Molecular evolution and diversification of snake toxin genes, revealed by analysis of intron sequences.

The genes encoding erabutoxin (short chain neurotoxin) isoforms (Ea, Eb, and Ec), LsIII (long chain neurotoxin) and a novel long chain neurotoxin pseudogene were cloned from a Laticauda semifasciata genomic library. Short and long chain neurotoxin genes were also cloned from the genome of Laticauda laticaudata, a closely related species of L. semifasciata, by PCR. A putative matrix attached region (MAR) sequence was found in the intron I of the LsIII gene. Comparative analysis of 11 structurally relevant snake toxin genes (three-finger-structure toxins) revealed the molecular evolution of these toxins. Three-finger-structure toxin genes diverged from a common ancestor through two types of evolutionary pathways (long and short types), early in the course of evolution. At a later stage of evolution in each gene, the accumulation of mutations in the exons, especially exon II, by accelerated evolution may have caused the increased diversification in their functions. It was also revealed that the putative MAR sequence found in the LsIII gene was integrated into the gene after the species-level divergence.

Complete nucleotide sequences of cDNAs encoding long chain alpha-neurotoxins from sea krait, Laticauda semifasciata.

This paper presents the nucleotide sequences of the cDNAs encoding two long chain alpha-neurotoxins from Laticauda semifasciata venom gland. The deduced amino acid sequences are Ls-III and its iso-neurotoxin.

New actions of parathyroid hormone through its degradation.

Parathyroid hormone degradation is intimately connected with its action. By the action of the unique renal neutral cytosolic PTH ase, PTH is split into 1-34 and 35-84 fragments, and further into 35-70 and 71-84 fragments. Amino-terminal 1-34 peptide was found to participate in the autoregulation of PTH secretion, suppressing the intact PTH secretion both in vivo in humans and in vitro in the dispersed bovine parathyroid cells. C-terminal fragment 35-84 and N-terminal fragment 1-34 both suppress the alkaline phosphatase production by ROS 17/2.8 cells to a lesser extent than the intact PTH 1-84, and the sum of the effects of the two fragments approximately equaled that of the intact hormone. Fragments 35-70 and 71-84 were devoid of such activity. Intracellular free calcium of human vascular endothelial cells was raised by intact 1-84, lowered on the contrary by C-terminal 35-84 fragment, but fragments 1-34, 35-70 and 71-84 had no effect. Fragments generated by the actions, supporting the physiological significance of PTH degradation by its target cells.

Calcium treatment of essential hypertension in elderly patients evaluated by 24 H monitoring.

We used 24-h monitoring of blood pressure (BP) to evaluate the effect of calcium supplementation on mild to moderate essential hypertension in elderly hospitalized patients for the first time in a controlled crossover study. The mean systolic and diastolic BP over a period of 24 h declined by 13.6 mm Hg (P less than .005) and 5.0 mm Hg (P less than .05) respectively in patients whose diet was supplemented with 1 g of elemental calcium in the form of oystershell electrolysate (AA calcium). Serum ionized calcium and urinary calcium and sodium excretion increased (serum Ca2+ 0.16 +/- 0.03 mEq/L, P less than .05; FECa 0.5 +/- 0.2%, P less than .05; FENa 0.4 +/- 0.1%, P less than .05) and plasma parathyroid hormone was suppressed (12.2 +/- 2.3 pg/mL, P less than .005). These data suggest that supplementation of dietary calcium may contribute to a reduction of BP in elderly patients with essential hypertension.

Direct inhibitory effect of amino-terminal parathyroid hormone fragment [PTH(1-34)] on PTH secretion from bovine parathyroid primary cultured cells in vitro.

