Phase-dependent locomotor activity in first-stadium nymphs of the desert locust, Schistocerca gregaria: effects of parental and progeny rearing density

This study examined the effects of parental and progeny rearing density on locomotor activity of 1st-stadium nymphs of the desert locust, Schistocerca gregaria, using an actograph. Progeny obtained from solitarious (isolated-reared) or gregarious (crowd-reared) locusts were reared in isolation or in a group of 30 nymphs. Crowding after hatching had a slight influence on mean activity shortly after the start of measurements, but no clear effect was detected until day 2, when maximum activity during the 6-24 h of observation was significantly higher than that of the nymphs kept in isolation. On the other hand, the effects of parental rearing density on locomotor activity manifested at all ages examined (0-2 days old). Progeny of gregarious locusts showed consistently higher activity than those of solitarious locusts. In newly hatched nymphs, the effect of parental rearing density was explained by variation in body size at hatching, one of the phase-dependent characteristics. Hatchling body color was also correlated with locomotor activity and body weight. Similar levels of locomotor activity were exhibited when green, solitarious and black, gregarious nymphs were similar in body weight. These results suggested that parental rearing density indirectly influences locomotor activity in the progeny shortly after hatching by affecting their body size as eggs or hatchlings.     

Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome.

The ribosome-releasing factor (RRF) gene was localized at a position between 2 and 6 min on the Escherichia coli chromosome by measuring the gene-dosage-dependent production of RRF in various E. coli F' merozygotes. This position was confirmed and refined by using a nucleotide probe corresponding to a 16-amino-acid sequence in RRF. It was found that the RRF gene was contained in pLC 6-32 of the Clark-Carbon Gene Bank. Restriction enzyme mapping of E. coli genomic DNA with the above probe led us to conclude that the RRF gene is situated in the 4-min region, somewhere downstream (clockwise) of the elongation factor Ts gene, tsf. A pLC 6-32-derived DNA fragment which carries the RRF gene was found to contain a partial sequence of tsf. The exact location of the translational initiation site of the RRF gene was determined to be 1.1 kilobases downstream from the translational termination site of tsf. The RRF gene is designated frr.     

Hypoxia-induced apoptosis and tube breakdown are regulated by p38 MAPK but not by caspase cascade in an in vitro capillary model composed of human endothelial cells

In order to improve medical treatment of ischemic injury such as myocardial infarction, it is important to elucidate hypoxia-induced changes to endothelial cells. An in vitro blood vessel model, in which HUVECs are stimulated to form a network of capillary-like tubes, was used to analyze hypoxia-induced morphological and biochemical changes. When exposed to hypoxia, the network of capillary tubes broke down into small clusters. This tube breakdown was accompanied by chromatin condensation and cell nuclear fragmentation, morphological markers of apoptosis, and activation of two apoptotic signals, caspase-3 and p38. We investigated what roles caspase cascade and p38 play in hypoxia-induced apoptosis and tube breakdown by using zVAD-fmk and SB203580, specific inhibitors of these two apoptotic signals, respectively. Chromatin condensation and cell nuclear fragmentation and tube breakdown were effectively inhibited by SB203580, but not by zVAD-fmk. SB203580 caused dephosphorylation of p38, which indicates that p38 was autophosphorylated. Inhibition by zVAD-fmk caused slight MW increase in p17 and emergence of p19, which indicates that the inhibitor caused partial processing of caspase-3. Inhibition of p38 suppressed activation of caspase-3 but not vice versa. In addition, these two inhibitors were shown to differentially inhibit cleavage of so-called caspase substrates. SB203580 inhibited cleavage of PARP and lamin A/C, while zVAD-fmk inhibited cleavage of lamin A/C but not that of PARP. Taken together, these results show that p38 is located upstream of caspase cascade and that, although caspase-3 is activated, a p38-regulated caspase-independent pathway is crucial for the execution of hypoxia-induced apoptosis and tube breakdown.     

