Purification and characterization of a prolyl aminopeptidase from Debaryomyces hansenii

A prolyl aminopeptidase (PAP) (EC 3.4.11.5) was isolated from the cell extract of Debaryomyces hansenii CECT12487. The enzyme was purified by selective fractionation with protamine and ammonium sulfate, followed by two chromatography steps, which included gel filtration and anion-exchange chromatography. The PAP was purified 248-fold, with a recovery yield of 1.4%. The enzyme was active in a broad pH range (from 5 to 9.5), with pH and temperature optima at 7.5 and 45 degrees C. The molecular mass was estimated to be around 370 kDa. The presence of inhibitors of serine and aspartic proteases, bestatin, puromycin, reducing agents, chelating agents, and different cations did not have any effect on the enzyme activity. Only iodoacetate, p-chloromercuribenzoic acid, and Hg(2+), which are inhibitors of cysteine proteases, markedly reduced the enzyme activity. The K(m) for proline-7-amido-4-methylcoumarin was 40 micro M. The enzyme exclusively hydrolyzed N-terminal-proline-containing substrates. This is the first report on the identification and purification of this type of aminopeptidase in yeast, which may contribute to the scarce knowledge about D. hansenii proteases and their possible roles in meat fermentation.     

Flavopiridol induces mitochondrial-mediated apoptosis in murine glioma GL261 cells via release of cytochrome c and apoptosis inducing factor

Glioblastoma (GBM) remains one of the most challenging solid cancers to treat due to its highly proliferative, angiogenic and invasive nature. The small molecule CDK inhibitor, flavopiridol, has demonstrated antitumor activity in human xenograft models and is currently in clinical trials showing efficacy in patients with advanced disease. We have developed an experimental animal model using the murine glioma GL261 cells as a novel in vivo system to screen potential therapeutic agents for GBM. Results of in vitro testing demonstrate that flavopiridol has several relevant clinical characteristics such as its ability to: 1. inhibit cell growth; 2. inhibit cell migration; 3. decrease expression of cyclin D1, CDK4 and p21; 4. induce apoptosis in cells with high levels of p27 expression; and 5. decrease the expression of the anti-apoptotic protein Bcl-2. The mechanism by which flavopiridol induces apoptosis is mitochondrial-mediated. We demonstrate by electron microscopy and immunohistochemistry that drug treatment induces mitochondrial damage that was accompanied by the release of cytochrome c into the cytosol together with the translocation of apoptosis inducing factor (AIF) into the nucleus. This finding in murine glioma cells differs from the mechanism of flavopiridolinduced cell death reported by us for human glioma cells (Alonso et al., Mol Cancer Ther 2003; 2:139) where drug treatment induced a caspase- and cytochrome c-independent pathway in the absence of detectable damage to mitochondria. In apoptotic human glioma cells only translocation of AIF into the nucleus occurred. Thus, the same drug kills different types of glioma cells by different mitochondrial-dependent pathways.     

Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by multisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-alpha, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-alpha levels were also measured. Production of hepatic IL-18 and TNF-alpha and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-alpha in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.     

Cell death in the nervous system: lessons from insulin and insulin-like growth factors

Programmed cell death is an essential process for proper neural development. Cell death, with its similar regulatory and executory mechanisms, also contributes to the origin or progression of many or even all neurodegenerative diseases. An understanding of the mechanisms that regulate cell death during neural development may provide new targets and tools to prevent neurodegeneration. Many studies that have focused mainly on insulin-like growth factor-I (IGF-I), have shown that insulin-related growth factors are widely expressed in the developing and adult nervous system, and positively modulate a number of processes during neural development, as well as in adult neuronal and glial physiology. These factors also show neuroprotective effects following neural damage. Although some specific actions have been demonstrated to be anti-apoptotic, we propose that a broad neuroprotective role is the foundation for many of the observed functions of the insulin-related growth factors, whose therapeutical potential for nervous system disorders may be greater than currently accepted.     

