Detection of ICAM-1 in experimentally induced colitis of ICAM-1-deficient and wild-type mice: an immunohistochemical study

Adhesion molecules (e.g. ICAM-1, CD 54) are known to be upregulated on activated vascular endothelial cells during inflammatory reactions. To study the role of ICAM-1 in intestinal inflammation in vivo, we induced acute experimental colitis in wild-type (C57BL/6) mice and ICAM-1-deficient mice, by feeding the animals with 3% dextran sodium sulphate (DSS) in drinking water for 7 days. In the control strain the immunohistochemical staining showed a very pronounced endothelial upregulation of ICAM-1 after the DSS treatment observed in areas of inflammatory infiltrate, especially in venules or arterioles of the propria and submucosa, and partly in the mesocolon. DSS-fed ICAM-1-deficient mice showed no endothelial enhancement and only faint staining of venules or capillaries approaching that encountered in the control ICAM-1-deficient animals. Our data indicate that ICAM-1 may play a crucial role in the development of acute intestinal inflammation, consistent with our finding that ICAM-1 deficiency can obviate severe forms of experimentally induced colitis in mice.     

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Vallon and S. Ghanegaonkar contributed equally to this work.     

Photosynthetic mutants of the cyanobacteria Synechocystis sp. strains PCC 6714 and PCC 6803: sodium p-hydroxymercuribenzoate as a selective agent.

Among a wide range of potential selective agents examined, sodium p-hydroxymercuribenzoate successfully enriched for mutants of Synechocystis sp. strains PCC 6714 and 6803 defective in photosynthesis. When both photosystems I and II were operating, viability of wild-type cells decreased to between 5 X 10(-5) and 1 X 10(-6) after 5 h of incubation with 500 microM p-hydroxymercuribenzoate (strain 6714), and after 8 h with 200 microM (strain 6803). Between 0.1 and 0.5% of the survivors were stable mutants defective in different steps of photosynthesis. The compound was not mutagenic. It was less toxic to cells grown chemoheterotrophically in the dark or photoheterotrophically in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. p-Hydroxymercuribenzoate therefore killed only cells which were performing photosynthesis at high rates, thereby specifically selecting for mutants deficient in this process.     

Characterization of novel mutants with an altered gibberellin spectrum in comparison to different wild-type strains of Fusarium fujikuroi

The rice pathogen Fusarium fujikuroi is known for producing a wide range of secondary metabolites such as pigments, mycotoxins, and a group of phytohormones, the gibberellic acids (GAs). Bioactive forms of these diterpenes are responsible for hyperelongation of rice stems, yellowish chlorotic leaves, and reduced grain formation during the bakanae disease leading to severely decreased crop yields. GAs are also successfully applied in agriculture and horticulture as plant growth regulators to enhance crop yields, fruit size, and to induce earlier flowering. In this study, six F. fujikuroi wild-type and mutant strains differing in GA yields and the spectrum of produced GAs were cultivated in high-quality lab fermenters for optimal temperature and pH control and compared regarding their growth, GA production, and GA gene expression levels. Comparative analysis of the six strains revealed that strain 6314/ΔDES/ΔPPT1, holding mutations in two GA biosynthetic genes and an additional deletion of the 4'-phosphopantetheinyl transferase gene PPT1, exhibits the highest total GA amount. Expression studies of two GA biosynthesis genes, CPS/KS and DES, showed a constantly high expression level for both genes under production conditions (nitrogen limitation) in all strains. By cultivating these genetically engineered mutant strains, we were able to produce not only mixtures of different bioactive GAs (GA₃, GA_4, and GA₇) but also pure GA₄ or GA₇. In addition, we show that the GA yields are not only determined by different production rates, but also by different decomposition rates of the end products GA₃, GA₄, and GA₇ explaining the varying GA levels of genetically almost identical mutant strains.     

