Molecular heterogeneity of D-end products detected by anti-H-2.28 sera

Immunoprecipitation of NP-40 lysates of 125I-labeled lymph-node cells with different anti-H-2 sera and with anti-Qa-2 serum has shown that the BALB/cByA strain (H-2d, Qa-2-negative) expresses, besides H-2Ld, another molecule that is not detectable in the BALB/c-H-2dm2 strain. Electrophoresis in SDS polyacrylamide gels indicated that this molecule, provisionally designated Lq, has an apparent molecular weight of 41000 daltons, in contrast to approximately 49000 daltons for H-2Kd and H-2Ld, and 47000 daltons for H-2Dd molecules. The anti-Qa-2 serum precipitated from the Qa-2-positive strains BALB/cHeA but not from the Qa-2-negative strains BALB/cByA and BALB/c-H-2dm2 a protein that gave a very strong band corresponding to the molecular weight 41000 daltons in the gel electrophoresis. The biochemical characteristics of the Lq molecule are thus more similar to those of Qa-2 than of H-2 antigens.     

Expression patterns of loricrin in various species and tissues

Abstract. In this study we analyzed the expression patterns of loricrin in various species and tissues using immunohistochemistry, immunoblotting and Northern blots. Loricrin is a glycine-, serine- and cysteine-rich protein expressed very late in epidermal differentiation in the granular layers of normal mouse and human epidermis. Later on in differentiation, loricrin becomes cross-linked as a major component into the cornified cell envelope by the formation of N^ɛ -(γ-glutamyl)lysine isopeptide bonds. This process either occurs directly or by the intermediate accumulation in L-keratohyaline granules of mouse epidermis and human acrosyringia. Loricrin was identified in all mammalian species analyzed by virtue of its highly conserved carboxy-terminal sequences revealing an electric mobility of ∼60 kDa in rodents, rabbit and cow and of ∼35 kDa in lamb and human on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Loricrin is expressed in the granular layer of all mammalian orthokeratinizing epithelia tested including oral, esophageal and fore-stomach mucosa of rodents, tracheal squamous metaplasia of vitamin A deficient hamster and estrogen induced squamous vaginal epithelium of ovary ectomized rats. Loricrin is also expressed in a few parakeratinizing epithelia such as BBN [N-butyl-N-(4–hydroxybutyl)nitrosamine]-induced murine bladder carcinoma and a restricted subset of oral and single vaginal epithelial cells in higher mammals. Our results provide further evidence that the program of squamous differentiation in internal epithelia of the upper alimentary tract in rodents and higher mammals differ remarkably. In addition, we also have noted the distinct distribution patterns of human loricrin and involucrin, another major precursor protein of the cornified cell envelope.     

A membrane-associated form of C-reactive protein is the galactose-specific particle receptor on rat liver macrophages

Rat liver macrophages express a galactose-specific receptor which mediates endocytosis of particles or neuraminidase-treated blood cells. From rat serum we now have isolated and purified a galactose-specific lectin by affinity chromatography. Comparative analysis of this serum galactose-binding protein with the galactose-particle receptor protein purified from rat liver macrophages and with C-reactive protein (CRP) reveals close relation or identity of these proteins. An apparent m.w. of 30,000 was determined for all three proteins by SDS-PAGE under reducing conditions and m.w. of about 130,000 by native PAGE. All three proteins exhibit the same pentameric, ring-shaped structure in electron microscopy after negative staining. Antibodies raised against the serum galactose-binding protein or against the macrophage receptor cross-react. A mAb specific for rat neo-CRP labels liver macrophages but not hepatocytes and reacts with the isolated protein in a Western blot assay. Furthermore, the galactose-particle receptor can be functionally replaced by purified CRP: the binding capacity for neuraminidase-treated E of receptor-depleted liver macrophages can be restored by preincubation with purified rat CRP. We therefore conclude that CRP occurs as a membrane-associated protein constitutively expressed on liver macrophages functioning as a receptor mediating galactose-specific binding of particulate ligands.     

Sarcomere length uniformity determined from three-dimensional reconstructions of resting isolated heart cell striation patterns.

