Mutational analysis of upstream AUG codons of poliovirus RNA.

The 5' untranslated region of poliovirus type 2 Lansing RNA consists of 744 nucleotides containing seven AUG codons which are followed by in-frame termination codons, thus forming short open reading frames (ORFs). To determine the biological significance of these small ORFs, all of the upstream AUG codons were mutated to UUG. The point mutations were introduced into an infectious poliovirus cDNA clone, and RNA transcribed in vitro from the altered cDNA was transfected into HeLa cells to recover the virus. Mutation of AUG 7 resulted in a virus (called R2-5NC-14) with a small-plaque phenotype, whereas mutation of the other six AUG codons produced virus with a wild-type plaque morphology. To determine whether the small-plaque phenotype of R2-5NC-14 was due to altered translational efficiency of the viral mRNA, we constructed chimeric mRNAs containing the 5' noncoding region of poliovirus mRNA fused to the chloramphenicol acetyltransferase (CAT) coding sequence. mRNA containing a mutated AUG 7 codon showed decreased translational efficiency in vitro. The results indicate that the upstream ORFs of poliovirus RNA are not essential for viral replication and do not act as barriers to the translation of poliovirus mRNA. AUG 7 and flanking sequences may play a positive acting role in poliovirus RNA translation.     

Seasonal variations on water relations ofAmygdalus communis L. under drip irrigated and non irrigated conditions

Almond plants (Amygdalus communis L.) of the Garrigues variety were grown in the field drip irrigated and rainfed. Leaf water potential (Ψ) and leaf conductance (g₁) were determined throughout one growing season. Pre-dawn measurement for Ψ in the irrigated treatment was consistent through the growing season, whereas in the rainfed treatment it decreased gradually. Ψ values at midday (Ψ minimum) was closely dependent on atmospheric evaporative demand, and their recovery was quicker in the wet treatment than in the dry. The g₁ values were higher in the wet than dry treatments, decreasing in both cases by leaf ageing. Maximum values for g₁ were reached when evaporative demand was highest in the day. The relationship between Ψ and g₁ revealed a decrease in the hysteresis throughout the growing season, being most marked in the dry treatment. The results highlight the close dependence of Ψ and g₁ on evaporative demand, leaf ageing and irrigtion treatment during the growing season.     

Stomatal response to leaf water potential in almond trees under drip irrigated and non irrigated conditions

Almond plants (Amygdalus communis L. cv. Garrigues) were grown in the field under drip irrigated and non irrigated conditions. Leaf water potential () and leaf conductance (g₁) were determined at three different times of the growing season (spring, summer and autumn). The relationships between and g₁ in both treatments showed a continuous decrease of g₁ as decreased in spring and summer. Data from the autumn presented a threshold value of (approx. –2.7 MPa in dry treatment, and approx. –1.4 MPa in wet treatment) below which leaf conductance remained constant.     

Characterization of rat skeletal muscle sarcolemmal insulin receptors and a sarcolemmal insulin binding inhibitor

When insulin receptors of rat skeletal muscle sarcolemmal vesicles were solubilized with Triton X-100, the specific binding of 125I-labeled insulin increased by more than 10-fold over that seen in the intact vesicles. Partial purification of the skeletal muscle insulin receptors on wheat germ agglutinin affinity columns increased the total insulin binding activity by 7-fold and reduced the Kd for insulin binding from 1.92 to 0.20 nM, suggesting that an inhibitor of insulin binding was removed by this purification step. This was confirmed when the unbound fractions of the affinity column were dialyzed and reconstituted with the insulin receptors. The inhibitory activity in the sarcolemmal extract could not be accounted for by the presence of Triton X-100. The skeletal muscle inhibitor was more potent in inhibiting insulin binding to skeletal muscle insulin receptors than to liver or adipose receptors. The inhibitor was very effective in inhibiting insulin binding to wheat germ agglutinin-purified IM-9 receptors, but had negligible effects on insulin binding to intact IM-9 cells. The properties of the alpha and beta subunits of the skeletal muscle insulin receptors appear to be the same as those of insulin receptors of other tissues: cross-linking of 125I-labeled insulin to the receptor revealed a band of 130,000 daltons, and insulin stimulated the phosphorylation of bands of 90,000 and 95,000 daltons in the receptor preparation. The skeletal muscle insulin binding inhibitor elutes from molecular sieves in a major 160,000-dalton peak and minor 75,000-dalton peak. The binding inhibitor is not inactivated by heat, by mercaptoethanol, or by trypsin, pepsin, or proteinase K. Collectively, these data suggest that the inhibitor may be a small molecule that aggregates with itself, with larger proteins, or with detergent micelles.     

