Quantitative Isolation of Undegraded Polysomes from Aged Pea Tissue in the Absence of Contaminants and Artefacts

Undegraded polysomes were isolated successfully from aged peaepicotyls by grinding frozen tissue in at least 10 volumes ofbuffer A (0.2 M Tris-HCl, pH 8.5; 0.2 M sucrose; 60 mM KCl;30 mM MgCl₂), taking care to prevent the tissue from thawingprior to homogenization. Supposedly pure polysomes, derivedfrom the membrane-bound polysome fraction, were apparently contaminatedwith membranes, and contained polysomes clumped together vianascent chains. Problems with contaminants and artefacts werepartially alleviated by the use of polyoxyethylene tridecylether as a detergent replacing Triton X-100; further alleviatedby the use of large volumes of detergent-containing buffer toresuspend the membrane-bound polysome; and almost completelyeliminated by brief treatment of resuspended polysomes withprotease K. Optimal conditions for isolating polysomes fromaged tissue are given. ¹ Present address: Institute of Agricultural Environment Control,College of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama790, Japan. (Received April 24, 1985; Accepted September 2, 1985)     

Phylogenetic Survey of Proteins Related to Synapsin I and Biochemical Analysis of Four Such Proteins from Fish Brain

A phylogenetic survey of proteins immunologically related to Synapsin I, a major synaptic vesicle-associated phosphoprotein in mammals was carried out. Proteins antigenically related to Synapsin I were found by use of radioimmunoassay and other radioimmunochemical techniques in the nervous systems of several vertebrate and invertebrate species, which included birds, reptiles, amphibians, fish, echinoderms, arthropods, and mollusks. Four proteins present in fish brain, antigenically related to Synapsin I, were further studied and found to resemble mammalian Synapsin I in several respects. Like Synapsin I, the fish proteins were present in high amounts in nervous tissue, were enriched in synaptosomal fractions of brain where they were substrates for endogenous protein kinases, were acid extractable, and were sensitive to digestion by collagenase. In addition, two-dimensional peptide-mapping analysis revealed some homology between major phosphopeptide fragments of Synapsin I and the fish proteins. The results indicate that proteins related to Synapsin I are wide-spread in the animal kingdom.     

Substitution of selenocysteine for cysteine in a reticulocyte lysate protein synthesis system

Selenocysteine occurs in the peptide backbone of several selenoenzymes. The mechanism, of selenocysteine incorporation has not been well characterized. The incorporation of selenocysteine into protein in a rabbit reticulocyte lysate (RRL) was studied at high levels of selenocysteine. [⁷⁵Se]Selenocysteine incorporation was inhibited by cycloheximide and by nuclease treatment. Random RNA copolymers were tested for protein synthesis activity in the messenger RNA-dependent RRL system. Of the active polymers, poly CIU and GU most strongly stimulated the incorporation of selenocysteine. In a series of four polymers with different ratios of U to G, incorporation of selenocysteine and cysteine increased with increasing percentages of U, suggesting that selenocysteine and cysteine responded to the same codon, presumably UGU. Of the 20 protein amino acids, only cysteine and cystine competed with selenocysteine incorporation. Selenocysteine was charged to cysteine-accepting tRNA in RRL. These results show that at supraphysiological concentrations selenocysteine can substitute for cysteine in RRL protein synthesis. Misincorporation of selenocysteine could be important when animal tissues contain high levels of selenium.     

Effects in chicks of arsenic,arginine, and zinc and their interaction on body weight,plasma uric acid,plasma urea,and kidney arginase activity

The interaction among arsenic, zinc, and arginine was studied in chicks using two fully crossed, three-way, two-by-two-by-two experiments. Arsenic at levels of 0 and 2 μg/g zinc at levels of 2.5 (zinc-deficient) and 25 (zinc-adequate) μg/g, and arginine at levels of 0 and 16 mg/g were supplemented to the diet. After 28 d in both experiments, growth was depressed in chicks fed diets either supplemented with arginine or deficient in zinc. Arsenic deprivation depressed growth of chicks fed diets containing the basal level of arginine and 25 μg supplemental Zn/g. Arsenic deprivation had little or no effect on growth of zinc-deprived chicks fed diets containing the basal level of arginine, or in zinc-deprived or zinc-adequate chicks fed supplemental arginine. Zinc-deficiency elevated urea in plasma and arginase activity in kidney. Those elevations, however, were more marked in arsenic-supplemented than in arsenic-deprived chicks. Also, plasma urea and kidney arginase activity were markedly elevated in chicks fed supplemental arginine; the elevations were more marked in zinc-deficient chicks. These findings support the concept that arsenic has a physiological role, associated with zinc, that can influence arginine metabolism in the chick.     

