Molecular cloning of groESL locus, and purification and characterization of chaperonins, GroEL and GroES, from Bacillus brevis

The groESL locus of a protein-hypersecreting bacterium, Bacillus brevis, was cloned by PCR using primers designed based on the DNA sequence of a B. subtilis homolog. GroEL protein was purified to apparent homogeneity and its ATPase activity was characterized: it hydrolyzed ATP, CTP, and TTP in this order of reaction rate, and its specific activity for ATP was 0.1 micromole/min/mg protein. Purified GroEL forms a tetradecamer. GroEL was estimated to contain 22% alpha-helix, 24% beta-sheet, and 19% turn structures, by CD measurement. GroES protein was also highly purified to examine its chaperonin activity. GroEL protected from thermal inactivation of and showed refolding-promoting activity for malate dehydrogenase, strictly depending on the presence of ATP and GroES.     

Role of cadherins in maintaining the compartment boundary between the cortex and striatum during development

In ventricular cells of the mouse telencephalon, differential expression of cadherin cell adhesion molecules defines neighbouring regions; R-cadherin delineates the future cerebral cortex, while cadherin-6 delineates the lateral ganglionic eminence. By using cell labelling analyses in the whole embryo culture system, we demonstrated that the interface between R-cadherin and cadherin-6 expression is a boundary for cell lineage restriction at embryonic day 10.5. Interestingly, when a group of cells with exogenous cadherin-6 were generated to straddle the cortico-straital boundary by electroporation at embryonic day 11.0, ectopic cadherin-6-expressing cortical cells were sorted into the striatal compartment, and the reverse was the trend for ectopic R-cadherin-expressing striatal cells. Although cadherin-6 gene knockout mice engineered in this study showed no obvious phenotype in telencephalic compartmentalisation, the preferential sorting of ectopic cadherin-6-expressing cells was abolished in this mutant background. Thus, the differential expression pattern of cadherins in the embryonic telencephalon is responsible for maintaining the cortico-striatal compartment boundary.     

Development of lateral line organs in leptocephali of the freshwater eel Anguilla japonica (Teleostei,Anguilliformes)

A study of the ontogeny of the lateral line system in leptocephali of the Japanese eel Anguilla japonica reveals the existence of three morphologically different types of lateral line organs. Type I is a novel sensory organ with hair cells bearing a single kinocilium, lacking stereocilia, distributed mainly on the head of larvae, and morphologically different from typical superficial neuromasts of the lateral line system. Its developmental sequence suggests that it may be a presumptive canal neuromast. Type II is an ordinary superficial neuromast, common in other teleost larvae, which includes presumptive canal neuromasts that first appear on the trunk and accessory superficial neuromasts that later appear on the head and trunk. Type III is a very unusual neuromast located just behind the orbit, close to the otic vesicle, with radially oriented hair cells, suggesting that these serve as multiple axes of sensitivity for mechanical stimuli. The behavior of larval eels suggests that the radially oriented neuromasts may act as the sole mechanosensory organ until the ordinary superficial neuromasts develop. The finding that larval eels possess a well-developed mechanosensory system suggests the possibility that they are also capable of perceiving weak environmental mechanical stimuli, like other teleost larvae.     

Recombinant Rhodococcus sp. strain T09 can desulfurize DBT in the presence of inorganic sulfate

The putative Rhodococcus rrn promoter region was cloned from the benzothiophene desulfurizing Rhodococcus sp. strain T09, and the dibenzothiophene desulfurizing gene, dsz, was expressed under the control of the putative rrn promoter in the strain T09 using a Rhodococcus–E.coli shuttle vector. Strain T09 harboring the expression vector, pNT, could desulfurize dibenzothiophene in the presence of inorganic sulfate, methionine, or cysteine, while the Dsz phenotype was completely repressed in recombinant cells carrying the gene under the control of the native dsz promoter under the same conditions. Among the sulfur sources examined, no intermediates were detected and only the final desulfurized product, 2-hydroxy-biphenyl, was produced using ammonium sulfate as the sulfur source. Received: 4 December 2001 / Accepted: 7 January 2002     

Fasting-induced suppression of pulsatile luteinizing hormone secretion is related to body energy status in ovariectomized goats

