Studies on the association of NIDDM in Japanese patients with hyperthyroid Graves' disease.

We attempted to analyze the association of hyperthyroid Graves' disease with non-insulin-dependent diabetes mellitus (NIDDM). Forty-nine patients (23 males and 26 females; 7.6%) of a total of 647 patients with hyperthyroid Graves' disease had NIDDM, several years before or after Graves' disease was diagnosed. Only 1 patient had insulin-dependent diabetes mellitus. Compared with the general Japanese population (n = 9,133), the incidence of NIDDM (n = 348; 3.9%) in patients with Graves' disease was higher in all age groups. Only 4 patients (8.2%) of the 49 hyperthyroid patients with NIDDM had a history of being overweight (body mass index > 25). In contrast, 276 (79.9%) of the 348 diabetic patients were currently or previously overweight. Moreover, the incidence of a family history of diabetes (13 of the 49 hyperthyroid Graves' patients with NIDDM; 26.5%) was also lower in the patients with NIDDM in the general Japanese population (50% incidence). The male:female ration in patients with Graves' disease and NIDDM was 1:1.1; much different from that in the total Graves' disease population (1:4.1). Analysis of the HLA loci A, B, C, DR and DQ (35 determinations) in 35 hyperthyroid patients with NIDDM and in 386 subjects from the general population revealed a highly significant difference between them in the incidence of HLA-Cw4, -DR2, -DQw1, -DQw3 and -DQw4. This study suggests that there was an association of Graves' disease with NIDDM. A significant association of HLA-DR and -DQ loci was observed in hyperthyroid Graves' patients with NIDDM.     

Formation and Some Properties of the Acid Phosphatase in Aspergillus terreus

Substrate specificity of purified preparations of phytase from Asp, terreus was examined. The enzyme showed broad specificity. It was found that Asp, terreus produced only one kind of acid phosphatase and it had phytase activity.

Effective materials for the enzyme formation were examined. The formation of the enzyme occurred only during times that mycelia was in contact with inositol.

By differential centrifugation and electron-microscopic autoradiography, it was determined that inositol was incorporated into the mycelia and that it was located at almost the same point as where the active enzyme was located.     

Chemical and Physicochemical Properties of Phytase from Aspergillus terreus

Some chemical and physicochemical properties of the purified phytase preparation produced by Asp. terreus were investigated. From the results of the examination of amino acid analysis, it was suggested that there existed some components other than amino acids in the purified enzyme. Examination of the neutral sugar analysis, therefore, was made by gaschromatography, and it was found that the purified enzyme preparation contained mannose, galactose and a small amount of inositol.

The molecular weight of the enzyme was found to be 214,000 by the Archibald method, and 2.2~2.3×10⁵ by gel-filtration on a Sephadex G–200 column. It was found that by guanidine hydrochloride or by urea, the purified enzyme preparation was dissociated into only one kind of subunit. The native enzyme was supposed to be a homohexamer of the subunits whose molecular weight is 37,000.     

Direct measurement of renal sympathetic nervous activity in high-fat diet-related hypertensive rats

The elevation of renal sympathetic nervous activity (SNA) is a possible cause of blood pressure (BP) elevation. Although a high-fat diet (FAT) often induces BP elevation in animals, the effect of FAT on renal SNA in animals is not consistent between studies. Thus, we compared the basal levels of efferent renal SNA and BP in FAT- or high-carbohydrate diet (CHO)-fed rats. Twenty-four male Sprague-Dawley rats were fed FAT (P/F/C=20/45/35% cal) or CHO (20/5/75) from 5 weeks of age. After 20-21 weeks of feeding, a 24-h urine sample was collected to measure sodium excretion. The next day, blood (0.2 ml) was withdrawn from a femoral artery, and basal efferent renal nerve discharges and mean arterial pressure (MAP) were recorded under anesthesia. Immediately after the experiment, abdominal (epididymal, perirenal and mesenteric) adipose tissues were dissected. Total abdominal fat weight was significantly greater in the FAT group than in the CHO group. The plasma level of leptin was significantly higher in the FAT group, but blood glucose and plasma insulin levels did not differ between the two groups. MAP and renal SNA were significantly higher in the FAT group. In addition, the ratio of urinary sodium excretion to dietary sodium intake was significantly lower in the FAT group than in the CHO group. The data suggest that the increased renal SNA may contribute to BP elevation in FAT-fed rats. The present study firstly demonstrated that renal SNA was elevated with FAT-related BP elevation.     

