Genetics of cell-surface receptors for bioactive polypeptides: A variant of mouse BALBc/3T3 fibroblasts possessing altered insulin-binding ability

An insulin-nonresponsive variant was isolated from mutagenized mouse BALBc/3T3 fibroblasts. Selection was based on the insulin's mitogenic action upon quiescent cells and subsequent arrest at mitosis by vinblastine sulfate to remove insulin-responsive cells. Among four surviving colonies, one, designated IN-2, exhibited no binding for [¹²⁵I] insulin at 2× 10^–10 M and at 4° C. The binding ability, however, recovered substantially at 15° C and increased with higher temperature and at higher ligand concentrations. The binding profiles, Scatchard plot analysis, and the dissociation kinetics indicated that the receptors expressed on IN-2 cells possess lower affinity than the parental 3T3 cells. The IN-2 cells were negative for stimulating effects of insulin on 2-deoxyglucose uptake, thymidine incorporation, and cell growth. The IN-2 cells were also negative for cross-reactivity to antibodies which react with insulin receptors on 3T3 cells and for the susceptibility to a cytotoxic chimeric insulin which was cross-linked to diphtheria toxin fragment A. This negative response of IN-2 cells can be attributed to a deficiency in high-affinity receptors for insulin. The insulin bound to the low-affinity binding sites of IN-2 cells, however, undergoes internalization and intracellular degradation. Therefore, such processing by itself does not account for insulin's mitogenic action.     

Juvenile hyaline fibromatosis: A report of two unrelated adult sibling cases and a literature review

Two unrelated adult sibling cases (36- and 32-year-old females) of Juvenile hyaline fibromatosis are presented. The parents of one of these patients were non-consanguineous but natives of a small Island, and one elder sister among four siblings was affected with the same disease. The parents of the other patient were consanguineous, and one other sibling suffered from the identical disease. Both patients presented with multiple subcutaneous nodules, which they had had since infancy, and had undergone numerous surgical excisions. Light microscopy examination of skin lesions from both patients showed identical histology; an abundance of a homogenous, amorphous, eosinophlllc extracellular matrix in which spindle-shaped cells were embedded. Electron microscopically, the spindle-shaped cells had hypertrophic Golgi apparatus and dilated, rough endoplasmlc reticulum. Fine flbrillar and granular material-filled structures, the contents of which were occasionally released into the extracellular matrix, were also seen, immunohistochemically, the spindle-shaped cells were vlmentin-positive but negative for α-smooth muscle actln and S-100 protein, and the hyaline ground substance was positive for type I and type III collagen but negative for type II and type IV collagen and tenascin. Matrix metalloprotelnase-1, -2, and -9, and tissue inhibitor of matrix metalloproteinase (TlMP)-2 was positive but TIMP-1 was negative. A review of 39 cases of juvenile hyaline fibromatosis In the literature is also presented. In summary, skin lesions may be the most outstanding symptoms of juvenile hyaline fibromatosis, but joint contracture and gingival hypertrophy precede the skin manifestation.     

Autoimmune hepatitis associated with bile duct injury resembling chronic non-suppurative destructive cholangitis

Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are representative autoimmune liver diseases in which hepatocytes and intrahepatic bile ducts, respectively, are selectively damaged by autoimmune mechanisms. Bile duct injury and loss is characteristic of PBC and chronic non-suppurative destructive cholangitis (CNSDC), in particular, is a histological hallmark of PBC. In this report, we present an unusual case of AIH accompanied by CNSDC-like bile duct injury in a 46-year-old woman. The patient's serum aminotransferase level was abnormally high. The serum levels of alkaline phosphatase, gamma-GTP and IgG were also elevated, but the IgM level was within normal limits. The titer of antismooth muscle antibody (SMA) was 1:80, while antinuclear autoantibody (ANA) and the M2 fraction of antimitochondrial antibody (AMA) were both negative. Liver biopsy disclosed CNSDC-like bile duct injuries and severe interface hepatitis and lobular hepatitis with perivenular zonal necrosis were observed. The aggregate score of the International Autoimmune Hepatitis Group corresponded to the category of probable AIH. Immunohistochemically, histocompatibility leukocyte antigen-DR, which is aberrantly expressed in the damaged bile ducts of PBC, was not found in the injured bile ducts of this case. Laboratory data were much improved by treatment with prednisone, but ursodeoxycholic acid was not effective. Although the possibility of an overlapping syndrome of AIH- and AMA-negative PBC could not be excluded, this case was diagnosed as AIH with CNSDC-like bile duct lesions.     

