CD44 standard and variant isoform expression in normal human skin appendages and epidermis

 CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. The sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed. Accepted: 10 May 1996     

Qualitative mathematical discussion of different evolutionary states in water transport systems of plants

We shall present several qualitative mathematical models to describe the early evolution of water transport systems in plants. To perform this in a systematic way we apply methods which have been developed in phenomenological synergetics. These methods rest on the fact that it becomes possible to describe the macroscopic behavior of a complex system by a set of control and order parameters when they are suitably identified. Our presentation is addressed to community with interdisciplinary interests.     

Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase.

In the present study, insulin is shown to rapidly stimulate by 8- to 12-fold the enzymatic activity of RAC-PK alpha, a pleckstrin homology domain containing ser/thr kinase. In contrast, activation of protein kinase C by phorbol esters had almost no effect on the enzymatic activity of RAC-PK alpha. Insulin activation was accompanied by a shift in molecular weight of the RAC-PK alpha protein, and the activated kinase was deactivated by treatment with a phosphatase, indicating that insulin activated the enzyme by stimulating its phosphorylation. This insulin-induced shift in RAC-PK was also observed in primary rat epididymal adipocytes, as well as in a muscle cell line called C2C12 cells. The insulin-stimulated increase in RAC-PK alpha activity was inhibited by wortmannin (an inhibitor of phosphatidylinositol 3-kinase) in a dose-dependent manner with a half-maximal inhibition of 10 nM, but not by 20 ng/ml of rapamycin. Activation of RAC-PK alpha activity was also observed in a variant RAC lacking the pleckstrin homology domain. These results indicate that RAC-PK alpha activity can be regulated by the insulin receptor. RAC-PK alpha may therefore play a general role in intracellular signaling mediated by receptor tyrosine kinases.     

Recombination between Chromosomal Is200 Elements Supports Frequent Duplication Formation in Salmonella Typhimurium

Spontaneous tandem chromosomal duplications are common in populations of Escherichia coli and Salmonella typhimurium. They range in frequency for a given locus from 10(-2) to 10(-4) and probably form by RecA-dependent unequal sister strand exchanges between repetitive sequences in direct order. Certain duplications have been observed previously to confer a growth advantage under specific selective conditions. Tandem chromosomal duplications are unstable and are lost at high frequencies, representing a readily reversible source of genomic variation. Six copies of a small mobile genetic element IS200 are evenly distributed around the chromosome of S. typhimurium strain LT2. A survey of 120 independent chromosomal duplications (20 for each of six loci) revealed that recombination between IS200 elements accounted for the majority of the duplications isolated for three of the loci tested. Duplications of the his operon were almost exclusively due to recombination between repeated IS200 elements. These data add further support to the idea that mobile genetic elements provide sequence repeats that play an important role in recombinational chromosome rearrangements, which may contribute to adaptation of bacteria to stressful conditions.     

A subset of SR proteins activates splicing of the cardiac troponin T alternative exon by direct interactions with an exonic enhancer.

The cardiac troponin T pre-mRNA contains an exonic splicing enhancer that is required for inclusion of the alternative exon 5. Here we show that enhancer activity is exquisitely sensitive to changes in the sequence of a 9-nucleotide motif (GAGGAAGAA) even when its purine content is preserved. A series of mutations that increased or decreased the level of exon inclusion in vivo were used to correlate enhancer strength with RNA-protein interactions in vitro. Analyses involving UV cross-linking and immunoprecipitation indicated that only four (SRp30a, SRp40, SRp55, and SRp75) of six essential splicing factors known as SR proteins bind to the active enhancer RNA. Moreover, purified SRp40 and SRp55 activate splicing of exon 5 when added to a splicing-deficient S100 extract. Purified SRp30b did not stimulate splicing in S100 extracts, which is consistent with its failure to bind the enhancer RNA. In vitro competition of SR protein splicing activity and UV cross-linking demonstrated that the sequence determinants for SR protein binding were precisely coincident with the sequence determinants of enhancer strength. Thus, a subset of SR proteins interacts directly with the exonic enhancer to promote inclusion of a poorly defined alternative exon. Independent regulation of the levels of SR proteins may, therefore, contribute to the developmental regulation of exon inclusion.     

Partial restoration of impaired alpha1-adrenergic responsiveness in parotid cells of aged rats by S-adenosylmethionine treatment

The age related decrease in alpha₁-adrenergic stimulated inositol 1,4,5 trisphosphate (IP₃) production in parotid cells of aged rats can be partially restored by treatment with S-adenosylmethionine (SAM). This effect is completely blocked by S-adenosyl homocysteine (SAH) and occurs in association with an increase in the conversion of phosphatidylethanolamine to phosphatidylcholine and a decrease in membrane viscosity. In contrast, SAM treatment actually inhibits stimulated IP₃ production in cells of young rats. The membrane viscosity of these cells is lower than that of those from aged rats. Although conversion of phosphatidylethanolamine to phosphatidylcholine is enhanced, no further decrease in membrane viscosity is elicited in young cell preparations. These findings suggest that age changes in the membrane environment may result in impaired alpha₁-adrenergic signal transduction and that such alterations may be at least partially reversible by SAM treatment.     

