Comparative studies on the adhesiveness of granulocytes of guinea pig and man

Summary The high affinity of granulocytes of guinea pig and man to glass surfaces is modified by serum. Native serum contains both an adherence-promoting activity, which is related to complement, and components which reduce the adhesiveness of granulocytes. These components are stable at 56°C for 30 min and are tightly bound to the glass surface. -Lipoproteins are candidates for this adherence reducing ability of serum. Adherence promotion by native serum is mediated by coating the glass surface with C3b/C3bi. Human granulocytes from the peripheral blood adhered to glass surfaces coated by native human or guinea pig serum with C3b/C3bi to almost the same extent as in the presence of native serum, but on guinea pig granulocytes elicited in the peritoneal cavity, a cell surface metalloproteinase degraded the C3b/C3bi, thus reducing the adhesiveness of these cells. This proteinase was inhibited by MgEDTA, DTT, and 1,10-phenanthroline, whereby the high adhesiveness of granulocytes was restored to C3b/C3bi-coated glass.Abbreviations BA benzamidine hydrochloride - BTS Bacillus thuringiensis subtoxicus - DTT dithiothreitol - EAC -amino-caproic acid - gp guinea pig - LDL low density lipoproteins - SEM scanning electron microscopy     

Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

A new method for quenching correction leads to revisions of data in receptor autoradiography

Summary Differential quenching of -emission affects strongly the analysis of receptor distribution patterns in quantitative receptor autoradiography with tritiated ligands. Different methods for the quenching correction have been described in the past, but some of these are of limited value, if a detailed anatomical parcellation is necessary. Other methods correct exclusively local variations in lipid concentration, which is an important, but only one of several factors causing quenching. A new method for the measurement of quenching (or autoradiographic efficiency) is presented, which permits an anatomically detailed and direct determination of the total quenching without lipid extraction procedures. This method is based on the measurement of autoradiographic efficiency in cryostat sections homogeneously labeled with tritiated formaldehyde by an underlying gelatine section containing this labeled compound. Regional and layer specific measurements of autoradiographic efficiency in cortical and subcortical regions of the human and rat brain are reported. A significant correlation was found between the density of myelin and autoradiographic efficiency but other factors were also shown to influence differential quenching. The use of the here presented correction procedure leads to revisions of the laminar distribution patterns reported for different receptors in human and rat cortical areas. Our results show, that a complete quenching correction is necessary for the mapping of receptor distributions with tritiated ligands.     

Actin-binding proteins are conserved from slime molds to man

DNA clones encoding the actin-binding proteins alpha-actinin and severin from Dictyostelium discoideum were isolated and sequenced. Comparisons of the deduced amino acid sequences with proteins from other species showed striking similarities at distinct regions. The F-actin cross-linking molecule alpha-actinin carries two characteristic EF-hand structures highly homologous to the Ca2+-binding loops of proteins from the calmodulin superfamily. An N-terminal region that is conserved in alpha-actinin from D. discoideum and vertebrates is also related to parts of the dystrophin sequence and might represent the F-actin binding site. Severin, gelsolin, villin, and fragmin share homologous sequences that are believed to participate in the severing activity of these proteins.     

The determination of the local cerebral glucose utilization with the 2-deoxyglucose method

Summary In the adult mammalian brain, the energy metabolism is almost entirely dependent on glucose. Furthermore, a close relationship between the energy metabolism and the functional activity could be shown. Thus, the functional activity of the brain or parts thereof can be quantified by measuring the cerebral metabolic rate for glucose. Studying in vivo the fate of a radioactive labeled analogue of glucose, the 2-deoxy-d-[1-¹⁴C]glucose, and using quantitative autoradiographic techniques, it is possible to estimate the cerebral glucose utilization of every discrete brain region. The advantage of the 2-deoxyglucose method is, that the local cerebral glucose utilization represents a metabolic encephalography (Sokoloff 1982).     

Quantitative receptor autoradiography in the human brain

Summary Quantitative receptor autoradiography on sections of the human brain raises methodical problems of which some are relevant also for studies in animal tissue, but others are unique in studies of human brain tissue. Procedures for the following methodical aspects are discussed image analysis for quantitation of the regional distribution of receptor densities, saturation analysis on autoradiographs, influence of age and post-mortem delay and quenching of -radiation in brain tissue. The solutions proposed to these problems make receptor autoradiography in the human brain to a reliable method for studies of chemical neuroanatomy.     

The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

Limited nonenzymatic glucosylation of low-density lipoprotein does not alter its catabolism in tissue culture

This study examines the effects of various degrees of chemical modification of low-density lipoprotein (LDL) on its catabolism by various cell types. Moderate glucosylation of LDL does not alter its interaction with the high-affinity receptor present on human fibroblasts at concentration of 5-2000 micrograms LDL-cholesterol/ml. Only heavily glucosylated LDL (more than 12 lysine residues glucosylated per apolipoprotein B) or LDL glucosylated in the presence of Na(CN)BH3, i.e., conditions not expected to occur in diabetes, inhibit receptor-mediated internalisation and degradation. Moderately glucosylated LDL is also readily recognized by cultured rat hepatocytes and porcine endothelial cells. Human monocyte-derived macrophages accumulate cholesteryl ester when incubated with acetylated LDL for 12 days but no enhanced cholesteryl ester formation was found when native or glucosylated LDL (3.3 lysines glucosylated per apolipoprotein B) were used.     

Differential distribution of3H dihydrotestosterone and3H estradiol nuclear binding sites in mouse male accessory sex organs

Summary The distribution of specific nuclear binding sites for androgens and estrogens in the male accessory sex organs of the mouse was assessed by autoradiography with³H dihydrotestosterone (³H DHT) and³H estradiol (³H E₂). With³H DHT nuclear labeling differed among the epithelia of the organs. It was high in seminal vesicle and ampullary gland, moderate in ventral prostate, urethral gland, prostatic excretory ducts and the ampulla ductus deferentis, low in dorsal prostate and low or absent in coagulation gland. With³H E₂, in contrast, epithelial nuclear labeling was high only in coagulation gland, moderate or low in seminal vesicle, low or absent in ventral and dorsal prostate and absent in ampullary gland and ampulla ductus deferentis. In the lamina propria of all organs nuclear labeling with³H DHT was generally moderate and existed only in some cells, with the highest number in the ampulla ductus deferentis. With³H E₂, nuclear labeling in the lamina propria showed a high intensity in all organs, except in ventral and dorsal prostate which remained unlabeled. Many labeled cells were found in the deferent duct and its ampulla, while in the other organs only a few cells showed nuclear labeling with³H E₂. In the smooth muscle sheath of all organs, some muscle cells were moderately labeled with³H DHT, but not with³H E₂. The results indicate the presence of nuclear receptors in male accessory sex organs for both dihydrotestosterone and estradiol. The differential patterns of³H DHT and³H E₂ nuclear uptake suggest differential sensitivities of the individual organs and their tissue compartments for androgens and estrogens. Supported by PHS grant NSO9914 to W.E.S. and Deutsche Forschungsgemeinschaft Dr94/4 to U.D. The work of Dr. Schleicher and his stay in Chapel Hill were additionally sponsored by Studienstiftung des Deutschen Volkes and Boehringer-Ingelheim Fonds