Suppression of visually and memory-guided saccades induced by electrical stimulation of the monkey frontal eye field. I. Suppression of ipsilateral saccades

When a saccade occurs to an interesting object, visual fixation holds its image on the fovea and suppresses saccades to other objects. Electrical stimulation of the frontal eye field (FEF) has been reported to elicit saccades, and recently also to suppress saccades. This study was performed to characterize properties of the suppression of visually guided (Vsacs) and memory-guided saccades (Msacs) induced by electrical stimulation of the FEF in trained monkeys. For any given stimulation site, we determined the threshold for electrically evoked saccades (Esacs) at < or =50 microA and then examined suppressive effects of stimulation at the same site on Vsacs and Msacs. FEF stimulation suppressed the initiation of both Vsacs and Msacs during and about 50 ms after stimulation at stimulus intensities lower than those for eliciting Esacs, but did not affect the vector of these saccades. Suppression occurred for ipsiversive but not contraversive saccades, and more strongly for saccades with larger amplitudes and those with initial eye positions shifted more in the saccadic direction. The most effective stimulation timing for suppression was about 50 ms before saccade onset, which suggests that suppression occurred in the efferent pathway for generating Vsacs at the premotor rather than the motoneuronal level, most probably in the superior colliculus and/or the paramedian pontine reticular formation. Suppression sites of ipsilateral saccades were distributed over the classical FEF where saccade-related movement neurons were observed. The results suggest that the FEF may play roles in not only generating contraversive saccades but also maintaining visual fixation by suppressing ipsiversive saccades.     

Evidence of the monoclonal composition of human endometrial epithelial glands and mosaic pattern of clonal distribution in luminal epithelium

The endometrium is a highly regenerative tissue that plays a crucial role in implantation. We examined the clonal constitution of glandular cells as well as the luminal epithelium of this unique tissue. Using collagenase-based digestion techniques with microscopic manipulation, we isolated individual human endometrial glands and examined their clonality using a polymerase chain reaction-based assay for nonrandom X chromosome inactivation with an X-linked androgen receptor gene. Most of the glands analyzed were composed of monoclonal populations of epithelial cells and one of the glands exhibited a loss of heterogeneity in the androgen receptor gene. In addition, adjacent glands within a 1-mm(2) area shared clonality, suggesting that clonality of the luminal epithelium is regionally defined. The clonality of endometrium was further confirmed in a study of female mice that harbor the green fluorescent protein gene on either the maternal or paternal X chromosome. Fluorescent microscopy of uterine sections revealed that individual endometrial glands consisted completely of either fluorescent or nonfluorescent cells and that the surface epithelium exhibited a clear boundary between these cell types. These findings suggest that single or multiple stem cells with uniform clonality exist on the bottom of each endometrial gland and genetic alterations occurring in such cells may play a critical role in endometrial carcinogenesis. The possible association between area-specific X inactivation of the endometrial surface and the endometrial receptivity of embryo implantation remains to be clarified.     

Comparative sequence analysis of the M gene among rabies virus strains and its expression by recombinant vaccinia virus

Nucleotide sequences and the deduced amino acid sequences of the gene encoding the matrix (M) protein of the Nishigahara and the CVS strains of rabies virus have been determined. The M gene is 609 nucleotides long and is capable of coding for a peptide composed of 202 amino acids. Sequence comparison of these M genes with those of other stains [Pasteur (PV), ERA, Avol] revealed that there is 89.7–91.5% homology at the nucleotide level, and 90.1–92.1% homology at amino acid level, between almost all combinations of these strains. However, in the combinations of the PV and ERA strains, and the virulent CVS and the avirulent CVS-derived Avol strains, much higher homology was observed both at the nucleotide and amino acid levels. The predicted secondary structure and hydropathy profiles also exhibited similar features. Recombinant vaccinia virus containing the M gene was constructed. Sodium dodecyl sulfate (NaDodSO₄)-polyacrylamide gel electrophoresis of the precipitates obtained by immune reaction of the recombinant virus-infected cell lysate with a monoclonal antibody against the M protein revealed that electrophoretic mobility of the expressed protein is indistinguishable from that of the authentic M protein from rabies virions.The nucleotide sequence data of the M genes of the CVS and Nishigahara (RCEH) strains reported in this paper will appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers D90450 and D90451.     

Three-dimensional distribution patterns of PRL,GH and ACTH cells in the house musk shrew ( Suncus murinus)

Despite a multitude of reports on the classification and distribution of anterior pituitary cells, no previous study has attempted to obtain the three-dimensional (3D) and computer-graphic distribution pattern of each cell type in the whole pituitary. Therefore, we mapped the anterior pituitary cells of the house musk shrew ( Suncus murinus) and found a distinct cellular distribution pattern. Serial horizontal sections of whole shrew pituitaries were stained immunohistochemically for prolactin (PRL), growth hormone (GH), and adrenocorticotropic hormone (ACTH) cells. The contours of positive cells and the anterior, intermediate, and posterior lobes in each section were digitized for 3D visualization by a volume rendering method. The reconstructed images and virtual frontal and sagittal slices were examined in detail. On the 3D reconstructed images, the PRL and GH cells had similar distribution patterns, although the former were concentrated in the dorsolateral and ventrocentral portions, and the latter in the dorsocentral portions of the anterior lobe. On both sides of the pituitary stalk, there lay portions that were conspicuous by scarcity of PRL and GH cells. ACTH cells were widely scattered throughout the whole anterior lobe, but they were very few in the above portions and the dorsocentral portions where GH cells were concentrated. No sex difference in the distribution patterns of each cell type was observed. However, PRL cells in females were more numerous than in males, whereas the opposite was true for GH and ACTH cells. We discuss the relationship between the formation of the spatial distribution patterns and anterior pituitary ontogeny.     

