A single polypeptide acts both as the beta subunit of prolyl 4-hydroxylase and as a protein disulfide-isomerase

A single polypeptide is shown to act both as the beta subunit of the proline hydroxylase (EC 1.14.11.2) and as a protein disulfide-isomerase (EC 5.3.4.1). When isolated from chick embryos or rat liver, the beta subunit of prolyl 4-hydroxylase and the enzyme protein disulfide-isomerase have identical molecular weights and peptide maps as produced by digestion with Staphylococcus aureus V8 protease. The apparent molecular weights of both proteins isolated from human placental tissue are slightly higher, and the human beta subunit and one of its peptides have molecular weights about Mr 500 higher than the protein disulfide-isomerase and its corresponding peptide. Experiments with polyclonal and monoclonal antibodies also suggest a structural identity between the two proteins. The beta subunit isolated from the prolyl 4-hydroxylase tetramer has protein disulfide-isomerase activity similar to protein disulfide-isomerase itself, and even the beta subunit when present in the prolyl 4-hydroxylase tetramer has one-half of this activity.     

Serotonin receptor genes 5HT1A and 5HT2A modify the relation between childhood temperament and adulthood hostility

We examined a modifying role of 5HT1A and 5HT2A receptors in the relation between childhood difficult temperament and adulthood hostility in 729 subjects derived from a population-based sample. Subjects were 3–12 years when their childhood temperaments consisting of hyperactivity, low sociability and negative emotionality (i.e. the difficult temperament), were assessed by their mothers. Their adulthood hostility comprising anger, cynicism and paranoia, was measured twice, 17 and 21 years later. It was found that the 5HT1A and 5HT2A receptors were not related to childhood temperament or to adult hostility, but they modified the association between childhood hyperactivity and adult hostility in men. Male carriers of T/T genotype of 5HTR2A who were rated hyperactive by their mothers expressed a high level of hostility, especially that of cynicism, in adulthood. For men with other genetic variants, such an association was not seen. This finding was consistent across the two follow-ups 4 years apart. Further research is needed to clarify whether mother-related hyperactivity adequately describes the temperament of the child or is a reflection of mother's hostile child-rearing attitudes.     

Interchain disulfide bond formation in types I and II procollagen. Evidence for a protein disulfide isomerase catalyzing bond formation

The assembly of reduced pro-alpha chains of type I and type II procollagen into the native triple-helical molecule was examined in vitro in the presence and absence of pure protein disulfide isomerase. The data clearly indicates that protein disulfide isomerase is able to accelerate the formation of native interchain disulfide bonds in these procollagens. It takes about 6 min after disulfide bonding before triple-helical molecules exist, while the time required to produce triple-helical type I procollagen in the presence of protein disulfide isomerase is 9.4 min and that for type II procollagen 17.2 min. These values agree with those obtained for type I and II procollagen in vivo suggesting that protein disulfide isomerase is also an enzyme catalyzing interchain disulfide bond formation in procollagen in vivo. The formation of native disulfide bonds can proceed without any enzyme catalysis but then requires the presence of reduced and oxidized glutathione. Bonding is rather slow in such a case, however, resulting in a delay in the formation of the triple helix.     

Protein disulfide-isomerase retains procollagen prolyl 4-hydroxylase structure in its native conformation

Protein disulfide-isomerase was isolated as a homogeneous protein from 15-day-old chick embryos. The enzyme has a molecular weight of 56,000 in SDS-polyacrylamide gel electrophoresis. Its Km value for randomly cross-linked ribonuclease, a protein used as a substrate for the enzyme, was 0.3 microM, and the Km value for DTT was 1.0 microM. Its optimum pH was 7.5 and its optimum temperature, 33 degrees C. The maximal velocity of pure protein disulfide-isomerase from chick embryos under optimal conditions was about 29,000 units/g. Protein disulfide-isomerase was able to activate purified prolyl 4-hydroxylase 2- to 3-fold, the activation being higher for enzyme stored for a longer time. This activation is probably due to the repairing of disulfide exchanges occurring in the prolyl 4-hydroxylase structure during purification and storage. Prolyl 4-hydroxylase activity was very stable in microsomes, however, and protein disulfide-isomerase was unable to increase the microsomal prolyl 4-hydroxylase activity, suggesting that prolyl 4-hydroxylase retains its native conformation in microsomes. Protein disulfide-isomerase was able to reactivate prolyl 4-hydroxylase inactivated by mild H2O2 treatment. The activity obtained after this treatment and protein disulfide-isomerase incubation corresponded to the amount of prolyl 4-hydroxylase tetramer found after H2O2 treatment. The data suggest that protein disulfide-isomerase is able to activate only the tetramer part of the enzyme preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Apolipoprotein E and A-IV Polymorphisms in Ethnic Russians Living in Estonia