Testing an 'aging gene' in long-lived drosophila strains: increased longevity depends on sex and genetic background

Molecular advances of the past decade have led to the discovery of a myriad of 'aging genes' (methuselah, Indy, InR, Chico, superoxide dismutase) that extend Drosophila lifespan by up to 85%. Despite this life extension, these mutants are no longer lived than at least some recently wild-caught strains. Typically, long-lived mutants are identified in relatively short-lived genetic backgrounds, and their effects are rarely tested in genetic backgrounds other than the one in which they were isolated or derived. However, the mutant's high-longevity phenotype may be dependent on interactions with alleles that are common in short-lived laboratory strains. Here we set out to determine whether one particular mutant could extend lifespan in long-lived genetic backgrounds in the fruit fly, Drosophila melanogaster. We measured longevity and resistance to thermal stress in flies that were transgenically altered to overexpress human superoxide dismutase (SOD) in the motorneurones in each of 10 genotypes. Each genotype carried the genetic background from a different naturally long-lived wild-caught Drosophila strain. While SOD increased lifespan on average, the effect was genotype- and sex-specific. Our results indicate that naturally segregating genes interact epistatically with the aging gene superoxide dismutase to modify its ability to extend longevity. This study points to the need to identify mutants that increase longevity not only in the lab strain of origin but also in naturally long-lived genetic backgrounds.     

Chronic gamma-irradiation results in increased cell killing and chromosomal aberration with specific breakpoints in fibroblast cell strains derived from non-Hodgkin's lymphoma patients.

Cultured skin fibroblast cells from 6 patients with non-Hodgkin's lymphoma (NHL) and 2 clinically normal subjects were compared for cell survival and chromosomal aberration after chronic gamma-irradiation. Fibroblasts from an ataxia telangiectasia (AT) homozygote and an AT heterozygote were used as positive controls. Following irradiation, fibroblasts from all 6 NHL patients showed an increase in both cell death and chromosomal aberration (breaks and rearrangements) compared to the normal subjects. The difference in the frequency of chromosomal aberration between the normals and the NHL patients remained virtually unchanged over a period of 24-72 h post irradiation incubation of the cells. Cell cycle analysis by flow cytometry carried out in 1 normal and 1 NHL fibroblast cell strain showed that more cells representing the NHL patient were in G2/M phase compared to the normal at various times of cytogenetic analysis. While the AT homozygote appeared to be the most radiosensitive, the AT heterozygote showed a slightly higher incidence of cell death and chromosomal aberration than the normals. The cellular and chromosomal radiosensitivity of fibroblast cell lines from the NHL patients differed slightly from that of the AT heterozygote but clearly occupied an intermediate position between the AT homozygote and the normal subjects. Cells from 3 of the NHL patients showed radiation-induced specific chromosomal breaks involving chromosomes 1, 2, 6, 8, 10 and 11 which correspond to known fragile sites. Such breakpoints associated with increased radiosensitivity may be indicative of predisposition to malignancy in the patients studied.     

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.     

Ferrochelatase activity in wild-type and mutant strains of Spirillum itersonii. Solubilization with chaotropic reagents

New low-angle X-ray diffraction data have been obtained from nerve myelin after rehydration. The X-ray patterns show the first six orders of diffraction of a lamellar repeat unit of about 100 Å. Direct methods of structure analysis have been used to determine uniquely the phases of the first three orders of diffraction. The electron density profile of rehydrated nerve myelin has been obtained on an absolute electron density scale and is compared with the electron density profile of normal nerve myelin at the same resolution of 16–17 Å. Possible electron-density profiles of rehydrated nerve myelin at a resolution of 8 Å are shown.     