Characterisation of SMN hybrid genes in Spanish SMA patients: de novo, homozygous and compound heterozygous cases

Autosomal recessive spinal muscular atrophy (SMA) is classified, by age of onset and maximal motor milestones achieved, into type I (severe form), type II (intermediate form) and type III (mild/moderate form). SMA is caused by mutations in the survival motor neuron telomeric gene (SMN1) and a centromeric functional copy of this gene (SMN2) exists, both genes being located at 5q13. Homozygous deletion of exons 7 and 8 of SMN1 has been detected in approx 85% of Spanish SMA patients regardless of their phenotype. Nineteen cases with the sole deletion of exon 7 but not exon 8 (2 cases of type I, 13 cases of type II, four cases of type III) were further analysed for the presence of SMN2-SMN1 hybrid genes. We detected four different hybrid structures. Most of the patients were carriers of a hybrid structure: centromeric intron 6- centromeric exon 7- telomeric exon 8 (CCT), with or without neuronal apoptosis-inhibitor protein (NAIP). In two patients, a different hybrid structure, viz. telomeric intron 6- centromeric exon 7- telomeric exon 8 (TCT), was detected with or without NAIP. A phenotype-genotype correlation comparing the different structures of the hybrid alleles was delineated. Type I cases in our series are attributable to intrachromosomal deletion with a smaller number of SMN2 copies. Most cases with hybrid genes are type II occurring by a combination of a classical deletion in one chromosome and a hybrid gene in the other. Type III cases are closely associated with homozygozity or compound heterozygozity for hybrid genes resulting from two conversion events and have more copies of hybrid genes and SMN2 than type I or II cases.     

Systematic revision of Jorunna Bergh, 1876 (Nudibranchia: Discodorididae) with a morphological phylogenetic analysis

The genus Jorunna is characterized by a dorsum covered withcaryophyllidia, a prostate with two sections, a penis usuallyunarmed but occasionally armed with hooks, a copulatory spine,the presence of an accessory gland and a labial cuticle smoothor armed with jaw elements. The examination of 216 non-typespecimens, 30 types, and a review of the literature show thatthere are 16 valid species of the genus Jorunna: J. tomentosa(Cuvier, 1804); J. funebris (Kelaart, 1859); J. pantherinaAngas,1864; J. rubescens (Bergh, 1876); J. labialis (Eliot, 1908);J. parva (Baba, 1938); J. spazzola (Marcus, 1955); J. hartleyi(Burn, 1958); J. alisonaeMarcus, 1976; J. lemchei (Marcus, 1976);J. evansi (Eliot, 1906); J. pardusBehrens & Henderson, 1981;J.ramicolaMiller, 1996 and J. onubensis Cervera, García-Gómez& García, 1986. In addition, two new species fromthe Eastern Pacific are described: J. osae n. sp. and J. tempisquensisn. sp. We propose two new combinations: Jorunna parva and J.evansi. New records for the genus Jorunna are provided fromItaly, Algeria, Seychelles, Madagascar, Thailand, Marshall Islands,New Caledonia, Île de la Réunion, Sudan, PapuaNew Guinea, Indonesia, Panama, Costa Rica, Bahamas, and SouthernMexico. We present the first preliminary phylogenetic analysisof this cryptobranch dorid genus, based on morphological anatomicaldata, and discuss the biogeography and evolution of severalcharacters in this group. The phylogeny supports the hypothesisthat the genus Jorunna is a monophyletic group and shows thatKentrodoris is nested within it. (Received 31 December 2004; accepted 10 January 2008)     

Influence of queen weight and colony origin on worker response in Solenopsis geminata

Abstract. The influence of weight and colony origin of the queen of Solenopsis geminata (F.) (Hymenoptera: Formicidae) on worker attraction is studied under laboratory conditions. In the first experiment, worker response to individual queens of different weight from the same colony is evaluated. Heavier queens are more attractive than smaller queens to their own workers. In subsequent experiments, the colony origin effect is investigated and worker response to a pair of queens of the same weight from the same or different colonies is compared. When queens are from the same colony, workers do not show a significant preference between queens. However, when queens are from a different colony, workers are significantly more attracted to their own queen than to the foreign queen. Finally, the response of workers to queens of different weight from the same or different colonies is investigated. In both cases, workers are significantly more attracted to a heavier queen than a lighter queen, even if the lighter queen is their own queen. A putative pheromonal component (E)‐6‐(1‐pentenyl)‐2H‐2‐pyranone, is not positively correlated with queen weight.