To determine the distribution of genetic variations in apolipoprotein E (apoE) and apolipoprotein A-IV (apoA-IV) genes, 137 Russians living in Estonia was screened by isoelectric focusing and immunoblotting procedures. The apoA-IV-2 allele and apoE4 allele frequency of the Russians tended to be lower than in most other European populations.     

Disulfide bonding as a determinant of the molecular composition of types I, II and III procollagen

Procollagen molecules have amino-terminal and carboxy-terminal propeptides at the respective ends of the collagenous triple helix. The carboxy-terminal propeptides enhance and direct the association of pro alpha-chains into procollagen molecules, but the mechanism of this registration function is still obscure. A hypothesis concerning the function of disulfide bonding in the assembly of types I, II and III procollagen is put forward here.     

Identification of disulfide bonds in carboxy-terminal propeptides of human type I procollagen

Intra-chain and inter-chain disulfide bonds within the carboxy-terminal propeptides of human type I procollagen were studied using cyanogen bromide cleavage of the propeptides and electrophoresis on SDS-polyacrylamide/glycerol gels. The results could provide a better understanding of the assembly of pro alpha-chains into procollagen.     

Cloning of new rubisco promoters from <Emphasis Type="Italic">Brassica rapa</Emphasis> and determination of their activity in stably transformed <Emphasis Type="Italic">Brassica napus</Emphasis> and <Emphasis Type="Italic">Nicotiana tabacum</Emphasis> plants

The aim of our study was to identify the highest expressing rubisco small subunit (RbcS) promoters (pRbcS) from the cotyledons of germinating seedlings of Brassica rapa var. oleifera to drive high-level and preferably stage-specific transgenic protein expression in Brassicaceae plants. We cloned four new pRbcS promoters using several approaches, including the construction of a cDNA library and use of genome walking technique. Real-time PCR analysis of RbcS mRNA expression clearly showed that two of these promoters exhibited the highest activity on the germination stage of plant development. We used gusA expression as a reporter of promoter activity in Brassica napus and Nicotiana tabacum plants that were transformed with the constructs using an Agrobacterium-mediated transformation strategy. The mRNA level of RbcS and of gusA was quantified in transformed plants. The data obtained demonstrate that the promoter most active in seedlings under native conditions was also most active in transgenic constructs at the same stage of plant development. The fine structure of the promoters is discussed herein.     

Phylogenetic analysis of Acacia (Mimosaceae) as revealed from chloroplast RFLP data

 Chloroplast DNA of 22 species of Acacia (Tourn.) Miller was digested with ten restriction endonucleases, Southern-blotted and probed with cloned fragments covering the chloroplast genome of tobacco (Nicotiana tabacum L.). Phyletic and phenetic analyses of the resulting 176 polymorphic bands recorded among the 22 species were performed. The phylogram was reconstructed using heuristic search and Wagner parsimony. The resulting most parsimonious consensus phylogram displayed three major phyletic lineages, consistent with the previously established three subgenera of Acacia. The 10 species of subgenus Acacia and the 6 species of subgenus Heterophyllum formed two monophyletic sister clades. The 5 species of subgenus Aculeiferum studied and Acacia albida (Syn. Faidherbia albida) grouped together and were basal to the clades of subgenera Acacia and Heterophyllum. The phylogram indicated that subgenus Heterophyllum diverged earlier from subgenus Aculeiferum than did subgenus Acacia; however, the phenogram indicated the reverse. The study indicated that A. nilotica and A. farnesiana are sister species, though A. nilotica is Afro-Asiatic and A. farnesiana is American. The phenogram separated the three subgenera in agreement with the phylogram, but the two dendrograms differed regarding the topologies of the species and the distance of evolution between subgenera Acacia and Heterophyllum. Received: 8 July 1998 / Accepted: 24 July 1998