Description of complete DNA sequence of two plasmids from the nukacin ISK-1 producer, Staphylococcus warneri ISK-1

We report the whole DNA sequence of two plasmids, pPI-1 (30.2 kb) and pPI-2 (2.8 kb). These plasmids are from Staphylococcus warneri ISK-1, which produces a lantibiotic, nukacin ISK-1. Curing of pPI-1 resulted in a loss of bactericidal activity in the culture supernatant and the host's immunity to nukacin ISK-1, suggesting that the biosynthetic genes of the bacteriocin are encoded by pPI-1. Based on the results of a homology search of each open reading flame, pPI-1 is comprised of the following four distinct regions: (1) the nukacin ISK-1 biosynthesis and immunity gene cluster, (2) the thioredoxin gene cluster, (3) the replication region, and (4) a region of Staphylococcus epidermidis ATCC 12228, highly homologous to pSE-12228-05. Gene organization in the nukacin ISK-1 biosynthesis and immunity gene cluster is different from that in other lacticin-481 type gene clusters. The features of the replication protein encoded in the replicating region are somewhat different from other staphylococcus theta-replicating plasmids. pPI-2 comprised a disinfectant resistant gene, qacC, and the whole DNA sequence showed significant similarity to those of other qacC plasmids such as pSK108, suggesting that pPI-2 belongs to the qacC plasmid group.     

Cloning and expression of type XII collagen isoforms during bovine adipogenesis

In order to isolate candidate genes involved in bovine adipocyte differentiation, we have constructed a subtraction library from a clonal bovine intra-muscular pre-adipocyte (BIP) cell line using the suppression subtractive hybridization method. We have isolated a set of subtracted cDNA fragments whose respective mRNA levels are up-regulated during the adipogenic differentiation of BIP cells, and cloned cDNAs from a differentiated BIP-lambda ZAP II cDNA library. Two cDNA clones were highly homologous to the sequence of mouse and human type XII collagen alpha-1, determined by a BLAST homology search. As type XII collagen has been reported to have four types of splicing isoform, two clones were determined to be XII-1 and XII-2 splicing isoforms, respectively, because of a difference in the C-terminal NC1 domain. From the expression analysis of type XII collagen, the XIIA-2 isoform was mainly expressed in differentiated BIP cells and adipose tissues. Although the function of type XII collagen has not been established as yet, these results suggest that type XII collagen may be associated with adipocyte differentiation and adipose formation in cattle and is a potentially useful marker for adipogenesis.     

Proteomic studies of isoforms of the P25 component of Bombyx mori fibroin

It is recognized that P25 is one of three polypeptide components of the fibroin synthesized in the larval silk gland (SG) of silkworm, having two glycosylated isoforms. In the present study, however, eight P25 isoforms were separated by proteomics, including two-dimensional gel electrophoresis of whole SG proteins, and were identified by the peptide mass fingerprinting method. Four of the eight isoforms were identified as Bombyx mandarina P25s, although the SG of Bombyx mori has never been considered to contain the P25 from B. mandarina. It is suggested that this diversity of P25 isoforms depends on phosphorylation modification in addition to glycosylation.     

Usefulness and limitation of measurement of insulin-like growth factor binding protein-3 (IGFBP-3) for diagnosis of growth hormone deficiency.

To analyze the utility of insulin-like growth factor binding protein-3 (IGFBP-3) radioimmunoassay for diagnosis of growth hormone deficiency (GHD) we measured IGFBP-3 in sera from normal children, short children and patients with GHD. The sensitivity (true positive ratio) of IGFBP-3 for complete GHD (cGHD) was 93%, while the specificity (true negative ratio) for normal short children (NS) was 88%. In contrast, the sensitivity of IGFBP-3 for partial GHD (pGHD) was only 43%. The poor discrimination between patients with pGHD and NS may be the result of their relatively similar GH level, as compared to cGHD, or due to the limitations of GH stimulation tests. The specificity of IGFBP-3 for NS was excellent in children of all ages: less than 10 years old (87%) and older than 10 (88%). However, sensitivity for GHD was good for children less than 10 years old (84%) but poor for children older than 10 (64%). IGFBP-3 may be less sensitive for diagnosing GHD in older children because IGFBP-3 levels may also increase during puberty due to mechanisms independent of the GH-IGF-I axis.     

Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound

After oral administration of an organic germanium compound, Ge-132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat- or acid-treatment, suggesting that the induced IFN is of gamma-nature. The molecular weight of this IFN was estimated to be 50,000 daltons by gel filtration. The NK activity of spleen cells was increased 24 hr after the oral administration of Ge-132, and cytotoxic macrophages were induced in the peritoneal cavity by 48 hr. In the mice receiving an intraperitoneal (ip) injection of trypan blue or carrageenan 2 days before oral administration of Ge-132, neither induction of IFN nor augmentation of NK activity occurred, and X-ray irradiation of mice also rendered the mice incapable of producing IFN, all indicating that both macrophages and lymphocytes are required for this IFN induction. Both NK and cytotoxic macrophages appeared 18 hr after ip administration of the induced IFN with a titer as low as 20 U/ml. These facts suggest that both the augmentation of NK activity and activation of macrophages in mice after oral administration of Ge-132 are mediated by the induced IFN.     

PEG-papain catalyzed synthesis of a Kyotorphin derivative in aqueous organic media

Summary PEG-papain catalyzed synthesis of a Kyotorphin derivative has been investigated in aqueous organic solvents using Bz-Tyr-OEt and Arg-OMe as substrates. PEG-papain has shown increased resistance to high pH and high ethanol concentration; 42 % of Bz-Try-OEt has been converted to Bz-Tyr-Arg-OH under the optimized reaction condition (pH 7.0–9.0; 40 % EtOH).     

Steady-state approximation of enzyme activation and inhibition

This article deals with the effects of the initial concentration of effector (inhibitor or activator) on the steady-state approximation of enzyme kinetics. The results could be summarized as follows: (1) In competitive inhibition, the increase in the initial concentration of inhibitor led to the reduction of steady state time. (2) In noncompetitive and uncompetitive inhibitions, the steady state time was not changed with the increase in the initial concentration of inhibitor. (3) In nonessential activation, the increase in the initial concentration of activator led to the reduction of steadystate time. (4) It was specially noted that in nonessential activation, even if the reaction is in the steady-state, activation constant (K(A)) can not be determined exactly unless the initial concentration of activator is very small.     

Further study on gas phase acid hydrolysis of protein: improvement of recoveries for tryptophan, tyrosine, and methionine

A novel method for vapor phase acid hydrolysis of protein suitable for quantitative analysis of tryptophan is presented. The hydrolysis is carried out in vapor of a mixture made of 7 M HCl, 10% trifluoroacetic acid, and 20% thioglycolic acid in the presence of indole. Reasonably good recoveries of common amino acids, including tryptophan (above 75%), were achieved.