A PIF-dependent recombinogenic signal in the mitochondrial DNA of yeast

From their recombination properties, tandem rho^- mutants of the mitochondrial genome of Saccharomyces cerevisiae were divided into two categories. In crosses between PIF-independent rho^- and rho⁺ strains, the recombination frequency is low and similar in PIF/pif and pif/pif diploids. In crosses between PIF-dependent rho^- and rho⁺ strains, the recombination frequency is stimulated 10-50 times in PIF/pif diploids and is drastically decreased in pif/pif diploids. These results suggest that a recombinogenic signal is present in the mitochondrial (mt) DNA of PIF-dependent rho^- clones. This signal is not recognized in pif mutants. Sequence analysis of a series of small (<300 bp) overlapping tandem rho^- genomes located in the ery region of the 21S rRNA gene led us to identify an essential element of this signal within a 41-bp A+T sequence exhibiting over 26 bp a perfect dyad symmetry. However the recombinogenic signal is not sequence-specific since the sequence described above does not characterize PIF-dependent rho^- clones located in the oli1 region. Our results rather suggest that the recombinogenic signal is related to the topology of rho^- DNA. Denaturated sites in the double helix or cruciform structures elicited by local negative supercoiling might be preferred sites of the initiation of recombination.     

Double blind comparative study of omeprazole 10 mg and 30 mg daily for healing duodenal ulcers.

Healing of duodenal ulcers was assessed in 66 patients who received omeprazole either 10 mg or 30 mg daily for four weeks in a double blind study. Healing was rapid in both groups. At two weeks the ulcers in 15 of the 30 patients taking 10 mg daily had healed compared with 28 of the 36 (78%) taking 30 mg daily (p less than 0.03). At four weeks the respective proportions had risen to 83% (25/30) and 94% (33/35) (p greater than 0.05). In non-smokers the proportion of ulcers healed did not differ significantly with the two doses, although there was a trend for less healing at two weeks with 10 mg daily; in smokers significantly fewer ulcers (p less than 0.05) were healed with 10 mg than 30 mg daily at two weeks (7/16 (44%) v 17/21 (81%] and at four weeks (12/16 (75%) v all 21 (100%]. Adverse reactions were few and transient and were considered unlikely to be due to omeprazole.     

Intergenic suppression of spoO phenotypes by the Bacillus subtilis mutation rvtA

Summary A collection of intergenic suppressors of the Bacillus subtilis spoOF221 mutation has been isolated. One of these suppressors, rvtA, has been mapped between lys-1 and aroD. The rvtA suppressor restores spoOF sporulation to wild type levels and substantially improves the sporulation efficiencies of spoOB and spoOE strains. The rvtA gene does not affect the Spo phenotype of spoOH, spoOJ or spoOK mutants. The rvtA gene also prevents the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.     

A monoclonal antibody to the embryonic myosin heavy chain of rat skeletal muscle

A monoclonal antibody, 2B6, has been prepared against the embryonic myosin heavy chain of rat skeletal muscle. On solid phase radioimmunoassay, 2B6 shows specificity to myosin isozymes known to contain the embryonic myosin heavy chain and on immunoblots of denatured contractile proteins and on competitive radioimmunoassay, it reacts only with the myosin heavy chain of embryonic myosin and not with the myosin heavy chain of neonatal or adult fast and slow myosin isozymes or with other contractile or noncontractile proteins. This specificity is maintained with cat, dog, guinea pig, and human myosins, but not with chicken myosins. 2B6 was used to define which isozymes in the developing animal contained the embryonic myosin heavy chain and to characterize the changes in embryonic myosin heavy chain in fast versus slow muscles during development. Finally, 2B6 was used to demonstrate that thyroid hormone hastens the disappearance of embryonic myosin heavy chain during development, while hypothyroidism retards its decrease. This confirmed our previous conclusion that thyroid hormones orchestrate changes in isozymes during development.     

Electron microscopic localization of neuron-specific enolase in rat and mouse brain

The cellular distribution and intracellular localization of neuron-specific enolase (NSE) has been studied by electron microscopic immunocytochemistry in the brain of the rat and of the mouse. Although the intensity of staining was less in the mouse, the same structures were positive in both species. In the cerebrum, the neuronal perikarya and dendrites were intensely stained, but staining was almost entirely absent in the presynaptic terminals. The deep neurons of the brain stem were also positive. In the cerebellum, perikarya, axons, and parallel fibers of the granule cell neurons were stained as were the synaptic vesicles and presynaptic membranes of the synapses between the parallel fibers and the Purkinje cell dendrites. Golgi cell dendrites, basket cells and their axons, and mossy fibers were also positive. In contrast, the Purkinje cells including their dendrites, and the climbing fibers that formed synapses with the Purkinje cell dendrites were not stained. The majority of the myelinated axons in both the cerebrum and the cerebellum did not stain, but the fibrillary astrocytic processes between myelinated axons in the white matter did. Oligodendroglia, protoplasmic astrocytes, Bergmann glia, astrocytes investing capillaries, and vascular endothelial cells were negative for reaction product. In the positively staining cells and their processes, the positivity was dispersed throughout the cytoplasm and corresponded most closely to the distribution of ribosomes, the granular endoplasmic reticulum, and microtubules. Nuclei, mitochondria, the cisternae of the Golgi complex, myelin lamellae, and most membranes were not stained.