Immunocytochemical location of vitellin in the egg of the silkworm,Bombyx mori,during early developmental stages

Summary Vitellin was purified from eggs of the silkworm,Bombyx mori, by a new method in which vitellin was extracted from isolated yolk granules. The purified vitellin had a molecular weight of 540,000. An antibody against purified vitellin was prepared in rabbits. It reacted with the hemolymph vitellogenin as well as with purified vitellin, but not with other proteins in the hemolymph or in the extract from yolk granules. The anti-vitellin IgG was used to immunocytochemically locate vitellin in theBombyx non-diapause egg during early developmental stages. In the egg, just after oviposition, vitellin was located in internal yolk granules and in small yolk granules of the periplasm. During the early developmental stages studied, vitellin was not metabolized uniformly throughout the egg. The vitellin of the internal yolk granules located at the posterior-dorsal part and of the small peripheral yolk granules was utilized in 16 h and 2 days, respectively, after oviposition. A thin, very vitellin-poor layer was located between the periplasm and the vitellin-rich interior in the newly laid egg. it was always in close contact with the periphery where blastoderm and germ-band cells developed.     

Metabolism of lipid and carbohydrate in sea urchin spermatozoa

When the dry sperm of the sea urchin, Hemicentrotus pulcherrimus, were diluted 100 times in artificial sea water at 0°C and at 20°C, they became motile and the levels of ATP and creatine phosphate decreased rapidly. The level of ADP hardly changed, and the AMP level increased after the dilution. After the dilution, the respiratory rate at 2°C was almost one fifth that of 20°C. Both phospholipid and glycogen were used for the energy sources in sea urchin sperm. The level of phospholipid was 10-fold higher than that of glycogen in the dry sperm. The phospholipid level decreased after dilution at 20°C, though the level hardly changed at 0°C, suggesting that phospholipid was hardly metabolized the lower temperature. The level of α -glycerophosphate increased at 20°C after the dilution but did not change at 0°C. The level of glycogen decreased after the dilution, regardless of the temperature. The glycolysis was also activated after the dilution. Of the intermediates of the tricarboxylic acid cycle, the citrate concentration increased at 0°C and the malate concentration also increased at 0°C and especially strongly at 20°C.     

Localization and characterization of phosphatidylcholine in sea urchin spermatozoa.

Sea urchin spermatozoa obtain energy for movement through oxidation of endogenous phospholipids, particularly phosphatidylcholine (PC). This study was undertaken to determine the localization of PC available for utilization in energy metabolism in spermatozoa of the sea urchin, Hemicentrotus pulcherrimus. Following incubation with sea water, the PC content in sperm heads decreased significantly, while that in sperm tails did not change. PC was abundant in sperm heads, particularly the midpieces. PC composed of unsaturated fatty acids was consumed to a greater extent during incubation than that consisting of saturated fatty acids. Analysis by gas-liquid chromatography indicated most of fatty acid moieties in the midpieces PC to be unsaturated. Phospholipase A2 activity was also distributed in sperm heads, particularly the midpieces. It thus appears that PC as a substrate for energy metabolism is located in the midpieces of sea urchin spermatozoa.     

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Karyology of four land-planarian species of the genus Bipalium from Japan

We have employed a new scale for characterizing chromosomal forms in the karyotypes of four species of Bipalium from five localities in Japan. Specimens of Bipalium nobile Kawakatsu et Makino, 1982, from Yokohama had a diploid chromosome number of 2x = 10 (2m + 2sm + 2sm + st & sm + 2sm); specimens of the same species from Toyonaka had this number as well but with slightly different chromosomal form (2m + 2sm + sm & st + 2st + m & sm). An undescribed species from Sanjô, Bipalium sp. 2, with two dorsal stripes and a yellow head crescent, had 2x = 10 (2m + 2sm + 2sm + 2sm + 2m); and another undescribed species from Chichijima Island, Bipalium sp. 3, with five dorsal stripes, had 2x = 10 (2m + 2sm + 2sm + 2sm + 2m). A non-sexual bipaliid tentatively identified as Bipalium kewense Moseley, 1878, from Chichijima Island had 2x = 18 (2m + 2m + 2m + 2sm + 2st + 2sm + 2sm + 2sm + 2sm).     

