Urine and plasma metabolites predict the development of diabetic nephropathy in individuals with Type 2 diabetes mellitus.

AIMS: Early detection of individuals with Type 2 diabetes mellitus or hypertension at risk for micro- or macroalbuminuria may facilitate prevention and treatment of renal disease. We aimed to discover plasma and urine metabolites that predict the development of micro- or macroalbuminuria. METHODS: Patients with Type 2 diabetes (n = 90) and hypertension (n = 150) were selected from the community-cohort 'Prevention of REnal and Vascular End-stage Disease' (PREVEND) and the Steno Diabetes Center for this case-control study. Cases transitioned in albuminuria stage (from normo- to microalbuminuria or micro- to macroalbuminuria). Controls, matched for age, gender, and baseline albuminuria stage, remained in normo- or microalbuminuria stage during follow-up. Median follow-up was 2.9 years. Metabolomics were performed on plasma and urine. The predictive performance of a metabolite for albuminuria transition was assessed by the integrated discrimination index. RESULTS: In patients with Type 2 diabetes with normoalbuminuria, no metabolites discriminated cases from controls. In patients with Type 2 diabetes with microalbuminuria, plasma histidine was lower (fold change = 0.87, P = 0.02) and butenoylcarnitine was higher (fold change = 1.17, P = 0.007) in cases vs. controls. In urine, hexose, glutamine and tyrosine were lower in cases vs. controls (fold change = 0.20, P < 0.001; 0.32, P < 0.001; 0.51, P = 0.006, respectively). Adding the metabolites to a model of baseline albuminuria and estimated glomerular filtration rate metabolites improved risk prediction for macroalbuminuria transition (plasma integrated discrimination index = 0.28, P < 0.001; urine integrated discrimination index = 0.43, P < 0.001). These metabolites did not differ between hypertensive cases and controls without Type 2 diabetes. CONCLUSIONS: Type 2 diabetes-specific plasma and urine metabolites were discovered that predict the development of macroalbuminuria beyond established renal risk markers. These results should be confirmed in a large, prospective cohort.

Regiospecific phosphohydrolases from Dictyostelium as tools for the chemoenzymatic synthesis of the enantiomers D-myo-inositol 1,2,4-trisphosphate and D-myo-inositol 2,3,6-trisphosphate: non-physiological, potential analogues of biologically active D-myo-inositol 1,3,4-trisphosphate.

A new de novo synthesis of the enantiomeric pair D-myo-inositol 1,2,4-trisphosphate and D-myo-inositol 2,3,6-trisphosphate is described. Starting from enantiopure dibromocyclohexenediol, several C2 symmetrical building blocks were synthesized which gave access to D-myo-inositol 1,2,4,5-tetrakisphosphate and D-myo-inositol 1,2,3,6-tetrakisphosphate. Exploiting the high regiospecificity of two partially purified phosphohydrolases from Dictyostelium, a 5-phosphatase and a phytase, the inositol tetrakisphosphates were converted enzymatically to the target compounds. Their potential to modulate the activity of Ins3,4,5,6P4 1-kinase was investigated and compared with the effects of D-myo-inositol 1,3,4-trisphosphate.

Homologous transformation of Trichoderma hamatum with an endochitinase encoding gene, resulting in increased levels of chitinase activity.

A 42-kDa endochitinase encoding gene, Tham-ch, was cloned by screening the genomic library of Trichoderma hamatum strain Tam-61 with a PCR-amplified chitinase sequence from the same fungus. Tham-ch with its own regulatory sequences was reintroduced into the host strain. The integration of the transforming construct was stable only in one copy. Homologous integration occurred in nine transformants, while non-homologous integration was detected in one transformant. All but one transformant expressed higher levels of chitinase activity in comparison to the wild-type recipient strain; the maximum level of increase was 5-fold. Duplicating the copy number of the highly conserved approximately 42-kDa endochitinase encoding gene appears to be one potential means by which the biocontrol capability of the Trichoderma species might be improved.

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca(2+)-inhibition and metal competence in the double mutant D254E/D256E.

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg2+ as well as ternary complexes with bivalent metal ions and the open-chain inhibitor xylitol were crystallized for x-ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 A resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme.

