重组人骨形态形成蛋白2在家蚕幼虫中表达及产物纯化

将编码人ＢＭＰ２ｃＤＮＡ基因插入昆虫杆状病毒转移载体ｐＢａｃＰＡＫ１,与修饰的家蚕核形多角体病毒Ｂｍ－ＢａｃＰＡＫＤＮＡ共转染家蚕细胞,通过同源重组得到含有在多角体蛋白基因启动子控制下的ＢＭＰ２ｃＤＮＡ基因的重组病毒Ｂｍ－ＢａｃＰＡＫ－ＢＭＰ２。用重组病毒感染家蚕幼虫,第五天ＢＭＰ２表达率最高,每毫升蚕血淋巴中约１０μｇ表达产物;表达产物在在体内被加工成Ｃ－端１６ｋＤ片段,以二硫键连结成分子量为３０ｋＤ的同源二聚体;经纯化获得９０％以上纯度的成熟ＢＭＰ２,与骨基质胶原结合后植入大鼠皮下,７天后在局部诱导生成软骨组织。     

Entamoeba histolytica extrachromosomal circular ribosomal DNA: analysis of clonal variation in a hypervariable region.

The ribosomal RNA genes of the protozoan parasite Entamoeba histolytica are highly repeated and display restriction fragment length polymorphism. Using a set of four DNA probes spanning the coding region and part of the flanking region of the E. histolytica ribosomal RNA genes, an analysis of the DNA bands generated by EcoRI digestion of Entamoeba DNA is presented. This analysis included five strains of E. histolytica, four strains of E. moshkovskii, and one strain each of E. invadens and E. terrapinae. No common bands were observed between E. histolytica and the other Entamoeba. Within E. histolytica, two bands were conserved in all strains while the others were polymorphic. Detailed analysis of DNA from independently isolated clones of the strain HM-1:IMSS of E. histolytica showed two bands to be highly polymorphic. Of these, the 4.4-kb band of clone 6 was further analyzed. Polymorphism in this band could even be demonstrated in cells of the same clone. Restriction enzyme analysis of this DNA band from two clones of HM-1:IMSS showed that the polymorphism may be due to variable numbers of DraI repeat units present in this DNA stretch.     

Entamoeba histolytica: is conversion of "nonpathogenic" amebae to the "pathogenic" form a real phenomenon?

Entamoeba histolytica isolates have been shown to fall into two groups based on isoenzyme analysis. These groupings ("pathogenic" and "nonpathogenic") correlate well with the clinical course of the infection. A controversy exists over whether isoenzyme patterns are stable or whether under certain circumstances an isolate can convert from one form to the other. Resolution of this uncertainty is of importance since the nonpathogenic pattern has never been observed in amebae isolated from cases of active disease. This implies that, if the patterns are stable, carriers of amebae with this nonpathogenic pattern may never develop invasive disease. Although we set out to study isoenzyme conversion, we have been unable to replicate the two published accounts of this phenomenon. We have examined all of the variables proposed to be involved in the triggering of conversion, both individually and in combination. In none of the experiments was an alteration in the isoenzyme pattern observed. We now believe that isoenzyme patterns are stable and that all available evidence, other than the reported conversions, points to pathogenic and nonpathogenic E. histolytica being distinct species.     

Effect of chemical modifications on the susceptibility of collagen to proteolysis. II. Dehydrothermal crosslinking.

Collagen was dehydrothermally treated (heat cured) by heating dry under vacuum at 60, 80, 100 and 120 degrees C. The change in stability was determined by subjecting to measurement of gross crosslinking, content of lysino-alanine and naturally occurring collagen crosslinks, shrinkage temperature (TM), susceptibility to digestion by lysosomal thiol proteases, and susceptibility to pepsin and trypsin. Morphological changes were examined by electron microscopy. The in vivo biodegradation of dehydrothermally treated collagen sponges was investigated using a rat lumbar muscle implantation model for up to 28 days. For all heat-cured collagens, the data strongly indicated that both crosslinking and denaturation/degradation was present in increasing quantities with increasing temperature of treatment, its level was too low (maximum 179 pmol mg-1) to account for the decreased solubility and increased molecular weight gross changes observed. Increasing resistance of treated collagen to both lysosomal cathepsins and pepsin correlated well with increased crosslinking and increasing temperature of the heat-curing process. However, increased denaturation/degradation of the collagen at higher temperatures was revealed by electrophoretic analysis, trypsin hydrolysis data and by electron microscopy. Differential scanning calorimetry (d.s.c.) correlated well with these results showing an increased level of denaturation in heated samples. The in vivo study showed little difference between control and heat-cured samples except for the material treated at 120 degrees C which was biodegraded in vivo at a significantly faster rate. The data shows, therefore, that crosslinking induced by the dehydrothermal treatment of collagen decreases its rate of proteolysis at acid pH in vitro. However, the simultaneous denaturation/degradation of the protein during the heat-cure process appears to be a more important factor in determining the fate of the material implanted into rat muscle.     

