Protein extraction and comparison of stain protocols for analysis of two-dimensional electrophoresis gels

Protein extraction and the proteome of Lactobacillus delbrueckii subsp. bulgaricus were studied using different stains. The reversible silver staining technique was shown to be more sensitive than the irreversible silver stain. Coomassie colloidal was demonstrated to be as sensitive as reversible silver stain; however, the Coomassie colloidal blue solution developed a higher background and for sample preparation was more time-consuming.     

Population genetic studies of the pancreatic amylase (AMY2, E.C. 3.2.1.1) in Bulgaria

The pancreatic amylase (AMY2, E.C. 3.2.1.1) polymorphism has been studied in 2346 individuals from south-central and south-eastern Bulgaria. The allele frequencies have been determined as AMY2*1 = 0.9520 and AMY2*2 = 0.0480. The neighbor joining tree of seven subpopulations revealed only small genetic distances. Compared with other populations, the Bulgarian sample clustered with samples from Romania, Hungary, Germany and Switzerland, with larger distances to Albania, Greece and Macedonia.     

Enhancement of vitamin E production in sunflower cell cultures

The most biologically active component of vitamin E, -tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural -tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that -tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in -tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of -tocopherol was reached by these sunflower cell cultures.     

CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile

The chemokine receptor CCR9 is expressed on most small intestinal lamina propria and intraepithelial lymphocytes and on a small subset of peripheral blood lymphocytes. CCR9-expressing lymphocytes may play an important role in small bowel immunity and inflammation. We studied the phenotype and functional characteristics of CCR9(+) lymphocytes in blood from normal donors. A subset of CCR9(+) T cells have a phenotype of activated cells and constitutively express the costimulatory molecules CD40L and OX-40. In contrast to CCR9(-), CCR9(+)CD4(+) peripheral blood T cells proliferate to anti-CD3 or anti-CD2 stimulation and produce high levels of IFN-gamma and IL-10. IL-10-producing cells were exclusively detected within the CCR9(+) subset of CD4(+) T cells by intracellular staining and were distinct from IL-2- and IFN-gamma-producing cells. Moreover, memory CCR9(+)CD4(+) lymphocytes respond to CD2 stimulation with proliferation and IFN-gamma/IL-10 production, whereas memory CCR9(-)CD4(+) cells were unresponsive. In addition, memory CCR9(+)CD4(+) T cells support Ig production by cocultured CD19(+) B cells in the absence of prior T cell activation or addition of exogenous cytokines. Our data show that the memory subset of circulating CCR9(+)CD4(+) T cells has characteristics of mucosal T lymphocytes and contains cells with either Th1 or T-regulatory 1 cytokine profiles. Studies on the cytokine profile and Ag specificity of this cell subset could provide important insight into small intestinal immune-mediated diseases and oral tolerance in humans.     

The spatial organization of centromeric heterochromatin during normal human lymphopoiesis: evidence for ontogenically determined spatial patterns

It is believed that pericentromeric heterochromatin may play a major role in the epigenetic regulation of gene expression. We have previously shown that centromeres in human peripheral blood cells aggregate into distinct "myeloid" and "lymphoid" spatial patterns, suggesting that the three-dimensional organization of centromeric heterochromatin in interphase may be ontogenically determined during hematopoietic differentiation. To investigate this possibility, the spatial patterns of association of different centromeres were analyzed in hematopoietic progenitors and compared with those in early-B and early-T cells, mature B and T lymphocytes, and, additionally, mature granulocytes and monocytes. We show that those patterns change during lymphoid differentiation, with major spatial arrangements taking place at different stages during T and B cell differentiation. Heritable patterns of centromere association are observed, which can occur either at the level of the common lymphoid progenitor, or in early-T or early-B committed cells. A correlation of the observed patterns of centromere association with the gene content of the respective chromosomes further suggests that the variation in the composition of these heterochromatic structures may contribute to the dynamic relocation of genes in different nuclear compartments during cell differentiation, which might have functional implications for cell-stage-specific gene expression.     

