Electron microscopy evidence that cytoplasmic localization of the p16INK4A "nuclear" cyclin-dependent kinase inhibitor (CKI) in tumor cells is specific and not an artifact. A study in non-small cell lung carcinomas

It is well established that p16^INK4A protein acts as a cell cycle inhibitor in the nucleus. Therefore, cytoplasmic localization of p16 ^INK4A usually is disregarded by investigators as nonspecific. Three recent studies reported findings that differ from the current view concerning p16^INK4A immunohistochemical localization. All three demonstrated that breast and colon cancers expressing cytoplasmic p16^INK4 represent distinct biological subsets. We previously detected in a percentage of non-small cell lung carcinomas simultaneous nuclear and cytoplasmic p16^INK4A staining. In view of the reports concerning breast and colon carcinomas, we conducted an ultrastructural re-evaluation of our cases to clarify the specificity of p16^INK4A cytoplasmic expression. We observed p16 ^INK4A immunolocalization in both the nucleus and the cytoplasm of a proportion of tumor cells. Diffuse dense nuclear staining was detected in the nucleoplasm, whereas weaker granular immunoreactivity was observed in the cytoplasm near the rough endoplasmic reticulum. Negative tumor cells also were visible. In the tumor-associated stromal, cells p16^INK4A immunoreactivity was detected only in the nuclei. We have demonstrated that p16^INK4A cytoplasmic staining is specific and suggest that it represents a mechanism of p16^INK4A inactivation similar to that observed in other tumor suppressor genes.     

Evaluation of an entomopathogenic fungus, Paecilomyces fumosoroseus (Wize) Brown and Smith (Deuteromycota: Hyphomycetes) obtained from Formosan subterranean termites (Isop., Rhinotermitidae)

Abstract:  An isolate of Paecilomyces fumosoroseus was obtained from Coptotermes formosanus collected in Hong Kong, and a commercially available isolate of Metarhizium anisopliae , were both tested against C. formosanus shipped live from China. Survivorship of termites treated with a suspension of 5 × 10⁵ M. anisopliae conidia/ml and kept alone declined more rapidly than for those treated at the same concentration of P. fumosoroseus conidia. At a 5 × 10⁶ conidia/ml concentration, no significant differences in terms of termite survivorship were observed between the two fungal species. However, among termites kept in groups of 10 after treatment, those sprayed with P. fumosoroseus conidia at either 5 × 10⁵ or 5 × 10⁶ conidia/ml had significantly lower survivorship than those sprayed with M. anisopliae conidia. All the cadavers of termites treated with P. fumosoroseus and kept alone sporulated and among grouped termites 29% of the cadavers sporulated. By comparison, 53% of the cadavers of termites treated with M. anisopliae and kept alone sporulated, and only 4% of the cadavers of treated termites kept in groups sporulated.     

Alteration of Endoplasmic Reticulum Lipid Rafts Contributes to Lipotoxicity in Pancreatic β-Cells

Chronic saturated fatty acid exposure causes β-cell apoptosis and, thus, contributes to type 2 diabetes. Although endoplasmic reticulum (ER) stress and reduced ER-to-Golgi protein trafficking have been implicated, the exact mechanisms whereby saturated fatty acids trigger β-cell death remain elusive. Using mass spectroscopic lipidomics and subcellular fractionation, we demonstrate that palmitate pretreatment of MIN6 β-cells promoted ER remodeling of both phospholipids and sphingolipids, but only the latter was causally linked to lipotoxic ER stress. Thus, overexpression of glucosylceramide synthase, previously shown to protect against defective protein trafficking and ER stress, partially reversed lipotoxic reductions in ER sphingomyelin (SM) content and aggregation of ER lipid rafts, as visualized using Erlin1-GFP. Using both lipidomics and a sterol response element reporter assay, we confirmed that free cholesterol in the ER was also reciprocally modulated by chronic palmitate and glucosylceramide synthase overexpression. This is consistent with the known coregulation and association of SM and free cholesterol in lipid rafts. Inhibition of SM hydrolysis partially protected against ATF4/C/EBP homology protein induction because of palmitate. Our results suggest that loss of SM in the ER is a key event for initiating β-cell lipotoxicity, which leads to disruption of ER lipid rafts, perturbation of protein trafficking, and initiation of ER stress.     

