Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence

The relationship between the mechanical properties of cells and their molecular architecture has been the focus of extensive research for decades. The cytoskeleton, an internal polymer network, in particular determines a cell's mechanical strength and morphology. This cytoskeleton evolves during the normal differentiation of cells, is involved in many cellular functions, and is characteristically altered in many diseases, including cancer. Here we examine this hypothesized link between function and elasticity, enabling the distinction between different cells, by using a microfluidic optical stretcher, a two-beam laser trap optimized to serially deform single suspended cells by optically induced surface forces. In contrast to previous cell elasticity measurement techniques, statistically relevant numbers of single cells can be measured in rapid succession through microfluidic delivery, without any modification or contact. We find that optical deformability is sensitive enough to monitor the subtle changes during the progression of mouse fibroblasts and human breast epithelial cells from normal to cancerous and even metastatic state. The surprisingly low numbers of cells required for this distinction reflect the tight regulation of the cytoskeleton by the cell. This suggests using optical deformability as an inherent cell marker for basic cell biological investigation and diagnosis of disease.     

The effects of the insulin-like growth factor-I aptamer, NBI-31772, on glucose homeostasis in the mouse

The majority of insulin-like growth factor-I (IGF-I) in the adult rodent circulation is bound to high affinity IGF binding proteins. We investigated the changes in IGF-I clearance, blood glucose and plasma insulin levels, and tissue 2-deoxyglucose uptake after intravenous administration of the IGF aptamer, NBI-31772, which selectively competes with IGF-I for binding to the IGFBPs, but has no effect at the IGF-I receptor. Clearance of 125I-IGF-I was significantly increased in NBI-31772-treated mice compared with vehicle-treated mice (t1/2 = 45.0 +/- 1.9 vs. 56.3 +/- 3.9 min, respectively; p = 0.021). However, NBI-31772 had no significant effect on glucose levels, and no insulin sparing effect was apparent neither under basal conditions nor during an intravenous glucose challenge. The decline in the specific activity after 3H-2-deoxyglucose administration was significantly less rapid in NBI-31772-treated mice compared with controls, suggesting that the IGF-I aptamer had an inhibitory effect on hepatic gluconeogenesis. In contrast, no insulin-like effect was apparent in other tissues examined. 3H-2-deoxyglucose accumulation was similar in all tissues analyzed, including skeletal muscle, which is thought to be particularly sensitive to IGF-I. These data suggest that the IGF-I aptamer affects clearance of radiolabeled IGF-I from the circulation, but has no marked effects on glucose nor insulin homeostasis. The search for hydrophilic IGF aptamers with longer duration of action that could be used in the treatment of diabetes may be rewarding.     

Stability of proteins: temperature, pressure and the role of the solvent

We focus on the various aspects of the physics related to the stability of proteins. We review the pure thermodynamic aspects of the response of a protein to pressure and temperature variations and discuss the respective stability phase diagram. We relate the experimentally observed shape of this diagram to the low degree of correlation between the fluctuations of enthalpy and volume changes associated with the folding-denaturing transition and draw attention to the fact that one order parameter is not enough to characterize the transition. We discuss in detail microscopic aspects of the various contributions to the free energy gap of proteins and put emphasis on how a cosolvent may either enlarge or diminish this gap. We review briefly the various experimental approaches to measure changes in protein stability induced by cosolvents, denaturants, but also by pressure and temperature. Finally, we discuss in detail our own molecular dynamics simulations on cytochrome c and show what happens under high pressure, how glycerol influences structure and volume fluctuations, and how all this compares with experiments.     

Immunohistochemical analysis of steroid receptors and glycodelin A (PP14) in isolated glandular epithelial cells of normal human endometrium

Highly purified fractions of isolated endometrial cells can be useful for investigating endometrial function. After a first collagenase digestion, normal human endometrial stromal and epithelial cells were separated by filtration. Glands were purified further by two collagenase digestion steps, filtration, differential sedimentations, and Ficoll gradient centrifugation. