Allatostatins of the tiger prawn,Penaeus monodon (Crustacea: Penaeidea)

More than 40 peptides belonging to the -Y/FXFGL-NH(2) allatostatin superfamily have been isolated and identified from the central nervous system (CNS) of the tiger prawn, Penaeus monodon (Crustacea: Penaeidea). The peptides can be arranged in seven sub-groups according to the variable post-tyrosyl residue represented by Ala, Gly, Ser, Thr, Asn, Asp, and Glu. Two of the residues (Thr and Glu) have not been observed in this position previously in either insects or crustaceans. Also reported for the first time for allatostatins, two of the peptides are N-terminally blocked by a pyroglutamic acid residue. The yields of certain peptides with similar amino acid sequences to each other were, in some instances, very different. As an example, the yield of ANQYTFGL-NH(2) was 2pmol, compared with ASQYTFGL-NH(2), with a yield of 156 pmol. There are several possibilities to account for this. If, as in all species so far investigated, there is a single allatostatin gene in P. monodon, then it would appear that different sub-populations have contributed mutant forms of particular peptides to the extract. Another, less likely possibility is that this species has more than one allatostatin gene, producing a variable array of peptides albeit in different molar ratios. Several peptides were present apparently as a result of the loss of one or more residues at the N-terminus of a larger form, either due to N-terminal degradation or specific post-translational processing. The number of peptides identified exceeds that for any other insect or crustacean species previously investigated. None is identical to any of the 60-70 insect allatostatins so far identified, and only three are common to other crustaceans. Immunohistochemical study of the CNS of P. monodon, with the same antisera as used to monitor the purification, confirms the widespread nature and complexity of allatostatinergic neural pathways in arthropods. Thus, all neuromeres of the brain, and all except one of the ventral cord ganglia, possess allatostatin neurons and extensive areas of allatostatin-innervated neuropile. In addition to the cytological evidence that the allatostatins act as neurotransmitters, associated with tissues as varied as eyes and legs, their presence in neurohemal areas such as the sinus gland and the perineural sheath of the thoracic ganglia suggests a neuroendocrine function. As well as posing a challenge to physiologists assigning specific functions to the allatostatins, their extensive intra-species multiplicity, linked to their inter-species variability, also presents a complex problem to geneticists and evolutionists.     

Effects of heat and high-pressure treatments on antigenicity of beef extract

The sera of bovine gamma globulin (BGG) positive beef allergic patients were used in this study in order to investigate changes in IgE-specific binding activity with regard to beef extract altered by heat or high-pressure treatment. In inhibition-ELISA, the sample treated at 60 degrees C did not show any significant changes in the antigenicity of BGG, but the sample treated at 100 degrees C showed a decrease of the antigenicity. In the case of the treatment with heating at 100 degrees C, heat-coagulation occurred in the beef extract. The resulting supernatant and precipitate of the sample by centrifugation were analyzed by immunoblotting. Only the fraction of precipitate showed a specific binding activity with the sera. Based on this result, it was speculated that the persistent antigenicity found even after the treatment at 100 degrees C in inhibition-ELISA remained principally in the heat-coagulated fraction, which indicated the importance of the method of handling the heat-coagulation in heat treatment. High-pressure treatments (200 MPa-600 MPa) of beef extract did not show any significant changes in the binding with the sera.     

Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas

Proteomics and immunohistochemistry were used to reveal tumor heterogeneity among urothelial papillomas (UPs) with the long term goal of predicting their biological potential in terms of outcome. First, we identified proteins that were deregulated in invasive fresh lesions as compared with normal urothelium, and thereafter we immunostained UPs with a panel of antibodies against some of the markers. Twenty-two major proteins showing variations of 2-fold or more in at least one-third of the invasive lesions were selected. Specific antibodies against several of the proteins were obtained, but only a few reacted positively in immunostaining. A panel consisting of antibodies against keratinocytes (CKs) 5, 13, 18, and 20 and markers of squamous metaplasia (CKs 7, 8, and 14) was used to probe normal urothelium and 30 UPs collected during a period of five years. Four UPs showed a normal phenotype, whereas the rest could be grouped in five major types that shared aberrant staining with the CK20 antibody. Type 1 heterogeneity (n = 4) showed preferred staining of the umbrella cells with the CK8 antibody. Type 2 (n = 11) was typified by the staining of the basal and intermediate layers with the CK20 antibody. Type 3 (n = 7) was characterized by the predominant staining of the basal cell layer with the CK5 antibody. Type 4 (n = 1) showed areas of CK7 negative cells, whereas type 5 (n = 3) showed loss of staining of the basal cells with the CK20. 29% of the patients experienced recurrences, but none progressed to invasive disease. Patients harboring phenotypic alterations in the basal cell compartment (types 3 and 5) showed the highest number of recurrences (4/7 and 2/3, respectively), and all type 3 lesions progressed to a higher degree of dedifferentiation. Even though a long term prospective study involving a larger sample size is required to assess the biological potential of these lesions, we believe that this approach will prove instrumental for revealing early phenotypic changes in different types of cancer.     

