Sequence of an intestinal cDNA encoding human motilin precursor

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is identical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.     

Serum free thyroxine and thyroxine-binding globulin concentrations in early phase of subacute thyroiditis

Serum total thyroxine (T4), total triiodothyronine (T3), T4-binding globulin (TBG), free T4(FT4) and free T3(FT3) concentrations and the T3-uptake(T3-U) value were estimated in 11 patients with subacute thyroiditis, and compared with the same parameters in 11 patients with Graves' disease, whose serum T4 concentrations were similar to the former group. Seven patients with subacute thyroiditis, who were treated with dicrofenac sodium alone, were investigated as to the sequential changes in serum parameters during their clinical courses. The mean serum T3-U value and FT4, T3 and FT3 concentrations in patients with subacute thyroiditis were increased, but all were significantly lower than those in patients with Graves' disease (p less than 0.01, p less than 0.001, p less than 0.001 and p less than 0.001, respectively). Three patients with subacute thyroiditis, who showed shorter duration of symptoms than 10 days, had serum TBG excess. Thus the mean (+/- SD) serum TBG concentration (26.5 +/- 8.4 micrograms/ml) was significantly higher than that (18.3 +/- 2.9 micrograms/ml) in patients with Graves' disease (p less than 0.02). The ratios of serum T3 to T4 and FT3 to FT4 in patients with subacute thyroiditis were also significantly lower than those in patients with Graves' disease (p less than 0.001 and p less than 0.001, respectively). The serum FT4 in 7 patients treated with dicrofenac sodium alone decreased to the normal range after 3 to 8 weeks from the onset of the illness. In 3 patients with TBG excess and one patient (TBG; 29.0 micrograms/ml), serum TBG declined in consequence of the serum FT4 normalization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human IFN-gamma production is inhibited by a synthetic peptide homologous to retroviral envelope protein

A synthetic 17 amino acid peptide (CKS-17) homologous to a highly conserved region of human and animal retroviral transmembrane proteins was investigated for its influence on the in vitro production of IFN-gamma from human peripheral mononuclear cells. The results showed that CKS-17 coupled to a carrier protein, BSA, inhibited production of IFN-gamma in a dose-dependent manner. Controls, consisting of BSA, which had undergone the coupling procedure or neurotensin coupled to BSA in an identical manner as CKS-17, showed no such inhibition. Reduction in IFN-gamma production could not be attributed to decreased viability of cells, delay of IFN-gamma production or to involvement of suppressor cells. Moreover, inhibition of IFN-gamma production was not related to the inhibition of DNA synthesis. The inhibition appeared to be a direct effect of CKS-17 on IFN-gamma-producing cells. Kinetic studies revealed that this suppression occurred when CKS-17 was introduced to the culture concurrent with or within 48 h after introduction of IFN inducers. Preincubation experiments showed that the presence of CKS-17 in the culture medium was not necessary to exert its inhibitory effect. These results suggest that a portion of retroviral envelope proteins possess important immunomodulatory actions.     

Rotation and protein-protein interactions of cytochrome P-450 in the inner membrane of adrenocortical mitochondria

Rotational diffusion of the total cytochrome P-450 (P-450scc plus P-45011 beta) in bovine adrenocortical mitochondria was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the hemo.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The measurements were used to investigate intermolecular interactions of cytochrome P-450 with other membrane proteins. The absorption anisotropy decayed within 1 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the presence and absence of deoxycorticosterone (DOC), a substrate for cytochrome P-45011 beta. The observed value for the normalized time-independent anisotropy r(infinity)/r(0) and the average rotational relaxation time phi are r(infinity)/r(0) = 0.88 and phi = 233 microseconds when DOC is absent, and r(infinity)/r(0) = 0.65 and phi = 350 microseconds when DOC is present. Judging from the phi value, rotating P-450 is not a monomeric molecule, but would be a small microaggregate with an average diameter of about 120 A. A significantly high value of r(infinity)/r(0) implies co-existence immobile populations of cytochrome P-450. Based on the assumption that the heme angle tilts 55 degrees from the membrane plane (Gut et al. (1983) J. Biol. Chem. 258, 8588-8594), 65% (when DOC is present) or 88% (when DOC is absent) of cytochrome P-450 in mitochondria is immobilized within the experimental time range of 2 ms due to the presence of immobile protein microaggregates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.

Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.     

Voiceprint identification and its application to sociological studies of wild Japanese monkeys (Macaca fuscata yakui)

Japanese monkeys often exchange the particular vocal sound, “coo,” especially when they feed or move as a group. It was considered that the “coo” sound had no positive social meaning, perhaps because the “coo” sound network and its function were hidden behind other behavioral observations. For identification of the vocalizer only from hearing the “coo” sound, three phonetic values, i.e., the “fundamental,” “duration,” and “formants,” plus other characteristics were used as indices of voiceprints. The results indicated that these were effective for identifying the vocalizer in two-thirds of the adults in the study troop which was composed of 12 adults and 16 immature members. The “coo” sound exchange network among the troop members (adults) was drawn on the basis of the voiceprint identification. The network showed three characteristics as follows: (1) matriarchs of the kin-groups frequently exchanged “coo” sounds with each other; (2) the other females exchanged “coo” sounds mostly within their own kin-groups; and (3) males seldom participated in the “coo” sound exchange. This suggests that “coo” sound exchange plays a central role for the matriarch of kin-groups in binding each kin-group and, ultimately, in binding all members together into an organized troop.     

Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina

Mitani, Michiko (National Institute of Genetics, Mishima, Japan), and Tetsuo Iino. Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina. J. Bacteriol. 90:1096-1101. 1965.-The arrangement of flagella was observed by dark-field and electron microscopy in three strains of Salmonella abortivoequina, namely, normal flagellar, curly flagellar, and paralyzed curly flagellar strains. With dark-field microscopy, bundled flagella could be seen in 5 to 10% of actively moving normal or curly mutant cells. Under the electron microscope, a great many bundled flagella were observed in the curly mutant strain, but in the normal strain most of the flagella were dissociated or the bundles were rather loose and irregular. Normal flagella seem to separate easily during the process of preparation, but not the curly ones. Single flagella were found to run parallel with each other and to form a bundle consisting of five or more flagella; the bundle was spirally gyrating, with the characteristic flagellar wave. It is thought that the bundle observed with the electron microscope corresponds to that observed under the dark-field microscope. Further, the marked decrease of bundle formation in the paralyzed curly mutant cells suggests that bundle formation is not caused by curly flagellar structure per se, but corresponds to the mode of locomotion of peritrichously flagellated bacteria.     

Accessory cells induce a polyphosphatidylinositol response when cultured with mitogen-activated T lymphocytes

Monocytes (MO) influenced phosphoinositide metabolism when human T lymphocytes, isolated from peripheral blood, were activated by polyclonal mitogens. In the 3 hr immediately following mitogenic challenge, the synthesis of phosphatidylinositol (PI) was augmented and the synthesis of PI-4-phosphate (PIP) and PI-4,5-bisphosphate (PIP2) was induced in cultures of T lymphocytes and MO. In addition, MO induced a rapid and transient degradation of PIP and PIP2 in T cells prelabeled with [32P]PL and subsequently activated by mitogen. Induction of a PIP/PIP2 response correlated well with induction of DNA replication by MO when T cells were activated by phytohemagglutinin or by neuraminidase plus galactose oxidase. MO did not influence polyphosphoinositide metabolism when T cells were stimulated by the nonmitogenic lectin wheat germ agglutinin. Interleukin 1 could not substitute for monocytes in inducing a polyphosphoinositide response. By causing a rapid and transient release of the second messengers diacylglycerol and inositol phosphates and by subsequently increasing their cellular precursors, MO may induce the interleukin 2 responsive state in T lymphocytes.     

A New Enzymatic Cycling Technique for Glutamate Determination in Brain Microdialysates

A quantitative analysis of glutamate in brain dialysate was made by using an enzymatic cycling technique. This method made it possible to measure the concentration of glutamate in dialysate collected at 30-s intervals. Dialysates were collected from Mongolian gerbil hippocampus before, during, and after two 90-s ischemic insults at an interval of 5 min. An extracellular increase in levels of glutamate was already observed in samples collected during a 30-60 s period after the onset of each ischemia, and the levels of glutamate were maximal at the end of each period of ischemia (approximately a fourfold increase). The increased levels of glutamate rapidly returned almost to preischemic levels by 30 s of recirculation. This method will provide more precise information about temporal changes in the extracellular glutamate concentration in the brain during ischemia.     

Isolation of aldosterone synthase cytochrome P-450 from zona glomerulosa mitochondria of rat adrenal cortex

A cytochrome P-450 capable of producing aldosterone from 11-deoxycorticosterone was purified from the zona glomerulosa of rat adrenal cortex. The enzyme was present in the mitochondria of the zona glomerulosa obtained from sodium-depleted and potassium-repleted rats but scarcely detected in those from untreated rats. It was undetectable in the mitochondria of other zones of the adrenal cortex from both the treated and untreated rats. The cytochrome P-450 was distinguishable from cytochrome P-45011 beta purified from the zonae fasciculata-reticularis mitochondria of the same rats. Molecular weights of the former and the latter cytochromes P-450, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were 49,500 and 51,500, respectively, and their amino acid sequences up to the 20th residue from the N terminus were different from each other at least in one position. The former catalyzed the multihydroxylation reactions of 11-deoxycorticosterone giving corticosterone, 18-hydroxydeoxycorticosterone, 18-hydroxycorticosterone, and a significant amount of aldosterone as products. On the other hand, the latter catalyzed only 11 beta- and 18-hydroxylation reactions of the same substrate to yield either corticosterone or 18-hydroxydeoxycorticosterone. Thus, at least two forms of cytochrome P-450, which catalyze the 11 beta- and 18-hydroxylations of deoxycorticosterone, exist in rat adrenal cortex, but aldosterone synthesis is catalyzed only by the one present in the zona glomerulosa mitochondria.