Proteins encoded by the mre gene cluster in Streptomyces coelicolor A3(2) cooperate in spore wall synthesis

It is still an open question how an intracellular cytoskeleton directs the synthesis of the peptidoglycan exoskeleton. In contrast to MreB of rod-shaped bacteria, which is essential for lateral cell wall synthesis, MreB of Streptomyces coelicolor has a role in sporulation. To study the function of the S. coelicolor mre gene cluster consisting of mreB, mreC, mreD, pbp2 and sfr, we generated non-polar replacement mutants. The individual mutants were viable and growth of substrate mycelium was not affected. However, all mutants produced enlarged spores, which frequently germinated prematurely and were sensitive to heat, high osmolarity and cell wall damaging agents. Protein-protein interaction assays by bacterial two-hybrid analyses indicated that the S. coelicolor Mre proteins form a spore wall synthesizing complex, which closely resembles the lateral wall synthesizing complex of rod-shaped bacteria. Screening of a genomic library identified several novel putative components of this complex. One of them (sco2097) was deleted. The Δsco2097 mutant formed sensitive spores with an aberrant morphology, demonstrating that SCO2097 is a new player in cell morphogenesis of Streptomyces. Our results suggest that all Mre proteins cooperate with the newly identified proteins in the synthesis of the thickened spore wall required to resist detrimental environmental conditions.     

Reduced activity of a sensory neuron during a sleep-like state in Caenorhabditis elegans

Sleep-like states occur in the life of all animals carefully studied and are characterized by reduced behavioral and neural activity as well as reduced responsiveness to stimulation [1]. How is reduced responsiveness to stimulation generated? We used calcium imaging to investigate a sleep-like state in larvae of the nematode Caenorhabditis elegans. We found that overall spontaneous neural activity was reduced during the sleep-like state in many neurons, including the mechanosensory neuron ALM. Stimulus-evoked calcium transients and behavior were reduced in ALM during the sleep-like state. Thus, reduced activity of ALM may contribute to reduce responsiveness during a sleep-like state.     

TNF-α -308 genotypes are associated with TNF-α and TGF-β₁ mRNA expression in blood leucocytes of humans

AimTumor necrosis factor α (TNF-α) influences the pathogenesis of lung-fibrosis and carcinogenesis in normal cells. Polymorphisms of this gene are suggested to be associated with susceptibility to lung-diseases. Additionally TNF-α is postulated to play a significant role in regulating. Transforming growth factor (TGF-β₁) expression Therefore we investigated if the TNF-α or TGF-β₁ gene expression level is different within the ?308 TNF-α genotypes.MethodsQuantitative Real-time PCR of TNF-α and TGF-β₁ was performed in 178 Germans. Calculations of expression were made with the 2^?ΔΔCT method. Detection of the ?308 promoter polymorphism of the TNF-α gene was performed by rapid capillary PCR with melting curve analysis.ResultsThe relative TNF-α mRNA expression revealed significant differences between the TNF-α ?308 homozygote wild-type G/G (0.00079 ± 0.00011; n = 113) and the heterozygote genotype G/A (0.0005 ± 0.00008; n = 52; p = 0.030) as well as between homozygote wild-type G/G and the homozygote mutant A/A (0.00029 ± 0.00009; n = 5; p = 0.004). The relative TGF-β mRNA expression showed, similar to TNF-α, the highest mRNA expression was seen within the TNF-α ?308 homozygote wild-types, while the lowest mRNA expression lay within the homozygote mutant-types.ConclusionOur findings suggest that the G-allele of TNF-α ?308 is associated with a significantly higher TNF-α mRNA expression compared to the A-allele and that this also reflects in TGF-β expression. Therefore we support the thesis that TGF-β is regulated by TNF-α.     

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.     