The genes of the lysosomal cysteine proteinases cathepsin B, H, L, and S map to different mouse chromosomes

The mouse genes for the lysosomal cysteine proteinases cathepsin B, H, L, and S were mapped to Chromosomes (Chrs) 14, 9, 13, and 3, respectively. Two of the DNA probes used in this study detected an additional, independently segregating locus. The cathepsin B-specific probe hybridized to a locus on Chr 2, and the cathepsin H probe to a locus on the X Chr. These loci either correspond to pseudogenes or to cathepsin B- and cathepsin H-related genes. The four cysteine proteinases mapped in this study lie within known regions of conserved synteny between mouse and human chromosomes, when compared with the corresponding positions of their human homologs. Assuming that the genes of the cysteine proteinase gene family arose from a common ancestral gene, our results suggest that these four cysteine proteinases had been dispersed over different chromosomes before separation of mouse and human in evolution. Received: 22 August 1996 / Accepted: 20 November 1996     

The impact of the Scheldt input on the trace metal distribution in the Belgian coastal area (results of 1981–1983 and 1995–1996)

Offshore fluxes of Cu, Zn, Cd, Pb and Hg were calculated based onresidual flow patterns and salinity gradients along the Belgian coast. Theresidual flow lines along the Belgian coast are more or less parallel to thecoast except in the area where the north-easterly flowing watermass comingfrom the Channel encounters the south-westerly-oriented Scheldt outflow,forming a residual hydrodynamical front. From the steady-state salinitypattern, diffusion coefficients perpendicular to the residual flow werededuced; they ranged from 21 to 108 m² s^-1. Offshore fluxes of dissolved and particulate trace metals based on diffusiveand mixing processes are calculated. The steady state profiles of dissolvedmetals show a dilution effect in the coastal waters, reaching an almostconstant concentration in the marine watermass in the 1981–1983dataset. The ratios of the Scheldt input of trace metals to the totaldissolved offshore flux vary from 38 to 55% (1981–1983),depending on the kind of metal, and from 55 to 91% (1995–1996).The ratio of the Scheldt input to the dissolved metal flow parallel to thecoast, is in both periods (1981–1983 and 1995–1996), smallerthan 1%. The steady-state concentration profiles of particular metalsversus salinity are fairly constant in the coastal-estuarine and marinewatermasses, but decrease very abruptly from the first to the secondwatermass. Assuming a conservative behaviour of the particular metals,offshore fluxes and the resulting concentration increases agree fairly wellwith the observed values. The ratios of the Scheldt input to the particulatetrace metal offshore flux vary between 30 to 46% (1981–1983)and 13 to 37% (1995–1996). The contribution of the Scheldtestuary to the flows parallel to the coast ranges from 1.6 to 2.9%(1981–1983) and from 0.6 to 1.6% (1995). This revised version was published online in July 2006 with corrections to the Cover Date.     

Transposition without transposase: a spontaneous mutation in bacteria.

Transposition mutations are typically associated with the activities of transposable elements such as transposons and insertion sequences, whose mobility is dependent upon transposase enzymes that catalyze exchanges between element ends and target sites. We describe a single transposition event in which a block of donor sequence is inserted at a target site without the involvement of any known transposase or the ends of any known transposable element. We propose that this is a new type of spontaneous mutation which may be difficult to detect in standard mutant hunts but may be of evolutionary importance.     

Calcium concretions in the pineal gland of aged rats: an ultrastructural and microanalytical study of their biogenesis

The genesis of calcium concretions in aged rats was studied by means of transmission and scanning electron microscopy. The potassium pyroantimonate method, combined with X-ray microanalysis, allowed us to study the distribution of cations and calcium. Notable accumulations of calcium (associated with phosphorus) were localized in vesicles, vacuoles, lipid droplets, lipopigments, and mitochondria of dark pinealocytes. The results obtained in the present investigation suggest that these organelles are involved in the genesis of the concretions. The presence of sulfur indicates the existence of an organic matrix. We propose that genesis takes place in dark pinealocytes, which contain more calcium than light pinealocytes. Mineralization foci are some-times associated with cellular debris and enlarge by further apposition of material. Two types of concretions, as determined by electron microscopy and confirmed by electron diffraction, could be observed: the amorphous type with concentric layers and the crystalline type with needle-shaped crystals. Once formed, the concretions reach the extracellular space and the cell breaks down. Possible extracellular calcification is suggested in the extracellular calcium-rich floculent material. The mineralization process is interpreted as being an age-related phenomenon and mainly a consequence of the degeneration of pinealocytes.