Three-Dimensional Labeling Program for Elucidation of the Geometric Properties of Biological Particles in Three-Dimensional Space

For all biological particles such as cells or cellular organelles, there are three-dimensional coordinates representing the centroid or center of gravity. These coordinates and other numerical parameters such as volume, fluorescence intensity, surface area, and shape are referred to in this paper as geometric properties, which may provide critical information for the clarification ofin situmechanisms of molecular and cellular functions in living organisms. We have established a method for the elucidation of these properties, designated the three-dimensional labeling program (3DLP). Algorithms of 3DLP are so simple that this method can be carried out through the use of software combinations in image analysis on a personal computer. To evaluate 3DLP, it was applied to a 32-cell-stage sea urchin embryo, double stained with FITC for cellular protein of blastomeres and propidium iodide for nuclear DNA. A stack of optical serial section images was obtained by confocal laser scanning microscopy. The method was found effective for determining geometric properties and should prove applicable to the study of many different kinds of biological particles in three-dimensional space.     

Acute and chronic effects of glucose and carbachol on insulin secretion and phospholipase C activation: studies with diazoxide and atropine

The acute and chronic effects of 20 mM glucose and 10 microM carbachol on beta-cell responses were investigated. Acute exposure of rat islets to 20 mM glucose increased glucose usage rates and resulted in a large insulin-secretory response during a dynamic perifusion. The secretory, but not the metabolic, effect of 20 mM glucose was abolished by simultaneous exposure to 100 microM diazoxide. Glucose (20 mM) significantly increased inositol phosphate (IP) accumulation, an index of phospholipase C (PLC) activation, from [(3)H]inositol-prelabeled islets. Diazoxide, but not atropine, abolished this effect as well. Unlike 20 mM glucose, 10 microM carbachol (in the presence of 5 mM glucose) increased IP accumulation but had no effect on insulin secretion or glucose (5 mM) metabolism. The IP effect was abolished by 50 microM atropine but not by diazoxide. Chronic 3-h exposure of islets to 20 mM glucose or 10 microM carbachol profoundly reduced both the insulin-secretory and PLC responses to a subsequent 20 mM glucose stimulus. The adverse effects of chronic glucose exposure were abolished by diazoxide but not by atropine. In contrast, the adverse effects of carbachol were abolished by atropine but not by diazoxide. Prior 3 h of exposure to 20 mM glucose or carbachol had no inhibitory effect on glucose metabolism. Significant secretory responses could be evoked from 20 mM glucose- or carbachol-pretreated islets by the inclusion of forskolin. These findings support the concept that an early event in the evolution of beta-cell desensitization is the impaired activation of islet PLC.     

Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.     

Structures of Gibberellins A39, A48, A49 and a New Kaurenolide in Cucurbita pepo L.

The structures of three new gibberellins A₃₀, A₄₈ and A₄₉ and a new kaurenolide, isolated from seeds of Cucurbita pepo L., were elucidated. The structures of GA₃₉, GA₄₈ and GA₄₉ were shown to be ent-3α,12β-dihydroxygibberell-16-ene-7,19,20-trioic acid (1), ent-2α,3α,10,12α-tetrahydroxy-20-norgibberell-16-ene-7,19-dioic acid 19,10-lactone (5) and the epimer at C–12 of GA₄₈ (8), respectively. The kaurenolide was shown to have the structure: ent-6β,7α,12β-trihydroxykaur-16-en-19-oic acid 19,6-lactone (14).     

Complete Nucleotide Sequence and Genetic Organization of Aichi Virus, a Distinct Member of the Picornaviridae Associated with Acute Gastroenteritis in Humans

The complete nucleotide sequence of a novel enteric virus, Aichi virus, associated with nonbacterial acute gastroenteritis in humans was determined. The Aichi virus genome proved to be a single-stranded positive-sense RNA molecule with 8,251 bases excluding a poly(A) tail; it contains a large open reading frame with 7,302 nucleotides that encodes a potential polyprotein precursor of 2,433 amino acids. The genome contains a 5′ nontranslated region (NTR) with 712 bases and a 3′ NTR with 240 bases followed by a poly(A) tail. The structure of the genome, VPg–5′ NTR–leader protein–structural proteins–nonstructural proteins–3′ NTR–poly(A), was found to be typical of a picornavirus. The VP0-VP3 and VP3-VP1 cleavage sites were determined to be Q-H and Q-T, respectively, by N-terminal amino acid sequence analyses using purified virion proteins. Possible cleavage sites, Q-G, Q-A, and Q-S, which cleave P2 and P3 polyproteins were found to be similar to those of picornaviruses. A dendrogram based on 3D^pol proteins indicated that Aichi virus is genetically distinct from the known six genera of picornaviruses including entero-, rhino-, cardio-, aphtho-, and hepatovirus and echovirus 22. Considering this together with other properties of the virus (T. Yamashita, S. Kobayashi, K. Sakae, S. Nakata, S. Chiba, Y. Ishihara, and S. Isomura, J. Infect. Dis. 164:954–957, 1991), we propose that Aichi virus be regarded as a new genus of the family Picornaviridae.     

