Effect of gastric distention on RNA synthesis in neonatal chick liver.

RNA synthesis in the nuclei of liver from newly hatched chicks was enhanced 1.25 fold at 10 min after intragastric administration of water. Differential inhibition of RNA synthesis by alpha-amanitin indicated that the enhancement mainly represented rRNA synthesis; the synthesis of mRNA and tRNA was scarcely affected. Enhanced RNA synthesis was accompanied by greater susceptibility of nuclei to digestion by micrococcal nuclease, indicating that the chromatin structure was modified. It was further shown that the "water effect" was mimicked by distention of the stomach by raising the pressure in the intragastric balloon. Since the prior administration of atropine abolished the "water effect", the enhancement of hepatic RNA synthesis may be mediated by hepatic nervous system.     

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.     

Unfolding and refolding of the constant fragment of the immunoglobulin light chain

The kinetics of reversible unfolding and refolding by guanidine hydrochloride of the constant fragment of the immunoglobulin light chain are described. The kinetic measurements were made at pH 7.5 and 25 °C using tryptophyl fluorescence and farultraviolet circular dichroism.The kinetics of unfolding of the constant fragment showed two phases in the conformational transition zone and a single phase above the transition zone. A double-jump experiment confirmed the presence of two forms of the unfolded molecule. These results were thoroughly explained on the basis of the three-species mechanism, U₁

U₂

N, where U₁ and U₂ are the slow-folding and fast-folding species, respectively, of unfolded protein and N is native protein. The equilibrium constant for the process of U₂ to U₁ was estimated to be about 10 and was independent of the conditions of denaturation. These findings were consistent with the view that the U₁

U₂ reaction is proline isomerization. The rates of interconversion between N and U₂ changed greatly with the concentration of guanidine hydrochloride. On the other hand, the refolding kinetics below the transition zone showed behavior unexpected from the three-species mechanism. Whereas the apparent rate constant of the slow phase of refolding was independent of the refolding conditions, its amplitude decreased markedly with the decrease in the final concentration of guanidine hydrochloride. On the basis of this and other results, formation of an intermediate during refolding was ascertained and the refolding kinetics were consistently explained in terms of a more general mechanism involving a kinetic intermediate probably containing non-native proline isomers. The intermediate seemed to have a folded conformation similar to native protein. Comparison of the refolding kinetics of the constant fragment with those of other domains of the immunoglobulin molecule suggested that Pro143 is responsible for the appearance of the slow phase.     

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.     

New polymorphisms of esterase 7 and esterase 8 in inbred strains of rats: Tissue expression and linkage studies

Two new esterase polymorphisms (Es-7 and Es-8) were identified in the testis homogenate of laboratory rats, Rattus norvegicus, by using discontinuous gradient polyacrylamide gel electrophoresis. Es-7 expressed two phenotypes: ES-7A (fast) and ES-7B (slow). Es-8, which migrated in the cathodal region rather than the ES-7 region, also expressed two phenotypes: ES-8A (fast) and ES-8B (slow). Linkage tests among Es-2, Es-7, and Es-8 were made from backcross progeny of the mating (LEJ/Hkm × T/Hok)F₁ × LEJ/Hkm. One recombinant in 51 progeny tested was observed between Es-2 and Es-7; however, recombination between Es-2 and Es-8 was not observed in the same progeny. In addition, we show that the esterase polymorphisms of Es-5 in liver homogenate and Es-3 in small intestine homogenate are identical.