Glucose-induced production of a Penicillium purpurogenum xylanase by Aspergillus nidulans

The heterologous secretion of xylanase B from Penicillium purpurogenum using glucose as inducer was performed in Aspergillus nidulans. For this purpose, plasmid pEVXB, containing the xylanase B cDNA (including its own signal peptide) under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter, was constructed and used to transform A. nidulans. Analysis of transformed clones showed that several of them secreted extracellular xylanase activity when grown in a medium containing glucose. The clone showing the highest xylanase activity was chosen for further work. When this clone was grown on glucose, xylanase activity (0.72 U/ml), was detected in the culture supernatant. This was confirmed by a zymogram analysis and by the amplification of xynB cDNA from this clone. To our knowledge, this is the first example of the production of a xylanase from Penicillium in heterologous fungal hosts using glucose as inducer.     

CHARACTERIZATION OF PUTATIVE AMINO ACID TRANSMITTER RELEASE FROM SLICES OF RAT DENTATE GYRUS

Abstract— Superfused slices of the rat dentate gyrus were employed to study the release of GABA, glutamate and aspartate, which are considered strong neurotransmitter candidates in this region. The introduction of Ca²⁺ to a Ca²⁺-free superfusion medium containing a depolarizing agent augmented the efflux of all three amino acids. The response to application of Ca²⁺ nearly always occurred within 30 s, the shortest interval tested in these studies. The efflux rate reached a peak within 90 s and then declined to a level slightly greater than the prestimulation baseline. The failure to maintain the maximal rate with continued exposure to Ca²⁺ and depolarizing influences appeared not to result from a reduction in Ca²⁺ permeability caused by continuous depolarization. Ca²⁺ also stimulated the efflux of exogenously loaded radiolabeled GABA, glutamate and aspartate, but not proline. Exogenously loaded GABA was more readily released than endogenous GABA. Otherwise the effects of various treatments on their efflux rates were qualitatively similar. Mg²⁺ inhibited Ca²⁺-dependent efflux. Ba²⁺, but not Mg²⁺, stimulated amino acid efflux in the absence of Ca²⁺. Extracellular Na⁺ was not required to support Ca²⁺-dependent efflux. Addition of Ca²⁺ to a Ca²⁺-free medium in the absence of a depolarizing agent released GABA from the slices, but not glutamate or aspartate. K⁺-enriched medium and the depolarizing alkaloid, veratridine, stimulated both Ca²⁺-dependent and Ca²⁺-independent release processes. Na⁺-free medium enhanced the Ca²⁺-independent releasing action of elevated K⁺. Ca²⁺-independent release was inhibited by raising the Mg²⁺ concentration by 15 or 30 mM and appeared to be inhibited by Ca²⁺ as well. Amino acid output in the absence of Ca²⁺ is probably not directly related to transmission and is considered to result partially from a general increase in membrane permeability induced by depolarization in a Ca²⁺-free medium and partially from stimulation of carrier-mediated amino acid efflux. These results support previously suggested transmitter roles for GABA, glutamate and aspartate in the rat dentate gyrus.     

<Emphasis Type="Italic">Gallibacterium</Emphasis> elongation factor-Tu possesses amyloid-like protein characteristics,participates in cell adhesion,and is present in biofilms

Gallibacterium, which is a bacterial pathogen in chickens, can form biofilms. Amyloid proteins present in biofilms bind Congo red dye. The aim of this study was to characterize the cell-surface amyloid-like protein expressed in biofilms formed by Gallibacterium strains and determine the relationship between this protein and curli, which is an amyloid protein that is commonly expressed by members of the Enterobacteriaceae family. The presence of amyloid-like proteins in outer membrane protein samples from three strains of G. anatis and one strain of Gallibacterium genomospecies 2 was evaluated. A protein identified as elongation factor-Tu (EF-Tu) by mass spectrometric analysis and in silico analysis was obtained from the G. anatis strain F149^T. This protein bound Congo red dye, cross-reacted with anti-curli polyclonal serum, exhibited polymerizing properties and was present in biofilms. This protein also reacted with pooled serum from chickens that were experimentally infected with G. anatis, indicating the in vivo immunogenicity of this protein. The recombinant EF-Tu purified protein, which was prepared from G. anatis 12656-12, polymerizes under in vitro conditions, forms filaments and interacts with fibronectin and fibrinogen, all of which suggest that this protein functions as an adhesin. In summary, EF-Tu from G. anatis presents amyloid characteristics, is present in biofilms and could be relevant for the pathogenesis of G. anatis.     

The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE(C103S) and wild-type penDE genes in P. chrysogenum (Wis54-DE(C103S) strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (beta subunit formation) were observed in the Wis54-DE(C103S) strain, suggesting a regulatory role of the unprocessed IAT(C103S) in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT(C103S) was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing.     

Synthesis and antigenic properties of reduced peptide bond analogues of an immunodominant epitope of the melanoma MART-1 protein.