A- and I-band striation positions have been obtained, three-dimensionally reconstructed, and statistically analyzed from the volumes of resting isolated heart cells. Striation patterns from optically discrete subvolumes are imaged along the length of these myocytes with a computer-interfaced optical microscope imaging system. Planar striation maps are reconstructed by the computer from sequentially obtained striation pattern images displaced across the width or depth of the cell in controlled steps. Multiple planar maps are combined to form full three-dimensional (3-D) reconstructions that illustrate the sarcomeric structure and ordering throughout the volume of the cell. These reconstructions demonstrate a high degree of striation registration throughout most regions of cardiac cells. The striation registration is often slightly (less than 10 degrees) skewed across the width or depth of nearly every cell and is occasionally disrupted between adjacent groups of sarcomeres. These disruptions in registration are always associated with the locations of the nuclei. Rigorous statistical analyses indicate small volumetric regions of the cell delineated by these disruptions can have significantly (0.014-0.113 micron) shorter or longer average sarcomere length periodicities. Unlike skeletal muscle "fibrillenstruktur," these data from cardiac cells exhibit no evidence of helical packing schemes for sarcomere order. These observations suggest that the relatively large nuclei displace and disrupt the normal registration of the sarcomeres, which is probably mediated by internal cytoskeletal structures.     

Effects of stormwater sewer discharges on the aquatic community in urban canals in Lelystad

In urban areas with a separate sewerage system, the stormwater runoff is discharged into surface waters. A study on the effects of stormwater sewer discharges on the composition of the aquatic community in urban waters was carried out in two areas with a primarily residential use in the new town of Lelystad. The aquatic organisms considered included hydrophytes, epiphytic diatoms, filamentous algae and macroinvertebrates.The results indicate that the stormwater sewer discharges cause a slight change in the composition of the aquatic community. The water in the urban surface waters can be characterized as eutrophic and - toa-mesosaprobic. Near stormwater sewer outfalls the water tends to the more polluteda-mesosaprobic state. The shifts in the composition of the aquatic community could be traced primarily on the basis of a number of epiphytic diatoms and macroinvertebrates (in particular some Diptera and Trichoptera). Most hydrophytes, filamentous algae and the remaining epiphytic diatoms and macroinvertebrates (in particular water beetles and water mites) turned out to be of little or no use in this respect.Because the investigation was carried out in an unusually dry summer, the results probably underestimate the effects of stormwater discharges on the aquatic community.     

Structure-function analysis of plasma cholesteryl ester transfer protein by protease digestion and expression of cDNA fragments in Escherichia coli

In an attempt to define an active domain of the protein, fragments of cholesteryl ester transfer protein (CETP) were obtained by limited digestion of the native, plasma-derived protein with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease or by expression of CETP cDNA restriction fragments in Escherichia coli. Although digestion of native CETP with these proteases resulted in extensive fragmentation of the protein and loss of the intact 74-kDa molecule as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, CE transfer activity was unaffected (trypsin or chymotrypsin treatment) or only partially lost (V8 protease treatment). Analysis by molecular sieve chromatography showed that the CE transfer-active product of this proteolysis consisted of polypeptide fragments which remained associated, retaining the native molecular weight of CETP. These proteolyzed complexes were resistant to dissociation by dithiothreitol, 8 M urea, or delipidating agents. As shown by CE transfer activity, native CETP was found to possess a stable conformation which remained unchanged in buffers containing up to 4.5 M urea, or following exposure to even higher (8 M) urea concentrations. CETP polypeptides from bacterially expressed cDNA fragments were found to be catalytically inactive although they contained the epitope for an inhibitory anti-CETP monoclonal antibody and had emulsion binding properties similar to native CETP. Selected synthetic CETP peptides (including the peptide containing the inhibitory monoclonal antibody epitope) were also devoid of CE transfer activity. Thus, no evidence was found for an independently active subunit of the CETP. Together, the results indicate that the CETP possesses a distinct and highly stable tertiary structure which is required for CE transfer catalytic activity.