We have examined the possibility of direct inhibitory effect of PTH(1-34) on PTH secretion in bovine parathyroid cells. As low as 10(-12) M PTH(1-34) completely inhibited low calcium (0.5 mM Ca2+)-stimulated PTH secretion by these cells. In the presence of 1.25 mM Ca2+, 10(-12) M PTH(1-34) inhibited PTH secretion by about 14.3% of the basal value, while 10(-11) M or higher concentration of PTH(1-34) showed potent inhibitory effects equivalent to the inhibitory action of high calcium concentration (2.5 mM Ca2+) on PTH secretion. At 2.5 mM Ca2+, as much as 10(-9) M PTH(1-34) failed to inhibit PTH secretion further. These results suggest that PTH(1-34) might directly, not via calcium concentration, inhibit PTH secretion by parathyroid cells and that a cooperative mechanism could exist between calcium and PTH(1-34) to inhibit PTH secretion.

Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the clonal osteogenic UMR-106 cell.

Parathyroid hormone (PTH) -degrading activity was studied using osteoblast-like UMR-106 cells. PTH-degrading activity was assessed by the amount of PTH fragments produced in the medium after exposure of intact human PTH-(1-84) to UMR-106 cells. PTH immunoreactivity recovered in trichloroacetic acid-soluble products of the medium and in fractions eluted from reverse-phase high-performance liquid chromatography (HPLC) was measured by radioimmunoassay using an antibody specific for the mid-region and C-terminus of PTH. In this study, intact UMR-106 cells but not extracellular enzymes cleaved human PTH(1-84) into fragments which were released into the medium (in a time- and temperature-dependent fashion). HPLC analysis of the PTH fragments depicted three immunoreactive peaks (peaks 1, 2 and 3) besides intact PTH, indicating a limited PTH-hydrolyzing activity of the cells. Furthermore, a 1000-fold molar excess of either hPTH-(3-34) or [Nle8,Nle18,Tyr34]hPTH-(3-34)amide inhibited PTH-degrading activity by 63% and 80% of control, respectively, whereas neither calcitonin, vasopressin nor growth hormone suppressed it. Additionally, HPLC analysis of the samples treated with [Nle8,Nle18,Tyr34]hPTH-(3-34)amide showed a reduction of the three peaks, suggesting an involvement of PTH receptor in the production of PTH fragments. This PTH-degrading activity was strongly inhibited by phenylmethylsulfonyl fluoride and chymostatin, but not by soybean trypsin inhibitor, elastatinal or inhibitors of cysteine, aspartic or metalloproteinases, indicating that it is due to a seryl chymotrypsin-like endopeptidase. Chymotrypsin-like activity seems to be solely responsible for PTH-degrading activity in intact UMR-106 cells, since all three PTH fragments were predominantly suppressed in the presence of chymostatin. Further analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by ultraviolet absorbance at 210 nm, three of which were measurable by PTH radioimmunoassay, each corresponding to the three PTH fragments produced by UMR-106 cells. To explore the cleavage sites of PTH further, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the view that the chymotrypsin-like enzyme in UMR-106 cells cleaved the hormone between residues 23-24 and 34-35, to produce, at least, hPTH-(24-84) and -(35-84). Our present study indicates that a chymotrypsin-like endopeptidase is solely responsible for limited hydrolysis of PTH by intact UMR-106 cells.

Parathyroid hormone degradation by chymotrypsin-like endopeptidase in the opossum kidney cell.

