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.     

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): Evidence for gene duplication

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red mombim) was also higher than that of the host fruit infested with A. fraterculus (guava).This research was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnologico (CNPq-PIG 40.2486/82).     

Physical, chemical and spectral properties of leaf surfaces of Berberis aquifolium (Pursh.)

Abstract The leaves of Berberis aquifolium (Pursh.) exhibit either diffuse or specular (shiny) reflection, depending on the variety, but in no case are the leaves obviously glaucous. The dull-surfaced leaves were less wettable than the glossy ones. Using scanning electron microscopy it was determined that the diffuse reflection was due to tubular crystals of wax 250 nm in diameter. The crystals were primarily composed of 19-nonacosanol, a 29-carbon secondary alcohol, as determined by gas chromatography-mass spectrometry. The chemical constituents of the wax underlying the tubes appeared to be the same as those of the wax from glossy leaves, with 29-carbon and 31-carbon n-alkanes and n-heptacosanol as major constituents. The reflection spectra of dull-surfaced (diffuse reflection) or glossy (specular reflection) leaves were the same, as were those of leaves with different amounts of epicuticular wax. Removing the epicuticular wax with chloroform did not change the spectrum.     

Metabolic,physiological and kinetic aspects of the alcoholic fermentation of whey permeate by Kluyveromyces fragilis NRRL 665 and Kluyveromyces lactis NCYC 571

Optimum growth conditions for the fermentation of non-concentrated whey permeate by Kluyveromyces fragilis NRRL 665 have been defined. Use of 3.75 g yeast extract l^?1, a growth temperature of 38°C and a pH of 4.0 allowed a maximum productivity of 5.23 g ethanol l^?1 h^?1 in continuous culture with a yield 91% of theoretical. Complete batch fermentation of permeate with 100 g lactose l^?1 was possible with a maximum specific growth rate of 0.276 h^?1 without any change in ethanol yield. Fermentation of concentrated permeate resulted, however, in a general decrease of specific substrate consumption rate, demonstrated by the inability to completely convert an initial 90 or 150 g lactose l^?1 in continuous culture, even at dilution rates as low as 0.05 and 0.08 h^?1, respectively. The decrease could be related to substrate inhibition, to an increase in osmotic pressure caused by lactose and salts, and to ethanol inhibition of both alcohol and biomass yield. The decrease in specific productivity could be counterbalanced by use of high cell density cultures, obtained by cell recycle of K. fragilis. Fermentation of a non-concentrated permeáte at a dilution rate of 1 h^?1 resulted in a productivity of 22 g l^?1 h^?1 at 22 g ethanol l^?1. Cell recycle using flocculating Kluyveromyces lactis NCYC 571 was also tested. With this strain a productivity of 9.3 g l^?1 h^?1 at 45 g product l^?1 was attained at a dilution rate of 0.2 h^?1, with an initial lactose concentration of 95 g l^?1.     

Distorted segregation and linkage of alcohol dehydrogenase genes in Camellia japonica L. (Theacease)

Alcohol dehydrogenase isozymes in Camellia japonica are encoded by two genes, Adh-1 and Adh-2. Both loci are expressed in seeds, and their products randomly associate into intragenic and intergenic dimers. Electrophoresis of leaf extracts reveals only the products of Adh-2. Formal genetic analysis indicated that the two Adh loci are tightly linked (combined estimate of r=0.004). Most segregations fit expected Mendelian ratios, but in some families distorted segregation was observed at Adh-1, Adh-2, or both loci. The deficient progeny class varied across families, and in two apparent backcrosses three rather than two phenotypic classes were recovered. The mechanism underlying these distortions is not known, but evidence is presented that suggests that the phenomenon is genic or segmental in nature. Plausible hypotheses include linkage of the Adh structural genes with a gametophytic self-incompatibility locus, translocation heterozygosity involving the segment bearing Adh-1 and Adh-2, or a combination of these two mechanisms.     

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

Effects of anoxia and flooding on alcohol dehydrogenase in roots of Glyceria maxima and Pisum sativum

Flood tolerant Glyceria maxima and intolerant Pisum sativum were compared in respect of the effects of anoxia and flooding on the maximum catalytic activities of alcohol dehydrogenase in their roots. Small (<73%) increases in enzyme activity occurred when excised roots of both species were incubated in nitrogen for up to 2 days. Further incubation in nitrogen rapidly and permanently damaged the roots of both species. Enzyme activity in flooded roots of Glyceria was about double that in corresponding non-flooded roots. A marginally greater difference was found for roots of Pisum. It was concluded that the two species respond so similarly to the above treatments that variation in the extent of induction of alcohol dehydrogenase is unlikely to be a significant factor in determining their ability to tolerate flooding.     

In vivo interactions of acrylonitrile with macromolecules in rats

The irreversible binding of [2,3-14C]acrylonitrile (VCN) to proteins, RNA and DNA of various tissues of male Sprague-Dawley rats after a single oral dose of 46.5 mg/kg (0.5 LD50) has been studied. Proteins were isolated by chloroform-isoamyl alcohol-phenol extraction. RNA and DNA were separated by hydroxyapatite chromatography. Binding of VCN to proteins was extensive and was time dependent. Radioactivity in nucleic acids was registered in the liver and the target organs, stomach and brain. DNA alkylation, which increased by time, was significantly higher in the target organs, brain and stomach (119 and 81 pmol/mg, respectively, at 24 h) than that in the liver. The covalent binding indices for the liver, stomach and brain at 24 h after dosing were, 5.9, 51.9 and 65.3, respectively. These results suggest that VCN is able to act as a multipotent carcinogen by alkylation of DNA in the extrahepatic target tissues, stomach and brain.     

A new bacterial alcohol dehydrogenase active on degraded lignin and several low molecular weight aromatic compounds

A new intracellular bacterial dehydrogenase has been purified. It was active in the reversible reduction by NADH of conjugated carbonyl groups in partially degraded lignin. It was also active on various aromatic aldehydes such as vanillin, syringaldehyde and cinnamaldehyde, but had no effect on acetovanillone and lignin models carrying a conjugated ketone. It is proposed that this enzyme functions as a broadly specific lignin dehydrogenase at the level of aldehydic groups that are present in the lignin preparations.