Ultrastructural localization of keratinocyte surface associated heparan sulphate proteoglycans in human epidermis

Fixation and staining procedures were developed for the electron microscopic demonstration of glycosaminoglycans (GAGs) in human epidermis. En bloc staining with cuprolinic blue (CB), ruthenium red (RR) and tannic acid (TA) in the primary fixative were applied for the localization of the GAGs. Removal of the epidermal basal lamina and underlying dermis was a prerequisite for stain penetration. In CB-fixed specimens 50 nm long, rod-like granules were found attached to keratinocyte cell surfaces, while the RR- and TA-fixed specimens contained round granules (luminal diameter 10 and 30 nm, respectively). The stainability of the CB-positive granules in the presence of 0.3 mol/l MgCl2 indicated that they contained sulphated GAGs. Prefixation digestions of epidermal sheets with chondroitinase ABC. Streptomyces hyaluronidase, and heparitinase showed that the RR-positive granules also contained sulphated GAGs, mostly heparan sulphate. The granules visualized with TA on keratinocytes were susceptible to heparitinase treatment, but the abundance of TA-staining suggested that TA also stained structures other than heparan sulphate. The EM data was in accordance with the 35SO4 labelling experiments showing that heparan sulphate was the major sulphated GAG synthesized in epidermis, whereas chondroitin/dermatan sulphates comprised about one fifth of the total activity incorporated. The distributions of the CB-, RR- and TA-positive granules on cell surfaces were similar. The morphology of the proteoglycan granules was probably determined by the extent of the GAG-chain collapse following binding to each of the dyes.     

Local Climate Experts: The Influence of Local TV Weather Information on Climate Change Perceptions

Individuals who identify changes in their local climate are also more likely to report that they have personally experienced global climate change. One way that people may come to recognize that their local climate is changing is through information provided by local TV weather forecasters. Using random digit dialing, 2,000 adult local TV news viewers in Virginia were surveyed to determine whether routine exposure to local TV weather forecasts influences their perceptions of extreme weather in Virginia, and their perceptions about climate change more generally. Results indicate that paying attention to TV weather forecasts is associated with beliefs that extreme weather is becoming more frequent in Virginia, which in turn is associated with stronger beliefs and concerns about climate change. These associations were strongest for individuals who trust their local TV weathercaster as a source of information about climate change, and for those who identify as politically conservative or moderate. The findings add support to the literature suggesting that TV weathercasters can play an important role in educating the public about climate change.     

Antibodies against recombinant heat shock protein 65 of Tropheryma whipplei in patients with and without Whipple's disease.

Tropheryma whipplei is the causative agent of Whipple's disease (WD), a chronic, life-threatening infection. Laboratory diagnosis is mainly based on PCR and histopathological analysis in duodenal biopsies and other specimens requiring invasive procedures. We have examined the presence of antibodies to recombinant heat shock protein (Hsp65) of T. whipplei in patients with Whipple's disease as well as in control subjects by Western blot and enzyme-linked immunosorbent assay (ELISA). A recombinant plasmid carrying the entire T. whipplei hsp65 gene was constructed, and the expression yielded a 65-kDa histidine-tagged protein. Among four patients with Whipple's disease, two showed an IgG- and one an IgA-response, respectively, when analyzed by Western blotting, whereas from 10 patients without Whipple's disease, only two patients showed a positive IgG-response. The differences between the sera from patients and controls were thus not significant. Successful purification of the protein was achieved by Ni-NTA affinity chromatography. Quantitative analysis of serum antibodies by ELISA demonstrated that antibody levels in the sera of 14 patients were not significantly higher than in those of 89 control subjects. The established ELISA test is not useful to clinical diagnostics.     

Post-genomic Pseudomonas

A report on the 19th Whitehead Institute Symposium, Cambridge, USA, 14-16 October 2001.

     

Post-genomic Pseudomonas

A report on the Pseudomonas 2001 Meeting, Brussels, Belgium, 17-21 September 2001.     

Specific localization of scallop gill epithelial calmodulin in cilia

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.     

Aerobic microbial flora of intertrigenous skin.

The incidence and density of intertrigenous microflora were determined in subjects using nonmedicated soap. The axilla, groin, toe web, and finger web were examined. The incidence of gram-negative rods was 17% for the axilla, 13% for the groin, 10% for the toe web, and 9% for the finger web. Klebsiella, Proteus, and Enterobacter were the predominant organisms, in that order. The highest incidence of Staphylococcus aureus was in the groin (12%) and toe web (11%). Lipophilic diphtheroids were the most prevalent bacteria in the groin (1.1 X 10(6)/cm2) and toe web (1.2 X 10(6)/cm2). Nonlipophilic diphtheroids were the predominant flora in the axilla (1.3 X 10(7)/cm. Micrococci had the highest counts in the toe web (7.6 X 10(5)/cm2). The incidence of coagulase-negative staphylococci was highest in the finger web, but the major flora were those of micrococci.