Cytokeratin immunoreactivity in lobular intraepithelial neoplasia.

Eighteen commercially available antibodies reactive against different cytokeratin proteins were tested on classic examples of lobular intraepithelial neoplasia (LIN) and of ductal intraepithelial neoplasia (DIN) of the breast. About 90% of higher-grade DIN (AIDH and DCIS) show no or substantially diminished reaction with clone 34betaE12 (specified as reactive against keratins 1, 5, 10, and 14 as determined by the manufacturer), while the cells of LIN were found to express the antigen reactive with this antibody. To determine which of these four keratins are present in the cells of LIN, antibodies reactive against these individual four keratins were tested. None of the four antibodies to keratins 1, 5, 10, or 14 reacted with the cells of LIN. To investigate this further, 13 additional monoclonal antibodies to various other keratin proteins were tested on the cells of LIN. Those that successfully reacted with the cells of LIN were further tested on the cells of DIN. All of the individual antibodies reactive with the cells of LIN were also reactive with the cells of DIN to a degree, with clone RCK108 (reactive against keratin 19) coming the closest to demonstrating the reactivity seen with 34betaE12. We conclude that the reactivity seen in the cells of LIN with 34betaE12 is due to either (a) a crossreaction with keratin 19 that is slightly less prominent than the reaction of the individual clone RCK108, (b) a crossreaction with a keratin protein that was not tested (3, 11, 12), (c) a crossreaction with a protein closely resembling keratin in formalin-fixed, paraffin-embedded tissue, or (d) the detection of a mutated or truncated form of keratin 1, 5, 10, or 14 that cannot be detected by the individual monoclonal antibody.     

RUNAWAY COEVOLUTION: ADAPTATION TO HERITABLE AND NONHERITABLE ENVIRONMENTS

Populations evolve in response to the external environment, whether abiotic (e.g., climate) or biotic (e.g., other conspecifics). We investigated how adaptation to biotic, heritable environments differs from adaptation to abiotic, nonheritable environments. We found that, for the same selection coefficients, the coadaptive process between genes and heritable environments is much faster than genetic adaptation to an abiotic nonheritable environment. The increased rate of adaptation results from the positive association generated by reciprocal selection between the heritable environment and the genes responding to it. These associations result in a runaway process of adaptive coevolution, even when the genes creating the heritable environment and genes responding to the heritable environment are unlinked. Although tightening the degree of linkage accelerates the coadaptive process, the acceleration caused by a comparable amount of inbreeding is greater, because inbreeding has a cumulative effect on reducing functional recombination over generations. Our results suggest that that adaptation to local abiotic environmental variation may result in the rapid diversification of populations and subsequent reproductive isolation not directly but rather via its effects on heritable environments and the genes responding to them.     

Pore-forming Activity of the Escherichia coli Type III Secretion System Protein EspD

Enterohemorrhagic Escherichia coli is a causative agent of gastrointestinal and diarrheal diseases. Pathogenesis associated with enterohemorrhagic E. coli involves direct delivery of virulence factors from the bacteria into epithelial cell cytosol via a syringe-like organelle known as the type III secretion system. The type III secretion system protein EspD is a critical factor required for formation of a translocation pore on the host cell membrane. Here, we show that recombinant EspD spontaneously integrates into large unilamellar vesicle (LUV) lipid bilayers; however, pore formation required incorporation of anionic phospholipids such as phosphatidylserine and an acidic pH. Leakage assays performed with fluorescent dextrans confirmed that EspD formed a structure with an inner diameter of ∼2.5 nm. Protease mapping indicated that the two transmembrane helical hairpin of EspD penetrated the lipid layer positioning the N- and C-terminal domains on the extralumenal surface of LUVs. Finally, a combination of glutaraldehyde cross-linking and rate zonal centrifugation suggested that EspD in LUV membranes forms an ∼280–320-kDa oligomeric structure consisting of ∼6–7 subunits.     

