Mass fragmentographic determination of lofepramine and its metabolites in human plasma and urine using deuterated internal standards

A sensitive and specific method for the determination of lofepramine and its metabolites, desipramine and 2-hydroxydesipramine, in human plasma and urine is described. Lofepramine, desipramine and 2-hydroxydesipramine were derivatized to ethyl p-chlorobenzoate, the bis(heptafluorobutyryl) derivative and the N,O-bis(trifluoroacetyl) derivative, respectively, and then analysed by gas chromatography—mass fragmentography. Corresponding deuterated compounds were used as internal standards. Determination was possible at levels as low as 2 ng/ml for lofepramine and desipramine and 20 ng/ml for 2-hydroxydesipramine.     

The asparagine-linked sugar chains of the glycoproteins in rat erythrocyte plasma membrane—Fractionation of oligosaccharides liberated by hydrazinolysis and structural studies of the neutral oligosaccharides

The asparagine-linked sugar chains of the plasma membrane glycoproteins of rat erythrocytes were released as oligosaccharides by hydrazinolysis and labeled by NaB³H₄ reduction. The radioactive oligosaccharides were separated into a neutral and at least four acidic fractions by paper electrophoresis. The neutral oligosaccharide fraction was separated into at least 11 peaks upon Bio-Gel P-4 column chromatography. Structural studies of them by sequential exoglycosidase digestion in combination with methylation analysis revealed that they were a mixture of three high mannose-type oligosaccharides and at least 11 complex type oligosaccharides with Manα1 → 6(Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4(±Fucα1 → 6)GlcNAc as their cores and Galβ1 → 4GlcNAc, Galβ1 → 3Galβ1 → 4GlcNAc, and various lengths of Galβ1 → 4GlcNAc repeating chains in their outer chain moieties. Most of the complex-type Oligosaccharides were biantennary, and the tri- and tetraantennary Oligosaccharides contain only the Galβ1 → 3Galβ1 → 4GlcNAc group in their outer chain moieties.     

Different responses of CR(−) and CR(+) B cells to LPS stimulation

Proliferative response of B cells with or without CR [CR(+) or CR(?) B cells] was compared in their polyclonal response when they were stimulated with lipopolysaccharide (LPS). CR(+) B cells responded better in proliferation and more poorly in polyclonal antibody formation than did CR(?) B cells. The dissociation between proliferation and antibody formation in LPS response was not due to the shift of the time kinetics nor the exhaustion of the culture medium. T cells and macrophages did not take part in the dissociation, since macrophage depletion from nu/nu mouse spleen cells could not modify the dissociation. The polyclonal antibody response of CR(?) B cells was more resistant to irradiation than that of CR(+) B cells. These results suggest that among LPS-responsive B cells there are CR(?) B-cell subset(s) more mature than CR(+) B cells.     

Identification of Elastase as a Secretory Protease from Cultured Rat Microglia

In the course of studying the secretory products of microglia, we detected protease activity in the conditioned medium. Various proteins (casein, histone, myelin basic protein, and extracellular matrix) were digested. The protease activity was characterized by using purified myelin basic protein as a substrate. Maximal activity was observed at neutral pH levels (7-8), which was different from the optimum pH level of proteolytic activity observed in the cell homogenate. The activity was inhibited approximately 60 and 50% by 1 mM phenylmethylsulfonyl fluoride and 40 microM elastatinal, respectively. In gel filtration, the major activity, which was inhibited in the presence of N-methoxysuccinyl-Ala-Ala-Pro-Val-methyl chloride, eluted at a position corresponding to a molecular mass of approximately 25 kDa. These results suggest that the major protease present in microglial conditioned medium is elastase or an elastase-like protease. This suggestion was confirmed by the finding that the 25-kDa protein band was stained with anti-elastase antiserum by western blotting. De novo synthesis of elastase in microglia was supported by [35S]methionine incorporation. In the presence of lipopolysaccharide, the secretory elastase decreased. These results demonstrate that microglia secrete proteases, one of which was identified as elastase. The significance of this enzyme production in physiological and pathological conditions is discussed.     

