Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.     

Fast, efficient and selective deprotection of the tert-butoxycarbonyl (Boc) group using HCl/dioxane (4 m).

Fast, efficient and selective deprotection of the tert-butoxycarbonyl (Boc) group of various amino acids and peptides was achieved by using hydrogen chloride (4 m) in anhydrous dioxane solution for 30 min at room temperature. In the cases studied in our laboratory, this protocol provided superior selectivity to deprotect Nalpha-Boc groups in the presence of tert-butyl esters and tert-butyl ethers, including thio-tert-butyl ethers, but not phenolic tert-butyl ethers.     

Presence of pepsinogens immunoreactive to anti-embryonic chicken pepsinogen antiserum in fish stomachs: possible ancestor molecules of chymosin of higher vertebrates

1. Stomachs of adult teleosts and elasmobranchs reacted to an anti-embryonic chicken pepsinogen antiserum (anti-ECPg) as well as to an anti-adult chicken pepsinogen antiserum (anti-ACPg). 2. Zymograms and immunoblots of stomach extracts revealed that anti-ECPg- and anti-ACPg-reactive substances possess peptic activity. 3. The possible relationship between anti-ECPg-reactive pepsinogens in fish and prochymosins in higher vertebrates is discussed.     

Further studies on the genetic control of chicken plasma alkaline phosphatase isozyme.

The effects of neuraminidase treatment on the electrophoretic pattern of alkaline phosphatase (AP) isozymes and AP activity were investigated in chicken plasma. AP comprised three isozymes. The zymogram of an individual chicken plasma had two bands, either the faster (F) or the slower (S) moving band by isozyme types and the B band irrespective of isozyme types. Mobility of the S band and AP activity in chicken plasma were not affected by neuraminidase treatment. The treatment has a reduced migration rate of the F band equal to that of the S band and the B band of both types closer to the origin. The genetic control of these bands is discussed.     

Repeated streptozotocin injections cause early onset of glomerulosclerosis in mice.

Diabetic nephropathy (DN), a major cause of end-stage chronic renal failure, is histologically characterized by glomerulosclerosis. To investigate the molecular mechanisms of DN, it is important to establish a stable model of glomerulosclerosis in mice, because genomic manipulation techniques (such as gene destruction or transgene insertion) are well established in rodent species. In this study, we found that repeated administrations of streptozotocin led to early onset of glomerular sclerotic lesions in C57BL/6 mice, accompanied with renal dysfunction. During the natural course of DN, glomerular endothelial cells decreased at 10 weeks after the start of streptozotocin-injections, whereas myofibroblastic mesangial cells became evident. Our results provide an animal tool to elucidate the molecular mechanisms of DN, for example to investigate vascular pathology in diabetic glomerular diseases.     

Molecular cloning and sequencing of cDNA encoding goat pre alpha-lactalbumin

In poly(A)+RNA extracted from a lactating goat mammary gland, mRNA of about 750 nucleotides was shown to encode pre alpha-lactalbumin by using in vitro translation and immunoprecipitation. From the total poly(A)+RNA, the cDNA library was constructed using the Escherichia coli plasmid pUC18; it was screened with the oligodeoxyribonucleotide probe corresponding to the amino acid sequence of Trp60-Gln65 of goat alpha-lactalbumin. A plasmid containing almost full-length cDNA of goat pre alpha-lactalbumin, pGLA-1, was identified. The cDNA insert of pGLA-1 comprises 727 base pairs and contains the signal peptide and mature protein sequence.     

Decreased transport of D-glucose and L-alanine across brush-border membrane vesicles from small intestine of rats treated with mitomycin C

To elucidate the mechanisms underlying the dysfunctions of intestinal absorption induced by antitumor drugs, the effect of pretreatment with mitomycin C on sodium gradient-dependent D-glucose and L-alanine transports was studied in rat brush-border membrane vesicles. 24, 48, 96, or 120 h following a single intravenous injection of mitomycin C, brush-border membrane vesicles were prepared from rat small-intestines. The uptake of D-glucose and L-alanine was shown to be Na+ gradient-dependent even in the case of vesicles obtained from mitomycin C-treated rats, but uptake rates measured at 15 s and magnitude of overshooting effect in uptake of both solutes were decreased in vesicles maximally from 48 h mitomycin C-treated rats. The rate of D-glucose uptake calculated at 15 s recovered to the control level in vesicles prepared at 96 h and 120 h after mitomycin C-treatment, indicating that the effect of mitomycin C on Na+ gradient-dependent D-glucose transport would be fully reversible. Tracer exchange experiments under Na+ and D-glucose equilibrated conditions indicated that the Na+/D-glucose transporters were similarly operative in the vesicles from control and 48 h mitomycin C-treated rats. Rates of 22Na+ uptake measured at 15 s in vesicles from 48 h mitomycin C-treated rats, however, were increased. The increased permeability to Na+ might bring about a more rapid dissipation of the Na+ gradient in these vesicles and this would secondarily cause the decrease in Na+-dependent D-glucose uptake in vesicles from mitomycin C-treated rats.     

Random cloning of bent DNA segments from Escherichia coli chromosome and primary characterization of their structures.

A simple method for the selective detection of DNA segments containing a sequence-directed static bend was developed. Two-dimensional polyacrylamide gel electrophoresis performed at two different temperatures (60 degrees C and 10 degrees C) can effectively separate a bent DNA from a mixture of normal DNA. Using this method, a bank of plasmids carrying bent DNA inserts from the E. coli total chromosome was constructed. The primary characterization of a set of bent DNA segments randomly cloned from E. coli was presented.     

Prostaglandin D2 receptor of mastocytoma P-815 cells--possible regulation by phosphorylation and dephosphorylation

The 3H-labeled prostaglandin D2 [( 3H]PGD2) binding protein in the membrane fraction of mastocytoma P-815 cells was characterized. The specific binding of [3H]PGD2 to the cells or the membranes reached a maximum at pH 5.6, and was saturable, displaceable and of high affinity when incubated at 0 or 37 degrees C. The Bmax values for [3H]PGD2 binding in the two preparations at pH 5.6 were much higher at 0 degrees C than at 37 degrees C, whereas the Kd values were almost equal (85.3 nM for the cells and 80.5 nM for the membranes, respectively). High specific [3H]PGD2 binding activity in the mildly acid-treated cells was still observed when the external pH was raised from 5.6 to 7.2. Furthermore, specific [3H]PGD2 binding to the membranes (at 0 degrees C, pH 5.6) increased on addition of phosphatase inhibitors (NaF and molybdate) in the presence of 10 microM ATP, but practically disappeared on pretreatment of the membranes with phosphatase. On incubation of the membrane with [gamma-32P]ATP and molybdate, the stimulated incorporation of the [32P]phosphate into several peptides, including ones having an Mr of around 100,000-120,000, was observed. These results suggest that [3H]PGD2 binding in the mastocytoma P-815 cell membrane is controlled through phosphorylation-dephosphorylation of the receptor itself.