Interaction with type IV pili induces structural changes in the bacterial outer membrane secretin PilQ

Type IV pili are cell surface organelles found on many Gram-negative bacteria. They mediate a variety of functions, including adhesion, twitching motility, and competence for DNA uptake. The type IV pilus is a helical polymer of pilin protein subunits and is capable of rapid polymerization or depolymerization, generating large motor forces in the process. Here we show that a specific interaction between the outer membrane secretin PilQ and the type IV pilus fiber can be detected by far-Western analysis and sucrose density gradient centrifugation. Transmission electron microscopy of preparations of purified pili, to which the purified PilQ oligomer had been added, showed that PilQ was uniquely located at one end of the pilus fiber, effectively forming a "mallet-type" structure. Determination of the three-dimensional structure of the PilQ-type IV pilus complex at 26-angstroms resolution showed that the cavity within the protein complex was filled. Comparison with a previously determined structure of PilQ at 12-angstroms resolution indicated that binding of the pilus fiber induced a dissociation of the "cap" feature and lateral movement of the "arms" of the PilQ oligomer. The results demonstrate that the PilQ structure exhibits a dynamic response to the binding of its transported substrate and suggest that the secretin could play an active role in type IV pilus assembly as well as secretion.     

cTnT1, a cardiac troponin T isoform, decreases myofilament tension and affects the left ventricular pressure waveform

Four isoforms of cardiac troponin T (cTnT), a protein essential for calcium-dependent myocardial force development, are expressed in the human; they differ in charge and length. Their expression is regulated developmentally and is affected by disease states. Human cTnT (hcTnT) isoform effects have been examined in reconstituted myofilaments. In this study, we evaluated the modulatory effects of overexpressing one cTnT isoform on in vitro and in vivo myocardial function. A hcTnT isoform, hcTnT(1), expressed during development and in heart disease but not in the normal adult heart, was expressed in transgenic (TG) mice (1-30% of total cTnT). Maximal active tension measured in skinned myocardium decreased as a function of relative hcTnT(1) expression. The pCa at half-maximal force development, Hill coefficient, and rate of redevelopment of force did not change significantly with hcTnT(1) expression. In vivo maximum rates of rise and fall of left ventricular pressure decreased, and the half-time of isovolumic relaxation increased, with hcTnT(1) expression. Substituting total cTnT charge for hcTnT(1) expression resulted in similar conclusions. Morphometric analysis and electron microscopy revealed no differences between wild-type (non-TG) and TG myocardium. No differences in isoform expression of tropomyosin, myosin heavy chain, essential and regulatory myosin light chains (MLC), TnI, or in posttranslational modifications of mouse cTnT, cTnI, or regulatory MLC were observed. These results support the hypothesis that cTnT isoform amino-terminal differences affect myofilament function and suggest that hcTnT(1) expression levels present during human development and in human heart disease can affect in vivo ventricular function.     

Mini ecosystem in space--preliminary experiment on board STS-77.

An enclosed ecosystem which is stable on Earth will behave differently when brought into space. Micro-gravity and radiation will affect the dynamics of material circulation or the activities of small creatures of the ecosystem. One series of space experiments aiming to address such issues was planned in the United States (It is termed as ABS--Autonomous Biological System) and Japanese group has been involved with cooperating with the analysis of the flight samples. Before the ecosystem will be on board Russian Space Station "Mir" later 1996 for 3 months, a preliminary flight was carried out in May 1996 on Space Shuttle (STS-77) for 10 days flight. It was the first of such experiments to fly one whole ecosystem in space.     

Decomposition of seawater-irrigated halophytes: implications for potential carbon storage

Plants were cultivated in a nutrient solution containing increasing cadmium concentrations (i.e. 0.001–25 µM), under strictly controlled growth conditions. Changes in both growth parameters and enzyme activities, directly or indirectly related to the cellular free radical scavenging systems, were studied in roots and leaves of 14-day-old maize plants (Zea mays L., cv. Volga) as a result of Cd uptake. A decrease in both shoot length and leaf dry biomass was found to be significant only when growing on 25 µM Cd, whereas concentrations of chlorophyll pigments in the 4th leaf decreased from 1.7 µM Cd on. Changes in enzyme activities occurred at lower Cd concentrations in solution leading to lower threshold values for Cd contents in plants than those observed for growth parameters. Peroxidase (POD; E.C. 1.11.1.7) activity increased in the 3rd and 4th leaf, but not in roots. In contrast, glucose-6-phosphate dehydrogenase (G6PDH; E.C. 1.1.1.49), isocitrate dehydrogenase (ICDH; E.C. 1.1.1.42) and malic enzyme (ME; E.C. 1.1.1.40) activities decreased in the 3rd leaf. According to the relationship between the POD activity and the Cd content, a toxic critical value was set at 3 mg Cd per kg dry matter in the 3rd leaf and 5 mg Cd per kg dry matter in the 4th. Anionic POD were determined both in root and leaf protein extracts; however, no changes in the isoperoxidase pattern were detected in case of Cd toxicity. Results show that in contrast with growth parameters, the measurement of enzyme activities may be included as early biomarkers in a plant bioassay to assess the phytotoxicity of Cd-contaminated soils on maize plants.     