Mass spectrometry in the study of lysosomal storage disorders.

Lysosomal storage disorders represent a group of over 45 distinct genetic diseases, each one resulting from a deficiency of a particular lysosomal protein or, in a few cases, from non-lysosomal proteins that are involved in lysosomal biogenesis. A common biochemical feature of this group of disorders is the accumulation within lysosomes of undegraded or partially degraded substrates that are normally degraded within, and transported out of the lysosome. The particular substrates stored and the site(s) of storage vary with disease type and enzyme/protein deficiency. The nature of the substrate can be used to group the disorders into broad categories including the mucopolysaccharidoses, lipidoses, glycogenoses and oligosaccharidoses. These categories show many clinical similarities within groups as well as significant similarities between groups. For most lysosomal storage disorders the relationship between the stored substrates (type, amount and location) and the disease pathology is not well understood. The use of mass spectrometry and in particular tandem mass spectrometry provides a powerful tool for the investigation of stored substrates in this group of disorders. In this review we will describe the use of mass spectrometry for the analysis of stored substrates. We will discuss progress in the field, limitations of current methods, and summarise issues relating to the diagnosis and treatment of some of the more prevalent lysosomal storage disorders.     

Population genomics of Sinorhizobium medicae based on low-coverage sequencing of sympatric isolates

We investigated the genomic diversity of a local population of the symbiotic bacterium Sinorhizobium medicae, isolated from the roots of wild Medicago lupulina plants, in order to assess genomic diversity, to identify genomic regions influenced by duplication, deletion or strong selection, and to explore the composition of the pan-genome. Partial genome sequences of 12 isolates were obtained by Roche 454 shotgun sequencing (average 5.3 Mb per isolate) and compared with the published sequence of S. medicae WSM 419. Homologous recombination appears to have less impact on the polymorphism patterns of the chromosome than on the chromid pSMED01 and megaplasmid pSMED02. Moreover, pSMED02 is a hot spot of insertions and deletions. The whole chromosome is characterized by low sequence polymorphism, consistent with the high density of housekeeping genes. Similarly, the level of polymorphism of symbiosis genes (low) and of genes involved in polysaccharide synthesis (high) may reflect different selection. Finally, some isolates carry genes that may confer adaptations that S. medicae WSM 419 lacks, including homologues of genes encoding rhizobitoxine synthesis, iron uptake, response to autoinducer-2, and synthesis of distinct polysaccharides. The presence or absence of these genes was confirmed by PCR in each of these 12 isolates and a further 27 isolates from the same population. All isolates had rhizobitoxine genes, while the other genes were co-distributed, suggesting that they may be on the same mobile element. These results are discussed in relation to the ecology of Medicago symbionts and in the perspective of population genomics studies.     

Genetic estimates of immigration and emigration rates in relation to population density and forest patch area in <Emphasis Type="Italic">Peromyscus leucopus</Emphasis>

An emerging pattern is that population densities of generalist rodents are higher in small compared to large forest patches in fragmented landscapes. We used genetically based measures of migration between patches to test two dispersal-based hypotheses for this negative density-area relationship: (1) emigration rates from small patches should be relatively lower compared to large patches (“inhibited dispersal hypothesis”), or (2) immigration rates should be higher into small than large patches (“immigration hypothesis”). Neither hypothesis was supported using data on dispersal inferred from eight microsatellite loci for 12 populations of Peromyscus leucopus in six small (1.3–2.7 ha) and six large (8–150 ha) forest patches. Emigration rates were not lower from and immigration rates were not higher into small than large patches. In fact, contrary to both hypotheses, emigration rates were higher from populations of P. leucopus in small compared to large patches. Based on a combination of genetic and field data, we speculate that higher reproduction in smaller patches resulted in higher densities which led to higher emigration rates from those patches. Rates of reproduction (presumably driven by better habitat conditions in smaller patches), rather than dispersal, seems to drive density differences in forest patches. We conclude that smaller forest patches within an agricultural matrix act as a source of individuals, and that migration rates are fairly high among forest patches regardless of size.