Relationship of cholera incidence to El Niño and solar activity elucidated by time-series analysis.

Using time-series analysis, we investigated the monthly cholera incidence in Dhaka, Bangladesh during an 18-year period for its relationship to the sea surface temperature (SST) linked to El Niño, and to the sunspot number. Dominant periodic modes identified for cholera incidence were 11.0, 4.8, 3.5, 2.9, 1.6, 1.0 and 0.5 years. The majority of these modes, e.g. the 11.0-, 4.8-, 3.5-, 1.6- and 1.0-year modes, were essentially consistent with those obtained for the SST data (dominant modes: 5.1, 3.7, 2.5, 2.1, 1.5, 1.0 years) and the sunspot number data (dominant modes: 22.1, 11.1, 7.3, 4.8, 3.1 years). We confirmed that the variations of cholera incidence were synchronous with SSTs, and were inversely correlated to the sunspot numbers. These results suggest that the cholera incidence in Bangladesh may have been influenced by the occurrence of El Niño and also by the periodic change of solar activity.

Two mammalian cell systems for propagation of the hepatitis B virus genome in extrachromosomal and chromosomally integrated states: production of the surface and e antigens.

A recombinant plasmid consisting of (i) the entire genome of hepatitis B virus (HBV) DNA, (ii) the replication origin of SV40 virus, and (iii) a deletion derivative of pBR322 was introduced either into COS cells of monkey origin which constitutively express SV40 large T antigen, or into thymidine kinase(TK)-deficient mouse L cells together with the TK DNA of Herpes simplex virus. In the COS cell system, the transfecting recombinant DNA replicates via SV40 origin and is maintained in an autonomously replicating state. The cells carrying these extrachromosomal elements express the hepatitis B surface antigen gene at moderate rate, and release the products into the culture medium. However, neither core antigen nor e antigen expression was detected in this system. In the L cell system, the transformed L cells carry the recombinant DNA in a chromosomally integrated state. Such cells express the surface antigen gene at high rate, and release the products into the culture medium. This system also excretes the e antigen into the culture medium. The core antigen was not detected.

Expression of herpes simplex virus glycoprotein B gene in yeast.

The DNA sequence coding for herpes simplex virus type 1 glycoprotein B was placed under control of the acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable of replicating in both yeast and Escherichia coli. Yeast transformed by the plasmid synthesized immunologically active glycoprotein B polypeptide.

New technique for time series analysis combining the maximum entropy method and non-linear least squares method: its value in heart rate variability analysis.

A new technique for time series analysis, which is a combination of the maximum entropy method (MEM) for spectral analysis and the non-linear least squares method (LSM) for fitting analysis, is described. In this technique, the MEM power spectral density (MEMPSD) is calculated using a very large lag that could diminish the lag dependence of dominant periods estimated by the MEM analysis. The validity of this large lag is confirmed by the LSM, given that the ten dominant MEM periods are known quantities. To validate the MEM plus LSM technique, it is compared with autoregressive (AR) modelling, by analysing heart rate variability under pharmacological interventions (phenylephrine and trinitroglycerine), using 16 young males. The results indicate that the MEMPSD, when compared with the ARPSD, has numerous periods that could reproduce the original time series much more accurately, as revealed by the LSM analysis. However, both the low- and high-frequency powers with MEMPSD and ARPSDs shift in the expected directions in accordance with the pharmacological effects on the cardiovascular system. The implications of these results are discussed from the theoretical and practical standpoints of the MEM plus LSM technique, compared with AR modelling.

[Clinical study on intraoperative hyperketonemia in non-diabetic surgical patients under general anesthesia].

We measured plasma 3-hydroxybutyrate (3-OHBA) concentrations in 925 non-diabetic surgical patients who underwent various surgical procedures under various types of general anesthesia. Lactated Ringer's solution only was used as an intraoperative fluid. Among them, 46 patients (4.9%) developed high 3-OHBA levels of over 500 microM.l-1 during surgery. The causes of observed hyperketonemia would be surgical stress and preoperative fasting irrespective of 8 types of general anesthesia. Blood glucose, lactic acid, base excess and pH were not influenced by hyperketonemia. Prolonged recovery from anesthesia was not observed in any patients with hyperketonemia. Without any specific treatments, plasma 3-OHBA levels were unchanged or rather decreased in 70% of the patients whose 3-OHBA levels were over 500 microM. The results suggest that hyperketonemia below 500 microM.l-1 is not always disadvantageous in nondiabetic patients under general anesthesia.

[Clinical study on total intravenous anesthesia with droperidol, fentanyl, and ketamine--18. Effect on peripheral circulation as judged by core-peripheral temperature gradient].

Effect of total intravenous anesthesia with droperidol, fentanyl and ketamine (DFK) on peripheral circulation was studied by examining core-peripheral temperature gradient in twenty five patients who underwent abdominal surgery. A core temperature probe was attached on the forehead and peripheral probe on the palm of the hand of the side on which the blood pressure cuff was not applied. The temperature gradient was less than three degrees centigrade in 60% of the patients and the gradient was significantly less as compared with that of isoflurane anesthesia even at 300 minutes after the start of surgical operation. This advantage would have been caused by such factors as circulatory stimulating effect of ketamine, sympathetic blocking effect by droperidol and adequate postoperative analgesia by fentanyl and norketamine, a metabolite of ketamine. The results suggest that DFK would exert a beneficial effect on peripheral circulation, particularly during prolonged surgical procedures.

[Changes in rectal temperature during tympanoplasty under general anesthesia].