Study of the alcohol dehydrogenase-1 (Adh1) gene in tetraploid corn: expression in the pollen grains and restriction fragment length polymorphism.

The results of two separate experiment are presented in this paper. In the first experiment pollen assays were carried out in Wf9 tetraploid maize plants in order to decide whether the lack of the ADH enzyme activity in the pollen grains was caused by a 'O' allele or an active transposon. On the basis of the results we suppose that the reduced ADH enzyme activity in the pollen grains is the result of a transposon affecting only the gametophyte. In the second experiment a maize Adh1-S genomic clone was constructed by polymerase chain reaction (PCR) and used as a probe to detect polymoprhism around the Adh1 gene in W117 S18 tetraploid maize sublines. The PCR20-EcoRI clone-enzyme combination resulted in a monomorphic RFLP pattern. The PCR20-Bgl II probe-enzyme combination yielded a multiple-banded pattern.

Transcription of tobacco phytochrome-A genes initiates at multiple start sites and requires multiple cis-acting regulatory elements.

Promoter regions of the Nicotiana tabacum PHYA1 and PHYA2 genes display 89% sequence identity. Analysis of the 5' ends of both the PHYA1 and the PHYA2 transcript revealed multiple, distinct mRNA species, each differing in length and in abundance. The levels of the major PHYA1, PHYA2 transcripts were found to be auto-regulated by phytochrome. This auto-regulation was most efficient in 2-8-day old seedlings. Furthermore, we examined the expression pattern of the PHYA1-GUS reporter gene, containing a 4700 bp PHYA1 promoter fragment or its 5'-deletion derivatives, by GUS histochemistry and by RNase protection assays in transgenic tobacco plants. Our data indicate that the PHYA1 promoter contains three regions which are necessary for the maximum level and regulated expression. We show that a 264 bp promoter fragment contains a cis-regulatory element(s) responsible for expression in the root tips of transgenic seedlings. The major cis-regulatory elements required for high-level transcription and expression in other organs are located in separate regions of the PHYA1 promoter. These data indicate the contribution of multiple cis-regulatory elements for the maximum and regulated expression of tobacco genes coding for phytochrome A protein.

Phenotype of the fission yeast cell cycle regulatory mutant pim1-46 is suppressed by a tobacco cDNA encoding a small, Ran-like GTP-binding protein.

Mutations in which the onset of mitosis is uncoupled from the completion of DNA replication has recently been described. Characterization of these mutants led to the identification of Pim1/Spi1 in fission yeast and RCC1/Ran proteins in mammalian cells. Their Saccharomyces cerevisae homologues, the MTR1/CNR1 proteins, appear to be involved in controlling RNA metabolism and transport. Here the isolation and partial characterization of plant cDNA clones which encode proteins homologous to the mammalian/fission yeast/budding yeast Ran/Spi/CNR proteins are reported. Higher plants appear to contain more than one gene per haploid genome which codes for Ran proteins. These genes are expressed in different plant tissues, including root tips and stems, known to contain mitotically active cells. The tobacco Ran-like proteins, like their mammalian and yeast homologues, are soluble proteins which are found in the cytoplasm and in the nucleus. In addition, it has been shown that overexpression of the tobacco Nt-Ran-A1 cDNA suppressed the phenotype of the temperature-sensitive fission yeast pim1-46 mutant. These results suggest that the plant Ran genes can be functionally equivalent to the mammalian/fission yeast/budding yeast Ran/Spi/CNR genes and that they may play a role: (i) in maintaining a coordinated cell cycle; (ii) in controlling RNA metabolism and transport in higher plants; and/or (iii) in protein import into the nucleus.

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins.

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.

The isolated N-terminal DNA binding domain of the c repressor of bacteriophage 16-3 is functional in DNA binding in vivo and in vitro.

