13C NMR studies of glycogen turnover in the perfused rat liver

To assess whether hepatic glycogen is actively turning over under conditions which promote net glycogen synthesis we perfused livers from 24-h fasted rats with 20 mM D-[1-13C]glucose, 10 mM L-[3-13C]alanine, 10 mM L-[3-13C]lactate, and 1 microM insulin for 90 min followed by a 75-min "chase" period with perfusate of the same composition containing either 13C-enriched or unlabeled substrates. The peak height of the C-1 resonance of the glucosyl subunits in glycogen was monitored, in real time, using 13C NMR techniques. During the initial 90 min the peak height of the C-1 resonance of glycogen increased at almost a constant rate reflecting a near linear increase in net glycogen synthesis, which persisted for a further 75 min if 13C-enriched substrates were present during the "chase" period. However, when the perfusate was switched to the unenriched substrates, the peak height of the C-1 resonance of glycogen declined in a nearly linear manner reflecting active glycogenolysis during a time of net glycogen synthesis. By comparing the slopes of the curve describing the time course of the net [1-13C] glucose incorporation into glycogen with the rate of net loss of 13C label from the C-1 resonance of glycogen during the "chase" period we estimated the relative rate of glycogen breakdown to be 60% of the net glycogen synthetic rate. Whether this same phenomenon occurs to such an appreciable extent in vivo remains to be determined.     

Plasmid transformation of Streptomyces venezuelae: modified procedures used to introduce the gene(s) for p-aminobenzoate synthase

Sucrose was unsuitable as an osmotic stabilizer in buffer solutions and media used for transformation of Streptomyces venezuelae ISP5230. Its replacement with NaCl, together with other modifications in the procedure, allowed efficient formation and regeneration of protoplasts but did not support transformation of S. venezuelae ISP5230 by vectors pIJ41 and pIJ941. With pIJ702, transformants with a low plasmid-copy-number and altered growth characteristics were obtained. Both pIJ702 and pIJ941, but not pIJ41, transformed S. venezuelae 13s; when pIJ941 was used, the plasmid in 18 of 20 transformants contained a deletion in the region reported to code for replication and transfer. The modified plasmid transformed S. venezuelae ISP5230 efficiently and was used to introduce a fragment of DNA from the pab locus of the wild-type into a Cml-1 mutant of ISP5230 blocked in chloramphenicol formation. Transformants that overproduced p-aminobenzoic acid were obtained but they remained blocked in chloramphenicol production; thus, the cloned pab fragment did not contain genes able to complement the cml-1 mutation. The results also suggest that the Cml-1 phenotype is not due to a defective reaction common to the biosynthesis of p-aminobenzoic acid and chloramphenicol.     

Affinity purification of the novel cysteine proteinase papaya proteinase IV, and papain from papaya latex.

A procedure is described for the purification of a previously undetected cysteine proteinase, which we have called papaya proteinase IV, from spray-dried latex of the papaya (Carica papaya) plant. The purification involves affinity chromatography on Gly-Phe-aminoacetonitrile linked to CH-Sepharose 4B, with elution by 2-hydroxyethyl disulphide at pH 4.5. The product thus obtained is a mixture of almost fully active papain and papay proteinase IV, which are then separated by cation-exchange chromatography. A preliminary characterization of papaya proteinase IV showed it to be very similar to chymopapain in both molecular size and charge. However, the new enzyme is immunologically distinct from the previously characterized cysteine proteinases of papaya latex. It also differs in its lack of activity against the synthetic substrates of the other papaya proteinases, in its narrow specificity against protein substrates and its lack of inhibition by chicken cystatin. Papaya proteinase IV is abundant, contributing almost 30% of the protein in spray-dried papaya latex, and contamination of chymopapain preparations with this enzyme may account for some of the previously reported heterogeneity of chymopapain.     

Effects of light on circadian pacemaker development

1. The effects of raising cockroaches, Leucophaea maderae, in non-24 h light cycles on circadian rhythms in adults were examined. The average period (tau) of freerunning rhythms of locomotor activity of animals exposed to LD 11:11 (T22) during post-embryonic development was significantly shorter (tau = 22.8 +/- 0.47 SD, n = 85) than that of animals raised in LD 12:12 (T24) (tau = 23.7 +/- 0.20 h, n = 142), while animals raised in LD 13:13 (T26) had significantly longer periods (tau = 24.3 +/- 0.21 h, n = 65). Animals raised in constant darkness (DD) had a significantly shorter period (tau = 23.5 +/- 0.21 h, n = 13) than siblings raised in constant light (LL) (tau = 24.0 +/- 0.15 h, n = 10). 2. The differences in tau between animals raised in T22 and T24 were found to be stable in DD for at least 7 months and could not be reversed by exposing animals to LD 12:12 or LD 6:18. 3. Animals raised in either T24 or DD and then exposed as adults to T22 exhibited average freerunning periods that were not different from animals not exposed to T22. 4. Measurement of freerunning periods at different temperatures of animals raised in T22, T24, or T26 showed that the temperature compensation of tau was not affected by the developmental light cycle. These results indicate that the lighting conditions during post-embryonic development can permanently alter the freerunning period of the circadian system in the cockroach, but do not affect its temperature compensation.     

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.     

Molecular analysis of mutations in a patient with purine nucleoside phosphorylase deficiency.

Purine nucleoside phosphorylase (PNP) deficiency is an inherited autosomal recessive disorder resulting in severe combined immunodeficiency. The purpose of this study was to determine the molecular defects responsible for PNP deficiency in one such patient. The patient's PNP cDNA was amplified by PCR and sequenced. Point mutations leading to amino acid substitutions were found in both alleles. One point mutation led to a Ser-to-Gly substitution at amino acid 51 and was common to both alleles. In addition, an Asp-to-Gly substitution at amino acid 128 and an Arg-to-Pro substitution at amino acid 234 were found in the maternal and paternal alleles, respectively. In order to prove that these mutations were responsible for the disease state, each of the three mutations was constructed separately by site-directed mutagenesis of the normal PNP cDNA, and each was transiently expressed in COS cells. Lysates from cells transfected with the allele carrying the substitution at amino acid 51 retained both function and immunoreactivity. Lysates from cells transfected with PNP alleles carrying a substitution at either amino acid 128 or amino acid 234 contained immunoreactive material but had no detectable human PNP activity. In summary, molecular analysis of this patient identified point mutations within the PNP gene which are responsible for the enzyme deficiency.     

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.     

A catalytically active high-Mr form of human cathepsin B from sputum.

A cysteine proteinase from purulent sputum was partially purified by a method involving affinity chromatography on Sepharose-aminohexanoylphenylalanylglycinaldehyde semicarbazone. It was immunologically related to lysosomal cathepsin B from human liver and was similar in many, but not all, other aspects. It was catalytically active, as demonstrated by active-site-directed radioiodination, and hydrolysed three cathepsin B substrates, two with Km values similar to those of lysosomal cathepsin B. In addition, the rates of inactivation of the sputum and lysosomal forms of the enzyme by L-3-carboxy-2,3-transepoxypropionyl-leucylamido(4-guanidino) butane (Compound E-64) were very similar. However, the sputum enzyme differed from lysosomal cathepsin B in the following respects. Inhibition by chicken cystatin was much weaker for sputum cathepsin B than for the lysosomal enzyme. Sputum cathepsin B had greater stability at pH 7.5 and a higher apparent Mr, even after deglycosylation, than lysosomal cathepsin B. We conclude that the form of cathepsin B found in sputum is probably a truncated form of human procathepsin B, with some differences in properties that could be of physiological importance.