Structural studies of a human gamma 3 myeloma protein (Jir) bearing the allotypic marker Gm(st)

The authors established the amino acid substitutions determining G3m(s) and G3m(t) specificities, which characterize Mongoloid populations, by sequence analysis of the Fc region of a myeloma protein (Jir). By comparing the amino acid sequences of the IgG3 (Jir) and the other IgG subclasses analyzed to date, it was found that G3m(s) was an isoallotype specified by an amino acid substitution at position 435; i.e., whereas the subclasses IgG1, IgG2, and IgG4 had histidine in common, G3m(s-) had arginine in this position. This was also confirmed by the observation that the Fc fragment in question bound to protein A. It was also established that the amino acid at position 379 of G3m(t-) IgG3 and the other subclasses was valine, whereas methionine in this position was specific for G3m(t+). In addition, the amino acids at position 339 of G3m(u-) IgG3 Jir was threonine, and at position 296 of G3m(g-) IgG3 Jir was tyrosine. These findings are not in accord with the hitherto postulated relations of alanine and phenylalanine to G3m(u-) and G3m(g-), respectively. Finally, this study showed that a large number of substitutions occurred at positions 384 through 389, which suggests that many specificities of the G3m(b) group occur on IgG3 proteins.     

Activation of TIMP-2/progelatinase A complex by stromelysin.

Progelatinase A was purified as a complex with TIMP-2 from the conditioned medium of a human glioblastoma cell line. The TIMP-2/progelatinase complex was resistant to the activation by p-aminophenylmercuric acetic acid (APMA), and showed less than 10% of the activity of the TIMP-2-free active enzyme. When the complex was incubated with stromelysin in the presence of APMA, the 64-kDa progelatinase was effectively converted to the 57-kDa mature enzyme, increasing its gelatinolytic activity about 8-fold. These results suggest that stromelysin is a natural activator of TIMP-2-bound progelatinase A.     

Successful pregnancy in a woman with ovarian failure associated with mutation in the beta-subunit of luteinizing hormone.

BACKGROUND: We report a successful pregnancy in a woman with severe ovarian dysfunction and infertility associated with a variant beta-subunit of luteinizing hormone (LH). METHOD/OUTCOME: A 35-year-old woman consulted our unit for infertility. Laparoscopy and ultrasonography showed obstruction of the right tube and ovulation from the right ovary only. Human menopausal gonadotrophin (hMG) therapy was used for six subsequent cycles, but did not result in conception. Subsequently, marked elevation of follicle-stimulating hormone (FSH) and testosterone, together with polycystic ovary (PCO) were noted. The patient failed to respond to ovarian stimulation by hMG. Severe ovarian dysfunction such as premature ovarian failure (POF) was strongly suspected. Sequence analysis of the LH beta-subunit gene indicated heterozygosity for point mutations Trp(8) to Arg(8) and Ile(15) to Thr(15) in the coding sequence. LH hypersecretion resembling that seen in PCO syndrome was observed. Induction of ovulation by hMG was successful in the first cycle in which the basal LH and FSH were well controlled with gonadotrophin-releasing hormone analog following estrogen-progesterone replacement. She conceived and delivered a healthy male infant at term. CONCLUSION: Clinicians should be clinically aware of patients with immunologically anomalous LH variant who might be at risk of developing ovarian failure within a relatively short time span. Pertinent treatment should be applied without delay in such cases.     

Transformation of rat liver cell line by Rous sarcoma virus causes loss of cell surface fibronectin, accompanied with secretion of metallo-proteinase that preferentially digests the fibronectin

An epithelial cell line derived from the liver of a normal Buffalo rat (BRL) was transformed by Rous sarcoma virus (RSV). The RSV-transformed cells were separated into five clones (RSV-BRL1 through 5), which were morphologically different. RSV-BRL cells exhibited the following characteristics distinct from those of BRL cells: tumorigenicity, irregular cell arrangement, loose intercellular junction, growth in soft agar (anchorage-independent growth) except for RSV-BRL3 and 5, and loss of cell surface fibronectin. When BRL cells were cultured in the standard medium supplemented with the serum-free conditioned medium of RSV-BRL cells, the amount of the cell surface fibronectin decreased significantly. It was found that RSV-BRL cells secreted a proteinase capable of hydrolyzing the fibronectin, whereas BRL cells secreted hardly any of this proteinase. The fibronectin-hydrolyzing proteinase (FNase) could also hydrolyze plasma fibronectin added as an exogenous substrate. The hydrolysis of plasma fibronectin was inhibited by ethylenediamine tetraacetate, but stimulated by rho-chloromercuribenzoate and calcium ion. This indicates that FNase is a metallo-enzyme, but not a serine or thiol enzyme. In addition to the proteinase, RSV-BRL cells secreted plasminogen activator and a proteinase inhibitor which inhibited the activity of plasmin but not FNase.     

Effect of calcium overload on the phosphoinositide breakdown in the rat left ventricular papillary muscle

We investigated the effect of Ca²⁺ overload on the phospholipase C-catalyzed hydrolysis of phosphoinositides in the rat left ventricular papillary muscle. Ca²⁺ overload on the papillary muscle was induced by treatment with 0.3 mM ouabain in Ca²⁺-containing medium following either Ca²⁺-containing or Ca²⁺-free superfusion. The phosphoinositide breakdown was evaluated by determining accumulations of [³H]inositol phosphates ([³H]IPs) in the tissues prelabeled with [³H]inositol. Ca²⁺ repletion following Ca²⁺-free superfusion resulted in a rapid but small increase in resting tension that was not followed by contracture, nor was it associated with a significant increase in [³H]IPs accumulations. Treatment with ouabain following Ca²⁺-containing superfusion increased resting tension after a lag period of several minutes and produced contracture associated with an increase in [³H]IPs accumulations. The ouabain induced increases in resting tension, and accumulations of [³H]IPs were significantly potentiated by prior Ca²⁺-free superfusion instead of Ca²⁺-containing superfusion. There was a significant positive correlation between increases in resting tension and the phosphoinositide breakdown. The increased resting tension and the accumulations of [³H]IPs were not antagonized by treatments with prazosin plus atropine or indomethacin, but were abolished by superfusion with Ca²⁺-free buffer solution. Although the enhanced phospholipase C-catalyzed hydrolysis of phosphoinositides appears to be a consequence rather than a cause of increased intracellular Ca²⁺, such a biochemical change may provoke a positive feedback mechanism to develop the muscle contracture through the putative intracellular messenger action of inositol triphosphate and diacylglycerol.Abbreviations [³H]IPs [³H]Inositol Phosphates - IP Inositol Phosphate - IP₂ Inositol Bisphosphate - IP₃ Inositol Trisphosphate - PI Phosphatidylinositol - PI-4-P Phosphatidylinositol-4-phosphate - PI-4,5-P₂ Phosphatidylinositol 4,5-bisphosphate - PRZ Prazosin - ATR Atropine - INDO Indomethacin - min Minutes     

Proteinic prorenin-releasing-stimulator (PRS) in the rat submandibular gland

The release of prorenin as well as renin from rat renal slices was confirmed by a rat prorenin-prosegment ELISA system and an assay system for determining the renin activity. A significant increase of the prorenin release was found by adding rat submandibular gland extract to the slice medium, indicating the existence of a prorenin-releasing stimulator (PRS) in the extract. The pI and molecular mass of PRS were 8.5-8.7 and 28-30 kDa, respectively. The PRS was completely inactivated by boiling or a proteinase treatment.