Post-translational modification of therapeutic peptides by NisB, the dehydratase of the lantibiotic nisin

Post-translationally introduced dehydroamino acids often play an important role in the activity and receptor specificity of biologically active peptides. In addition, a dehydroamino acid can be coupled to a cysteine to yield a cyclized peptide with increased biostability and resistance against proteolytic degradation and/or modified specificity. The lantibiotic nisin is an antimicrobial peptide produced by Lactococcus lactis. Its post-translational enzymatic modification involves NisB-mediated dehydration of serines and threonines and NisC-catalyzed coupling of cysteines to dehydroresidues, followed by NisT-mediated secretion. Here, we demonstrate that a L. lactis strain containing the nisBTC genes effectively dehydrates and secretes a wide range of medically relevant nonlantibiotic peptides among which variants of adrenocorticotropic hormone, vasopressin, an inhibitor of tripeptidyl peptidase II, enkephalin, luteinizing hormone-releasing hormone, angiotensin, and erythropoietin. For most of these peptides, ring formation was demonstrated. These data show that lantibiotic enzymes can be applied for the modification of peptides, thereby enabling the biotechnological production of dehydroresidue-containing and/or thioether-bridged therapeutic peptides with enhanced stability and/or modulated activities.</p

Response of encapsulated rat pancreatic islets to hypoxia

Hypoxia contributes to encapsulated pancreatic islet graft failure. To gain insight into the mechanisms that lead to hypoxia-induced graft failure, encapsulated islet function, vitality, and cell replication were assessed after 2 and 5 days of hypoxic (1% O-2) and normoxic (20% O-2) culture. The mRNA expression levels of Bcl-2, Bax, inducible nitric oxide synthase (iNOS), and monocyte chemoattractant protein I (MCP-1) were assessed, as well as the amount of nitrite and MCP-1 in the culture medium. Hypoxia was associated with loss of encapsulated islet function and vitality, but not with an increase in islet cell replication. Loss of vitality was due to necrosis', and only modestly due to apoptosis. Hypoxia was not associated with changes in the Bcl-2/Bax mRNA ratio, but it did increase the expression of iNOS and MCP-1 mRNA. The increased mRNA levels were, however, not associated with elevated concentrations of nitrite nor with elevated levels of MCPI protein. The increased iNOS mRNA levels imply a role for NO in the completion of cell death by hypoxia. The increased MCP-1 mRNA levels suggest that encapsulated islets in vivo contribute to their own graft failure by attracting cytokine-producing macrophages. The discrepancy between iNOS mRNA and nitrite is explained by the longer half-life of NO during hypoxia. MCP-1 protein levels are underestimated as a consequence of the lower number of vital cells in combination with a higher proteolytic activity due to necrosis. Thus, strategies to eliminate hypoxia may not only improve islet function and vitality, but may also reduce the attraction of macrophages by encapsulated islets

De Novo Peroxisome Biogenesis in Penicillium Chrysogenum Is Not Dependent on the Pex11 Family Members or Pex16

We have analyzed the role of the three members of the Pex11 protein family in peroxisome formation in the filamentous fungus Penicillium chrysogenum. Two of these, Pex11 and Pex11C, are components of the peroxisomal membrane, while Pex11B is present at the endoplasmic reticulum. We show that Pex11 is a major factor involved in peroxisome proliferation. We also demonstrate that P. chrysogenum cells deleted for known peroxisome fission factors (all Pex11 family proteins and Vps1) still contain peroxisomes. Interestingly, we find that, unlike in mammals, Pex16 is not essential for peroxisome biogenesis in P. chrysogenum, as partially functional peroxisomes are present in a pex16 deletion strain. We also show that Pex16 is not involved in de novo biogenesis of peroxisomes, as peroxisomes were still present in quadruple Δpex11 Δpex11B Δpex11C Δpex16 mutant cells. By contrast, pex3 deletion in P. chrysogenum led to cells devoid of peroxisomes, suggesting that Pex3 may function independently of Pex16. Finally, we demonstrate that the presence of intact peroxisomes is important for the efficiency of ß-lactam antibiotics production by P. chrysogenum. Remarkably, distinct from earlier results with low penicillin producing laboratory strains, upregulation of peroxisome numbers in a high producing P. chrysogenum strain had no significant effect on penicillin production

Effective removal of alginate-poly-L-lysine microcapsules from pancreatic islets by use of trypsin-EDTA

Although the transplantation of alginate-poly-L-lysine-alginate encapsulated islets of Langerhans usually is successful, graft survival is still limited. Molecular analysis by RT-PCR of the encapsulated islets may provide insight into the mechanisms that affect islets during graft failure. However, RT-PCR on encapsulated islets is not possible because the poly-L-lysine of the capsule interferes with both cDNA synthesis and PCR amplification. We applied a method that mechanically removes the microcapsules from the islets after a short trypsin-EDTA treatment (decapsulation), thereby enabling RT-PCR analysis. The results of this study show that the decapsulation procedure does not affect islet vitality and has only minor effects on islet function and morphology. The decapsulation does not affect GAPDH, beta-actin, Bcl-2, or Bax gene expression. This method is an improvement over the time-consuming manual dissection of microcapsules because it allows for the rapid and relatively harmless removal of capsules on a larger scale. Decapsulation offers the possibility of applying RT-PCR, as well as other methods, which cannot be performed on encapsulated islets. (C) 2003 Wiley Periodicals, Inc

Development and validation of the short Use of Creative Cognition Scale in studying

This paper reports the development and validation of a short Use of Creative Cognition Scale in Studying (UCCS) that was inspired by the Cognitive Processes Associated with Creativity (CPAC) scale. In Study 1, items from two of the six subscales of the CPAC were excluded due to conceptual and psychometric issues to create a 21-item CPAC scale, which was administered to 517 university students. Exploratory factor analysis revealed that the 21-item CPAC scale is unidimensional. Five items were selected to create the new unidimensional UCCS. In Study 2, 696 students completed the UCCS and a set of scales measuring related constructs. Confirmatory factor analysis corroborated the unidimensional structure of the scale. The scale correlated positively with measures of flow, trait intrinsic motivation, adaptive metacognitive traits and positive affect, it correlated negatively with negative affect, and it did not correlate with core maladaptive metacognitive traits. The findings indicate that the scale is a valid and reliable tool for research and monitoring

Addressing Stress with Social Work Students: A Controlled Evaluation

This study used a classical pretest-posttest control group design to measure changes in participants\u27 responses after a 45-minute seminar provided 48 undergraduate social work students with techniques on how to better handle stressful situations. When compared to the control group, responses from the students who received the seminar showed significant changes, and these students reported levels of stress and apprehension that were significantly lowered. These findings suggest that formal efforts are important for short-term assistance to help students cope with stress encountered during the course of their studies. Recommendations for future research and intervention in this area are made. © by the Council on Social Work Education, Inc