4a in mesothelioma cells results in cell cycle arrest, cell death, tumor suppression and tumor regression

Absence of expression of the p16IKN4a gene product is commonly observed in mesothelioma tumors and cell lines, while wild-type pRB expression is maintained. We have examined the biologic and potential therapeutic role of re-expressing p16INK4a gene product in mesothelioma cells and tumors. Following transduction with a p16INK4a expressing adenovirus (Adp16), over-expression of p16INK4a in mesothelioma cells resulted in cell cycle arrest, inhibition of pRB phosphorylation, diminished cell growth, and eventual death of the transduced cells. Expression of p16INK4a protein was accompanied by decreased expression of pRB as detected by immunoblot and immunohistochemistry. Experiments in mesothelioma xenografts demonstrated inhibition of tumor formation, tumor growth arrest and diminished tumor size and spread. p16INK4a gene product expression was also demonstrated in intraperitoneal xenografts of human mesothelioma cells. These results demonstrate that p16INK4a gene transfer may play a therapeutic role in the treatment of mesothelioma.     

Subclavian artery pseudoaneurysm in type IV Ehlers-Danlos syndrome

We report case of a subclavian artery pseudoaneurysm in a patient with type IV Ehlers-Danlos Syndrome. A 16-year-old boy underwent successful repair of a subclavian artery pseudoaneurysm that occurred after a cervical hyperextension injury. Subsequent workup included skin biopsy and fibroblast culture, which were consistent with a diagnosis of type IV Ehlers-Danlos Syndrome. This condition is a dominantly inherited connective tissue disorder, which in this patient was found to be caused by a spontaneous point mutation in the COL3A1 gene that encodes the chains of type III procollagen. The clinical, genetic, and molecular characteristics of type IV Ehlers-Danlos Syndrome are briefly reviewed.     

Induced and synchronized estrus in cattle: dose titration of estradiol benzoate in peripubertal heifers and postpartum cows after treatment with an intravaginal progesterone-releasing insert and prostaglandin F2alpha

Seven cysteine-rich repeats form the ligand-binding region of the low-density lipoprotein (LDL) receptor. Each of these repeats is assumed to bind a calcium ion, which is needed for association of the receptor with its ligands, LDL and beta-VLDL. The effects of metal ions on the folding of the reduced N-terminal cysteine-rich repeat have been examined by using reverse-phase high-performance liquid chromatography to follow the formation of fully oxidized isomers with different disulfide connectivities. In the absence of calcium many of the 15 possible isomers formed on oxidation, whereas in its presence the predominant product at equilibrium had the native disulfide bond connectivities. Other metals were far less effective at directing disulfide bond formation: Mn2+ partly mimicked the action of Ca2+, but Ba2+, Sr2+, and Mg2+ had little effect. This metal-ion specificity was also observed in two-dimensional 1H NMR spectral studies; only Ca2+ induced the native three-dimensional fold. The two paramagnetic ions, Gd3+ and Mn2+, and Cd2+ did not promote adoption of a well-defined structure, and the two paramagnetic ions did not displace calcium ions. The location of calcium ion binding sites in the repeat was also explored by NMR spectroscopy. The absence of chemical shift changes for the side chain proton resonances of Asp26, Asp36, and Glu37 from pH 3.9 to 6.8 in the presence of calcium ions and their proximal location in the NMR structures implicated these side chains as calcium ligands. Deuterium exchange NMR experiments also revealed a network of hydrogen bonds that stabilizes the putative calcium-binding loop.