Functional relationship between bovine brain phosphodiesterase activator protein and bovine heart troponin C

Phosphodiesterase activator protein has been purified from bovine brain and its properties compared with that of bovine heart troponin C. While both proteins activate ‘activator depleted phosphodiesterase’ in the presence of Ca²⁺, a 200-fold greater concentration of troponin C was necessary and the maximal effect was less than that with the activator protein. The activator protein formed a Ca²⁺ -dependent complex with bovine heart troponin I during electrophoresis in 6 M urea-polyacrylamide gel. However, the mobility of this complex was different from that of troponin C · troponin I complex and the affinity between troponin C and troponin I was much stronger than that between the activator protein and troponin I. Ca²⁺ induced changes in the electrophoretic mobility of activator protein and the pattern of its elution during gel filtration which were similar to the Ca²⁺-dependent conformational changes observed with troponin C. Bovine heart troponin I reduced basal, troponin C and the activator protein stimulation of phosphodiesterase activity. These results are compatible with the concept that phosphodiesterase activator protein and troponin C might have a functional relationship.     

Biosynthesis of Thiamine Pyrophosphate in Escherichia coli

In Escherichia coli, thiamine pyrophosphate is synthesized from thiamine monophosphate. Free thiamine is not involved as an intermediate in de novo synthesis of thiamine pyrophosphate.     

Mutations in the leucine zipper motif and sterol-sensing domain inactivate the Niemann-Pick C1 glycoprotein.

Niemann-Pick type C (NPC) disease, characterized by accumulation of low density lipoprotein-derived free cholesterol in lysosomes, is caused by mutations in the NPC1 gene. We examined the ability of wild-type NPC1 and NPC1 mutants to correct the NPC sterol trafficking defect and their subcellular localization in CT60 cells. Cells transfected with wild-type NPC1 expressed 170- and 190-kDa proteins. Tunicamycin treatment resulted in a 140-kDa protein, the deduced size of NPC1, suggesting that NPC1 is N-glycosylated. Mutation of all four asparagines in potential N-terminal N-glycosylation sites to glutamines resulted in a 20-kDa reduction of the expressed protein. Proteins with a single N-glycosylation site mutation localized to late endosome/lysosomal compartments, as did wild-type NPC1, and each corrected the cholesterol trafficking defect. However, mutation of all four potential N-glycosylation sites reduced ability to correct the NPC phenotype commensurate with reduced expression of the protein. Mutations in the putative sterol-sensing domain resulted in inactive proteins targeted to lysosomal membranes encircling cholesterol-laden cores. N-terminal leucine zipper motif mutants could not correct the NPC defect, although they accumulated in lysosomal membranes. We conclude that NPC1 is a glycoprotein that must have an intact sterol-sensing domain and leucine zipper motif for cholesterol-mobilizing activity.     

Resonance Raman study of D-amino acid oxidase-inhibitor complexes

The resonance Raman (RR) spectra of the complexes of D-amino acid oxidase (DAO) with benzoate derivatives were measured. The RR spectra of complexes of DAO with benzoate derivatives excited at 514.5 nm are similar to one another and also similar to that of oxidized flavin. In the cases of DAO-o-NH2-benzoate and DAO-o-OH-benzoate complexes, however, the line at 568 or 565 cm-1, derived from the benzoate derivative, was intensified. In the case of DAO-o-NH2-benzoate complex, which has an intense charge-transfer absorption band, the resonance enhancement of the Raman lines at 1583 and 568 cm-1 in the RR spectrum excited at 632.8 nm is striking. The former line is known to involve the vibrational displacements of the N(5) and C(4a) atoms of isoalloxazine and the latter is considered to be derived from a ring deformation mode of o-NH2-benzoate. This suggests that the o-NH2-benzoate molecule lies along the N(5)-C(4a) bond and parallel to the flavin face. A Raman line derived from o-OH-benzoate in the RR spectrum of DAO-o-OH-benzoate complex excited at 514.5 nm was detected. This result supports the view that the complex has a charge-transfer band, as has been pointed out by Massey and Ganther. Also, the spectrum of quasi-DAO-o-OH-benzoate complex is identical with that of the complex of DAO, suggesting that the active sites of these two enzymes have similar structures.