The Chicago variant of clinical galactosemia

Summary A new variant of clinical galactosemia with two hitherto unidentified alleles on the transferase locus in one family is described. This new clinical variant of transferase has 25% of normal control activity in blood and in skin fibroblasts, and the patient accumulates galactose-1-phosphate in blood on an unrestricted galactose diet. Using starch gel electrophoresis on the hemolysate of the family members, a fast-moving transferase with mobility in between those of the normal control and of the Duarte variant is identified. This new allele is designated as (fast-moving Chicago variant). In addition, a second new allele was documented in this family by studying the instability of the transferase enzyme in hemolysates of family members at 50°C for various time intervals. This new allele is designated as (heat-labile Chicago variant). On the basis of the studies, the transferase genotype of this patients is thought to be a double heterozygote compound, /GALT^G.     

The genetics of phenotypic plasticity. IV. Chromosomal localization

The contributions of each chromosome to the traits thorax size and plasticity of thorax size as affected by temperature in Drosophila melanogaster were measured. A composite stock was created from lines previously subjected to selection on thorax size or plasticity of thorax size. A chromosome extraction was performed against a uniform background lacking genetic variation, provided by a stock of marked balancer flies. With regard to amount of plasticity, chromosome I and the balancer stock showed no plasticity, the composite stock showed the greatest plasticity, and chromosomes II and III were intermediate. Chromosome I showed significant genetic variation for thorax size at both 19° C and 25° C, but not for plasticity, while chromosome II showed significant genetic variation for plasticity, but not for thorax size. Chromosome III showed significant genetic variation for both thorax size and plasticity. We tested the predictions of three models of the genetic basis of phenotypic plasticity: overdominance, pleiotropy, and epistasis. The results support the epistasis model, in agreement with earlier work. The amount of developmental noise was correlated with phenotypic plasticity at 25° C, in agreement with earlier work. A negative correlation was found at 19° C for chromosome II, contrary to earlier work.     

Uncoupling activity and physicochemical properties of derivatives of fluazinam.

The physico-chemical properties and uncoupling activity of eight derivatives of N-phenyl-2-pyridinamines related to the fungicide fluazinam were analyzed using rat liver mitochondria. The uncoupling activity of these compounds relies on the deprotonable secondary amino group. One of the derivatives tested (B-3) was slightly more efficient than fluazinam. By phase-distribution analysis we could show that the N-phenyl-2-pyridinamines are chemicals with moderate hydrophobicity. Deprotonation of the compound reduces the water/octanol partition coefficient by about one order of magnitude. The pKA value of the deprotonable group is affected equally by electron withdrawing substituents of the phenyl- and the pyridinyl-ring, and could be predicted simply from the sum of the Hammett coefficients. The uncoupling efficiency was not dependent on the hydrophobicity of the compound, but appeared to be governed by the pKA of the deprotonable group. This structure/uncoupling characteristic is different from that of the generally more hydrophobic uncouplers of the salicylanilide-type. The pKA resulting in the most efficient uncoupling was found to lie in the range of the pH of the reaction medium. A model based on a solution complexation mechanism, which describes this behaviour, is presented. We conclude that the N-phenyl-2-pyridinamines uncoupled the mitochondria by a simple protonophoric cycle involving protonation/deprotonation in the bulk phase, and that the kinetics of uncoupling were primarily governed by the total concentration of the limiting uncoupler species.     

Mapping of the calpain proteolysis products of the junctional foot protein of the skeletal muscle triad junction

Summary The Ca²⁺ activated neutral protease calpain II in a concentration-dependent manner sequentially degrades the Junctional foot protein (JFP) of rabbit skeletal muscle triad junctions in either the triad membrane or as the pure protein. This progression is inhibited by calmodulin. Calpain initially cleaves the 565 kDa JFP monomer into peptides of 160 and 410 kDa, which is subsequently cleaved to 70 and 340 kDa. The 340 kDa peptide is finally cleaved to 140 and 200 kDa or its further products. When the JFP was labeled in the triad membrane with the hydrophobic probe 3-(trifuoromethyl) 3-(m) [¹²⁵I]iodophenyl diazirine and then isolated and proteolysed with calpain II, the [¹²⁵I] was traced from the 565 kDa parent to M _r, 410 kDa and then to 340 kDa, implying that these large fragments contain the majority of the transmembrane segments. A 70-kDa frament was also labeled with the hydrophobic probe, although weakly suggesting an additional transmembrane segment in the middle of the molecule. These transmembrane segments have been predicted to be in the C-terminal region of the JFP. Using an ALOM program, we also predict that transmembrane segments may exist in the 70 kDa fragment. The JFP has eight PEDST sequences; this finding together with the calmodulin inhibition of calpain imply that the JFP is a PEDST-type calpain substrate. Calpain usually cleaves such substrates at or near calmodulin binding sites. Assuming such sites for proteolysis, we propose that the fragments of the JFP correspond to the monomer sequence in the following order from the N-terminus: 160, 70, 140 and 200 kDa. For this model, new calmodulin sequences are predicted to exist near 160 and 225 kDa from the N-terminus. When the intact JFP was labeled with azidoATP, label appeared in the 160 and 140 kDa fragments, which according to the above model contain the GXGXXG sequences postulated as ATP binding sites. This transmembrane segment was predicted by the ALOM program. In addition, calpain and calpastatin activities remained associated with triad component organelles throughout their isolation. These findings and the existence of PEDST sequences suggest that the JFP is normally degraded by calpain in vivo and that degradation is regulated by calpastatin and calmodulin     