Iron,coronary artery calcification,and mortality in patients undergoing hemodialysis

ObjectiveA high coronary artery calcification score (CACS) may be associated with high mortality in patients undergoing hemodialysis (HD). Recently, effects of iron on vascular smooth muscle cell calcification have been described. We aimed to investigate the relationships between iron, CACS, and mortality in HD patients.MethodsWe studied 173 consecutive patients who were undergoing maintenance HD. Laboratory data and Agatston’s CACS were obtained at baseline for two groups of patients: those with CACS ≥400 (n = 109) and those with CACS <400 (n = 64). Logistic regression analyses for CACS ≥400 and Cox proportional hazard analyses for mortality were conducted.ResultsThe median (interquartile range) age and duration of dialysis of the participants were 67 (60–75) years and 73 (37–138) months, respectively. Serum iron (Fe) and transferrin saturation (TSAT) levels were significantly lower in participants with CACS ≥400 than in those with CACS <400, although the serum ferritin concentration did not differ between the groups. TSAT ≥21% was significantly associated with CACS ≥400 (odds ratio 0.46, p<0.05). TSAT ≥17%, Fe ≥63 µg/dL, and ferritin ≥200 ng/mL appear to protect against 5-year all-cause mortality in HD patients, independent of conventional risk factors of all-cause mortality (p < 0.05).ConclusionWe have identified associations between iron, CACS, and mortality in HD patients. Lower TSAT was found to be an independent predictor of CACS ≥400, and iron deficiency (low TSAT, iron, or ferritin) was a significant predictor of 5-year all-cause mortality in HD patients.     

Purification and Partial Characterization of an Indomethacin Hydrolyzing Enzyme from Pig Liver

Purpose. Indomethacin is well known to be metabolized via O-demethylation and N-deacylation. In this paper we found an enzyme involved in the hydrolysis of amide-linkage of indomethacin and partially characterized it as well as its substrate specificity. Methods. An indomethacin hydrolyzing enzyme was purified to homogeneity from pig liver microsomes using columns of Q-Sepharose, Red-Sepharose and Blue-Sepharose. The enzyme activity was assayed by measuring of -chlorobenzoic acid liberated from indomethacin by HPLC. Results. The purified enzyme effectively hydrolyzed the amide linkage in indomethacin but not those in -naphthylacetate and -nitrophenylacetate, which are typical substrates for carboxylesterase. The subunit molecular mass of the enzyme was 65 kDa according SDS-polyacrylamide gel electrophoresis. The Michaelis constant (Km) and maximum velocity (Vmax) values for indomethacin were 67.8 µM and 9.02 nmol/min/mg protein, respectively. The amino acid sequence analysis of the enzyme after cyanogen bromide cleavage showed high homology with a mouse carboxylesterase isozyme designated as ES-male. The activity of indomethacin hydrolysis was relatively high in the pig, rabbit and human liver homogenate, but not in those from rat and mouse. On the other hand, purified human liver carboxylesterases pl 5.3 and 4.5, and pig liver carboxylesterases have no catalytic activity for indomethacin. Conclusions. These results indicate that the hydrolysis of amide-linkage of indomethacin in humans would be associated with an enzyme similar to the indomethacin hydrolyzing enzyme from pig liver microsomes described here.     

Distribution of connectin (titin), nebulin and α-actinin at myotendinous junctions of chicken pectoralis muscles: an immunofluorescence and immunoelectron microscopic study

Summary The distribution of connectin (titin), nebulin and -actinin in the areas of myotendinous junctions of chicken pectoralis muscles was examined by immunocytochemical methods. Staining with antibodies against connectin (4C9, SM1 and P1200) and nebulin formed doublets flanking nonterminal Z-bands; near the end of muscle fibres singlets were seen within the terminal sarcomere on the side adjacent to the terminal Z-bands. The apical regions of muscle processes, where no myosin filaments are present although actin filaments exist, were reactive with anti-nebulin but not with anti-connection. Antibodies against pectoralis (skeletal muscle type) -actinin stained non terminal Z-bands and that against gizzard (smooth muscle type) the sarcolemma. Terminal Z-bands were unreactive with both of these antibodies. These findings indicate that, although terminal and nonterminal Z-bands differ in their molecular composition, connectin and nebulin filaments appear to link myosin and actin filaments, respectively, to both Z-band types.