Regulation of the Gts1p level by the ubiquitination system to maintain metabolic oscillations in the continuous culture of yeast

Yeast cells exhibit sustained ultradian oscillations of energy metabolism in coupling with cell cycle and stress resistance oscillations in continuous culture. We have reported that the rhythmic expression of Gts1p is important for the maintenance of ultradian rhythms. Structurally, Gts1p contains sequence motifs similar to N-degron and the ubiquitin association domain, raising the possibility that the Gts1p level is regulated by degradation via ubiquitination. When the lysine residue at the putative ubiquitination site of the N-degron was substituted with arginine, both the protein level and half-life of mutant Gts1p increased. During continuous culture, the protein level of the mutant Gts1p was elevated and did not fluctuate, leading to the disappearance of metabolic oscillation within a day. Furthermore, using three Gts1ps containing mutations in the ubiquitin association domain, we showed that the lower the binding activity of the mutant Gts1ps for polyubiquitin in vitro, the higher the protein level in vivo. Expression of the mutant Gts1ps in the continuous culture resulted in an increase in Gts1p and early loss of the oscillation. Therefore, Gts1p is degraded through conjugation with ubiquitin, and the UBA domain promoted the degradation of ubiquitinated Gts1p, causing a fluctuation in protein level, which is required for the maintenance of metabolic oscillations.     

Central,but not peripheral,glucagon-like peptide-1 inhibits crop emptying in chicks

We investigated the effect of central and peripheral glucagon-like peptide-1 (GLP-1) on crop emptying in growing chicks. Intracerebroventricular injection of two concentrations of GLP-1 (15 and 60 pmol) similarly suppressed crop emptying, compared with control chicks. The delay in crop emptying induced by GLP-1 (15 pmol) was partly attenuated by co-administration with exendin (5-39) (600 pmol), a GLP-1 receptor antagonist, although exendin (5-39) alone did not affect crop emptying. On the other hand, intraperitoneal administration of several doses of GLP-1 (120, 300 and 3000 pmol) did not alter crop emptying. The present study revealed that central, but not peripheral, GLP-1 inhibits crop emptying in chicks.     

Crystal structures of bacterial lipoprotein localization factors,LolA and LolB

Lipoproteins having a lipid-modified cysteine at the N-terminus are localized on either the inner or the outer membrane of Escherichia coli depending on the residue at position 2. Five Lol proteins involved in the sorting and membrane localization of lipoprotein are highly conserved in Gram-negative bacteria. We determined the crystal structures of a periplasmic chaperone, LolA, and an outer membrane lipoprotein receptor, LolB. Despite their dissimilar amino acid sequences, the structures of LolA and LolB are strikingly similar to each other. Both have a hydrophobic cavity consisting of an unclosed beta barrel and an alpha-helical lid. The cavity represents a possible binding site for the lipid moiety of lipoproteins. Detailed structural differences between the two proteins provide significant insights into the molecular mechanisms underlying the energy-independent transfer of lipoproteins from LolA to LolB and from LolB to the outer membrane. Furthermore, the structures of both LolA and LolB determined from different crystal forms revealed the distinct structural dynamics regarding the association and dissociation of lipoproteins. The results are discussed in the context of the current model for the lipoprotein transfer from the inner to the outer membrane through a hydrophilic environment.     

Immunoelectron microscopic study for histamine in the gastric enterochromaffin-like cells of rats treated with the proton pump inhibitor lansoprazole

The enterochromaffin-like (ECL) cells of the gastric mucosa in animals play an important role in gastric acid secretion. They contain few granules and numerous secretory vesicles and microvesicles. They operate under the control of circulating gastrin. In the present study, we conducted an immunoelectron microscopic study for histamine (HA) in the ECL cells of rats given the proton pump inhibitor lansoprazole (LP), which is known to induce hypergastrinemia. The pre-embedding indirect immunoperoxidase procedure utilized a mouse monoclonal antibody AHA-2 against glutaraldehyde-conjugated HA. Rats received LP (50 g/kg per day, subcutaneously) over a period of a month, and developed hypertrophy of the ECL cells in the stomach. It was clearly demonstrated that HA was located to a much higher degree in the cytoplasm of ECL cells of LP-treated rats than in normal rats. HA immunoreactivity was observed in the cores of the granules and secretory vesicles of the ECL cells in all the rats, but in the LP-treated rats it was observed in the cores of the newly developed vacuoles as well. These results may suggest that HA may be actively generated in the cytoplasm of the hypertrophic ECL cells of LP-treated rats. Also suggested in the present study is that HA is instrumental in the transformation of granules into secretory vesicles and in their consequent enlargement, and that vacuoles are formed by the fusion of large secretory vesicles. Furthermore, the finding that relatively little HA immunoreactivity existed in the vacuoles may suggest that the vacuoles actively degrade superfluous secretory products (for example, HA) through enhanced autophagocytosis and/or oxidative stress. Another possibility may be that the membrane-bounded structure regarded as the vacuoles in this study might actually be an invagination structure produced as a result of successive series of exocytosis through which the secretory vesicles actively and rapidly release HA.     