Molecular characterisation of inter and intra-specific somatic hybrids of potato using randomly amplified polymorphic DNA (RAPD) markers

Summary Protoplast fusion allows the transfer of both mono- and polygenic traits between species that are sexually incompatible. This approach has particular relevance for potato, and somatic hybridisation has been used to introduce a range of disease resistance genes from sexually incompatible wild species into the cultivated potato gene pool. In addition, protoplast fusion allows the resynthesis of tetraploid genotypes from pre-selected diploid or dihaploid donor parents. A limiting factor for the efficient exploitation of this technology in potato breeding is the difficulty of unequivocally identifying nuclear hybrids (heterokaryons). In order to facilitate the identification of hybrids at an early stage following fusion, Randomly Amplified Polymorphic DNA markers (RAPDs) have been used to characterise molecularly both inter- and intra-specific somatic hybrids of potato. RAPD markers detect naturally occurring polymorphism in the donor genotypes and utilise short oligonucleotide primers of arbitrary nucleotide sequence in combination with the polymerase chain reaction (PCR). The exploitation of RAPDs in the characterisation of both somatic and sexual hybrids is discussed.     

Assessment of inter- and intra-population variation in quinoline alkaloid profile of juvenile shoot cultures of Cinchona ledgeriana: effect of method of nutrient delivery on alkaloid profile

Intra-population quinoline alkaloid profiles surveying quinine, quinidine, cinchonine and cinchonidine were determined for each of five populations of Cinchona ledgeriana grown as shoot-culture for 125 days. No significant difference in respect of mean alkaloid content between populations was detected. In contrast, there was considerable between-seedling variation in alkaloid content within each population. When nutrients were delivered to shoot-cultures in droplet form by means of an aerosol spray (as compared to the supply of nutrients direct from agar-or liquid-based reservoirs) alkaloid profile was greatly perturbed; most notable in this respect was a four-fold increase in the production of cinchonidine concomitant with a four-fold decrease in the production of cinchonine. These data are discussed with reference to the optimisation of quinoline alkaloid production by juvenile shoot-cultures of Cinchona ledgeriana.     

An investigation of dye reduction by food-borne bacteria

S.A. LEAROYD, R.G. KROLL AND C.F. THURSTON. 1992. The rates of reduction of seven redox dyes by 13 bacterial strains were measured and found to vary greatly between different bacterium/dye combinations. Phenazine ethosulphate and toluidine blue were the most rapidly reduced dyes by the majority of bacteria and resorufin and 2-hydroxy-1,4-naphthoquinone were reduced slowly, if at all. There was also considerable variation in the rates of reduction with any single dye/organism combination. Glucose stimulated the rates of endogenous dye reduction in about half of the organisms. For Bacillus cereus, Pseudomonas fluorescens and Escherichia coli , dye reduction was stimulated by a range of exogenous substrates but lactose, the primary available carbon and energy source in milk, had little effect. In Lactococcus lactis , dye reduction was stimulated by sugars but not by organic acids. Oxygen successfully competed with dye reduction in organisms containing respiratory chains, but with membrane fractions, dye reduction was more rapid than oxygen consumption. All the organisms showed little cytosolic dye reduction, except L. lactis which showed substantial rates of reduction of some dyes by this fraction. With the membrane fraction of E. coli and Ps. fluorescens , cyanide inhibited NADH and succinate-dependent dye reduction, Antimycin A inhibited lactate and succinate and rotenone had no significant effect, but inhibition was not always observed with membrane from both organisms.     

A plant serpin gene. Structure, organization and expression of the gene encoding barley protein Z4

A 3133-bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5' upstream and 523 bp 3' downstream of the coding region. The 1079-bp 5' upstream region of the gene shows little similarity to 5' regions of other sequences genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334-bp-long intron (bases 1497-1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43,128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25-30% and pronounced in the carboxy-terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane-bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36-56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The 'high-lysine' allees, lys1 (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8-kbp fragment of the barley genome Paz psi, similar to the protein-Z4-coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.     

Isolation of a terminal cisterna protein which may link the dihydropyridine receptor to the junctional foot protein in skeletal muscle

The isolated dihydropyridine receptor and junctional foot protein were employed as protein ligands in overlay experiments to investigate the mode of interaction of these two proteins. As previously demonstrated by Brandt et al. [Brandt et al. (1990) J. Membr. Biol. 113, 237-251], the DHP receptor directly binds to an intrinsic terminal cisterna protein of Mr 95,000 (95-kDa protein). The junctional foot protein also binds to an Mr 95,000 protein showing similar organelle distribution to the 95-kDa protein which binds to the dihydropyridine receptor. The 95-kDa protein which binds to the dihydropyridine receptor was isolated to over 85% purity employing sequential column chromatography. Junctional foot protein and dihydropyridine receptor overlays of the column fractions at successive stages of isolation show an identical pattern of distribution, indicating that both probes bind to the same protein. When CHAPS-solubilized terminal cisterna/triads were passed through Sepharose with attached 95-kDa protein, the junctional foot protein was specifically retained, as evidenced by ryanodine binding. The junctional foot protein was incompletely released by 1 M NaCl. The alpha 1 subunit but not the beta subunit of the dihydropyridine receptor was also specifically retained, as evidenced by immunoblotting employing dihydropyridine receptor subunit-specific antibodies. A 170-kDa Stains-all blue staining protein, which appears to be bound to the luminal side of the terminal cisterna, was also retained on the 95-kDa protein column. From these findings, a model for the triad junction is proposed.