Molecular properties of the red cell calcium pump: II. Effects of calmodulin,proteolytic digestion and drugs on the calcium-induced membrane phosphorylation by ATP in inside-out red cell membrane vesicles

In inside-out human red cell membrane vesicles /IOV/, in the absence of Mg²⁺, the only calcium-induced labelling by γ³²P-ATP occurs in a 140–150 000 molecular weight protein fraction, representing the hydroxylamine-sensitive phosphorylated intermediate /EP/ of the calcium pump. In the presence of Mg²⁺ calcium-induced phosphorylation is accelerated but several other membrane proteins are also phosphorylated through protein kinase action forming hydroxylamine-insensitive bonds. Addition of calmodulin accelerates EP formation both in the absence and presence of Mg²⁺.Treatment of the membrane with SH-group reagents significantly reduces EP formation. Mild trypsin digestion of IOVs, stimulating active calcium transport, eliminates calmodulin action and decreases the steady-state level of EP. In trypsin-digested IOVs the molecular weight of the ³²P-labelled EP is shifted to lower values /110–120 000/ We suggest that trypsin digestion cleaves off a 20–40 000 molecular weight calmodulin-binding regulatory subunit of the calcium pump molecule.     

A study of the role of histidine side-chains at the active centre of amylolytic enzymes

The role of histidine side-chains in reactions catalysed by porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase has been studied by using diethyl pyrocarbonate, a specific protein reagent. Changes in the activity, binding affinity, and apparent kinetic parameters due to ethoxycarbonylation have been determined. For pancreas alpha-amylase, four of the eight histidine groups, for sweet-potato beta-amylase, six of the seven histidine groups, and for A. niger glucamylase, four of the six histidine groups were shown to be ethoxycarbonylated. Ethoxycarbonylation occurred as an apparent first-order reaction, with rate constants in the range 3.6–4.9 x 10^?2min^?1. Ethoxycarbonylation of the histidine group at the active centre rapidly inactivated alpha-amylase, whereas the other three groups are not located in the active centre, although activity and substrate binding are only slightly affected by their modification. For beta-amylase and glucamylase, only slight or no change in activity could be detected on ethoxycarbonylation, whereas significant changes were observed in the binding affinity.     

Evidence of the heterogeneity of mast cell population based on their biogenic amine content

The heterogeneity of the mast cells localized in various organs has been demonstrated by histochemical determination in their indole amine and histamine content. The strongest amine reaction was found in the mast cells in the mesenterium and peritoneal fluid where their maturation occurs. The reaction of the subcutaneous connective tissue is weaker; in the thyroid gland the reaction is weak as well as diffuse. In the thymus the mast cells localized along the vessels give a strong histamine reaction. The experiments support previously published data concerning the heterogeneity of mast cell populations.     

Nuclear totipotency of cultured rabbit morulae to support full-term development following nuclear transfer.

The rabbit was used as a model for nuclear transfer. A critical step in nuclear transfer is oocyte activation, which was evaluated in this research. Optimal field strength of an electric stimulus for activation was examined. A significantly higher activation rate in all criteria tested was achieved when oocytes were activated electrically with a field strength of 2.4 kV/cm versus 1.2 or 1.8 kV/cm. Also, electrical stimulation with combined alternating current (AC) and direct current (DC) was superior to DC stimulation alone for activation. In another study involving 586 oocytes, exposure of oocytes to cytochalasin B for 1 h followed by activation with electrical stimulation significantly improved development of the oocytes to blastocyst stage compared to oocytes without cytochalasin B pre-exposure (38% vs 26%, p less than 0.05). Cytochalasin B exposure alone (control), however, had no effect on activation. Exposing oocytes to activation medium without electrical stimulation also activated some oocytes. In the nuclear transfer experiment, blastomeres from 8-cell embryos cultured for 20-24 h to the 32-64-cell stage were used as nuclear donor cells. Of 491 oocytes used, 459 (93%) survived the enucleation and fusion procedure, 370 (81%) fused, and 284 (77%) developed into 2-4-cell embryos. A total of 243 of these 2-4-cell embryos were transferred to 15 pseudopregnant recipients and produced 8 young (3%). Although the efficiency is low, this study demonstrated that rabbit morulae cultured for 20-24 h to the 32-64-cell stage as nuclear donors for transfer remain totipotent.     

