Distribution and development of the serotonin-and RFamide-like immunoreactive neurons in the arrowworm,Paraspadella gotoi (Chaetognatha)

Summary The distribution and development of serotonin-and RFamide-like immunoreactivities in the nervous system of Chaetognatha, Paraspadella gotoi, were examined in whole-mount preparations. In adults, a single serotonin-like immunoreactive (5HTLI) neuron and numerous RFamide-like immunoreactive (RFaLI) neurons were found in the central nervous system. Based on the structure of the fins, hooks, and eyes, seven postembryonic developmental stages were recognized. The most obvious features of the stages are: stage 1, newly hatched young; stage 2, elongation of a continuous lateral tail fin; stage 3, separation of the lateral and tail fins; stage 4, appearance of hooks; stage 5, pigmentation of eyes, stage 6, attachment by tail adhesive fins; stage 7, prey capture. Stage 1 did not show any immunoreactivity. The 5HTLI neuron first appeared at stage 4 and its axonal pathway became similar to the adult at stage 6. On the other hand, the RFaLI neurons appeared at stage 3 in the ventral ganglion. Some of their somata disappeared at stage 5 and the neuronal architecture resembled the adult at stage 7 although the RFaLI neurons in the cerebral ganglion were complete at the juvenile stage.We are sad to announce that Dr. M. Yoshida died on 29 October 1988     

Properties of invertase immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber membrane

Invertase was ionically immobilized on the poly(ethylene-co-vinyl alcohol) hollow fiber inside surface, which was aminoacetalized with 2-dimethylaminoacetaldehyde dimethyl acetal. Immobilization and enzyme reaction were carried out by letting the respective solutions pass or circulate through the inside of the hollow fiber, and the activity of invertase was determined by the amount of glucose produced enzymatically from sucrose. Immobilization conditions were examined with respect to the enzyme concentration and to the time, and consequently the preferable conditions at room temperature were found to be 5 mug/mL of enzyme concentration and 4 h of immobilization time. Under those conditions the immobilization yield and the ratio of the activity of the immobilized invertase to that of the native one were 89 and 80%, respectively. For both repeating and continuous usages, the activity fell to ca. 60% of the initial activity in the early stage and after that almost kept that value. The apparent Michaelis constant K(m) (') for the immobilized invertase decreased with increasing the flow rate of the substrate solution, to be close to the value for the native one. Furthermore, the possibility of the separation of the enzymatically formed glucose from the reaction mixture through the hollow fiber membrane was preliminarily examined.     

Formation of giant protoplasts from protoplasts of Pleurotus cornucopiae by the cell wall lytic enzyme

Summary When purified protoplasts of Pleurotus cornucopiae IFO9614 were incubated with a mixture of cell wall lytic enzymes, they were found to increase their size. Their average diameter increased from 4.3 m to 31 m after 65 h incubation at 24° C. The presence of cellulase ONOZUKARS in the enzyme mixture had a significant effect on the formation of giant protoplasts. Regeneration frequency of giant protoplasts in a medium containing 0.5 M sucrose was 3.5%, approximately six times that of normal protoplasts.     

Immunoreactive glucagon in the vascular walls of the rat

We detected immunohistochemically immunoreactive glucagon (IRG) in the smooth muscle of blood vessels and the myoepithelial cell of sweat glands of rats using two antisera against pancreatic glucagon; OAL-123 and 30K. The content of IRG in the blood vessels was found to be 320-1, 270 pg per g wet tissue weight. Filtration of the extracted IRG through a Bio Gel P-30 column yielded a single peak of IRG at 3,500 daltons, the same elution volume of pancreatic glucagon. These findings suggest that the blood vessels of the rats is one of the extrapancreatic sources of IRG in plasma, although physiological role of the IRG is not known.     

Concentrations of essential elements after repeated administrations of tin and selenium

To study effects of simultaneous administration of tin (Sn) and selenium (Se) on concentrations of several essential elements, mice were injected with either SnCl2 (ip) or Na2SeO3 (sc), alone or both compounds at a daily dose of 5 mumol/kg each for 12 consecutive days. Mice were sacrificed at 20 h after the last injection and concentrations of Sn, Se, Na, Ca, Zn, P, Fe, K, and Mg in the liver, kidney, spleen, pancreas, testis, seminal vesicle, lung, femoral muscle, and femoral bone were determined. In the control mice, Sn and Se concentrations were the highest in bone (0.69 micrograms Sn and 6.93 micrograms Se/g dry wt). Administered Sn was found to accumulate in all organs except the testis. Among the essential elements determined, Na was the most affected in terms of concentration in the organs and Mg was the least affected element in these organs. Among the organs tested, each elemental concentration in the pancreas was most affected. Simultaneous injections of Sn and Se appeared to keep the correlation coefficients between elements similar to those found in the control mice.     

