Production of recombinant human glucagon in the form of a fusion protein in Escherichia coli; recovery of glucagon by sequence-specific digestion

Summary Recombinant human glucagon was succesfully produced with a high level of expression in Escherichia coli as a fusion protein with human interferon . The synthetic gene was designed to release glucagon, which does not contain glutamic acid residues, from fusion protein with the Staphylococcus aureus strain V8 protease that specifically cleaves the peptide bond on the carboxyl side of the glutamic acid residue. The resulting glucagon was purified to homogeneity by a combination of C₁₈ reverse-phase HPLC and ion-exchange HPLC. The yield of intact glucagon obtained from 11 of culture was approximately 12 mg. The structure of recombinant human glucagon was confirmed by HPLC and amino acid composition/sequence analyses. Offprint requests to: J. Ishizaki     

A novel brain-specific mRNA encoding nuclear protein (necdin) expressed in neurally differentiated embryonal carcinoma cells

A novel DNA sequence has been isolated from a subtraction cDNA library of P19 embryonal carcinoma cells treated with retinoic acid which induces neural differentiation of the stem cells. The cDNA insert (4B) hybridized with a single 1.7 kb mRNA, whose abundance was markedly increased in P19 cells after retinoic acid treatment. The 1.7 kb mRNA was also expressed in the brain, but not in other non-neuronal tissues. A 1.6 kb cDNA insert (4BFL), which was cloned by screening another cDNA library with the 4B probe, encodes a novel protein sequence of 325 amino acids (Mr 36,831). The protein expressed in 4BFL-transfected COS cells was translocated into the nuclei as detected with antibodies against subsequences of the predicted protein. The antibodies stained the nuclei of neurally differentiated P19 cells but not of the undifferentiated stem cells. This novel mRNA encoding the nuclear protein, termed necdin, may represent a useful marker for the differentiation and development of brain cells.     

Down-regulation of prostaglandin E2 receptors in regenerating rat liver and its physiological significance.

The properties of prostaglandin (PG) E2 receptors in regenerating liver were studied using rat hepatocytes in primary culture. The control cells possessed stereo-specific PGE2 receptors with Bmax and Kd values, at 4 degrees C, of 526 fmol/mg protein and 6.5 nM respectively. In cells from regenerating liver after 70% hepatectomy, Bmax was reduced to 42-43% that of the controls; Kd did not change. Administration of indomethacin before surgery prevented Bmax reduction. These results indicate that PGE2, produced during the regeneration process, evoked cellular events and regulated the density of its receptors.     

Activation of ATP hydrolysis by an uncoupler in mutant mitochondria lacking an intrinsic ATPase inhibitor in yeast

An intrinsic ATPase inhibitor and 9-kDa protein are regulatory factors of mitochondrial ATP synthase in Saccharomyces cerevisiae. A gene encoding the ATPase inhibitor was isolated from a yeast genomic library with synthetic oligonucleotides as hybridization probes and was sequenced. The deduced amino acid sequence showed that the precursor protein contains an amino-terminal presequence of 22 amino acid residues. Mutant strains that did not contain the inhibitor and/or the 9-kDa protein were constructed by transformation of cells with their in vitro disrupted genes. The disruption of the chromosomal copy in recombinant cells was verified by Southern blot analysis, and the absence of the proteins in the mutant cells was confirmed by Western blot analysis. All the mutants could grow on a nonfermentable carbon source and the oxidative phosphorylation activities of their isolated mitochondria were the same as that of normal mitochondria. However, an uncoupler, carbonylcyanide-m-chlorophenylhydrazone, induced marked ATP hydrolysis in the inhibitor-deficient mitochondria, but not in normal mitochondria. These observations suggest that the ATPase inhibitor inhibits ATP hydrolysis by F1F0-ATPase only when the membrane potential is lost.     

Isolation and characterization of plasmid-deletion derivatives from an Hfr made with Rts1 plasmid carrying a chromosomal mutation, plt

Summary Rts1 is a kanamycin-resistance plasmid and multiphenotypically thermosensitive. The detrimental host cell growth at 42° C is expressed only when it exitst autonomously but not in an integrated state. However, a cholate-resistant (plt) mutant of the Hfr with an integrated Rts1 plasmid was found to be thermosensitive like a strain with the same plasmid autonomoulsy. This thermosensitivity depends on the existence of the integrated plasmid. Deletion derivatives of integrated plasmid genome from this Hfr strain were isolated with or without thermal selective growth at 42° C and mapping of the plasmid was attempted by analyzing them. A total of 141 kanamycin-sensitive derivatives were independently isolated and examined for their thermosensitivity (genetic locus: tsg), incompatibility (genetic locus: incT), conjugal fertility (genetic locus: tra), restriction of T4 phase (two genetic loci: resA and resB) and for DNase activity (genetic locus: dns). On the basis of characterization of 141 deletion derivatives, they were divided into 15 patterns which would correspond to a linear map integrated into the chromosome: ... resA ... dns ... resB ... tra ... kan ... incT ... tsg ... It is noteworthy that restriction of T4D phage is determined by two distinct genes, resA and resB, intervened by dns and that propagation of T4D phage on a strain with a resA ⁺ resB ^- genome failed to produce modified progeny phages.     

Growth of measles virus in nervous tissues. III. Neurovirulence of SSPE virus in ferrets.

The Niigata-1 strain isolated from a patient with subacute sclerosing panencephalitis (SSPE) was inoculated intracerebrally into ferrets. Neurological signs developed in about 1 week in most of the animals. Histopathological examinations of the central nervous tissues revealed degenerative lesions in the parenchyma of the brains and inflammatory reactions predominantly in the meninges and choroid plexus. Virus antigen was demonstrated mainly in the nerve cells by immunofluorescent staining. The results indicated high affinity of the Niigata-1 strain to the nerve cells. In contrast, the Mantooth strain of SSPE virus in cell-free state did not exhibit neurovirulence in ferrets.     

Reinitiation of chromosome replication in the presence of chloramphenicol under an integratively suppressed state by R6K.

The autonomous replication of an R plasmid, R6K (amp, str) was shown not to be affected by chloramphenicol. It provoked integrative suppression and gave rise to Hfr strains when integrated into the chromosome of a strain of Escherichia coli K-12 with a temperature-sensitive mutation in the gene, dnaA. An Hfr strain designated as Hfr(R6K) no. 1 was thus obtained and characterized. It was not completely stable as shown by a plating efficiency of 0.6 at 42 C relative to that at 30 C. The density labeling and the ultracentrifugation analysis suggested that the deoxyribonucleic acid replication in this Hfr strain did not stop immediately after completion of the round already started before temperature shift-up and the addition of chloramphenicol. These observations are discussed in relation to a possibility that the chromosome replication of this Hfr strain is under the control of the integrated plasmid at a nonpermissive temperature.     

Amino acid sequence of an active fragment of potato proteinase inhibitor IIa.

The complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its tryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14.] at pH 6.5. yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.