Aquaporin expression correlates with freeze tolerance in baker's yeast,and overexpression improves freeze tolerance in industrial strains

Little information is available about the precise mechanisms and determinants of freeze resistance in baker's yeast, Saccharomyces cerevisiae. Genomewide gene expression analysis and Northern analysis of different freeze-resistant and freeze-sensitive strains have now revealed a correlation between freeze resistance and the aquaporin genes AQY1 and AQY2. Deletion of these genes in a laboratory strain rendered yeast cells more sensitive to freezing, while overexpression of the respective genes, as well as heterologous expression of the human aquaporin gene hAQP1, improved freeze tolerance. These findings support a role for plasma membrane water transport activity in determination of freeze tolerance in yeast. This appears to be the first clear physiological function identified for microbial aquaporins. We suggest that a rapid, osmotically driven efflux of water during the freezing process reduces intracellular ice crystal formation and resulting cell damage. Aquaporin overexpression also improved maintenance of the viability of industrial yeast strains, both in cell suspensions and in small doughs stored frozen or submitted to freeze-thaw cycles. Furthermore, an aquaporin overexpression transformant could be selected based on its improved freeze-thaw resistance without the need for a selectable marker gene. Since aquaporin overexpression does not seem to affect the growth and fermentation characteristics of yeast, these results open new perspectives for the successful development of freeze-resistant baker's yeast strains for use in frozen dough applications.     

Methylation of basic proteins in ribosomes from wild-type and thiostrepton-resistant strains of Bacillus megaterium and their electrophoretic analysis.

Ribosomes, radioactively labelled in vivo with both [1-14C]methionine and [methyl-3H]methionine, have been isolated from both wild-type and thiostrepton-resistant strains of Bacillus megaterium and their constituent proteins separated by two-dimensional gel electrophoresis. Ribosomes from the wild-type strain possess one basic protein that is extensively methylated. In contrast no such protein can be detected in ribosomes from the thiostrepton-resistant strain.     

Preliminary studies of cyanobacteria, picoplankton, and virioplankton in the Salton Sea with special attention to phylogenetic diversity among eight strains of filamentous cyanobacteria

Cyanobacterial diversity in the Salton Sea, a high-salinity, eutrophic lake in Southern California, was investigated using a combination of molecular and morphological approaches. Representatives of a total of 10 described genera (Oscillatoria, Spirulina, Arthrospira, Geitlerinema, Lyngbya, Leptolyngbya, Calothrix, Rivularia, Synechococcus, Synechocystis) were identified in the samples; additionally, the morphology of two cultured strains do not conform to any genus recognized at present by the bacteriological system. Genetic analysis, based on partial 16S rRNA sequences suggested considerable cryptic genetic variability among filamentous strains of similar or identical morphology and showed members of the form-genus Geitlerinema to be distributed among three major phylogenetic clades of cyanobacteria. Cyanobacterial mats, previously described from the Sea were, in fact, composed of both filamentous cyanobacteria and a roughly equivalent biomass of the sulfur-oxidizing bacterium Beggiatoa, indicating their formation in sulfide rich regions of the lake. Flow cytometric analysis of the water samples showed three striking differences between samples from the Salton Sea and representative marine waters: (1) phycoerythrin-containing unicells, while abundant, were much less abundant in the Salton Sea than they were in typical continental shelf waters, (2) Prochlorococcus appears to be completely absent, and (3) small (3–5 m) eukaryotic algae were more abundant in the Salton Sea than in typical neritic waters by one-to-two orders-of-magnitude. Based on flow cytometric analysis, heterotrophic bacteria were more than an order of magnitude more abundant in the Salton Sea than in seawater collected from continental shelf environments. Virus particles were more abundant in the Salton Sea than in typical neritic waters, but did not show increases proportionate with the increase in bacteria, picocyanobacteria, or eukaryotic algae.     

Alterations in electrocardiographic parameters induced by acute infection with Pseudomonas aeruginosa wild-type and mucoid strains in mice

Pseudomonas aeruginosa (PA) is a pathogen in patients with cystic fibrosis (CF) and burn wound infections. Few studies have investigated the role of PA in electrocardiogram parameters (ECGs) with acute infection. We hypothesized that PA induces adverse ECGs effects in mice. After inoculation with wild-type strain PA14, the mucoid CF isolate FRD1 (mucA22+), which produces extensive alginate and has a natural mucoid phenotype, and the PA14 algD^? strain that cannot produce alginate, were evaluated by computerized electrocardiographic. ECG conduction changes were present in the P wave, the R-R interval and heart frequency in all PA strains and the PR interval were significantly prolonged with PA infection with PA14 algD^? and FRD1, compared to the control (P < 0.05), indicating slowed atrial and atrial–ventricular conduction. We conclude that PA produces bradycardia and demonstrated dramatic decreases in other ECGs associated with exposure to wild-type and mutant from PA. Furthermore, the mechanisms by which PA affects cardiac conduction remain uncertain.     

