A VIP hybrid antagonist: From developmental neurobiology to clinical applications

Summary 1. The 28 amino acid vasoactive intestinal peptide, VIP, was originally isolated from the intestine, following a bioassay measuring vasodilating properties. Immunocytochemistry, receptor binding assays and in situ hybridizations have demonstrated VIP abundance in the nervous system, suggesting multiple bioactivities.2. A pharmacological approach was chosen to dissect VIP activities and a prototype VIP antagonist (Met-Hybrid) consisting of a carboxyl fragment of VIP_7–28 and a six amino acid fragment of neurotensin, neurotensin_6–11-VIP_7–28 was synthesized.3. This hybrid peptide was designed to maintain the binding capacity of one parent molecule (VIP), while loosing the agonistic properties, representing a classical competitive receptor antagonist. Furthermore, the new molecule exhibited increased specificity to central nervous system VIP receptors.4. The Met-Hybrid was originally discovered as a potent inhibitor of VIP functionin vivo. In the adult rodent, acute administration of the antagonist resulted in blockade of VIP-mediated potentiation of sexual behavior and chronic intracerebroventricular application impaired VIP-associated learning abilities. During ontogeny, chronic injections of the molecule resulted in neuronal damage, disruption of the diurnal rhythmicity of motor behavior, and retardation in the acquisition of neonatal reflexes in the rat.5. During gestation, severe microcephaly was induced by acute administration of the Met-Hybrid to pregnant mice. The hybrid antagonist inhibited VIP-stimulated mitosis in whole embryo cultures and in a variety of cancer cell linesin vitro andin vivo, suggesting therapeutical potential.     

Synthesis and comparative conformational energetics of D-phenylalanylsarcosine and its cyclic dehydration product, (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione

Summary Synthetic protocols are presented both for D-PheSar and the corresponding cyclised diketopiperazine, prepared from N-t-butoxycarbonylprotected D-PheSar. Deprotection conditions could be manipulated to yield either D-Phenylalanylsarcosine or (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione. Molecular modelling revealed several low energy conformers which contained a Z-peptide bond and which were readily amenable to cyclisation. Cyclisation was found by HPLC to be fastest in strongly acidic conditions.Abbreviation HBTU o-Benzotriazolyl-tetramethyluronium hexafluorophosphate     

Data Quality

A methodology is presented to develop and analyze vectors of data quality attribute scores. Each data quality vector component represents the quality of the data element for a specific attribute (e.g., age of data). Several methods for aggregating the components of data quality vectors to derive one data quality indicator (DQI) that represents the total quality associated with the input data element are presented with illustrative examples. The methods are compared and it is proven that the measure of central tendency, or arithmetic average, of the data quality vector components as a percentage of the total quality range attainable is an equivalent measure for the aggregate DQI. In addition, the methodology is applied and compared to realworld LCA data pedigree matrices. Finally, a method for aggregating weighted data quality vector attributes is developed and an illustrative example is presented. This methodology provides LCA practitioners with an approach to increase the precision of input data uncertainty assessments by selecting any number of data quality attributes with which to score the LCA inventory model input data. The resultant vector of data quality attributes can then be analyzed to develop one aggregate DQI for each input data element for use in stochastic LCA modeling.     

Characterization of SKM1, a Saccharomyces cerevisiae gene encoding a novel Ste20/PAK-like protein kinase

Ste20/PAK serine/threonine protein kinases have been suggested as playing essential roles in cell signalling and morphogenesis as potential targets of Cdc42 and Rac GTPases. We have isolated and characterized the Saccharomyces cerevisiae SKM1 gene, which codes for a novel member of this family of protein kinases. The amino acid sequence analysis of Skm1p revealed the presence of a PH domain and a putative p21-binding domain near its amino terminus, suggesting its involvement in cellular signalling or cytoskeletal functions. However, deletion of SKM1 produced no detectable phenotype under standard laboratory conditions. Moreover, disruption of each of the two other S. cerevisiae Ste20/PAK-like kinase-encoding genes, STE20 and CLA4 , in skm1 backgrounds, showed that Skm1p is not redundant with Ste20p or Cla4p. Interestingly, overexpression of SKM1 led to morphological alterations, indicating a possible role for this protein in morphogenetic control. Furthermore, overproduction of Skm1p lacking its N-terminus caused growth arrest. This effect was also seen when similarly truncated versions of Ste20p or Cla4p were overexpressed. We further observed that overproduction of this C-terminal fragment of Skm1p complements the mating defect of a ste20 mutant strain. These results suggest that the N-terminal domains of S. cerevisiae Ste20/PAK-like protein kinases share a negative regulatory function and play a role in substrate specificity.     

Isolation and characterization of the mycobacterial phagosome: segregation from the endosomal/lysosomal pathway

Mycobacteria have the ability to persist within host phagocytes, and their success as intracellular pathogens is thought to be related to the ability to modify their intracellular environment. After entry into phagocytes, mycobacteria-containing phagosomes acquire markers for the endosomal pathway, but do not fuse with lysosomes. The molecular machinery that is involved in the entry and survival of mycobacteria in host cells is poorly characterized. Here we describe the use of organelle electrophoresis to study the uptake of Mycobacterium bovis bacille Calmette Guerin (BCG) into murine macrophages. We demonstrate that live, but not dead, mycobacteria occupy a phagosome that can be physically separated from endosomal/lysosomal compartments. Biochemical analysis of purified mycobacterial phagosomes revealed the absence of endosomal/lysosomal markers LAMP-1 and β-hexosaminidase. Combining subcellular fractionation with two-dimensional gel electrophoresis, we found that a set of host proteins was present in phagosomes that were absent from endosomal/lysosomal compartments. The residence of mycobacteria in compartments outside the endosomal/lysosomal system may explain their persistence inside host cells and their sequestration from immune recognition. Furthermore, the approach described here may contribute to an improved understanding of the molecular mechanisms that determine the intracellular fate of mycobacteria during infection.     

