Physiology and pathophysiology of selectins, integrins, and IgSF cell adhesion molecules focusing on inflammation. A paradigm model on infectious endocarditis

The development of adhesion bonds, either among cells or among cells and components of the extracellular matrix, is a crucial process. These interactions are mediated by some molecules collectively known as adhesion molecules (CAMs). CAMs are ubiquitously expressed proteins playing a central role in controlling cell migration, proliferation, survival, and apoptosis. Besides their key function in physiological maintenance of tissue integrity, CAMs play an eminent role in various pathological processes such as cardiovascular disorders, atherogenesis, atherosclerotic plaque progression and regulation of the inflammatory response. CAMs such as selectins, integrins, and immunoglobulin superfamily take part in interactions between leukocyte and vascular endothelium (leukocyte rolling, arrest, firm adhesion, migration). Experimental data and pathologic observations support the assumption that pathogenic microorganisms attach to vascular endothelial cells or sites of vascular injury initiating intravascular infections. In this review a paradigm focusing on cell adhesion molecules pathophysiology and infective endocarditis development is given.     

A directed molecular evolution approach to improved immunogenicity of the HIV-1 envelope glycoprotein

A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences.     

Insight into the active-site structure and function of cytochrome oxidase by analysis of site-directed mutants of bacterial cytochromeaa 3 and cytochromebo

Cytochromeaa ₃ ofRhodobacter sphaeroides and cytochromebo ofE. coli are useful models of the more complex cytochromec oxidase of eukaryotes, as demonstrated by the genetic, spectroscopic, and functional studies reviewed here. A summary of site-directed mutants of conserved residues in these two enzymes is presented and discussed in terms of a current model of the structure of the metal centers and evidence for regions of the protein likely to be involved in proton transfer. The model of ligation of the hemea ₃ (oro)-Cu_B center, in which both hemes are bound to helix X of subunit I, has important implications for the pathways and control of electron transfer.     

Persistence of the nematophagous fungus Paecilomyces lilacinus strain 251 in soil under controlled conditions

The persistence of the nematophagous fungus Paecilomyces lilacinus Samson strain 251 (PL251) and the effect of application rate, substrate type, as well as the presence of the nematode host on its dynamics after application to the soil were investigated under controlled conditions. In all experiments, increase of P. lilacinus colony forming units after application was not found. In contrast, a gradual decline in fungal densities over time was observed. Application rate had no significant effect on the dynamics of the fungal population. Likewise, P. lilacinus density decline in soil was not significantly affected by the presence of the nematode host. Substrate type had a significant effect on P. lilacinus persistence in soil. The fungal agent persisted longer in silty loam and clay soil, with reduced persistence when sand was added to field soil. Conversely, when organic substrate was added to pure sand, persistence was significantly increased. Although persistence of fungal biocontrol agents in soil depends on various biotic and abiotic conditions, baseline data on persistence such as those reported in this study are helpful for biocontrol and environmental risk assessment and merit further study.     

Metabolic innovations towards the human lineage

Background  

We describe a function-driven approach to the analysis of metabolism which takes into account the phylogenetic origin of biochemical reactions to reveal subtle lineage-specific metabolic innovations, undetectable by more traditional methods based on sequence comparison. The origins of reactions and thus entire pathways are inferred using a simple taxonomic classification scheme that describes the evolutionary course of events towards the lineage of interest. We investigate the evolutionary history of the human metabolic network extracted from a metabolic database, construct a network of interconnected pathways and classify this network according to the taxonomic categories representing eukaryotes, metazoa and vertebrates.     

Tumorigenic Properties of Iron Regulatory Protein 2 (IRP2) Mediated by Its Specific 73-Amino Acids Insert

Iron regulatory proteins, IRP1 and IRP2, bind to mRNAs harboring iron responsive elements and control their expression. IRPs may also perform additional functions. Thus, IRP1 exhibited apparent tumor suppressor properties in a tumor xenograft model. Here we examined the effects of IRP2 in a similar setting. Human H1299 lung cancer cells or clones engineered for tetracycline-inducible expression of wild type IRP2, or the deletion mutant IRP2_Δ73 (lacking a specific insert of 73 amino acids), were injected subcutaneously into nude mice. The induction of IRP2 profoundly stimulated the growth of tumor xenografts, and this response was blunted by addition of tetracycline in the drinking water of the animals, to turnoff the IRP2 transgene. Interestingly, IRP2_Δ73 failed to promote tumor growth above control levels. As expected, xenografts expressing the IRP2 transgene exhibited high levels of transferrin receptor 1 (TfR1); however, the expression of other known IRP targets was not affected. Moreover, these xenografts manifested increased c-MYC levels and ERK1/2 phosphorylation. A microarray analysis identified distinct gene expression patterns between control and tumors containing IRP2 or IRP1 transgenes. By contrast, gene expression profiles of control and IRP2_Δ73-related tumors were more similar, consistently with their growth phenotype. Collectively, these data demonstrate an apparent pro-oncogenic activity of IRP2 that depends on its specific 73 amino acids insert, and provide further evidence for a link between IRPs and cancer biology.     