Characterization of the Rv3378c gene product, a new diterpene synthase for producing tuberculosinol and (13R, S)-isotuberculosinol (nosyberkol), from the Mycobacterium tuberculosis H37Rv genome

The Rv3377c and Rv3378c genes from Mycobacterium tuberculosis are specifically found in the virulent Mycobacterium species, but not in the avirulent species. The Rv3378c-encoded enzyme produced tuberculosinol 2 (5(6), 13(14)-halimadiene-15-ol), 13R-5a and 13S-isotuberculosinol 5b (5(6), 14(15)-halimadiene-13-ol) as its enzymatic products from tuberculosinyl diphosphate 3, indicating that the Rv3378c enzyme catalyzed the nucleophilic addition of a water molecule after the release of a diphosphate moiety. The three enzymatic products 2, 5a, and 5b were produced irrespective of the N- and C-terminal His-tagged Rv3378c enzymes, and of the maltose-binding protein fusion enzyme; the product distribution ratio was identical between the enzymes as 1:1 for 2:5, and 1:3 for 5a:5b. The successful separation of 5a and 5b by a chiral HPLC column provided the first complete assignments of 1H- and 13C-NMR data for 5a and 5b. The enzymatic mechanism for producing 2, 5a, and 5b is proposed here, and the optimal catalytic conditions and kinetic parameters, in addition to the divalent metal effects, are described. Site-directed mutagenesis of Asp into Asn, targeted at the DDXXD motif, resulted in significantly decreased enzymatic activity.     

Activation of slowly conducting medullary raphe-spinal neurons, including serotonergic neurons, increases cutaneous sympathetic vasomotor discharge in rabbit

Neurons in the rostral medullary raphe/parapyramidal region regulate cutaneous sympathetic nerve discharge. Using focal electrical stimulation at different dorsoventral raphe/parapyramidal sites in anesthetized rabbits, we have now demonstrated that increases in ear pinna cutaneous sympathetic nerve discharge can be elicited only from sites within 1 mm of the ventral surface of the medulla. By comparing the latency to sympathetic discharge following stimulation at the ventral raphe site with the corresponding latency following stimulation of the spinal cord [third thoracic (T3) dorsolateral funiculus] we determined that the axonal conduction velocity of raphe-spinal neurons exciting ear pinna sympathetic vasomotor nerves is 0.8 +/- 0.1 m/s (n = 6, range 0.6-1.1 m/s). Applications of the 5-hydroxytryptamine (HT)(2A) antagonist trans-4-((3Z)3-[(2-dimethylaminoethyl)oxyimino]-3-(2-fluorophenyl)propen-1-yl)-phenol, hemifumarate (SR-46349B, 80 microg/kg in 0.8 ml) to the cerebrospinal fluid above thoracic spinal cord (T1-T7), but not the lumbar spinal cord (L2-L4), reduced raphe-evoked increases in ear pinna sympathetic vasomotor discharge from 43 +/- 9 to 16 +/- 6% (P < 0.01, n = 8). Subsequent application of the excitatory amino acid (EAA) antagonist kynurenic acid (25 micromol in 0.5 ml) substantially reduced the remaining evoked discharge (22 +/- 8 to 6 +/- 6%, P < 0.05, n = 5). Our conduction velocity data demonstrate that only slowly conducting raphe-spinal axons, in the unmyelinated range, contribute to sympathetic cutaneous vasomotor discharge evoked by electrical stimulation of the medullary raphe/parapyramidal region. Our pharmacological data provide evidence that raphe-spinal neurons using 5-HT as a neurotransmitter contribute to excitation of sympathetic preganglionic neurons regulating cutaneous vasomotor discharge. Raphe-spinal neurons using an EAA, perhaps glutamate, make a substantial contribution to the ear sympathetic nerve discharge evoked by raphe stimulation.     