Development of the Formula-Detoxification System during Adaptation to Air of Submerged Rice Seedlings

Changes in activities of enzymes and levels of antioxidant substratesinvolved in the -detoxification system in seedlings of rice (Oryza sativa L. cv. Yamabiko) inresponse to variations in the oxygen environment were studied.Activities of superoxide dismutase, ascorbate peroxidase, monodehydroascorbatereductase, dehydroascorbate reductase, glutathione reductaseand catalase, expressed either on the basis of fresh weightof shoots or relative to levels of soluble protein were muchlower in seedlings germinated under water for 6 days than inthose germinated in air for the same period of time. When submergedseedlings were exposed to air, the activities of these six enzymesincreased to or exceeded the levels in aerobically grown controlsduring 24 h of adaptation to air. Ascorbate and glutathione,which act as antioxidant substrates in the -detoxification system, were present in submergedseedlings at nearly the same levels as those found in aerobicallygrown controls. On exposure of submerged seedlings to air, thelevel of ascorbate in creased slightly, but the level of glutathioneshowed a rapid increase, reaching 7 times that in aerobicallygrown controls within 12 h of adaptation to air. Levels of allsix antioxidative enzymes and of two substrates involved inthe detoxification of the superoxide radical increased withincreases in oxygen tension in the environment. Moreover, thedevelopment of this system consisted of two steps, namely, arapid increase in the level of glutathione and a subsequentslow increase in the activities of antioxidative enzymes. ¹ Present address: Research Institute for Bioresources, OkayamaUniversity, Kurashiki, 710 Japan.     

1H NMR studies on ferricytochromec 3 fromDesulfovibrio vulgaris Miyazaki F and its interaction with ferredoxin I

Summary The¹H NMR signals of the heme methyl, propionate and related chemical groups of cytochromec ₃ fromDesulfovibrio vulgaris Miyazaki F (D.v. MF) were site-specifically assigned by means of ID NOE, 2D DQFCOSY and 2D TOCSY spectra. They were consistent with the site-specific assignments of the hemes with the highest and second-lowest redox potentials reported by Fan et al. (Biochemistry,29 (1990) 2257–2263). The site-specific heme assignments were also supported by NOE between the methyl groups of these hemes and the side chain of Val¹⁸. All the results contradicted the heme assignments forD.v. MF cytochromec ₃ made on the basis of electron spin resonance (Gayda et al. (1987)FEBS Lett.,217 57–61). Based on these assignments, the interaction of cytochromec ₃ withD.v. MF ferredoxin I was investigated by NMR. The major interaction site of cytochromec ₃ was identified as the heme with the highest redox potential, which is surrounded by the highest density of positive charges. The stoichiometry and association constant were two cytochromec ₃ molecules per monomer of ferredoxin I and 10⁸ M^–2 (at 53 mM ionic strength and 25°C), respectively.     

Respiration and Motility in Starfish Spermatozoa at Various Times in the Breeding Season

In the spermatozoa of Asterias amurensis , patterns of changes in the respiratory rate and motility following dilution of dry sperm in sea water varied among batches and were classified into three types. The type I spermatozoa were immotile and exhibited quite low respiratory rate. Ethylenediamine tetraacetic acid made the type I spermatozoa motile and elevated their respiratory rate. The type II spermatozoa were also immotile and the respiratory rate remained quite low for about 5 min after the dilution. Thereafter, they spontaneously became motile and the respiratory rate increased. The type III spermatozoa became motile upon their dilution and exhibited high respiratory rate. Differences in the motility and respiratory rate of spermatozoa among batches probably result from degree of their maturation. In moving spermatozoa, the ADP and AMP levels increased at the expense of ATP. 2,4-Dinitrophenol elevated the respiratory rate only in immotile spermatozoa, which showed a high ATP level and quite low ADP level, but did not made them motile. Oligomycin inhibited the respiration of both motile and immotile spermatozoa. Probably, the respiratory rate is made low by a shortage of ADP in immotile spermatozoa and is enhanced by ADP production due to the initiation of their movement.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis: 3. Frequent occurrence of genetic variants in some abundant polypeptides of PHA-stimulated peripheral blood lymphocytes

Summary The 100 or so most intensely Coomassie blue-stained polypeptides from PHA-stimulated peripheral blood lymphocytes were analyzed by two-dimensional electrophoresis in combination with family and population studies. Besides polymorphic lymphocyte cytosol 64k polypeptide reported previously, genetic variants were frequently observed in three polypeptides with molecular weights of 100,000, 49,000, and 40,000. All of them occur in the cytosol. These variant polypeptides are charge variants, because they are separated in the isoelectric focusing dimension. It is indicated by family and population studies and cell distribution analysis that the polypeptide with a molecular weight of 100,000 shows a genetic polymorphism determined by two alleles at a new autosomal locus, as described in the following paper. Family and population studies also suggest that a genetic polymorphism defined by alleles at an autosomal locus is present in each of the polypeptides with molecular weights of 49,000 and 40,000. In contrast to the previous reports of the extremely restricted genetic variability of the 100 or so most abundant fibroblast polypeptides, the present data indicate that common genetic variants are present at least in four of the 100 or so most intensely Coomassie blue-stained lymphocyte polypeptides. The result also shows that careful side-by-side comparison of two-dimensional electrophoresis patterns among both parents and their children is an effective method to detect genetic variant polypeptides.     

Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.