alpha 1-Proteinase inhibitor, alpha 1-antichymotrypsin, and alpha 2-macroglobulin are the antiapoptotic factors of vascular smooth muscle cells

Serum depletion induces cell death. Whereas serum contains growth factors and adhesion molecules that are important for survival, serum is also likely to have antiapoptotic factor(s). We show here that the plasma proteinase inhibitors alpha1-proteinase inhibitor, alpha1-antichymotrypsin, and alpha2-macroglobulin function as critical antiapoptotic factors for human vascular smooth muscle cells. Cell survival was assured when serum-free medium was supplemented with any one or all of the above serine proteinase inhibitors. In contrast, the cells were sensitive to apoptosis when cultured in medium containing serum from which the proteinase inhibitors were removed. The antiapoptotic effect conferred by the proteinase inhibitors was proportional to proteinase inhibitory activity. Without proteinase inhibitors, the extracellular matrix was degraded, and cells could not attach to the matrix. Cell survival was dependent on the intact extracellular matrix. In the presence of the caspase inhibitor z-VAD, the cells detached but did not die. The activity of caspases was elevated without proteinase inhibitors; in contrast, caspases were not activated when medium was supplemented with one of the proteinase inhibitors. In conclusion, the plasma proteinase inhibitors prevent degradation of extracellular matrix by proteinases derived from cells. Presumably an intact cell-matrix interaction inhibits caspase activation and supports cell survival.     

Direct in situ viability assessment of bacteria in probiotic dairy products using viability staining in conjunction with confocal scanning laser microscopy

The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was approximately 10(8) bacteria/ml (equivalent to approximately 10(7) CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.     

Potent and selective [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines as retinoic acid metabolic blocking agents (RAMBAs)

A series of [2-imidazol-1-yl-2-(6-alkoxy-naphthalen-2-yl)-1-methyl-ethyl]-dimethyl-amines were designed and synthesized as CYP26 inhibitors, serving as retinoic acid metabolic blocking agents (RAMBA's).     

The influence of climate on the timing and rate of spring bird migration

Ecological processes are changing in response to climatic warming. Birds, in particular, have been documented to arrive and breed earlier in spring and this has been attributed to elevated spring temperatures. It is not clear, however, how long-distance migratory birds that overwinter thousands of kilometers to the south in the tropics cue into changes in temperature or plant phenology on northern breeding areas. We explored the relationships between the timing and rate of spring migration of long-distance migratory birds, and variables such as temperature, the North Atlantic Oscillation (NAO) and plant phenology, using mist net capture data from three ringing stations in North America over a 40-year period. Mean April/May temperatures in eastern North America varied over a 5°C range, but with no significant trend during this period. Similarly, we found few significant trends toward earlier median capture dates of birds. Median capture dates were not related to the NAO, but were inversely correlated to spring temperatures for almost all species. For every 1°C increase in spring temperature, median capture dates of migratory birds averaged, across species, one day earlier. Lilac (Syringa vulgaris) budburst, however, averaged 3 days earlier for every 1°C increase in spring temperature, suggesting that the impact of temperature on plant phenology is three times greater than on bird phenology. To address whether migratory birds adjust their rate of northward migration to changes in temperature, we compared median capture dates for 15 species between a ringing station on the Gulf Coast of Louisiana in the southern USA with two stations approximately 2,500 km to the north. The interval between median capture dates in Louisiana and at the other two ringing stations was inversely correlated with temperature, with an average interval of 22 days, that decreased by 0.8 days per 1°C increase in temperature. Our results suggest that, although the onset of migration may be determined endogenously, the timing of migration is flexible and can be adjusted in response to variation in weather and/or phenology along migration routes.     

Effects of cholera toxin and actinomycin on synthesis of [35S]methionine-labeled proteins during progesterone-induced maturation of Xenopus laevis oocytes

The incorporation of [³⁵S]methionine into polypeptides during progesterone-induced meiotic maturation of Xenopus laevis oocytes was studied by two-dimensional polyacrylamide gel electrophoresis. Five modifications were consistently observed: two polypeptides of an approximate molecular weight of 150K daltons and pI 5 were new proteins, two represent increased incorporation and one was decreased incorporation. Cholera toxin inhibited the appearance of the modifications induced by progesterone. Actinomycin and enucleation did not significantly alter the modifications. These data indicate that a good correlation exists between the modifications in protein synthesis induced by progesterone and the resumption of meiotic cell division.     

Flanking markers for the gene causing von Recklinghausen neurofibromatosis (NF1)

The defective gene causing von Recklinghausen neurofibromatosis (NF1), one of the most common inherited disorders affecting the human nervous system, was recently mapped to chromosome 17. We have used additional DNA markers to further narrow and bracket the NF1 defect. A multipoint linkage analysis suggests that the NF1 gene is flanked by D17Z1 on the centromeric side and by EW 207 on the telomeric side of the long arm of chromosome 17. The identification of closely linked flanking markers should allow us to develop a reliable prenatal and presymptomatic diagnostic test for this serious neurological disorder and provides the basis for applying chromosome-specific cloning techniques for the isolation and characterization of the mutant gene.     

Multipoint linkage analysis in neurofibromatosis type I: an international collaboration.

In addition to reporting, in accompanying papers, their individual analyses of mapping the neurofibromatosis type 1 (NF1) gene on chromosome 17, members of the International Consortium for NF1 Linkage contributed their data for our joint analysis to determine the exact sequence of flanking markers and to obtain precise estimates and confidence limits of the recombination fractions for the closest markers, in anticipation of clinical use. With specimens from 142 families and more than 700 affected persons, eight teams used 31 markers in the pericentric region of chromosome 17 to perform 13,838 genotypings. With the combined data, we used the computer program CRI-MAP to build the most likely sequence of loci by sequentially adding single loci to a fixed pair of loci and separately calculating the likelihood of all permutations of four consecutive loci. The best order is pter-pA10-41-EW301-centromere (p17H8)-pHHH202-NF1-EW206-EW207-EW203++ +-CRI-L581-CRI-L946-HOX2-NGFR-qter. The total genetic distance from pA10-41 to NGFR is 26 cM in males and 56 cM in females, and the overall difference in sex-specific maps is statistically significant (P = .006). The upper 99% confidence limits of the recombination fraction of the closest proximal marker, pHHH202, is 4%, and that for the closest distal marker, EW206, is 9%. These limits should decrease with the use of additional probes and the further evaluation of DNA from the six persons showing multiple recombinations within short genetic distances. Clinical application is technically feasible with currently available markers, although its appropriate use for prenatal and presymptomatic diagnosis requires further discussion and evaluation.