Isolation, characterization and expression of a cDNA encoding an elongation factor-1α from Porphyra yezoensis (Bangiales, Rhodophyta)

We report the isolation, characterization and expression of a cDNA encoding a polypeptide elongation factor‐1α (EF‐1α) from the marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and the sequence was analyzed. The clone contained an open reading frame for a protein of 449 amino acids which exhibits sequence similarity to the known EF‐1α. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra pur‐purea EF‐1αtef‐c (97%) than to the P. purpurea EF‐1αtef‐s (61%). The mRNA was detected both in the leafy gametophyte and the filamentous sporophyte.     

Microvascular changes of the liver preserved in UW solution. Pathological and immunohistochemical examination.

Rat livers preserved in University of Wisconsin (UW) solution for 24 h were compared with those preserved in Euro-Collins (EC) solution before and after liver transplantation using an immunohistochemical method. Tissue ATP and total tissue adenine nucleotide (TAN) were measured using HPLC. The levels of TAN in the UW group or the EC group were significantly low compared with the control group (no preservation) after 24-h storage. In the EC group, the levels of tissue adenine nucleotides (TAN) decreased 1 h after reperfusion and never reached control levels. In the UW group, the levels of TAN increased a little 1 h after reperfusion and increased more 3 h after reperfusion. After 24-h preservation, the expression of factor VIII-related antigen (FRA) in endothelial cells of central veins was weak in the EC group; in the UW group, FRA was clearly detected in these cells. After reperfusion, although severe endothelial cell damage to the central veins and numerous FRA-positive substances were observed in EC group, endothelial cells of central veins retained their normal structure and FRA-positive substances were rarely noted in the UW group. In both groups, no endothelial changes were detected in portal veins. From these results, it is concluded that UW solution prevents endothelial cell damage and microcirculatory injury in zone III during the preservation period resulting in prevention of initial graft nonfunction. Also, measurement of the TAN level after reperfusion is useful to predict the function of the graft.     

Comparison between two rat sympathetic pathways activated in cold defense

In cold defense and fever, activity increases in sympathetic nerves supplying both tail vessels and interscapular brown adipose tissue (iBAT). These mediate cutaneous vasoconstrictor and thermogenic responses, respectively, and both depend upon neurons in the rostral medullary raphé. To examine the commonality of brain circuits driving these two outflows, sympathetic nerve activity (SNA) was recorded simultaneously from sympathetic fibers in the ventral tail artery (tail SNA) and the nerve to iBAT (iBAT SNA) in urethane-anesthetized rats. From a warm baseline, cold-defense responses were evoked by intermittently circulating cold water through a water jacket around the animal's shaved trunk. Repeated episodes of trunk skin cooling decreased core (rectal) temperature. The threshold skin temperature to activate iBAT SNA was 37.3 +/- 0.5 degrees C (n = 7), significantly lower than that to activate tail SNA (40.1 +/- 0.4 degrees C; P < 0.01, n = 7). A fall in core temperature always strongly activated tail SNA (threshold 38.3 +/- 0.2 degrees C, n = 7), but its effect on iBAT SNA was absent (2 of 7 rats) or weak (threshold 36.9 +/- 0.1 degrees C, n = 5). The relative sensitivity to core vs. skin cooling (K-ratio) was significantly greater for tail SNA than for iBAT SNA. Spectral analysis of paired recordings showed significant coherence between tail SNA and iBAT SNA only at 1.0 +/- 0.1 Hz. The coherence was due entirely to the modulation of both signals by the ventilatory cycle because it disappeared when the coherence spectrum was partialized with respect to airway pressure. These findings indicate that independent central pathways drive cutaneous vasoconstrictor and thermogenic sympathetic pathways during cold defense.