Conversion of Tyrosine Hydroxylase to Stable and Inactive Form by the End Products

We have reported previously that tyrosine hydroxylase in the crude extract from rat striatum exists in the inactive form showing almost no activity at the physiological pH and that the inactive form is produced by the action of the end products of the enzyme, such as dopamine. The incubation of the enzyme with the end products resulted in not only the inactivation but also a remarkable stabilization of the enzyme. Catechols possessing amino groups but no negatively charged groups on the side chains (catecholamine-type catechols) were effective at a concentration as low as 10(-7) M in both the inactivation and stabilization of the enzyme. In contrast, catechols not possessing positively or negatively charged side chains (3,4-dihydroxyphenylethyleneglycol-type catechols) were ineffective at a concentration of 10(-7) M but effective at a concentration of 10(-6) M for both the inactivation and stabilization. Catechols possessing negatively charged groups (3,4-dihydroxyphenylacetic acid-type catechols) were ineffective even at a concentration of 10(-6) M. Thus, the end products of tyrosine hydroxylase appear to serve to keep the enzyme inactive and stable. The reaction mechanism of the conversion of the enzyme from the active/labile form to the inactive/stable form by dopamine was also investigated.     

Lan-1: A Human Neuroblastoma Cell Line With M1 and M3 Muscarinic Receptor Subtypes Coupled to Intracellular Ca2+ Elevation and Lacking Ca2+ Channels Activated by Membrane Depolarization

The LAN-1 clone, a cell line derived from a human neuroblastoma, possesses muscarinic receptors. The stimulation of these receptors with increasing concentrations of carbachol (CCh; 1-1,000 microM) caused a dose-dependent increase of the intracellular free Ca2+ concentration ([Ca2+]i). This increase was characterized by an early peak phase (10 s) and a late plateau phase. The removal of extracellular Ca2+ reduced the magnitude of the peak phase to approximately 70% but completely abolished the plateau phase. The muscarinic-activated Ca2+ channel was gadolinium (Gd3+) blockade and nimodipine and omega-conotoxin insensitive. In addition, membrane depolarization did not cause any increase in [Ca2+]i. The CCh-induced [Ca2+]i elevation was concentration-dependently inhibited by pirenzepine and 4-diphenylacetoxy-N-methylpiperidine methiodide, two rather selective antagonists of M1 and M3 muscarinic receptor subtypes, respectively, whereas methoctramine, an M2 antagonist, was ineffective. The coupling of M1 and M3 receptor activation with [Ca2+]i elevation does not seem to be mediated by a pertussis toxin-sensitive guanine nucleotide-binding protein or by the diacylglycerol-protein kinase C system. The mobilization of [Ca2+]i elicited by M1 and M3 muscarinic receptor stimulation seems to be dependent on an inositol trisphosphate-sensitive intracellular store. In addition, ryanodine did not prevent CCh-induced [Ca2+]i mobilization, and, finally, LAN-1 cells appear to lack caffeine-sensitive Ca2+ stores, because the methylxanthine was unable to elicit intracellular Ca2+ mobilization, under basal conditions, after a subthreshold concentration of CCh (0.3 microM), or after thapsigargin.     

Chemical modification of coenzyme B12-dependent diol dehydrase with pyridoxal 5′-phosphate: Lysyl residue essential for interaction between two components of the enzyme

The apoenzyme of diol dehydrase was inactivated by modification with pyridoxal 5′-phosphate (pyridoxal-P). The inactivation was accompanied by appearance of a new peak at 425 nm which was shifted to 325 nm by reduction with NaBH₄. ?-N-Pyridoxyl lysine was detected by paper chromatography and paper electrophoresis from the hydrolysate of the NaBH₄-reduced enzyme-pyridoxal-P complex. The relationship of inactivation vs pyridoxal-P incorporation as well as kinetic experiments suggests that one lysyl residue per enzyme molecule was essential for catalytic activity, although two to three pyridoxal-P molecules were introduced into the almost completely inactivated enzyme molecule. Both 1,2-propanediol (substrate) and adenosylcobalamin (coenzyme) completely protected the enzyme from inactivation. The result of disc gel electrophoresis showed that the inactivation of diol dehydrase by pyridoxal-P results from irreversible dissociation of the enzyme into subunits upon pyridoxal-P modification. Therefore, it is suggested that this modifiable lysyl residue is essential for subunit interaction to form an active oligomeric enzyme. The inactivated enzyme restored activity by addition of excess component F, but not by S, suggesting that the essential lysyl residue is located in component F of the enzyme. Pyridoxal-P-modified enzyme was no longer able to bind cyanocobalamin (a competitive inhibitor of adenosylcobalamin).     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.