The effect of energy status on the response of luteinizing hormone (LH) pulse frequency to acute short-term energy deficiency created by fasting in estradiol-treated ovariectomized Shiba goats was studied in two experiments. In experiment 1, eight goats whose mean body weight (BW) was 25.6 +/- 5.8 (mean +/- S.D.)kg were fed 500 g hay cubes daily for 1 week. Then they were fasted for 3 days. Blood samples were collected for 4 h at 6 min intervals on the last day of feeding, first, second and third day of fasting for LH analysis. The goats were divided into light (<24 kg, n = 4) and heavy (> or =24 kg, n = 4) groups for data analysis. There was no difference in LH pulse frequency between the last day of feeding and each day of fasting in the heavy group. LH pulse frequency was significantly (P < 0.05) suppressed on the second day (3.3 +/- 1.3 pulses/4 h) and on the third day (2.3 +/- 1.9 pulses/4 h) relative to the day prior to fasting (4.8 +/- 1.5 pulses/4 h) in the light group. In experiment 2, BW plus a body mass index (gBMI: (body weight (kg)/withers height (m)/body length (m)) x 10) were measured to define energy status. Nine goats (BW, 25.6 +/- 5.8 kg) were fed 500 g hay cubes daily for a week and then fasted for 3 days. Then they were divided into two groups offered either a maintenance (n = 4) or a restricted (n = 5) level of feeding for 4 weeks. The restricted level of feeding was 30% of maintenance requirement based on the BW recorded weekly. The feeding level was then adjusted to maintain BW for a further week followed by 3 day fasting for restricted animals. Blood samples were collected for 6 h at 10 min intervals on the day prior to fasting and on third day of fasting before and after the dietary manipulation. BW (26.6 +/- 2.2 to 26.8 +/- 3.8 kg) and gBMI (8.4 +/- 0.4 to 7.8 +/- 0.3) remained constant over the period prior to fasting for the maintenance animals but were significantly lower (P < 0.05) after 4 weeks for the restricted goats (BW, 26.3 +/- 2.1 to 21.5 +/- 2.4 kg; gBMI, 8.4 +/- 0.9 to 6.9 +/- 0.7). There was no significant difference in the LH pulse frequency between feeding and fasting day in both sampling periods in the maintenance group. In the restricted group, LH pulse frequency was not suppressed by fasting in the first sampling period (6.8 +/- 2.9 to 5.2 +/- 2.5 pulses/6 h), whereas it tended to be suppressed (4.8 +/- 3.1 to 1.6 +/- 2.3 pulses/6 h; P < 0.06) and was significantly (P < 0.05) correlated to body weight (r = 0.70) and gBMI (r = 0.81) after the dietary manipulation. These results suggest that the suppressive effect of short-term energy restriction (fasting) on pulsatile LH secretion is related to body energy status.     

Genetic analysis of rabies virus isolates in the Philippines

To determine the genetic characteristics of the rabies virus in the Philippines, 59 rabies virus isolates were obtained from domestic rabid dogs and their partial nucleotide sequences of nucleoprotein (N) gene were compared. Based on comparison with reported sequences, phylogenetic analysis revealed that all isolates from the Philippines had close genetic relations and formed two subgroups. The Philippines isolates belonged to a different lineage from other Asian isolates but were closer to them than to terrestrial isolates and laboratory strains. Several specific nucleotide and amino acid substitutions were observed among the Philippines isolates. Our results suggest that rabies viruses in the Philippines might have a characteristic evolution.     

Complete amino acid sequence and characterization of the reaction mechanism of a glucosamine-induced novel alcohol dehydrogenase from Agrobacterium radiobacter (tumefaciens)