Low-temperature sensors in bacteria

Bacteria are ubiquitous colonizers of various environments and host organisms, and they are therefore often subjected to drastic temperature alterations. Temperature alterations set demands on these colonizers, in that the bacteria need to readjust their biochemical constitution and physiology in order to survive and resume growth at the new temperature. Furthermore, temperature alteration is also a main factor determining the expression or repression of bacterial virulence functions. To cope with temperature variation, bacteria have devices for sensing temperature alterations and a means of translating this sensory event into a pragmatic gene response. While such regulatory cascades may ultimately be complicated, it appears that they contain primary sensor machinery at the top of the cascade. The functional core of such machinery is usually that of a temperature-induced conformational or physico-chemical change in the central constituents of the cell. In a sense, a bacterium can use structural alterations in its biomolecules as the primary thermometers or thermostats.     

Pharmacological characterization of cloned chicken neuropeptide Y receptors Y1 and Y5

The neuropeptide Y (NPY) receptor subtypes Y1 and Y5 are involved in the regulation of feeding and several other physiological functions in mammals. To increase our understanding of the origin and mechanisms of the complex NPY system, we report here the cloning and pharmacological characterization of receptors Y1 and Y5 in the first non-mammal, chicken (Gallus gallus). The receptors display 80-83% and 64-72% amino acid sequence identity, respectively, with their mammalian orthologues. The three endogenous ligands NPY, peptide YY (PYY) and pancreatic polypeptide (PP) have similar affinities as in mammals, i.e. NPY and PYY have subnanomolar affinity for both receptors whereas chicken PP bound with nanomolar affinity to Y5 but not to Y1. A notable difference to mammalian receptor subtypes is that the Y1 antagonist SR120819A does not bind chicken Y1, whereas BIBP3226 does. The Y5 antagonist CGP71863A binds to the chicken Y5 receptor. Anatomically, both Y1 and Y5 have high mRNA expression levels in the infundibular nucleus which is the homologous structure of the hypothalamic arcuate nucleus in mammals. These results suggest that some of the selective Y1 and Y5 antagonists developed in mammals can be used to study appetite regulation in chicken.     

Tobacco-smoke exposure indicators and urinary mutagenicity

The protective effect of calcium given orally by gavage with two doses (40 and 80mg/kg body weight) was evaluated against clastogenecity induced by lead acetate with two concentrations (200 and 400mg/kg diet) on bone marrow and spermatocyte cells of mice in vivo. The parameter screened was percentage of chromosomal aberrations with and without gaps and sperm abnormalities. Statistical analyses indicated the protection efficacy of calcium with the high dose rather than the other in both types of mouse cells.The observation from the laboratory tests, dealing that lead acetate can be considered as an environmental genotoxic material. We recommended that it must be administered of calcium (as calcium chloride) as a protective agent to reduce the genotoxic effect of lead in the somatic and germ cells.     

Tetrathiomolybdate inhibits angiogenesis and metastasis through suppression of the NFkappaB signaling cascade

Tetrathiomolybdate (TM), a specific copper chelator, has been shown to be a potent antiangiogenic and antimetastatic compound possibly through suppression of the NFkappaB signaling cascade. To further delineate the molecular mechanism of the anticancer effect of TM, we investigated whether TM has antineoplastic activity in the setting of genetic NFkappaB inhibition. In this study, SUM149 inflammatory breast carcinoma cells were transfected with a dominant-negative IkappaBalpha (S32AS36A) expression vector. Similar to TM-treated SUM149 cells, SUM149-IkappaBalphaMut clones secreted lower amounts of proangiogenic mediators, vascular endothelial growth factor, interleukin-1alpha, and interleukin-8 and exhibited a less invasive and motile phenotype. The reduction in the angiogenic and metastatic potential of SUM149-IkappaBalphaMut clones was not further affected by TM in vitro. SUM149-IkappaBalphaMut xenografts grew substantially slower and had less lung metastasis than SUM149 and SUM149-empty vector xenografts. The growth and metastatic potential of SUM149 and SUM149-empty tumors was significantly inhibited with systemic TM treatment, whereas TM had no further antitumor effect on the SUM149-IkappaBalphaMut tumors. Additionally, nuclear proteins isolated from TM-treated SUM149 tumors had lower NFkappaB binding activity, while AP1 and SP1 binding activities were unchanged. Taken together, these results strongly support that suppression of NFkappaB is the major mechanism used by TM to inhibit angiogenesis and metastasis.