Intermittent cold-induced hippocampal oxidative stress is associated with changes in the plasma lipid composition and is modifiable by vitamins C and E in old rats

This study primarily investigated the effects of intermittent cold exposure (ICE) on oxidative stress (OS) in the hippocampus(HC) and plasma lipid profile of old male rats. Secondly, it evaluated structural changes in the hippocampus region of the rat’s brain. Thirdly, it attempted an evaluation of the effectiveness of the combined supplement of vitamins C and E in alleviating cold stress in terms of these biochemical parameters. Thirty male rats aged 24 months were divided into groups of five each: control (CON), cold-exposed at 10 °C (C10), cold-exposed at 5 °C (C5), supplemented control (CON+S), and supplemented cold-exposed at either 5 °C (C5+S) or 10 °C (C10+S). The rats were on a daily supplement of vitamin C and vitamin E. Cold exposure lasted 2 h/day for 4 weeks. Rats showed increased levels of hydrogen peroxide (H₂O₂), and thiobarbituric acid reactive substances (TBARS) in the HC at 10 °C with further increase at 5 °C. Cold also induced neuronal loss in the hippocampus with concomitant elevations in total cholesterol (TCH), triglycerides (TG) and low-density lipoproteins (LDL-C) levels, and a depletion in high-density lipoprotein (HDL-C). A notable feature was the hyperglycaemic effects of ICE and depleted levels of vitamins C and E in the hippocampus and plasma while supplementation increased their levels. More importantly, a positive correlation was observed between plasmatic LDL-C, TCH and TG and hippocampal TBARS and H₂O₂ levels. Further, intensity of cold emerged as a significant factor impacting the responses to vitamin C and E supplementation. These results suggest that cold-induced changes in the plasma lipid profile correlate with OS in the hippocampus, and that vitamin C and E together are effective in protecting from metabolic and possible cognitive consequences in the old under cold exposures.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

Molecular Identification of Aeromonas and Coliform Bacteria Isolated on m Endo Media from Lake Erie Waters

Summary Bacteria from recreational waters collected from two Lake Erie beaches in Dunkirk, New York were plated onto m Endo LES media. The 16S rRNA gene was then amplified from coliform and non-coliform bacteria using the polymerase chain reaction. The PCR products were characterized by restriction fragment length polymorphism (RFLP) analysis. A total of 8 RFLP groups were identified from the analysis of 920 samples and selected PCR products from each group were sequenced. The DNA sequence analysis indicated that more than half of the bacteria identified as coliforms on the m Endo plates belonged to the genus Aeromonas from the family Aeromonadaceae. Most of the remaining coliforms were from the Enterobacteriaceae. The data indicate that m Endo agar plates allow the growth of non-coliform bacteria, especially Aeromonas species.     

Synthesis of apo-13- and apo-15-lycopenoids,cleavage products of lycopene that are retinoic acid antagonists

Consumption of the tomato carotenoid, lycopene, has been associated with favorable health benefits. Some of lycopene's biological activity may be due to metabolites resulting from cleavage of the lycopene molecule. Because of their structural similarity to the retinoic acid receptor (RAR) antagonist, β-apo-13-carotenone, the “first half” putative oxidative cleavage products of the symmetrical lycopene have been synthesized. All transformations proceed in moderate to good yield and some with high stereochemical integrity allowing ready access to these otherwise difficult to obtain terpenoids. In particular, the methods described allow ready access to the trans isomers of citral (geranial) and pseudoionone, important flavor and fragrance compounds that are not readily available isomerically pure and are building blocks for many of the longer apolycopenoids. In addition, all of the apo-11, apo-13, and apo-15 lycopenals/lycopenones/lycopenoic acids have been prepared. These compounds have been evaluated for their effect on RAR-induced genes in cultured hepatoma cells and, much like β-apo-13-carotenone, the comparable apo-13-lycopenone and the apo-15-lycopenal behave as RAR antagonists. Furthermore, molecular modeling studies demonstrate that the apo-13-lycopenone efficiently docked into the ligand binding site of RARα. Finally, isothermal titration calorimetry studies reveal that apo-13-lycopenone acts as an antagonist of RAR by inhibiting coactivator recruitment to the receptor.     

Pycnogenol® and vitamin E inhibit ethanol‐induced apoptosis in rat cerebellar granule cells

Pycnogenol® (PYC), a patented combination of bioflavonoids extracted from the bark of French maritime pine (Pinus maritima), scavenges free radicals and promotes cellular health. The protective capacity of PYC against ethanol toxicity of neurons has not previously been explored. The present study demonstrates that in postnatal day 9 (P9) rat cerebellar granule cells the antioxidants vitamin E (VE) and PYC (1) dose dependently block cell death following 400, 800, and 1600 mg/dL ethanol exposure (2) inhibit the ethanol‐induced activation of caspase‐3 in the same model system; and (3) reduce neuronal membrane disruption as assayed by phosphatidylserine translocation to the cell surface. These results suggest that both PYC and VE have the potential to act as therapeutic agents, antagonizing the induction of neuronal cell death by ethanol exposure. © 2004 Wiley Periodicals, Inc. J Neurobiol 59: 261–271, 2004