Backbone modifications have been introduced into the melanoma derived peptide MART-1(27-35) to increase its binding to class I major histocompatibility complex HLA-A2 molecule, and ultimately to enhance its immunogenicity. Each analogue was obtained by replacing one peptide bond at a time in the natural epitope by the aminomethylene (CH2-NH) surrogate. All analogues displayed an increased resistance to proteolysis. Interestingly, the comparative results showed that five analogues bound more efficiently to HLA-A2 than the parent peptide. On the other hand, two pseudopeptide/HLA-A2 complexes were recognized by one melanoma-specific T cell clone. Close examination of the impact of such modifications at the molecular level provides useful supports for the rational design of stable compounds with applications in anti-tumour specific immunotherapy and in vaccine development.     

Characteristics of HIV-1 Nef regions containing multiple CD8+ T cell epitopes: wealth of HLA-binding motifs and sensitivity to proteasome degradation

First and foremost among the many factors that influence epitope presentation are the degradation of Ag, which results in peptide liberation, and the presence of HLA class I molecules able to present the peptides to T lymphocytes. To define the regions of HIV-1 Nef that can provide multiple T cell epitopes, we analyzed the Nef sequence and determined that there are 73 peptides containing 81 HLA-binding motifs. We tested the binding of these peptides to six common HLA molecules (HLA-A2, -A3, -A24, -B7, -B8, and -B35), and we showed that most of them were efficient binders (54% of motifs), especially peptides associating with HLA-A3, -B7/35, and -B8 molecules. Nef peptides most frequently recognized by T cells of HIV-1-infected individuals were 90-97, 135-143, 71-81, 77-85, 90-100, 73-82, and 128-137. The frequency of T cell recognition was not directly related to the strength of peptide-HLA binding. The generation of Nef epitopes is crucial; therefore, we investigated the digestion by the 20S proteasome of a large peptide, Nef(66-100). This fragment was efficiently cleaved, and NH(2)-terminally extended precursors of epitope 71-81 were recognized by T cells of an HIV-1-infected individual. These results suggest that a high frequency of T cell recognition may depend on proteasome cleavage.     

Mutations in the S4 domain of a pacemaker channel alter its voltage dependence

In an attempt to study the functional role of the positively charged amino acids present in the S4 segment of hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channels, we have introduced single and sequential amino acid replacements throughout this domain in the mouse type 2 HCN channel (mHCN2). Sequential neutralization of the first three positively charged amino acids resulted in cumulative shifts of the midpoint voltage activation constant towards more hyperpolarizing potentials. The contribution of each amino acid substitution was approximately -20 mV. Amino acid replacements to neutralize either the first (K291Q) or fourth (R300Q) positively charged amino acid resulted in the same shift (about 20 mV) towards more hyperpolarized potentials. Replacing the first positively charged amino acid with the negatively charged glutamic acid (K291E) produced a shift of approximately -50 mV in the same direction. None of the above amino acid substitutions had any measurable effect on the time course of channel activation. This suggests that the S4 domain of HCN channels critically controls the voltage dependence of channel opening but is not involved in regulating activation kinetics. No channel activity was detected in mutants with neutralization of the last six positively charged amino acids from the S4 domain, suggesting that these amino acids cannot be altered without impairing channel function.     

The effects of stress on body weight: biological and psychological predictors of change in BMI

Objective: To investigate individual differences in the effects of stress on BMI. Research Methods and Procedures: Participants were 71 healthy women volunteers enrolled in a university‐based nurse practitioner program. Predictors of change in BMI were hypothesized to be cortisol secretion, dietary restraint, bingeing, mastery, mood, and eating attitudes. Measures were made at the beginning of the academic semester and 12 weeks later during the participants’ examination period. Results: The women were of normal weight (BMI 25.2 ± 4.3) for their age [43; standard deviation (SD), 7]. By the examination period, 40 had gained weight (mean, 5.5 pounds; SD, 2.2), 19 lost weight (mean, 2.5 pounds; SD, 1.5), and 12 had stable weight. BMI, salivary cortisol secretion, bingeing behavior, depression, and anxiety increased significantly, whereas scores on dietary restraint, weight, shape, and eating concerns, and mastery decreased significantly. Regression analysis showed that change in daily cortisol secretion significantly predicted change in BMI and that mastery significantly moderated this relationship. However, a reduction in dietary restraint was a perfect mediator of this relationship. Change in cortisol secretion also significantly predicted change in dietary restraint, and this was moderated by dietary restraint at the beginning of the academic semester. Reduction in dietary restraint was also predicted by a reduction in mastery and weight concern. Discussion: We identified individual differences that confer vulnerability to weight gain during stressful life events (dietary restraint and mastery). Given that women are exposed to daily stressors and use cognitive strategies to restrain their dietary intake, increasing awareness of the role of stress on eating behavior and weight is an important goal.