We measured rectal temperature of 35 patients who underwent tympanoplasty under various types of general anesthesia. They were allocated at random to three groups according to the types of general anesthesia; 10 patients of enflurane-N2O (enflurane group); 10 patients of neuroleptic anesthesia with droperidol, pentazocine-N2O (NLA group); 15 patients of total intravenous anesthesia with droperidol, fentanyl and ketamine (DFK group). After the induction of anesthesia, their rectal temperature was continuously monitored with an electric thermometer of NEC San-ei throughout the surgical procedure. Increase in rectal temperature was observed in all three groups, and increase in DFK group was significant compared with other two groups. It is possible that the significant increase in rectal temperature in DFK group is supposedly due to normally maintained hypothalamic thermoregulatory function as well as direct surgical stimulation to central nervous system.

Increase in intradermal vascular permeability caused by pertussis toxin from Bordetella pertussis.

Rabbits that were injected intradermally with pertussis toxin (PT), produced from Bordetella pertussis, showed slight edema and erythema at the injection sites, but not hemorrhage nor necrosis. The edema lesions were stained blue by the intravenous injection of Pontamine Sky Blue 6B dye, suggesting that PT caused increased vascular permeability, similarly to the permeability factor (PF) of cholera toxin. The reaction of the PF of PT could be determined by measuring the diameter of the blue area. The diameter of the blue area bore a good linear relationship to the logarithm of the dose of PT. The activity of the PF was neutralized by anti-PT rabbit serum. Detoxification of PT with formalin did not increase the vascular permeability, but reverted pertussis toxoid showed a PF reaction in proportion to the reverted leukocytosis-promoting and histamine-sensitizing activities of PT. The supernate of a Bordetella pertussis culture also induced a PF reaction and the reaction could be made clear by heating the supernate at 56 C for 30 min, but the supernate of Bordetella bronchiseptica did not induce the reaction at all.

Detection of cholera enterotoxin activity in suckling hamsters.

For detection of cholera enterotoxin activity, suckling hamster, mouse, and rat models were compared. It was found that not only suckling mice but also suckling rats and hamsters were sensitive to cholera enterotoxin. Among these models, suckling hamsters were the most sensitive and gave positive results with about 1/100 the cholera toxin seen in suckling mice and rats.

Distributions and recoveries of complement components during cold ethanol fractionation of human plasma.

The distributions and recoveries of the hemolytic activities and quantities of protein of complement components during cold ethanol fractionation were studied. Most of the components were recovered in Cohn's fraction III, with slight activities in fractions I and IV. The hemolytic activities of C1 and C5 were recovered in high yields in fraction III. The recoveries of activities of late components of complement (C6-C9) in fraction II + III were also high. These results suggest that many complement components can be isolated from Cohn's ethanol fractions.

Size and structure of the cholera toxin molecule and its subunits.

Cholera toxin (choleragen) dissociated into two types of subunit with molecular weights estimated to be 28,000 daltons (A) and 11,000 daltons (B); this dissociation was effected by gel filtration at acid pH with or without urea. Subunit A could be separated into two fragments, A1 (23,000 daltons) and A2 (about 2,500 daltons), after reduction and alkylation. Choleragenoid (68,000 daltons) appeared to be a polymerized form of subunit B. A-specific antigen was found in choleragen as well as in A1 and A2, while B-specific antigen was found in both choleragen and choleragenoid. In electron micrographs, the toxin molecule appeared as a particle of uniform size consisting of subunits in V- or Y-shape and in ring form. The dimensions coincided with the values calculated from the molecular weights.

Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.

Transforming activity of a synthetic c-Ha-ras gene containing O6-methylguanine in codon 12.

A mutagenic DNA-adduct, O6-methylguanine, was introduced into codon 12 of the synthetic c-Ha-ras gene by cassette mutagenesis. Transfection of this modified ras gene into normal NIH3T3 cells by the calcium phosphate procedure resulted in significant induction of focus formation. The ras gene inserted into the transformed cells was found to have a G to A transition at the position of the modified base. These results indicate that an O6-methylguanine residue in DNA may lead to a mutation and be one cause of activation of the ras gene.

Entrance of cholera enterotoxin subunits into cells.

Quantitative analysis of the staining of cholera enterotoxin on the surface of cells with specific antibodies against each subunit of cholera toxin, using a Fluorescence-Activated Cell Sorter, showed that not only subunit A but also subunit B penetrates the cell membrane. The detection of each subunit inside the cell was facilitated by the use of saponin, an agent which increases membrane permeability.

Incidence and clinical significance of HBe antigen and antibody in HBsAg-positive various liver diseases.

The HBeAg was detected in 5 of 24 patients with acute type B hepatitis (20.8%), 33 of 95 with chronic hepatitis (34.7%), 6 of 33 with liver cirrhosis (18.2%), and 3 of 39 with hepatocellular carcinoma (7.7%). On the other hand, anti-HBe was found in 4.2% of acute hepatitis, 18.9% of chronic hepatitis, 9.1% of liver cirrhosis, and 12.8% of hepatocellular carcinoma. We found that an early detection of HBeAg in patients with acute hepatitis is of no prognostic value, but its persistence may provide the earliest evidence of potential chronicity. In chronic liver diseases, HBeAg-positive cases showed remarkable fluctuations of serum transaminase levels, severe histological changes and poor responses to treatment. Many of the HBeAg-positive patients lost their initial positivity of HBeAg within six months or one year and in some cases serocoverted to anti-HBe after acute exacerbation. Follow-up study more than several years revealed that the presence of anti-HBe reflect an inactive stage and a more favorable outcome, whereas persistence of HBeAg may provide an active and continuing hepatocellular damage. From these results, we believed that serial measurements of HBeAg/anti-HBe system are useful prognostic marker in patients with HBsAg-positive liver disease.