The poison–antidote stability system of the broad-host-range Thiobacillus ferrooxidans plasmid pTF-FC2

In plasmid pTF-FC2, three small open reading frames (ORFs) are situated between the repB (primase) gene and the repA (helicase) gene of its IncQ-type replicon. Disruption of each of the three ORFs followed by tests for plasmid stability and host cell growth indicated that the ORFs encoded a poison–antidote plasmid stability system. The three genes were named pasA , pasB and pasC (plasmid addiction system), in which PasA is the antidote, PasB the toxin and PasC a protein that appears to enhance the ability of the antidote to neutralize the toxin. Disruption of the pasA gene resulted in two different spontaneous deletions, which inactivated the stability system but did not alter the host range or plasmid copy number. This indicated that the three small ORFs were not involved in plasmid replication. When placed behind a tac promoter, induction of pasB was found to be highly lethal to host cells, which suggests that the Pas system acts by killing plasmid-free host cells rather than by retarding the growth of plasmid-free segregants, as occurs in the ParD system of R1. In spite of this, the presence of the Pas poison–antidote system resulted in a relatively modest threefold stabilization of the pTF-FC2 host replicon and a similar increase in the stabilization of an unstable heterologous R1 plasmid replicon. The Pas system is a poison–antidote plasmid stability module, which appears to have become integrated within the pTF-FC2 replicon module.     

Human medulloblastoma gangliosides

To establish a model system for the study of ganglioside metabolismof the human brain tumor, medulloblastoma, we have chemicallycharacterized the gangliosides of the Daoy cell line. Thesecells contain a high concentration of gangliosides (143 ±13 nmol LBSA/10⁸ cells). The major species have been structurallyconfirmed to be G_M2 (65.9%), G_M3 (13.0%), and G_Dla (10.3%).Isolation of individual gangliosides homogeneous in both carbohydrateand ceramide moieties by reversed-phase HPLC and analysis bynegative-ion fast atom bombardment collisionally activated dissociationtandem mass spectrometry have allowed us to unequivocally characterizeceramide structures. In the case of G_M2, 10 major ceramide subspecieswere identified: d18:1-hC16:0, d18:1-C16:0, d18:0-C16:0, d18:1-C18:0,d18:1-C20:0, d18:1-C22:0, d18:2-C24:1, d18: 1-C23:1, d18:1-C24:1,and d18:1-C24:0. Taken together with previous studies, thesefindings in human medullo-blastoma cells support the view thathigh expression and marked heterogeneity of ceramide structureare general characteristics of tumor gangliosides, moleculeswhich are shed by the tumor cells and which are biologicallyactive in vivo. medulloblastoma gangliosides ceramide structure HPLC mass spectrometry     

Leilani Muir versus the Philosopher King: Eugenics on trial in Alberta

The Province of Alberta in Canada was the only jurisdiction in the British Empire where a eugenic sterilization law was passed (in 1928) and vigorously implemented. The pace of sterilization orders accelerated during the Nazi era and remained high after World War II, terminating only in 1972 when the Sexual Sterilization Act was repealed. The Alberta Eugenics Board operated away from public and legislative scrutiny, and many things done in the name of eugenics were clearly illegal. Eugenics was put on trial in Alberta in 1995 and a judge of the Court of Queen's Bench ruled in 1996 that the government had wrongly sterilized Leilani Muir. After hearing evidence about the history of the eugenics movement, the origins of Alberta's Sexual Sterilization Act, the operation of the Eugenics Board, and details of Muir's life, Madam Justice Joanne B. Veit found that ‘the damage inflicted by the operation was catastrophic’, the ‘wrongful stigmatization of Ms. Muir as a moron ⋯ has humiliated Ms. Muir every day of her life’, and ‘the circumstances of Ms. Muir's sterilization were so high-handed and so contemptuous of the statutory authority to effect sterilization, and were undertaken in an atmosphere that so little respected Ms. Muir's human dignity that the community's, and the court's, sense of decency is offended’. Veit awarded Muir damages of $740,780 CAD and legal costs of $230,000 CAD. The order for Muir's sterilization was signed by John M. MacEachran, founder of the Department of Philosophy and Psychology at the University of Alberta and chairman of the Eugenics Board from 1929 to 1965. An exponent of Platonic idealism, MacEachran believed sterilization of children with a low IQ test score was a means of ‘raising and safeguarding the purity of the race’. However, the Alberta Sterilization Act was passed and implemented with cavalier disregard for the principles of genetics as well as the rights of children. This revised version was published online in August 2006 with corrections to the Cover Date.     

Differential effects of five types of antipathogenic plant peptides on model membranes

The effects of five antipathogenic plant peptides, wheat α-thionin, potato PTH1 defensin, barley LTP2 lipid transfer protein, and potato tuber DL1 and DL2 defensins, have been tested against phospholipid vesicles (liposomes). Wheat thionin very actively induces aggregation and leakage of negatively charged vesicles. LTP2 displays the same activities, although to a limited extent. Under certain conditions PTH1 and DL2 induce vesicle aggregation, but not leakage. Potato defensin DL1 failed to show any effect on liposomes. The same peptides have been assayed against a plant pathogenic bacterium, both the membrane-active and -inactive compounds having efficient antibacterial action.