Genetic difference in susceptibility and fatality of three strains of mice experimentally infected with Neodiplostomum seoulense

The genetic influence on host susceptibility to Neodiplostomum seoulense infection and fatality of the host was studied in 3 inbred strains of mice (BALB/c [H-2d], C3H/He [H-2k], and C57BL/6 [H-2b]). The survival of the mice, worm expulsion kinetics, worm size, number of eggs produced per day (EPD), and number of uterine eggs were observed from day 1 to day 40 postinfection (PI) with 100 or 200 metacercariae per mouse. Infection with N. seoulense was highly lethal to all 3 strains of mice, but the lethality was dose-dependent and varied according to the genetic backgrounds of the mice. The C3H/He mice exhibited the highest mortality, the lowest worm burdens and EPD, and the quickest expulsion of worms. It is suggested that different genetic backgrounds of mice appear to affect the host's capacity to expel N. seoulense and the fatality of the hosts themselves.     

Inbred strains of brine shrimp derived from Artemia franciscana: lineage, RAPD analysis, life span, reproductive traits and mode, adaptation, and tolerance to salinity changes

Inbred strains of the brine shrimp were developed from dry dormant cysts of wild-type Artemia franciscana produced in the Great Salt Lake, U.S.A. The established strains were named GSL2, 4, and 7. They were raised in 2% natural sea salt solution at 28 degrees C under a long-day condition, and fed on food sold for Artemia. Ovoviviparous offspring (free-swimming nauplii) in each brood derived from full sib (sister x brother) matings were used for succeeding generations. The ordinal number of the filial generation increased at a rate of ten generations per year. The number was over 60, and the lineage was recorded. Random amplified polymorphic DNA (RAPD) analyses of the inbred strains revealed the uniqueness, homogeneity, and genetic similarity among them. Their life span, the time required to become sexually mature, brood size, mode of reproduction, and adaptation and tolerance to salinity changes were investigated. The inbred strains usually released free-swimming nauplii rather than spawning encysted gastrulae (dormant cysts). On the other hand, the opposite results were obtained from wild-type Artemia under the same conditions. Both adults and nauplii of the inbred strains appeared to be less adaptive and less tolerant to salinity changes compared to those of the wild type. The established inbred strains should provide a wider and deeper scope for Artemia biology in particular, and the life sciences in general.     

P signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge

This communication presents a short outline of the current knowledge on the molecular basis of P_II signal transduction in unicellular cyanobacteria with respect to the perception of environmental stimuli. First, the general characteristics of the P_II signalling system in unicellular cyanobacteria are presented, the hallmark of which is modification by serine-phosphorylation, as compared to the paradigmatic P_II signal transduction system in proteobacteria, which is based on tyrosyl-uridylylation. Then, the focus is turned on the signals controlling P_II phosphorylation state. Recently, the cellular phosphatase (termed PphA), which specifically dephosphorylates phosphorylated P_II (P_II-P) was identified in Synechocystis sp. strain PCC 6803. With the availability of a PphA-deficient mutant and the purified components for in vitro assay of PphA mediated P_II-P dephosphorylation, novel insights into the signals, to which P_II-P dephosphorylation responds, can be obtained. Here we present an investigation of the response of P_II-P dephosphorylation towards treatments that affect the redox-balance of the cells. Furthermore, a possible role of varying ATP/ADP ratios on P_II-P dephosphorylation was examined. From these studies, together with previous investigations, we conclude that P_II-P dephosphorylation specifically responds to changes in the levels of central metabolites of carbon metabolism, in particular 2-oxoglutarate.     

The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis. The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.