Rat lens gamma-crystallins. Characterization of the six gene products and their spatial and temporal distribution resulting from differential synthesis

We have isolated, purified and characterized six individual gamma-crystallin polypeptides present in the rat lens. Comparison of their amino acid compositions with the known structure of the six gamma-crystallin genes permits a one-to-one correspondence to be made between each protein synthesized and the encoding gene. This demonstrates that each of the six genes is actually expressed in vivo. Two classes of three gamma-crystallins each, which we have designated classes gamma ABC and gamma DEF, are known to exist, on the basis of internal sequence homology. We have measured the temperature-dependent phase-separation characteristics of solutions of the six purified gamma-crystallins, and find that the three members of the gamma DEF class (gamma 2-2, gamma 3-1 and gamma 4-1) are all cryo-proteins with relatively high phase-separation temperatures, whereas the three gamma ABC crystallins (gamma 1-1, gamma 1-2 and gamma 2-1) do not show phase separation above -7 degrees C. We have measured the spatial distribution in rat lens of each of the alpha-, beta- and gamma-crystallins as a function of age from 1 to 420 days, using size-exclusion and ion-exchange high-pressure liquid chromatography (HPLC). Our findings in the cortical layer permit us to establish the differential synthesis of each of the crystallins during lens development. Particular attention has been devoted to the spatial and temporal distribution of the six individual gamma-crystallins. Up to birth, synthesis of the three components of the gamma DEF class predominates, and in particular that of gamma 2-2. In subsequent development the three components of the gamma ABC class assume a greater proportion of monomeric crystallins synthesized, while beta s-crystallin synthesis predominates in late development. Our analysis of different layers within single lenses provides novel information on spatial gradients of the water-soluble and water-insoluble protein fractions as a function of age. We consider the consequences of these findings for lens transparency and opacity in both rat and mouse lens. We show that the high concentrations of gamma DEF-crystallins appear to be responsible for the opacity known to occur in young rat lenses. We conclude from these observations that close control of the differential synthesis of gamma-crystallins plays an important role in maintaining lens transparency during development.     

Persistent sympathetic nervous system arousal associated with tethering in cynomolgus macaques

The swivel-tether system has been used extensively in biomedical research involving nonhuman primates, yet there has been little or no investigation into potential adverse influences of this form of restraint on research results. In the study described here, a portable electrocardiographic telemetry system was used for continuous monitoring of the heart rate of 26 cynomolgus monkeys while: (a) pair-caged, 8 weeks prior to tethering; (b) singly-caged, tethered; (c) singly-caged, tethered, administered propranolol (30 mg/kg/day) in the diet; (d) group-housed (five monkeys per group), 1 week after group formation; and (e) group-housed (five monkeys per group), 4 weeks after group formation. Tethering resulted in persistent elevations in heart rate relative to the other conditions. Administration of propranolol, a beta-adrenergic antagonist, resulted in an abrupt, sustained decrease in heart rate indicating that the increase in heart rate associated with tethering was due to persistent stimulation of the sympathetic nervous system. Since multiple aspects of cardiovascular function are influenced by the sympathetic nervous system, and other organs and systems (e.g., pituitary-gonadal) also may be affected, investigators using the swivel-tether system should be cognizant of these potential effects when designing experiments and interpreting the results.     

Ten-Year-Replicated Circadian Profiles for 36 Physiological, Serological and Urinary Variables in Healthy Men

At 3-hr intervals over a 24-hr span, 36 systemic, serologic and urinary variables were examined in 7 men in their mid 20's in the Spring of 1969, and again in the same 7 men in the Spring of 1979 under a similar chronobiologic protocol, using the same chemical and numerical analytical procedures. The variables examined for rhythms by cosinor were: vital signs--blood pressure (systolic, diastolic, pulse pressure and mean arterial pressure), heart rate, intraocular pressure (left and right), oral temperature; serum components--albumin, albumin/globulin ratio, total bilirubin, calcium, carbon dioxide, chlorides, bilirubin, cholesterol, globulin, glucose, potassium, sodium, sodium/potassium ratio, transaminase, triglycerides, total protein, urea nitrogen; and urine components--calcium, calcium/magnesium ratio, creatinine, magnesium, pH, potassium, sodium, sodium/potassium ratio, urea clearance, urea nitrogen, volume and zinc. Although all subjects appeared clinically healthy in 1969 and in 1979, certain inter-study differences were observed in a number of rhythm parameters of different variables. Statistically significant increases in mesor for the group as a whole were observed for serum Ca, cholesterol, Cl, CO2, K, Na, and while statistically significant mesor decreases for a group as a whole were noted in serum glucose and transaminase. Statistically significant increases in amplitude for the group as a whole were observed in serum chloride and urinary Na/K ratio, while statistically significant decreases were observed in amplitude for blood pressure, heart rate, serum albumin, A/G ratio, globulin, glucose, protein, sodium and transaminase.(ABSTRACT TRUNCATED AT 250 WORDS)     

95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.     

The intron 7 donor splice site transition: a second Tay-Sachs disease mutation in French Canada

Mutations at the hexosaminidase A (HEXA) gene which cause Tay-Sachs disease (TSD) have elevated frequency in the Ashkenazi Jewish and French-Canadian populations. We report a novel TSD allele in the French-Canadian population associated with the infantile form of the disease. The mutation, a GA transition at the +1 position of intron 7, abolishes the donor splice site. Cultured human fibroblasts from a compound heterozygote for this transition (and for a deletion mutation) produce no detectable HEXA mRNA. The intron 7+1 mutation occurs in the base adjacent to the site of the adult-onset TSD mutation (G805A). In both mutations a restriction site for the endonuclease EcoRII is abolished. Unambiguous diagnosis, therefore, requires allele-specific oligonucleotide hybridization to distinguish between these two mutant alleles. The intron 7+1 mutation has been detected in three unrelated families. Obligate heterozygotes for the intron 7+1 mutation were born in the Saguenay-Lac-St-Jean region of Quebec. The most recent ancestors common to obligate carriers of this mutation were from the Charlevoix region of the province of Quebec. This mutation thus has a different geographic centre of diffusion and is probably less common than the exon 1 deletion TSD mutation in French Canadians. Neither mutation has been detected in France, the ancestral homeland of French Canada.