Huntingtin coordinates the dynein-mediated dynamic positioning of endosomes and lysosomes

Huntingtin (Htt) is a membrane-associated scaffolding protein that interacts with microtubule motors as well as actin-associated adaptor molecules. We examined a role for Htt in the dynein-mediated intracellular trafficking of endosomes and lysosomes. In HeLa cells depleted of either Htt or dynein, early, recycling, and late endosomes (LE)/lysosomes all become dispersed. Despite altered organelle localization, kinetic assays indicate only minor defects in intracellular trafficking. Expression of full-length Htt is required to restore organelle localization in Htt-depleted cells, supporting a role for Htt as a scaffold that promotes functional interactions along its length. In dynein-depleted cells, LE/lysosomes accumulate in tight patches near the cortex, apparently enmeshed by cortactin-positive actin filaments; Latrunculin B-treatment disperses these patches. Peripheral LE/lysosomes in dynein-depleted cells no longer colocalize with microtubules. Htt may be required for this off-loading, as the loss of microtubule association is not seen in Htt-depleted cells or in cells depleted of both dynein and Htt. Inhibition of kinesin-1 relocalizes peripheral LE/lysosomes induced by Htt depletion but not by dynein depletion, consistent with their detachment from microtubules upon dynein knockdown. Together, these data support a model of Htt as a facilitator of dynein-mediated trafficking that may regulate the cytoskeletal association of dynamic organelles.     

Full-length L1CAM and not its Δ2Δ27 splice variant promotes metastasis through induction of gelatinase expression

Tumour-specific splicing is known to contribute to cancer progression. In the case of the L1 cell adhesion molecule (L1CAM), which is expressed in many human tumours and often linked to bad prognosis, alternative splicing results in a full-length form (FL-L1CAM) and a splice variant lacking exons 2 and 27 (SV-L1CAM). It has not been elucidated so far whether SV-L1CAM, classically considered as tumour-associated, or whether FL-L1CAM is the metastasis-promoting isoform. Here, we show that both variants were expressed in human ovarian carcinoma and that exposure of tumour cells to pro-metastatic factors led to an exclusive increase of FL-L1CAM expression. Selective overexpression of one isoform in different tumour cells revealed that only FL-L1CAM promoted experimental lung and/or liver metastasis in mice. In addition, metastasis formation upon up-regulation of FL-L1CAM correlated with increased invasive potential and elevated Matrix metalloproteinase (MMP)-2 and -9 expression and activity in vitro as well as enhanced gelatinolytic activity in vivo. In conclusion, we identified FL-L1CAM as the metastasis-promoting isoform, thereby exemplifying that high expression of a so-called tumour-associated variant, here SV-L1CAM, is not per se equivalent to a decisive role of this isoform in tumour progression.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

Microarrays: how many do you need?

We estimate the number of microarrays that is required in order to gain reliable results from a common type of study: the pairwise comparison of different classes of samples. We show that current knowledge allows for the construction of models that look realistic with respect to searches for individual differentially expressed genes and derive prototypical parameters from real data sets. Such models allow investigation of the dependence of the required number of samples on the relevant parameters: the biological variability of the samples within each class, the fold changes in expression that are desired to be detected, the detection sensitivity of the microarrays, and the acceptable error rates of the results. We supply experimentalists with general conclusions as well as a freely accessible Java applet at www.scai.fhg.de/special/bio/howmanyarrays/ for fine tuning simulations to their particular settings.     

Lipolysis: pathway under construction

PURPOSE OF REVIEW: The lipolytic catabolism of stored fat in adipose tissue supplies tissues with fatty acids as metabolites and energy substrates during times of food deprivation. This review focuses on the function of recently discovered enzymes in adipose tissue lipolysis and fatty acid mobilization. RECENT FINDINGS: The characterization of hormone-sensitive lipase-deficient mice provided compelling evidence that hormone-sensitive lipase is not uniquely responsible for the hydrolysis of triacylglycerols and diacylglycerols of stored fat. Recently, three different laboratories independently discovered a novel enzyme that also acts in this capacity. We named the enzyme 'adipose triglyceride lipase' in accordance with its predominant expression in adipose tissue, its high substrate specificity for triacylglycerols, and its function in the lipolytic mobilization of fatty acids. Two other research groups showed that adipose triglyceride lipase (named desnutrin and Ca-independent phospholipase A2zeta, respectively) is regulated by the nutritional status and that it might exert acyl-transacylase activity in addition to its activity as triacylglycerol hydrolase. Adipose triglyceride lipase represents a novel type of 'patatin domain-containing' triacylglycerol hydrolase that is more closely related to plant lipases than to other known mammalian metabolic triacylglycerol hydrolases. SUMMARY: Although the regulation of adipose triglyceride lipase and its physiological function remain to be determined in mouse lines that lack or overexpress the enzyme, present data permit the conclusion that adipose triglyceride lipase is involved in the cellular mobilization of fatty acids, and they require a revision of the concept that hormone-sensitive lipase is the only enzyme involved in the lipolysis of adipose tissue triglycerides.