2-Amino-3-carboxy-1,4-naphthoquinone affects the end-product profile of bifidobacteria through the mediated oxidation of NAD(P)H

Glucose metabolism of bifidobacteria in the presence of 2-amino-3-carboxy-1,4-naphthoquinone (ACNQ), a specific growth stimulator for bifidobacteria, and ferricyanide (Fe(CN)(6)(3-)) as an extracellular electron acceptor was examined using resting cells of Bifidobacterium longum and Bifidobacterium breve. NAD(P)H in the cells is oxidized by ACNQ with the aid of diaphorase activity, and reduced ACNQ donates the electron to Fe(CN)(6)(3-). Exogenous oxidation of NADH by the ACNQ/Fe(CN)(6)(3-) system suppresses the endogenous lactate dehydrogenase reaction competitively, which results in the remarkable generation of pyruvate and a decrease in lactate production. In addition, a decrease in acetate generation is also observed in the presence of ACNQ and Fe(CN)(6)(3-). This phenomenon could not be explained in terms of the fructose-6-phosphate phosphoketolase pathway, but suggests rather that glucose is partially metabolized via the hexose monophosphate pathway. This was verified by NADP(+)-induced reduction of Fe(CN)(6)(3-) in cell-free extracts in the presence of ACNQ. Effects of the ACNQ/Fe(CN)(6)(3-) system on anaerobically harvested cells were also examined. Stoichiometric analysis of the metabolites from the pyruvate-formate lyase pathway suggests that exogenous oxidation of NADH is an efficient method to produce ATP in this pathway.     

Prevalence of antibodies against Simkania negevensis in a healthy Japanese population determined by the microimmunofluorescence test

Simkania negevensis has been associated with bronchiolitis in infants and community-acquired pneumonia in adults. Reports of exposure to this microorganism are only available from Israel, North America and Western Europe. Currently, no standard method for diagnosis of S. negevensis infection has been established nor have prevalence rates been shown in Japan. For the first time we demonstrated the ability of the microimmunofluorescence (MIF) test to detect S. negevensis-specific immunoglobulin G and exposure to S. negevensis in Japan. The positive rate in healthy volunteers was 4.3% (25/588), with rates increasing with age. Results indicate the usefulness of the MIF test as a serological method for detecting S. negevensis-specific antibodies. A standard serological test for infection with S. negevensis is needed.     

Three-year survey of the epidemiology of rotavirus, enteric adenovirus, and some small spherical viruses including "Osaka-agent" associated with infantile diarrhea

Studies were made by electron microscopy (EM) on the viruses associated with diarrhea of outpatients at a pediatric clinic in Osaka Prefecture during the three year period from 1980 through 1982. The viruses detected by EM by negative staining with phosphotungstic acid (PTA) were classified morphologically into 6 groups: rotavirus, adenovirus and four kinds of small spherical viruses, calicivirus, astrovirus, picornavirus/parvovirus (P/P)-like agent and Osaka-agent. Osaka-agent seems to be a newly identified small virus. It is 35-40 nm in diameter with a fringe of spike-like structures on its surface. Viruses were detected in 181 of the 395 cases of diarrhea (45.8%). Rotavirus was detected in 122 (30.9%) of the total cases and in 67.4% of the virus-positive cases, while other viruses were detected in 15% of the total cases; adenovirus in 23 (6%) and small agents in 36 (9%). Rotavirus infection showed a distinctive seasonal variation, being mainly restricted to cooler months, but infections with other viruses did not show any seasonal variation. The age distribution of patients suggested that infants of 0 to 2 years old are very susceptible to all viruses. Attempts to cultivate these viruses in vitro were successful with only two isolates of adenovirus type 5.     

Seroepidemiology of hepatitis A virus in Japan

A seroepidemiologic study to detect class-specific antibody against hepatitis A virus (HAV) was made with 831 randomly collected sera (415 in 1973 and 416 in 1984) from healthy Japanese. Competitive-inhibition, IgG, IgA, and IgM anti-HAV enzyme-linked immunosorbent assays (ELISA) were used. Both collections showed a low prevalence of IgG anti-HAV in young age groups and it increased rapidly at middle age and plateued at greater than or equal to 94% prevalence in the older age groups. However, two age groups spanning ages 25-34 demonstrated statistically lower IgG anti-HAV age prevalences in 1984 vs 1973 (P less than 0.001), with an average 10-year prevalence shift. These data suggest that there has been no significant level of HAV infection to alter antibody prevalences in Japan from 1973 to 1984. The markedly decreased incidence of HAV infection in Japan has created a presently large and growing population of HAV susceptibles.