A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites.

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.     

Staphylococcus aureus infection of human endothelial cells potentiates Fc receptor expression

Vasculitis, a recognized complication of staphylococcal-endovascular infections, may result in part, from the expression of FcR by Staphylococcus aureus-infected endothelial cells. FcR were measured using [51]Cr labeled SRBC preincubated with rabbit anti-SRBC IgG. FcR were not detected on uninfected endothelial cells, but were demonstrated on S. aureus infected cells using IgG, but not IgM labeled SRBC. FcR expression was dependent on the initial bacterial density (greater than or equal to 8 x 10(7) cfu/ml) and on phagocytosis of the staphylococci, but not on new protein synthesis. IgG labeled SRBC binding was blocked by aggregated IgG but not IgM. SRBC coated with the F(ab')2 portion of IgG did not bind, thus confirming that FcR were specifically involved in this interaction. FcR are expressed after S. aureus invasion of human endothelial cells and may contribute to the vasculitis which often accompanies S. aureus-endovascular infections.     

DNA-DNA hybridization assay for detection of Salmonella spp. in foods.

We have developed a DNA-DNA hybridization test for the presence of Salmonella spp. in foods. This test requires an initial pre-enrichment of food samples in nutrient broth but does not require selective enrichment. Samples of food cultures are collected on membrane filters and assayed by molecular hybridization to labeled probes. The probes consist of DNA sequences which are unique to the genus Salmonella and are widely distributed in the genus. A diverse panel of foods was assayed successfully by this methodology.     

Impedance analysis of a tight epithelium using a distributed resistance model.

This paper develops techniques for equivalent circuit analysis of tight epithelia by alternating-current impedance measurements, and tests these techniques on rabbit urinary bladder. Our approach consists of measuring transepithelial impedance, also measuring the DC voltage-divider ratio with a microelectrode, and extracting values of circuit parameters by computer fit of the data to an equivalent circuit model. We show that the commonly used equivalent circuit models of epithelia give significant misfits to the impedance data, because these models (so-called "lumped models") improperly represent the distributed resistors associated with long and narrow spaces such as lateral intercellular spaces (LIS). We develop a new "distributed model" of an epithelium to take account of these structures and thereby obtain much better fits to the data. The extracted parameters include the resistance and capacitance of the apical and basolateral cell membranes, the series resistance, and the ratio of the cross-sectional area to the length of the LIS. The capacitance values yield estimates of real area of the apical and basolateral membranes. Thus, impedance analysis can yield morphological information (configuration of the LIS, and real membrane areas) about a living tissue, independently of electron microscopy. The effects of transport-modifying agents such as amiloride and nystatin can be related to their effects on particular circuit elements by extracting parameter values from impedance runs before and during application of the agent. Calculated parameter values have been validated by independent electrophysiological and morphological measurements.     

BIOLOGY AND BEHAVIOUR OF FRIGATEBIRDS FREGATA SPP. ON ALDABRA ATOLL

Both species nested in mixed colonies in mangrove trees. The tops of trees were usually occupied exclusively by minor and the lower parts by ariel, but most nests of both species were in the intermediate parts of the canopy. The main laying season for both species was July to January. A census showed about 27 000 individuals present at the height of the season (1500 breeding pairs of minor, 5350 of ariel). Seasonal variation in numbers could be accounted for almost entirely by the changes in breeding activity of a resident population. Young of both species were fed at or near the nest-site for at least four months after fledging. A recovery near Bombay of a wing-tagged immature ariel shows that this species, at least, undergoes a post-fledging dispersal; it is suggested that young minor either do not disperse, or do so later than ariel. Food samples collected from chicks showed no overall difference between the species, but a seasonal analysis showed that ariel took more squid than minor in the wet season, and in the dry season the two species took different proportions of the two commonest species of flying-fish. Chicks of ariel received smaller meals than minor chicks in the wet season, but similar-sized meals in the dry season; ariel chicks grew more slowly than minor chicks. It is suggested that the timing of the breeding season is related to the need for adults to build up fat reserves to carry them through the courtship, nest-building and laying periods, when they are tied to the colony and so have little opportunity to feed. The evidence for non-annual breeding in frigatebirds is discussed. It is concluded that while successful breeders must breed at intervals of more than 12 months, they could theoretically nest in two successive seasons and that, since breeding success is low, most individuals probably do so. Existing knowledge of the biology of four of the five recognized species of frigatebirds is summarized, and shows that the family is at least as uniform as the tropicbirds and much more so than other Pelicaniformes.