Dialects of the DNA Uptake Sequence in Neisseriaceae

In all sexual organisms, adaptations exist that secure the safe reassortment of homologous alleles and prevent the intrusion of potentially hazardous alien DNA. Some bacteria engage in a simple form of sex known as transformation. In the human pathogen Neisseria meningitidis and in related bacterial species, transformation by exogenous DNA is regulated by the presence of a specific DNA Uptake Sequence (DUS), which is present in thousands of copies in the respective genomes. DUS affects transformation by limiting DNA uptake and recombination in favour of homologous DNA. The specific mechanisms of DUS–dependent genetic transformation have remained elusive. Bioinformatic analyses of family Neisseriaceae genomes reveal eight distinct variants of DUS. These variants are here termed DUS dialects, and their effect on interspecies commutation is demonstrated. Each of the DUS dialects is remarkably conserved within each species and is distributed consistent with a robust Neisseriaceae phylogeny based on core genome sequences. The impact of individual single nucleotide transversions in DUS on meningococcal transformation and on DNA binding and uptake is analysed. The results show that a DUS core 5′-CTG-3′ is required for transformation and that transversions in this core reduce DNA uptake more than two orders of magnitude although the level of DNA binding remains less affected. Distinct DUS dialects are efficient barriers to interspecies recombination in N. meningitidis, N. elongata, Kingella denitrificans, and Eikenella corrodens, despite the presence of the core sequence. The degree of similarity between the DUS dialect of the recipient species and the donor DNA directly correlates with the level of transformation and DNA binding and uptake. Finally, DUS–dependent transformation is documented in the genera Eikenella and Kingella for the first time. The results presented here advance our understanding of the function and evolution of DUS and genetic transformation in bacteria, and define the phylogenetic relationships within the Neisseriaceae family.     

Trafficking and localization studies of recombinant alpha1, 3- fucosyltransferase VI stably expressed in CHO cells

Peripheral alpha1,3-fucosylation of glycans occurs by the action of either one of five different alpha1,3-fucosyltransferases (Fuc-Ts) cloned to date. Fuc-TVI is one of the alpha1,3-fucosyltransferases which is capable to synthesize selectin ligands. The major alpha1, 3- fucosyltransferase activity in human plasma is encoded by the gene for fucosyltransferase VI, which presumably originates from liver cells. While the sequence, chromosomal localization, and kinetic properties of Fuc-TVI are known, immunocytochemical localization and trafficking studies have been impossible because of the lack of specific antibodies. Here we report on the development and characterization of a peptide-specific polyclonal antiserum monospecific to Fuc-TVI and an antiserum to purified soluble recombinant Fuc-TVI crossreactive with Fuc-TIII and Fuc-TV. Both antisera were applied for immunodetection in stably transfected CHO cells expressing the full-length form of this enzyme (CHO clone 61/11). Fuc-TVI was found to be a resident protein of the Golgi apparatus. In addition, more than 30% of cell-associated and released enzyme activity was found in the medium. Maturation and release of Fuc-TVI was analyzed in metabolically labeled CHO 61/11 cells followed by immunoprecipitation. Fuc-TVI occurred in two forms of 47 kDa and 43 kDa bands, while the secreted form was detected as a 43 kDa. These two different intracellular forms arose by posttranslational modification, as shown by pulse-chase experiments. Fuc-TVI was released to the supernatant by proteolytic cleavage as a partially endo-H resistant glycoform.      

Effects of Postnatal Trimethyltin or Triethyltin Treatment on CNS Catecholamine,GABA, and Acetylcholine Systems in the Rat

Abstract: The effects on brain neurochemistry of two neurotoxic tin compounds, trimethyltin (TMT) hydroxide and triethyltin (TET) sulfate, were examined. Long-Evans rats were treated with TMT hydroxide (1 mg/kg, i.p.) on alternate days from day 2 to 29 of life. These treatments caused a weight deficit of 10–20% by the time the animals were killed on day 55 by head-focused microwave irradiation. These TMT treatments are known to cause severe neuronal loss in the hippocampus and lesser damage in other brain regions. Accordingly, the concentration of γ-aminobutyric acid (GABA) was decreased in the hippocampus; however, acetylcholine and choline concentrations were unaffected. These data suggest that TMT-induced effects on GABA systems are greater than that due simply to generalized neuronal loss. The TMT treatments also caused a significant decrease in dopamine concentrations in the striatum, but did not alter the concentrations of dihydroxyphenylacetic acid or homovanillic acid, the acidic metabolites of dopamine. Conversely, concentrations of dopamine and norepinephrine in the brain stem and norepinephrine in the cerebellum were not altered. Despite reports in the literature of TMT-induced neuronal damage in areas of the cortex, no effects on GABA, acetylcholine, or choline levels were found in the cortical areas examined, or in the hypothalamus. TET sulfate (0.3 mg/kg/day) was administered for 6 consecutive days of every week during days 2–29 of life. This dose is lower than that needed to cause intramyelin edema, yet it does result in long-term behavioral changes. Despite this, no changes in the concentration of any of the measured neurotransmitters or their metabolites were detected. In concert, these data demonstrate that neurochemical methods should not be used as neurological “screens,” but rather to define specific mechanisms suggested by detailed behavior, pharmacological, and/or physiological studies.