The role of calcium ion in the L-glutamate-induced depolarization in the frog spinal cord

1. The role of Ca2+ in L-glutamate-induced depolarization was investigated in the isolated frog spinal cord. 2. The size of a depolarization induced by L-glutamate (3 mM) was inversely related to the extracellular Ca2+ concentration, but was reduced in a Ca2+-free medium containing EGTA (0.3 mM). 3. L-Glutamate caused a marked depolarization in both ventral and dorsal roots, even in a NaCl-deficient medium (Ca2+, 2.0 mM). The size of the depolarization was attenuated by a prolonged or repeated application of L-glutamate. Ca2+ can be replaced by Sr2+ or Mg2+. 4. Concanavalin A (1 microM) prevents the development of desensitization to L-glutamate. 5. Present results suggest that Ca2+ plays the role of a charge carrier for L-glutamate-induced depolarization and of a regulator of modulator for L-glutamate-receptor sensitivity. The roles are exaggerated in NaCl-free medium.     

Nucleotide sequence of the kanamycin resistance transposon Tn903

The entire nucleotide sequence of the kanamycin resistance transposon Tn903 was determined by analyzing a mini-ColE1 derivative carrying Tn903. Tn903 was 3094 base-pairs in length and at both extremities possessed two identical inverted 1057 base-pair sequences. Furthermore, 18 bases at the ends of the 1057 base-pair sequence are themselves present in an invertedly repeated order as has been described for various insertion sequences. Analysis of initiation and termination codons in the Tn903 sequence indicated that Tn903 could possibly code for at least three high molecular weight polypeptides. One in the region between the two 1057 base-pair sequences is suggested to be the kanamycin resistance determinant (aminoglycoside 3′-phosphotransferase) from its location and size. The other polypeptides were located within the 1057 base-pair sequence and may be associated with transposition functions of Tn903.     

Replication origin of the Escherichia coli K-12 chromosome: The size and structure of the minimum DNA segment carrying the information for autonomous replication

Summary A DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA ⁺ cells, whereas pBR322 replicates only in polA ⁺ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori⁺ and Ori^- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure.To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori^- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.     

Effect of glucose on protein phosphorylation in rat pancreatic islets

Isolated rat pancreatic islets, incubated in the presence of extracellular ³²Pi to steady state ³²P incorporation into cellular phosphopeptides, were exposed to glucose for 10 min. Glucose (16.7 mM) significantly stimulated the phosphorylation of six phosphoproteins with molecular weights of 15,000, 35,000, 49,000, 64,000, 93,000 and 138,000. Mannoheptulose (16.7 mM) markedly inhibited glucose-stimulated phosphorylation of these six phosphoproteins. This protein phosphorylation might be important in mediating glucose-stimulated insulin release.     

Stimulation of DNA synthesis by heated human serum in primary cultured normal adult rat hepatocytes

In primary culture of normal adult rat hepatocytes, human serum heated at 56°C for 30 min stimulated dose-dependently [³H]thymidine incorporation into trichloroacetic acid insoluble fraction of the cells, most of which was solubilized into hot trichloroacetic acid solution. The solubilized fraction was reduced when hydroxyurea was added to the culture. The heated serum also increased dose-dependently protein synthesis and cell viability determined from morphological findings. These results suggest that human serum has heat-stable factors stimulating DNA synthesis and maintaining cell viability of cultured rat hepatocytes.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Highly sensitive assay for tyrosine hydroxylase activity by high-performance liquid chromatography

A highly sensitive assay for tyrosine hydroxylase (TH) activity by high-performance liquid chromatography (HPLC) with amperometric detection was devised based on the rapid isolation of enzymatically formed DOPA by a double-column procedure, the columns fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminium oxide). DOPA was adsorbed on the second aluminium oxide column, then eluted with 0.5 M hydrochloric acid, and assayed by HPLC with amperometric detection. d-Tyrosine was used for the control. α-Methyldopa was added to the incubation mixture as an internal standard after incubation. This assay was more sensitive than radioassays and 5 pmol of DOPA formed enzymatically could be measured in the presence of saturating concentrations of tyrosine and 6-methyltetrahydropterin. The TH activity in 2 mg of human putamen could be easily measured, and this method was found to be particularly suitable for the assay of TH activity in a small number of nuclei from animal and human brain.     

Acyl migrations in diacyl derivatives of 2-methylmercaptobenzimidazole : A model of biotin

Diacyl derivatives of 2-methylmercaptobenzimidazole undergo the tautomerization 2 1 2′. Thermodynamic predominancy of one isomer over the others depends on the substituents on carbonyl groups. It has been found that electron-withdrawing substituents tend to favor 2-type compounds, whereas electron-releasing substituents make 1-type compounds more stable. The migration has been extended to include the carboethoxy group, and the results are discussed in relation to the mechanism of biotin-dependent enzymic carboxylation.