Walnut Consumption Increases Satiation but Has No Effect on Insulin Resistance or the Metabolic Profile Over a 4‐day Period

Obesity and diabetes have been associated with increased consumption of highly processed foods, and reduced consumption of whole grains and nuts. It has been proposed, mainly on the basis of observational studies, that nuts may provide superior satiation, may lead to reduced calorie consumption, and may decrease the risk of type 2 diabetes; but evidence from randomized, interventional studies is lacking. A total of 20 men and women with the metabolic syndrome participated in a randomized, double‐blind, crossover study of walnut consumption. Subjects had two 4‐day admissions to the clinical research center where they were fed an isocaloric diet. In addition, they consumed shakes for breakfast containing either walnuts or placebo (shakes were standardized for calories, carbohydrate, and fat content). Appetite, insulin resistance, and metabolic parameters were measured. We found an increased level of satiety (overall P value = 0.0079) and sense of fullness (P = 0.05) in prelunch questionnaires following the walnut breakfast as compared to the placebo breakfast, with the walnut effect achieving significance on day 3 and 4 (P = 0.02 and P = 0.03). We did not find any change in resting energy expenditure, hormones known to mediate satiety, or insulin resistance when comparing the walnut vs. placebo diet. Walnut consumption over 4 days increased satiety by day 3. Long‐term studies are needed to confirm the physiologic role of walnuts, the duration of time needed for these effects to occur, and to elucidate the underlying mechanisms.     

Emergence, development and diversification of the TGF- β signalling pathway within the animal kingdom

Background  

The question of how genomic processes, such as gene duplication, give rise to co-ordinated organismal properties, such as emergence of new body plans, organs and lifestyles, is of importance in developmental and evolutionary biology. Herein, we focus on the diversification of the transforming growth factor- β (TGF- β) pathway – one of the fundamental and versatile metazoan signal transduction engines.     

Glucose regulation of CDK7, a putative thiol related gene, in experimental diabetic nephropathy

We previously described the identification of the 3'end of an unknown gene CDK7 using differential display which appeared to be up-regulated in diabetic kidneys [R.A. Page, C.A. Morris, J.D. Williams, C.J. von Ruhland, A.N. Malik, Isolation of diabetes-associated kidney genes using differential display, Biochem. Biophys. Res. Commun. 232 (1997) 49-53]. Here we show that CDK7 is a putative thiol related gene which is regulated by glucose in human and rat renal cells. CDK7 mRNA increased by >threefold in cultured human mesangial cells grown in high glucose for 4 days. In the kidneys of the GK rat, a model of type II diabetes, CDK7 showed a steady age-related increase in mRNA, increasing to >sixfold in 40 week GK rats compared to normoglycemic age-matched Wistar rat kidneys, this increase correlates with progressive hyperglycemia. CDK7 mRNA is widely expressed, showing particularly high levels of expression in rat and human liver, and encodes a putative 338 amino acids highly conserved peptide with several conserved domains, including a cys-pro-arg-cys domain conserved in 15 diverse species which is similar to the catalytic centre of thioredoxin, suggesting a role in oxidative stress.     

Assay for the quantification of intact/fragmented genomic DNA

This study shows that the accuracy of the quantification of genomic DNA by the commonly used Hoechst- and PicoGreen-based assays is drastically affected by its degree of fragmentation. Specifically, it was shown that these assays underestimate by 70% the concentration of double-stranded DNA (dsDNA) with sizes less than 23 kb. On the other hand, DNA sizes greater and less than approximately 23 kb are commonly characterized as intact and fragmented genomic DNA, respectively, by the agarose electrophoresis DNA smearing assay and are evaluated only qualitatively by this assay. The need for accurate quantification of fragmented and total genomic DNA, combined with the lack of specific, reliable, and simple quantitative methods, prompted us to develop a Hoechst/PicoGreen-based fluorescent assay that quantifies both types of DNA. This assay addresses these problems, and in its Hoechst and PicoGreen version it accurately quantifies dsDNA as being either intact (>or=23 kb) or fragmented (<23 kb) in concentrations as low as 3 ng ml-1 or 5 pg ml-1 with Hoechst or PicoGreen, respectively, as well as the individual fractions of intact/fragmented DNA existing in any proportions in a total DNA sample in concentrations as low as 10 ng ml-1 or 15 pg ml-1 with Hoechst or PicoGreen, respectively. Because the assay discriminates total genomic DNA in the two size ranges (>or=23 and <23 kb) and quantitates them, it is proposed as the quantitative replacement of the agarose gel electrophoresis genomic DNA smearing assay.