The 197 amino acid c repressor of the temperate Rhizobium meliloti phage 16-3 still regulates the OR operator of the phage after removal of its carboxyl terminal region. When cloned in the low-copy-number plasmid pGA46, a severely truncated variant (R1-77), which retains only the first 77 amino acids of the intact protein, repressed in vivo transcription from the phage promoter PR. When the R1-77 repressor was fused to E. coli beta-galactosidase, the hybrid protein bound OR operator DNA in vitro. The behavior of fusion proteins derived from a point mutant is consistent with the assignment of DNA binding specificity to the amino-terminal region. Furthermore two repressor alleles bearing ts mutations that mapped in the R1-77 region (near a helix-turn-helix motif) were also temperature sensitive for regulation of the OR site, while an 18 bp "in frame" deletion mutant, which mapped in the carboxyl terminal segment, regulated the OR operator in wild-type fashion. The carboxyl terminal region of the repressor is however necessary for the control of lysogenic development of 16-3.

Does isoflurane lead to a higher incidence of myocardial infarction and perioperative death than enflurane in coronary artery surgery? A clinical study of 1178 patients.

To examine if the choice of volatile agents influences cardiac outcome in coronary artery surgery, 1178 patients undergoing elective coronary artery bypass grafting without additional operations received enflurane (608) or isoflurane (570) as their primary anesthetics. The inspired concentration of volatile agent (administered with 50% nitrous oxide) was adjusted depending on the level of blood pressure at the discretion of the anesthesiologist. In addition to the volatile agent assigned, each patient received small doses of fentanyl at induction and before sternotomy (total 0.006-0.008 mg/kg). The groups did not differ in preoperative and surgical characteristics except for a more frequent history of renal dysfunction in patients given isoflurane. The rates of postoperative myocardial infarction, administration of positive inotropic agents at the time of weaning from cardiopulmonary bypass, and in-hospital deaths in the enflurane and isoflurane groups were 1.8% and 4.0% (P less than 0.05), 4.9% and 8.1% (P less than 0.05%), and 0.3% and 2.1% (P less than 0.01), respectively. Although the mechanism of the adverse effects of isoflurane could not be clarified in this study, these results demonstrate that the use of isoflurane could be inappropriate in patients undergoing coronary artery bypass grafting.

A gene affecting accumulation of the RNA moiety of the processing enzyme RNase P.

The level of 10Sb (M1) RNA, the RNA of RNase P, is very low in growing cultures of rnpB mutants. Northern transfer experiments suggested that these strains accumulate no more than 10% of the wild-type level of 10Sb RNA. However, there is no indication that there is a limiting amount of RNase P activity in these mutants in vivo. A plasmid that directs the synthesis of 10Sb RNA does not complement the rnpB mutants, even though there is only a single gene for 10Sb RNA in the Escherichia coli genome. The 10Sb RNA synthesized from this plasmid is equivalent to wild-type 10Sb RNA since it can replace it in the reconstitution of RNase P. The 10Sb RNA, which is a rather stable molecule, is unstable in the presence of the rnpB mutation. This could explain why rnpB mutants do not accumulate 10Sb RNA. An F' plasmid that contains DNA from the rnpB region of the chromosome complements an rnpB mutant in vivo and in vitro, and it also contains the 10Sb RNA gene. A number of possible explanations for these phenomena are discussed.

The detailed physical map of the temperate phage 16-3 of Rhizobium meliloti 41.

Restriction cleavage maps for enzymes EcoRI, BamHI, PstI, PvuII, XbaI and EcoRV of Rhizobium meliloti temperate phage 16-3 have been established. Together with the earlier maps (HindIII, KpnI, HpaI, BglII) 98 restriction sites, 'evenly' distributed, have been mapped along the phage genome, including the so far unmarked silent region of the chromosome. All the restriction maps have been fitted to each other by computer optimalization. Beyond for conventional techniques a computer program (PMAP) for physical mapping of linear DNA has been employed which made the experimentation, in several cases, extremely efficient.

Heterozygosis of phage 16-3 of Rhizobium meliloti: moderate level of mismatch repair or gene conversion.

Analysis of clear/turbid mottled (heterozygotic plaques) of Rhizobium meliloti temperate phage 16-3 indicates that the efficiency of repair at three sites (ti3, ti4, and ti5) in the C cistron is 2 to 20-fold less than that observed in E. coli phage lambda. In agreement with this conclusion, heterozygotic plaques were observed at similar frequency in crosses where point and small deletion mutants were combined, suggesting that in Rhizobium, DNA molecules with short single-stranded loops can escape from repair as efficiently as the simple mismatches.

Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm.

The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI. The strategy employed for mapping included the analysis of double, triple and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage. Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.