Cathepsin-D has been previously reported to cleave intact PTH into PTH-(1-34) and -(35-84) in membranous fractions of rat and bovine kidney. Whether PTH degradation occurs by intact kidney cells, however, has not been examined in detail. We have, therefore, examined this possibility using an opossum kidney (OK) cell line which possesses the characteristics of proximal renal tubules and responds to PTH. PTH radioimmunoreactivity recovered in trichloroacetic acid-soluble products and in fractions eluted from reverse phase HPLC was measured using an antibody directed to the midregion and C-terminus of PTH. In this study, intact OK cells, but not extracellular enzymes, cleaved human (h) PTH-(1-84) into three discrete fragments which were released into the medium in a time- and temperature-dependent fashion. Half-maximal velocity of PTH-degrading activity (PTHDA) was observed at 9 nM hPTH-(1-84). A 1000-fold molar excess of PTH antagonists [hPTH-(3-34) and [Tyr34]hPTH-(7-34)amide] markedly inhibited PTHDA, whereas ACTH, glucagon, or big gastrin did not suppress it, suggesting an involvement of the PTH receptor in PTHDA. This PTHDA was strongly inhibited by phenylmethylsulfonylfluoride and chymostatin, but not by trypsin inhibitor, elastatinal, or inhibitors of aspartic, cysteine, or metalloproteinases, suggesting that it is due to a seryl chymotrypsin-like endopeptidase. Analysis of chymotrypsin-digested products of hPTH-(1-84) eluted from HPLC exhibited five fragments detected by UV absorbance (210 nm), three of which were measurable by PTH RIA, and each corresponded to the three PTH fragments produced by OK cells. All three fragments were predominantly suppressed in the presence of chymostatin, suggesting that chymotrypsin-like activity is solely responsible for PTHDA in intact OK cells. To further explore the cleavage sites of PTH by chymotrypsin, amino acid analysis of chymotrypsin-cleaved products was performed. The results strongly support the conclusion that a chymotrypsin-like enzyme in OK cells cleaved the hormone between residues 23-24, and 34-35 to produce, at least, hPTH-(24-84) and -(35-84). Lysosomal blockers (chloroquine, ammonium chloride, or monensin) did not affect this PTHDA. Our present study indicates that chymotrypsin-like endopeptidase, but not other endopeptidase or lysosomal enzymes, is responsible for the limited hydrolysis of PTH by intact OK cells.

Possible involvement of protein kinase C in parathyroid hormone degradation by osteoblast-like rat osteosarcoma cell line UMR106.

The effects of 12-O-tetraadecanoyl phorbol-13-acetate (TPA), 1-oleoyl-2-acetyl-glycerol (OAG), and 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) on the parathyroid hormone (PTH) degrading activity in a PTH-responsive osteoblast-like rat osteosarcoma cell line UMR106 were investigated to assess the role of Ca2+-activated. Phospholipid dependent protein kinase (protein kinase C) on the degradation of hormones. TPA and OAG, activators of protein kinase C, enhanced the PTH degrading activity dose-dependently, whereas H-7, an inhibitor of protein kinase C, exhibited a dose-dependent inhibition on this activity. These data suggest that protein kinase C activation may enhance PTH degrading activity by UMR106 cells as a possible regulator of PTH degradation.

Degrading activity for human parathyroid hormone [PTH-(1-84)] in rat osteoblast-like osteosarcoma cell line UMR106.

The degrading activity for human parathyroid hormone [hPTH-(1-84)] was studied in a rat osteoblast-like osteosarcoma cell line UMR106. At 37 C,UMR106 cells degraded hPTH-(1-84) into fragments in a time-dependent manner, which was shown by a radioimmunoassay with the use of antibody recognizing the C-terminal and middle regions of PTH molecule, whereas the degradation was completely suppressed at 4 C and failed to occur in the absence of the cells. The Lineweaver-Burk plot of this degrading activity at 37 C showed a fairly good linearity and gave a Km value of 5.1 X 10(-7) M. Reverse-phase high-performance liquid chromatography (HPLC) analysis of immunoreactive PTH fragments in the medium disclosed two peaks aside from intact PTH, indicating a limited PTH-hydrolyzing activity of UMR106 cells cleaving the molecule between at least two separate positions. This study suggests the possible involvement of osteoblasts on the metabolism of intact PTH.

Xanthogranuloma of the rectum.

A 46-year-old man with a suspected malignant submucosal tumor received a transabdominoperineal rectal amputation. The histologic diagnosis was xanthogranuloma. The differentiation of xanthogranuloma from malignant fibrous histiocytoma and other histiocytic tumors is difficult in the preoperative stage. Malignant fibrous histiocytoma can be diagnosed histologically by the presence of pleomorphism, mitotic activity, hyperchromatism, and a storiform pattern of cell arrangement. Moreover, some xanthogranulomas are also thought to have malignant potentiality and surgical resection is regarded as the preferred treatment. As for prognosis, the patient has lived for three years after the operation but further follow-up is thought to be necessary.