The cDNAs of two types of fatty acid-binding protein (FABP) present in human kidney, previously described as types A and B, were isolated using reverse transcriptase-PCR (RT-PCR) with human kidney mRNA and various sets of primers. The cDNA fragments were cloned and sequenced. Renal FABP type A and B cDNAs appeared to be completely identical to human liver- and heart-type FABP cDNAs respectively. In the second part of this study we demonstrated the presence of liver-type FABP in rat kidney by chromatography, e.l.i.s.a. and immunocytochemistry. The ratio and cellular distribution of the two FABP types varies markedly in human and rat kidney. Using RT-PCR we were also able to prepare and identify liver- and heart-type FABP cDNAs with mRNA from both male and female rat kidney.

Two types of fatty acid-binding protein (FABP) were isolated from human kidney by gel filtration and ion-exchange chromatography. Northern-blot analysis showed the presence of two FABP transcripts in total kidney RNA, hybridizing with cDNA of human liver and muscle FABP respectively. Characterisation based on molecular mass, isoelectric point, fluorescence with dansylaminoundecanoic acid and immunological cross-reactivity showed that one, type B, was fairly similar to human heart FABP. The other, type A, showed, like human liver FABP, a high fluorescence enhancement and a wavelength shift with dansylaminoundecanoic acid as well as the binding of a variety of ligands. Antibodies raised against FABP type A and against liver FABP markedly cross-reacted in e.l.i.s.a., in Western blotting and in indirect immunoperoxidase staining on kidney and liver sections. Differences in amino acid composition and isoelectric points, however, indicate that type A is a new kidney-specific FABP type. The FABP type A is more abundant in kidney than the B type and is predominantly localized in the cortex, especially in the cells of the proximal tubules. The FABP type B is mainly present in the cells of the distal tubules. In conclusion, this study shows the presence of two types of FABP in the kidney. One type seems to be related to heart FABP, while the other type resembles, but is not identical with, liver FABP. Both types have a characteristic cellular distribution along the nephron.

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A cDNA encoding a new form of the shared (3 subunit ((83) of the platelet integrin gpIlb/Ja and the vitronectin receptor was isolated from a placental cDNA library by screening with a (3 (gplIa) DNA probe. This (3 variant differs from the previously reported 3 in that the cytoplasmic domain is 8 amino adds shorter and has an alternative, 13-amino acid COOH-terminal peptide. The 3' untranslated region of the cDNA also differs from the previously reported sequence, while the region coding for the transmembrane domain and extracellular domain is identical to it. Reverse transcription combined with polymerase chain reaction was used to show that human placental tissue and two human cell lines contain the variant mRNA.

In order to contrast anionic sites in mouse lung alveoli, two staining procedures were applied: (a) staining with Ruthenium Red and Alcian Blue and (b) staining with Cuprolinic Blue in a critical electrolyte concentration method. The Ruthenium Red-Alcian Blue staining procedure revealed electron-dense granules in the alveolar basement membrane. The granules were closely associated with the epithelial cell membrane and continued to stain even when the procedure was carried out at a low pH, indicating the presence of sulphate groups in the granules. After staining with Cuprolinic Blue, electron-dense filaments, also closely associated with the cell membrane, became visible in the basement membrane of type I epithelial cells. Their length depended on the MgCl2 concentration used during staining. At 0.4 M MgCl2, the length was mostly within the range 100-180 nm. Using a modified Cuprolinic Blue method, the appearance of the filaments closely resembled that of spread proteoglycan monomers with their side-chains condensed. The basement membrane of type II epithelial cells also contained filaments positive towards Cuprolinic Blue; their length, however, was smaller in comparison with those of type I epithelial cells. The filaments lay in one plane and provided the whole alveolus with an almost continuous sheet of anionic sites. Cuprolinic Blue staining also revealed filaments in the basement membrane of the capillary endothelial cells. Furthermore, Cuprolinic Blue-positive filaments (average length about 40 nm) became apparent in close contact with collagen fibrils and separated from each other according to the main banding period of the collagen fibrils (about 60 nm), indicating a specific ultrastructural interaction between these two components. Filaments connecting collagen fibrils with each other were also detected.

Several connective tissues were stained for proteoglycans using the cationic dye Cuprolinic Blue according to the critical electrolyte concentration method. With this method, proteoglycans are visualized as electron-dense filaments. In most tissues, two types of proteoglycan filaments are present: a small (maximum length 60 nm), thin, collagen fibril-associated filament, and a thick, heavily-staining filament which is predominantly localized between bundles of collagen fibrils. Cartilage contains very large (about 300 nm) proteoglycan filaments while in cornea they are very small. Comparison with biochemical data from the literature suggests that the appearance of the proteoglycan filaments may be indicative for the glycosaminoglycan-protein ratio and for the molecular weight of the part of the protein core to which glycosaminoglycans are attached. The data thus obtained on the localization and structure of a proteoglycan may be useful when planning a strategy for its isolation.

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Since defective regulation of ion transport could initiate or contribute to the abnormal cellular function in myotonic dystrophy (MyD), Na+/K(+)-ATPase and sarcoplasmic reticulum (SR) Ca(2+)-ATPase were examined in skeletal muscle and cultured skeletal muscle cells of controls and MyD patients. Na+/K(+)-ATPase was investigated by measuring ouabain binding and the activities of Na+/K(+)-ATPase and K(+)-dependent 3-O-methylfluorescein phosphate (3-O-MFPase). SR Ca(2+)-ATPase was analysed by e.l.i.s.a., Ca(2+)-dependent phosphorylation and its activities with ATP and 3-O-methylfluorescein phosphatase (3-O-MFP). In MyD muscle the K(+)-dependent 3-O-MFPase activity and the activity and concentration of SR Ca(2+)-ATPase were decreased by 40%. In cultured muscle cells from MyD patients the activities as well as the concentration of both Na+/K(+)-ATPase and SR Ca(2+)-ATPase were reduced by about 30-40%. The ouabain-binding constant and the molecular activities, i.e. catalytic-centre activities with ATP or 3-O-MFP, of Na+/K(+)-ATPase and SR Ca(2+)-ATPase were similar in muscle as well as in cultured cells from both controls and MyD patients. Thus the decreased activity of both ATPases in MyD muscle is caused by a reduction in the number of their molecules. To check whether the deficiency of ATP-dependent ion pumps is a general feature of the pathology of MyD, we examined erythrocytes from the same patients. In these cells the Ca2+ uptake rate and the Ca(2+)-ATPase activity were lower than in controls, but the Ca(2+)-ATPase concentration was normal. Thus the reduced Ca(2+)-ATPase activity is caused by a decrease in the molecular activity of the ion pump. The Na+/K(+)-ATPase activity is also lower in erythrocytes of MyD patients. It is concluded that the observed alterations in ion pumps may contribute to the pathological phenomena in the muscle and other tissues in patients with MyD.