Fractionation of sugarcane bagasse by hydrothermal treatment

Hydrothermal treatment of sugarcane bagasse was conducted using a semi-batch reactor to develop a new biomass fractionation method that has low impact in the environment. A continuously increasing temperature was used in this treatment. It was found that hemicellulose and lignin could be mainly extracted as a water-soluble fraction at 200-230 degrees C, while the cellulose fraction was hydrolyzed at higher temperatures (230-280 degrees C) or recovered as solid residue from this treatment. Detailed analyses of the solid residue indicated that the crystal structure and the chemical composition of the residue were in good accordance with those of untreated crystalline cellulose. These experimental and analytical findings show that this method is promising for removal of hemicellulose and lignin from woody biomass without any catalyst and organic solvent.     

Antioxidant Peptides from the Protease Digest of Prawn (Penaeus japonicus) Muscle

The muscle of the prawn Penaeus japonicus was hydrolyzed by various proteases, and antioxidant activity of the hydrolysates was examined. Among the digests, pepsin digest showed the most potent antioxidant activity. Three antioxidant peptides have been isolated from the active peptidic fraction by ion-exchange chromatography, gel filtration, and ODS high-performance liquid chromatography. Their structures were identified as Ile-Lys-Lys, Phe-Lys-Lys, and Phe-Ile-Lys-Lys. Received October 16, 1998; Accepted January 8, 1999.     

Expression and Regulation of the Arsenic Resistance Operon of Acidiphilium multivorum AIU 301 Plasmid pKW301 in Escherichia coli

The arsenic resistance (ars) operon from plasmid pKW301 of Acidiphilium multivorum AIU 301 was cloned and sequenced. This DNA sequence contains five genes in the following order: arsR, arsD, arsA, arsB, arsC. The predicted amino acid sequences of all of the gene products are homologous to the amino acid sequences of the ars gene products of Escherichia coli plasmid R773 and IncN plasmid R46. The ars operon cloned from A. multivorum conferred resistance to arsenate and arsenite on E. coli. Expression of the ars genes with the bacteriophage T7 RNA polymerase-promoter system allowed E. coli to overexpress ArsD, ArsA, and ArsC but not ArsR or ArsB. The apparent molecular weights of ArsD, ArsA, and ArsC were 13,000, 64,000, and 16,000, respectively. A primer extension analysis showed that the ars mRNA started at a position 19 nucleotides upstream from the arsR ATG in E. coli. Although the arsR gene of A. multivorum AIU 301 encodes a polypeptide of 84 amino acids that is smaller and less homologous than any of the other ArsR proteins, inactivation of the arsR gene resulted in constitutive expression of the ars genes, suggesting that ArsR of pKW301 controls the expression of this operon.     

Development of juveniles of four flatheads (Perciformes, Platycephalidae) occurring in southern Japan

The juvenile development of four Japanese flatheads (Platycephalidae),Onigocia macrolepis. O. spinosa, Inegocia japonica andRogadius asper, is described and illustrated from specimens trawled in Tosa Bay, southern Japan. Juveniles of twoOnigocia species are diagnosed by an ocular flap and absence (O. macrolepis) or presence (O. spinosa) of distinct dark bars on the body.Inegocia japonica juveniles are distinctive in having a heavily pigmented body and 12 dorsal and anal fin soft rays.Rogadius asper has a unique antrorse preopercular spine prior to 15 mm SL. During the early juvenile stage, strongly negative allometric growth occurs in the interorbital region, accompanied by migration of the eyes from the lateral to dorsal surface of the head.     

Non-synonymous FGD3 Variant as Positional Candidate for Disproportional Tall Stature Accounting for a Carcass Weight QTL (CW-3) and Skeletal Dysplasia in Japanese Black Cattle

Recessive skeletal dysplasia, characterized by joint- and/or hip bone-enlargement, was mapped within the critical region for a major quantitative trait locus (QTL) influencing carcass weight; previously named CW-3 in Japanese Black cattle. The risk allele was on the same chromosome as the Q allele that increases carcass weight. Phenotypic characterization revealed that the risk allele causes disproportional tall stature and bone size that increases carcass weight in heterozygous individuals but causes disproportionately narrow chest width in homozygotes. A non-synonymous variant of FGD3 was identified as a positional candidate quantitative trait nucleotide (QTN) and the corresponding mutant protein showed reduced activity as a guanine nucleotide exchange factor for Cdc42. FGD3 is expressed in the growth plate cartilage of femurs from bovine and mouse. Thus, loss of FDG3 activity may lead to subsequent loss of Cdc42 function. This would be consistent with the columnar disorganization of proliferating chondrocytes in chondrocyte-specific inactivated Cdc42 mutant mice. This is the first report showing association of FGD3 with skeletal dysplasia.