Biochemical genetics of methylglyoxal dehydrogenases in the laboratory rat (Rattus norvegicus)

A genetic locus controlling the electrophoretic mobility of a methylglyoxal dehydrogenase (EC 1.2.1.23) in the rat is described. The locus, designatedMgd1, is expressed in liver and kidney. Inbred rat strains have fixed either alleleMgd1 ^a or alleleMgd1 ^b . Codominant expression is observed in heterozygotes, providing evidence for a tetrameric enzyme structure. Backcross progenies showed the expected 1:1 segregation ratio, and there is evidence thatMgd1 is linked toPep3 andFh1 on chromosome 13. There is also evidence for two additional methylglyoxal dehydrogenases:Mgd2, present in liver and kidney, andMgd3, present only in heart.Supported by the Deutsche Forschungsgemeinschaft (Grant Be 352/18-1).     

Extracellular metabolism of sucrose in a submerged culture of Claviceps purpurea: formation of monosaccharides and clavine alkaloids

Transformation of extracellular sucrose during cultivation of Claviceps purpurea led to the formation of mono- and oligosaccharides. Maltose was a suitable substrate for submerged fermentation of alkaloids. Fermentation in a medium with maltose was characterized by an insignificant formation of glucans, intensive sporulation, suspension growth of mycelium, and a higher formation of elymoclavine. Glucose alone yielded low levels of total alkaloids and high glucan formation; on the other hand, glucose promoted the formation of elymoclavine.     

Nuclear magnetic resonance studies of recombinant Escherichia coli glutaredoxin. Sequence-specific assignments and secondary structure determination of the oxidized form

Escherichia coli glutaredoxin (85 amino acid residues, Mr = 9100), the glutathione-dependent hydrogen donor for ribonucleotide reductase, was purified from an inducible lambda PL, expression system both with a natural isotope content and with uniform 15N labelling. This material was used for obtaining sequence-specific 1H magnetic resonance assignments and the identification of regular secondary structures in the oxidized form of the protein, which contains the redox-active disulfide Cys11-Pro-Tyr-Cys14. Oxidized glutaredoxin contains a four-stranded beta-sheet, with the peripheral strand 32-37 arranged parallel to the strand 2-7, which further combines with the two additional strands 61-64 and 67-69 in an antiparallel fashion. The protein further contains three helices extending approximately from residues 13-28, 45-54 and 72-84.     

Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: beta-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N'-triacetylchitobiose and 6'-O,N,N'-triacetylchitobiose

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (5a) was used as the glycosyl donor in a beta-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6'-O,N,N'-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N'-triacetylchitobiose (4).     

Nucleotide exchange in genomic DNA of rat hepatocytes using RNA/DNA oligonucleotides. Targeted delivery of liposomes and polyethyleneimine to the asialoglycoprotein receptor

Chimeric RNA/DNA oligonucleotides have been shown to promote single nucleotide exchange in genomic DNA. A chimeric molecule was designed to introduce an A to C nucleotide conversion at the Ser365 position of the rat factor IX gene. The oligonucleotides were encapsulated in positive, neutral, and negatively charged liposomes containing galactocerebroside or complexed with lactosylated polyethyleneimine. The formulations were evaluated for stability and efficiency in targeting hepatocytes via the asialoglycoprotein receptor. Physical characterization and electron microscopy revealed that the oligonucleotides were efficiently encapsulated within the liposomes, with the positive and negative formulations remaining stable for at least 1 month. Transfection efficiencies in isolated rat hepatocytes approached 100% with each of the formulations. However, the negative liposomes and 25-kDa lactosylated polyethyleneimine provided the most intense nuclear fluorescence with the fluorescein-labeled oligonucleotides. The lactosylated polyethyleneimine and the three different liposomal formulations resulted in A to C conversion efficiencies of 19-24%. In addition, lactosylated polyethyleneimine was also highly effective in transfecting plasmid DNA into isolated hepatocytes. The results suggest that both the liposomal and polyethyleneimine formulations are simple to prepare and stable and give reliable, reproducible results. They provide efficient delivery systems to hepatocytes for the introduction or repair of genetic mutations by the chimeric RNA/DNA oligonucleotides.     

Mapping of quantitative trait loci for seminal vesicle mass and litter size to rat chromosome 8

The spontaneously hypertensive rat (SHR) and the Brown Norway (BN) rat differ significantly in litter size (7.6 versus 4.5 pups). In the HXB and BXH sets of recombinant inbred (RI) strains derived from SHR and BN rats, heritability of litter size and of selected male reproductive parameters such as sperm production, sperm count, sperm morphology and motility, and the mass of the testis, epididymides, and seminal vesicles were estimated and a search was undertaken for quantitative trait loci (QTL) associated with these phenotypes. The mass of seminal vesicles was significantly associated with a QTL near the D8Cebr204S21 marker on chromosome 8 (LOD score = 4.1, P = 0.00001); this QTL was responsible for 46% of the genetic variability of the trait. The same gene marker on chromosome 8 also showed a suggestive association with the litter size. Litter size was significantly correlated with the mass of seminal vesicles (r = 0.58, P = 0.003). These findings indicate that the variability in litter size among RI strains may be due in part to differences in the mass of seminal vesicles and it is possible that both mass of seminal vesicles and litter size are determined by a pleiotropic effect of the same QTL on rat chromosome 8.     

Molecular characterization of binding of calcium and carbohydrates by an early activation antigen of lymphocytes CD69

CD69 is the earliest leukocyte activation antigen playing a pivotal role in cellular signaling. Here, we show that a globular C-terminal domain of CD69 belonging to C-type lectins binds calcium through Asp 171, Glu 185, and Glu 187 with K(d) approximately 54 microM. Closure of the calcium-binding site results in a conformational shift of Thr 107 and Lys 172. Interestingly, structural changes in all of these amino acids lead to the formation of high-affinity binding sites for N-acetyl-D-glucosamine. Similarly, a structural change in Glu 185 and Glu 187 contributes to a high-affinity site for N-acetyl-D-galactosamine. Site-directed mutagenesis and molecular modeling allowed us to describe the structural details of binding sites for both carbohydrates. These studies explain the importance of calcium for recognition of carbohydrates by CD69 and provide an important paradigm for the role of weak interactions in the immune system.     

Pharmacogenetic evidence that cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor gamma and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-type Cd36. In congenic and transgenic SHR with wild-type Cd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Induction and characterization of an unusual alpha-D-galactosidase from Talaromyces flavus

An extracellular alpha-d-galactosidase from Talaromyces flavus CCF 2686 with extremely broad and unusual acceptor specificity is produced exclusively in the presence of the specific inducer--6-deoxy-D-glucose (quinovose). The procedure for the preparation of this very expensive substance has been modified and optimized. Surprisingly, any of other common alpha-D-galactosidase inducers or substrates, e.g., D-galactose, melibiose and raffinose, did not stimulate its production. The crude alpha-D-galactosidase preparation was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The main isoenzyme (alphaGal1) was further purified by cation-exchange chromatography and fully characterized. When compared with other alpha-galactosidases and also with other isoenzymes produced by T. flavus, it showed a markedly different regioselectivity and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum, guar gum) and significantly inhibited by alpha-D-galactopyranosyl azide, D-galactose, D-xylose, melibiose, methyl alpha- and beta-D-galactopyranoside and lactose.