Acid alpha-glucosidase deficiency: Identification and expression of a missense mutation (S529V) in a Japanese adult phenotype

We report a missense mutation in an adult Japanese patient with acid alpha-glucosidase (GAA) deficiency. A TC to GT transition at nucleotides 1585–1586, was identified. This transition resulted in an amino acid substitution of Ser-529 to Val (S529V) in exon 11. We also have demonstrated that the S529V mutation abolishes the catalytic activity of the enzyme. Our data suggest that this mutation is the cause of the clinical manifestation known as adult-onset GAA deficiency. The missense mutation described here is a new mutation, and the first identified in Japanese patients with GAA deficiency. Received: 23 May 1995     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Metallothionein mRNA in the testis and prostate of the rat detected by digoxigenin-labeled riboprobe

Metallothionein (MT), a cysteine-rich heavy metal-binding protein, has been considered to play a role in the homeostatic control and detoxification of heavy metals, such as zinc, copper, and cadmium. In the present study, we have utilized a digoxigenin-labeled riboprobe to localize MT mRNA only by bright-field optics in the testis and prostate of the rat. In the rat testis, MT mRNA was found predominantly in primary spermatocytes and also in secondary spermatocytes and spermatids, but not in the spermatogonia, Sertoli cells, and Leydig cells. On the other hand, MT protein was present in these spermatogenic cells as well as in spermatozoa and Sertoli cells. In the prostate, MT mRNA was found predominantly in the epithelium of the dorsolateral lobes, but not in the ventral lobe, which is in agreement with the observed localization of MT protein. The utilization of both in situ hybridization and immunohistochemical staining on the same tissue specimens show MT gene expression in specific cell types in the male genital organs.     

Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency: Identification of point mutations in Japanese patients with Lesch-Nyhan syndrome and hereditary gout and their permanent expression in an HPRT-deficient mouse cell line

Two different single nucleotide transitions of hypoxanthine-guanine phosphoribosyltransferase (HPRT) were identified in a Japanese patient with Lesch-Nyhan syndrome (LNS) and a patient with hereditary gout. HPRT enzyme activities in the two patients were severely deficient, but the size and amount of mRNA were normal according to Northern analysis. Entire coding regions of HPRT cDNAs were amplified by PCR and sequenced. A G-to-A substitution at base 208 in exon 3, which predicted glycine 70 to arginine, was detected in the LNS patient (identical mutation with HPRT_Utrecht). A C-to-A substitution at base 73 in exon 2, which predicted proline 25 to threonine, was detected in the gout patient (designated HPRT_Yonago). We transfected normal HPRT cDNA, mutant cDNA with HRPT_Utrecht or mutant cDNA with HPRT_Yonago, respectively, to HPRT-deficient mouse cells and isolated permanent expression cell lines. The HPRT-deficient mouse cells had no detectable HPRT activity and a very low amount of HPRT mRNA. When the HPRT-deficient mouse cells were transfected with normal human cDNA, HPRT enzyme activity increased to 21.8% that of normal mouse cells. The mouse cells transfected with HPRT_Utrecht showed no increase in HPRT activity; however, when the mouse cells were transfected with HPRT_Yonago, the activity increased to 2.4% that of normal activity. The proliferative phenotypes of these cells in HAT medium and in medium containing 6-thioguanine were similar to those of skin fibroblasts from the patients. This series of studies confirmed that each of the two point mutations was responsible for the decreases in HPRT enzyme activity, and the proliferative phenotypes in HAT medium and medium containing 6-thioguanine.     

Is there an energy conservation “system” in brain that protects against the consequences of energy depletion?

A poorly understood marked decrease (circa 50% of control) in local cerebral glucose utilization is caused by sublethal doses of NaCN. The decrease is global, occurring in essentially all brain regions and is entirely reversible within hours, leaving no obvious pathology. This event is not unique to NaCN in so far as a strikingly similar pattern of decreased glucose utilization occus with some other toxins. Nor can it be attributed to a direct action of NaCN since local application by microdialysis to the striatum produces a global depression. These results imply that some widely distributed system or substance is involved. We speculate the existence of a system possibly related to the reticular activating system that senses a fall in energy production and acts globally to make cells quiescent and thus would give some protection from excitotoxic driven damage.Special issue dedicated to Dr. Sidney Ochs.