Crystal structure of human dipeptidyl peptidase IV/CD26 in complex with a substrate analog

Dipeptidyl peptidase IV (DPP-IV/CD26) is a multifunctional type II transmembrane serine peptidase. This enzyme contributes to the regulation of various physiological processes, including blood sugar homeostasis, by cleaving peptide hormones, chemokines and neuropeptides. We have determined the 2.5 A structure of the extracellular region of DPP-IV in complex with the inhibitor valine-pyrrolidide. The catalytic site is located in a large cavity formed between the alpha/beta-hydrolase domain and an eight-bladed beta-propeller domain. Both domains participate in inhibitor binding. The structure indicates how substrate specificity is achieved and reveals a new and unexpected opening to the active site.     

ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.     

Ca2+ uptake and cellular integrity in rat EDL muscle exposed to electrostimulation,electroporation, or A23187

We tested the hypothesis that increased Ca2+ uptake in rat extensor digitorum longus (EDL) muscle elicits cell membrane damage as assessed from release of the intracellular enzyme lactate dehydrogenase (LDH). This was done by using 1) electrostimulation, 2) electroporation, and 3) the Ca2+ ionophore A23187. Stimulation at 1 Hz for 120-240 min caused an increase in 45Ca uptake that was closely correlated to LDH release. This LDH release increased markedly with temperature. After 120 min of stimulation at 1 Hz, resting 45Ca uptake was increased 5.6-fold compared with unstimulated muscles. This was associated with an eightfold increase in LDH release, and this effect was halved by lowering extracellular Ca2+ concentration ([Ca2+]o). The poststimulatory increase in resting 45Ca uptake persisted for at least 120 min. An acute increase in sarcolemma leakiness induced by electroporation markedly increased 45Ca uptake and LDH leakage. Both effects depended on [Ca2+]o. A23187 increased 45Ca uptake. Concomitantly, LDH leakage increased 18-fold within 30 min, and this effect was abolished by omitting Ca2+ from the buffer. We conclude that increased Ca2+ influx may be an important cause of cell membrane damage that arises during and after exercise or electrical shocks. Because membrane damage allows further influx of Ca2+, this results in positive feedback that may further increase membrane degeneration.     

The use of cyclic voltammetry to detect biofilms formed by Pseudomonas fluorescens on platinum electrodes

The development of an electrochemical detector to monitor the in situ formation of biofilms is described. The detector consisted of an electrochemical cell containing three electrodes, whose response to the application of a potential profile to the working electrode was sensitive to the amount of biofilm present on the surface. The electrochemical technique used was repetitive cyclic voltammetry. Differences between the response of the uncolonised electrode and after Pseudomonas fluorescens biofilms of different ages were grown on its surface were determined. The results show that cyclic voltammetry applied to platinum electrodes can be used to detect young biofilms. The development of the shape of the voltammogram as the potential is cycled may constitute a means of providing information on the coverage of the surface. Observation of the platinum electrodes before and after the electrochemical measurements showed that even after 30 min of recycling, most of the cells were still adhered to the surface, although some may have lost viability.     

Molecular analysis of hereditary nonpolyposis colorectal cancer in the United States: high mutation detection rate among clinically selected families and characterization of an American founder genomic deletion of the MSH2 gene

The identification of germline mutations in families with HNPCC is hampered by genetic heterogeneity and clinical variability. In previous studies, MSH2 and MLH1 mutations were found in approximately two-thirds of the Amsterdam-criteria-positive families and in much lower percentages of the Amsterdam-criteria-negative families. Therefore, a considerable proportion of HNPCC seems not to be accounted for by the major mismatch repair (MMR) genes. Does the latter result from a lack of sensitivity of mutation detection techniques, or do additional genes underlie the remaining cases? In this study we address these questions by thoroughly investigating a cohort of clinically selected North American families with HNPCC. We analyzed 59 clinically well-defined U.S. families with HNPCC for MSH2, MLH1, and MSH6 mutations. To maximize mutation detection, different techniques were employed, including denaturing gradient gel electrophoresis, Southern analysis, microsatellite instability, immunohistochemistry, and monoallelic expression analysis. In 45 (92%) of the 49 Amsterdam-criteria-positive families and in 7 (70%) of the 10 Amsterdam-criteria-negative families, a mutation was detected in one of the three analyzed MMR genes. Forty-nine mutations were in MSH2 or MLH1, and only three were in MSH6. A considerable proportion (27%) of the mutations were genomic rearrangements (12 in MSH2 and 2 in MLH1). Notably, a deletion encompassing exons 1-6 of MSH2 was detected in seven apparently unrelated families (12% of the total cohort) and was subsequently proven to be a founder. Screening of a second U.S. cohort with HNPCC from Ohio allowed the identification of two additional kindreds with the identical founder deletion. In the present study, we show that optimal mutation detection in HNPCC is achieved by combining accurate and expert clinical selection with an extensive mutation detection strategy. Notably, we identified a common North American deletion in MSH2, accounting for approximately 10% of our cohort. Genealogical, molecular, and haplotype studies showed that this deletion represents a North American founder mutation that could be traced back to the 19th century.