Measurement of adrenolutin as an oxidation product of catecholamines in plasma

Using the reverse phase high-performance liquid chromatography (HPLC) with mobile phases composed of simple acids, we have developed an assay technique for the measurement of adrenolutin, one of the oxidation products of catecholamines, in rat plasma. Ion-pairing chromatography permits the separation and quantitation of plasma adrenolutin (M) in a linear manner. Sample preparation involved the precipitation of plasma proteins with perchloric acid and it is easier to handle a large number of samples at a time. However, we were unable to demonstrate the presence of adrenochrome, another oxidation product of catecholamines, in plasma since adrenochrome was rapidly destroyed in acid as well as in blood and was quickly changed, into adrenolutin. Adrenolutin peak in HPLC was confirmed by 1) the retention time; 2) co-injection of adrenolutin and; 3) the appearance of ³H-adrenolutin after injection of ³H-norepinephrine. Administration of different catecholamines as well as adrenochrome and adrenolutin in rats also increased the level of adrenolutin in plasma. Adrenolutin was found to be present in plasma in other species including dog, rabbit and pig. High level of adrenolutin, which may represent total concentration of aminolutin in plasma, suggests the presence of an efficient mechanism for the oxidation of catecholamines under in vivo conditions.     

Effect of water activity on enzymatic synthesis of cephalexin

Summary In enzymatic synthesis of cephalexin (CEX) from 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) and D--phenylglycine methyl ester (PGM) using an acylase fromXanthomonas citri, it was found that the synthetic activity and conversion yield were enhanced markedly by depressing the water activity (a _w ) of reaction system. This enhancement was probably resulted from the change of thermodynamic equilibrium and maximized at a range ofa _w from 0.96 to 0.97.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Two subunits of mannose permease, II-PMan and II-MMan, of Escherichia coli mediate coliphage N4 infection

Mutant strains of Escherichia coli that lack one or all of the intact components of mannose permease do not support the growth of phage N4. Complementation experiments using three recombinant plasmids containing DNA fragments coding for the subunits of mannose permease revealed that among the three component subunits, II-PMan and II-MMan alone are sufficient to confer N4 sensitivity.     

Low density lipoprotein and apoprotein B induce increases in inositol trisphosphate and cytosolic free Ca2+ via pertussis toxin-sensitive GTP-binding protein in vascular smooth muscle cells

Low density lipoprotein (LDL), a major cholesterol-carrying lipoprotein in the plasma, binds to its receptor through apoprotein B (Apo-B). The addition of LDL and Apo-B induced rapid (5 s), but transient increase in the inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) level with K0.5 values of 1.1 and 0.07 microgram/ml, accompanied by increases of cytosolic free Ca2+ concentration [( Ca2+]i), in vascular smooth muscle cells (VSMC). The increases by LDL and Apo-B were both reduced by pretreatment of the VSMC with pertussis toxin. The early change in Ins-1,4,5-P3 involving a GTP-binding protein may function as an initial signal for the action of LDL in VSMC.     

Fructose production by Zymomonas mobilis in fed-batch culture with minimal sorbitol formation

Summary Fed-batch cultures of Zymomonas mobilis (UQM 2864), a mutant unable to metabolise fructose, grown on diluted sugar cane syrup (200 g/l sucrose) achieved yields of 90.5 g/l fructose and 48.3 g/l ethanol with minimal sorbitol formation and complete utilization of the substrate. The effect of inoculum size on sorbitol formation in the batch stage of fed-batch fermentation are reported. Fermentation of sucrose (350 g/l) supplemented with nutrients yielded 142 g/l fructose and 76.5 g/l ethanol. Some fructose product loss at high fructose concentrations was observed. The fed-batch fermentation process offers a method for obtaining high concentrations of fructose and ethanol from sucrose materials.     

Distribution of FMRFamide-like immunoreactivity in the brain and neurohypophysis of the lamprey,Lampetra japonica

Summary Distribution of molluscan cardio-excitatory tetrapeptide Phe—Met—Arg—Phe—NH₂ (FMRFamide) was determined by means of immunohistochemistry in the brain and neurohypophysis of the lamprey, Lampetra japonica. Many FMRFamide-like immunoreactive neurons were found in the periventricular nuclear region and in a region near the mammillary recess. Neurons situated in the former region were larger. The immunoreactive cell groups were shown to be located at sites differing from those of the AF-positive cell groups. The fibers of immunoreactive neurons extended in all directions within the brain and towards the spinal cord, some reaching the third ventricle and capillaries. Thus, FMRFamide-like immunoreactive peptides appear to function as neurotransmitters or neuromodulators and possibly also as neurohormones. FMRFamide-like immunoreactive material was rarely observed in the posterior neurohypophysis (neural lobe), but was noted to be present to a limited extent in the caudal part of the anterior neurohypophysis (median eminence). It would thus follow that FMRFamide-like immunoreactive neurons may not necessarily be related to the hypothalamo-neural lobe system, but may possibly be associated with the hypothalamoadenohypophysial system. The pineal body showed no FMRFamide-like immunoreactivity.     

Selective adsorption/desorption of nucleic acids on submicron-sized polymeric particles

DNA can be removed or separated by the selective adsorption/desorption on positively charged submicronsized polymeric particles (SSPP). The selective adsorption of DNA, in the presence of protein, on positively charged SSPP was accomplished by increasing the concentration of potassium phosphate or sodium phosphate. The adsorption of DNA was not affected by the concentration of potassium phosphate or sodium phosphate up to 1.2M. On the other hand, the adsoprtion of a protein (bovine serum albumin) was completely impeded by 170mM potassium phosphate. DNA adsorbed on SSPP could be desorbed by increasing the concentration of NaCl or KCl, thus it can be recovered. DNA desorbed from SSPP when the concentration of NaCl or KC was higher than 0.6M. A complete desorption of DNA was achieved at the concentration of NaCl or KCl above 1.2M.