A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

A Comparison of the Efficacy of Nuclear Polyhedrosis and Granulosis Viruses in Spray and Bait Formulations for the Control of Agrotis segetum (Lepidoptera: Noctuidae) in Maize

Agrotis segetum nuclear polyhedrosis virus (AsNPV) and granulosis virus (AsGV), propagated in laboratory cultures of A. segetum in England and A. ipsilon in Spain, respectively, were applied to plots of maize plants at the one‐ to four‐leaf stage of growth. Plots were arranged in a 6 x 6 Latin square design and infested with second‐instar A. segetum larvae (the common cutworm). Each virus was applied in separate treatments by two application methods; as an aqueous spray containing 0.1% Agral as a wetting agent, and as a bran bait. The NPV was applied at a rate of 4 X 10¹² polyhedra/ha, and the GV at 4 X 10¹³ granules/ha. Soil and plants were sampled for larvae on three occasions following virus treatment: 24 h, 4 days and 11 days. The larvae were reared on diet in the laboratory, until death or pupation, to examine the rate and level of viral infection. Infection data showed 87.5% and 91% NPV infection and 12.5% and 55% GV infection in spray and bait treatments, respectively, in larvae sampled 24 h after treatment. In larvae sampled 4 days after treatment, the results were 78% and 100% NPV infection, and 13% and 6% GV infection. A total of only six larvae were retrieved on day 11. In both treatments larvae infected with AsNPV died significantly more rapidly and at an earlier instar than those infected with AsGV, indicating that AsNPV appears to have better potential as a control agent for A. segetum.     

Metabolic regulation of rice α-amylase and sucrose synthase genes in planta

Isolated rice embryos were used to investigate the regulatory effects of endosperm extracts and pure sugars on the expression of alpha-amylase gene RAmy3D and a sucrose synthase gene homologous to the maize isozyme Ss2. The high-level expression of RAmy3D in the scutella of isolated embryos could be inhibited by a variety of sugars as well as endosperm extracts from germinated rice grains. Glucose, at a concentration of 250 mM, was most effective in repressing RAmy3D mRNA accumulation. Furthermore, this repression was reversible. Interestingly, RAmy3D repression was always accompanied by the induction of sucrose synthase gene expression. These results support a model in which the expression of alpha-amylase and sucrose synthase genes in the rice scutellum are counter-regulated by the influx of sugars from the endosperm.     

Characterization of envelope proteins of infectious bovine rhinotracheitis virus (bovine herpesvirus 1) by biochemical and immunological methods.

Ten glycoproteins of molecular weights of 180,000, 150,000, 130,000, 115,000, 97,000, 77,000, 74,000, 64,000, 55,000, and 45,000 (designated as 180K, 150K, etc.) and a single nonglycosylated 107,000-molecular-weight (107K) protein were quantitatively removed from purified bovine herpesvirus 1 (BHV-1) virions by detergent treatment. Immunoprecipitations with monospecific and monoclonal antibodies showed that three sets of coprecipitating glycoproteins, 180K/97K, 150K/77K, and 130K/74K/55K, were the major components of the BHV-1 envelope. These glycoproteins were present in the envelope of the virion and on the surface of BHV-1-infected cells and reacted with neutralizing monoclonal and monospecific antibodies. Antibodies to 150K/77K protein had the largest proportion of virus-neutralizing antibodies, followed by antibodies to 180K/97K protein. Monoclonal antibodies to 130K/74K/55K protein were neutralizing but only in the presence of complement; however, monospecific antisera produced with 55K protein did not have neutralizing activity. Analysis under nonreducing conditions showed that the 74K and 55K proteins interact through disulfide bonds to form the 130K molecule. Partial proteolysis studies showed that the 180K protein was a dimeric form of the 97K protein and that the 150K protein was a dimer of the 77K protein, but these dimers were not linked by disulfide bonds. The 107K protein was not glycosylated and induced antibodies that did not neutralize BHV-1. The 64K protein was not precipitated by anti-BHV-1 convalescent antisera, and monospecific antisera to this protein precipitated several polypeptides from uninfected cell lysates, suggesting that 64K is a protein of cellular origin associated with the BHV-1 virion envelope.     

The distribution of N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid alpha-naphthyl acetate esterase and alpha-naphthyl acetate esterase in subpopulations of thymocytes, bone marrow cells and other lymphoid organs in mice

Thymocytes, bone marrow lymphocytes, as well as lymphocytes from spleen, lymphoid nodes and peripheral blood were obtained from BALB/c mice. Subpopulations of BALB/c bone marrow T-lymphocyte precursors and immature (small) and mature (large) thymocytes, as established by the percentage of terminal deoxynucleotidyl transferase (TdT) and peanut agglutinin (PNA) positive cells, were obtained by centrifugation on discontinuous density gradients. The activities of N-acetyl-beta-glucosaminidase (NAG), beta-glucuronidase (BG), acid alpha-naphthyl acetate esterase (ANAE) and alpha-naphthyl acetate esterase (NAE) were determined by enzymatic assays of cell extracts of the diverse T-lymphocyte subpopulations, in order to follow their evolution with the maturation of the T-lymphocytes in the thymus. These activities were compared with that determined in lymphocytes from spleen, lymphoid nodes and peripheral blood. The glucidases BG and NAG and the esterases ANAE and NAE present a high decrease in their activities from bone marrow T-lymphocyte progenitors to immature thymocytes. BG, NAG and ANAE activities undergo an about 3-fold increase with the evolution of the thymocytes from small to large cells. Whereas the level of the NAE activity decreases (2-fold) with that evolution of the thymocytes. Lymphocytes from spleen and lymphoid nodes exhibit activities of the glucidases and, specially, the esterases marked by higher than those of thymocyte populations. Peripheral blood lymphocytes also present NAG, ANAE and NAE activities higher than in thymocytes, but their BG activity is lower.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.     

Correlation between ventilation and brain blood flow during hypoxic sleep

Ventilation and brain blood flow (BBF) were simultaneously measured during carbon monoxide (CO) inhalation in awake and sleeping goats up to HbCO levels of 40%. Unilateral BBF, which was continuously measured with an electromagnetic flow probe placed around the internal maxillary artery, progressively increased with CO inhalation in the awake and both sleep stages. The increase in BBF with CO inhalation during rapid-eye-movement (REM) sleep (delta BBF/delta arterial O2 saturation = 1.34 +/- 0.27 ml X min-1 X %-1) was significantly greater than that manifested during wakefulness (0.87 +/- 0.14) or slow-wave sleep (0.92 +/- 0.13). Ventilation was depressed by CO inhalation during both sleep stages but was unchanged from base-line values in awake goats. In contrast to slow-wave (non-REM) sleep, the ventilatory depression of REM sleep was primarily due to a reduction in tidal volume. Since tidal volume is more closely linked to central chemoreceptor function, we believe that these data suggest a possible role of the increased cerebral perfusion during hypoxic REM sleep. Induction of relative tissue alkalosis at the vicinity of the medullary chemoreceptor may contribute to the ventilatory depression exhibited during this sleep period.