Involvement of the phosphoinositol (PI) system in the mechanism of hormonal imprinting

Certain components of the phosphoinositol (PI) system are present in the unicellular Tetrahymena. Treatment of Tetrahymena with insulin did not alter the relative proportions of the examined six phospholipid components (PIP2, PIP, phosphatidylcholine, phosphatidylethanolamine, PI, PA), but the primary interaction (imprinting) with insulin accounted for an about 75% decrease in the PIP2-level and an about 20% increase in the phosphatidylethanolamine level. The experimental results strongly suggest that hormonal imprinting accounted for adjustment of the second messenger systems of Tetrahymena to an energy saving level.     

The effect of cytokinins on nitrate reductase activity

Cytokinins in addition to nitrate induce nitrate reductase activity (NRA) in some plants. Effects of cytokinins onNRA was investigated in stem pith parenchyma of kale, intact wheat and barley seedlings and isolated cucumber cotyledons. The most profound effect onNRA was found in barley and wheat seedlings.NRA in seedlings sprayed with 100 μM 6-benzylaminopurine (BAP) for three subsequent days was increased in leaves and decreased in roots. These changes were further enhanced in seedlings grown in nutrient solution lacking nitrate:NRA in wheat and barley leaves was increased by 57% and 202%, respectively, in plants supplied with nitrate theNRA increase was not significant: in wheat and barley leaves by 22% and 9%, respectively. Similar effect of BAP and kinetin was found in kale stem parenchyma and cucumber cotyledons. The cytokinin kinetin or BAP alone increasedNRA about twice in kale and three times in cucumber. Addition of nitrate to the medium enhanced the effect of kinetin in kale discs, but the two effects were not additive. Additive effect of nitrate and BAP onNRA was found in cucumber cotyledons in light. In general NRA was more affected by cytokinins in intact seedlings of wheat and barley as compared to explanted tissue of kale and cucumber, and lack of nitrogen made their effect more expressive.     

Complex cytophotometric analysis of hormone-induced alterations in the glucose utilisation of Tetrahymena.

Insulin and adrenaline were both found to stimulate the glucose utilization of Tetrahymena, although somewhat differently. With exogenous sugar added, adrenaline always drops the glucose level in all regions of the unicellular, while the effect of insulin was less unequivocal.     

Conservation and chromosomal localization of DNA satellites in balenopterid whales

DNA satellites were isolated from three balenopterid species, viz. the minke, sei, and fin whales. In each of them at least two DNA satellites were recognizable with buoyant densities in neutral CsCl of =1.702/1.703 and =1.710/1.711, respectively. cRNAs from each satellite group were used for filter and in situ hybridisations. Homo- and heterologous DNA-cRNA hybrids within each satellite group yielded virtually identical melting curve profiles showing conservation of at least a considerable part of the DNA satellite sequences. There was no evident sequence homology between the =1.702/1.703 and the =1.710/1.711 satellites by filter hybridisation. — The in situ hybridisation showed that in each species the =1.702/1.703 satellite was located in centromeric-paracentromeric C-bands in a few pairs, whereas the =1.710/1.711 satellite was located in terminal C-bands throughout the karyotypes. — The data on the whale DNA satellites indicate that the quantitative evolution of the satellite DNA sequences preceded species divergence of the balenopterids and that the satellite sequences have remained relatively unaltered since the divergence took place. The function of satellite DNA is considered to imply the introduction of both chromosomal and genic polymorphisms and thus being of great importance in speciation. Based upon these concepts a model is postulated for the function of satellite DNA. According to this model at meiotic pairing euchromatin-heterochromatin overlapping between homologous chromosomes is considered to be of a general occurrence. This overlapping is presumed to be accentuated by the size heteromorphism frequently observed between homologous heterochromatic segments (C-bands). In the region of such euchromatin-heterochromatin overlapping, crossing-over would be excluded. The overlapping is suggested to be rectified progressively in the chromosome arms, leaving unaffected crossing-over distant to the euchromatin-heterochromatin junctions. The consequence of this will be that genes in the proximity of the junctions are collectively inherited and selected, whereas genes distant to the heterochromatin will be independently assorted and selected.