A glucosamine-induced novel alcohol dehydrogenase has been isolated from Agrobacterium radiobacter (tumefaciens) and its fundamental properties have been characterized. The enzyme catalyzes NAD-dependent dehydrogenation of aliphatic alcohols and amino alcohols. In this work, the complete amino acid sequence of the alcohol dehydrogenase was determined by PCR method using genomic DNA of A. radiobacter as template. The enzyme comprises 336 amino acids and has a molecular mass of 36 kDa. The primary structure of the enzyme demonstrates a high homology to structures of alcohol dehydrogenases from Shinorhizobium meliloti (83% identity, 90% positive) and Pseudomonas aeruginosa (65% identity, 76% positive). The two Zn(2+) ion binding sites, both the active site and another site that contributed to stabilization of the enzyme, are conserved in those enzymes. Sequences analysis of the NAD-dependent dehydrogenase family using a hypothetical phylogenetic tree indicates that these three enzymes form a new group distinct from other members of the Zn-containing long-chain alcohol dehydrogenase family. The physicochemical properties of alcohol dehydrogenase from A. radiobacter were characterized as follows. (1) Stereospecificity of the hydride transfer from ethanol to NADH was categorized as pro-R type by NMR spectra of NADH formed in the enzymatic reaction using ethanol-D(6) was used as substrate. (2) Optimal pH for all alcohols with no amino group examined was pH 8.5 (of the C(2)-C(6) alcohols, n-amyl alcohol demonstrated the highest activity). Conversely, glucosaminitol was optimally dehydrogenated at pH 10.0. (3) The rate-determining step of the dehydrogenase for ethanol is deprotonation of the enzyme-NAD-Zn-OHCH(2)CH(3) complex to enzyme-NAD-Zn-O(-)CH(2)CH(3) complex and that for glucosaminitol is H(2)O addition to enzyme-Zn-NADH complex.     

Possible involvement of ecdysteroids in photoperiodically induced suppresion of ovarian development in a Japanese strain of the migratory locust,Locusta migratoria

In a Japanese population of Locusta migratoria, adult females become reproductively inactive under crowding and long days (LD) and reproductively active under crowding and short days (SD). The identity and titre of ecdysteroids in the haemolymph and ovaries from adult females reared under SD and LD were investigated by RIA/HPLC. The effects of exogenous juvenile hormone (JH) III treatments on the termination of such reproductive arrest and ecdysteroid contents in LD females were also examined. In general, ecdysteroid titres in both haemolymph and ovaries were significantly higher in reproductively active SD females than in reproductively inactive LD females. A clear difference was also observed in oocyte growth between SD and LD individuals. JH III applications (three consecutive topical applications, 150 μg per insect per day from day 3) stimulated ovarian development in LD females and significantly increased the haemolymph and ovarian ecdysteroids to a level comparable to that of reproductively active SD adult females.     

An Insight into the pathway of the amyloid fibril formation of hen egg white lysozyme obtained from a small-angle X-ray and neutron scattering study

It is known that hen egg white lysozyme (HEWL) forms amyloid fibrils. Since HEWL is one of the proteins that have been studied most extensively and is closely related to human lysozyme, the variants of which form the amyloid fibrils that are related to hereditary systemic amyloidosis, this protein is an ideal model to study the mechanism of amyloid fibril formation. In order to gain an insight into the mechanism of amyloid fibril formation, systematic and detailed studies to detect and characterize various structural states of HEWL were conducted. Since HEWL forms amyloid fibrils in highly concentrated ethanol solutions, solutions of various concentrations of HEWL in various concentrations of ethanol were prepared, and the structures of HEWL in these solutions were investigated by small-angle X-ray and neutron scattering. It was shown that the structural states of HEWL were distinguished as the monomer state, the state of the dimer formation, the state of the protofilament formation, the protofilament state, and the state towards the formation of amyloid fibrils. A phase diagram of these structural states was obtained as a function of protein, water and ethanol concentrations. It was found that under the monomer state the structural changes of HEWL were not gross changes in shape but local conformational changes, and the dimers, formed by the association at the end of the long axis of HEWL, had an elongated shape. Circular dichroism measurements showed that the large changes in the secondary structures of HEWL occurred during dimer formation. The protofilaments were formed by stacking of the dimers with their long axis (nearly) perpendicular to and rotated around the protofilament axis to form a helical structure. These protofilaments were characterized by their radius of gyration of the cross-section of 2.4nm and the mass per unit length of 16,000(+/-2300)Da/nm. It was shown that the changes of the structural states towards the amyloid fibril formation occurred via lateral association of the protofilaments. A pathway of the amyloid fibril formation of HEWL was proposed from these results.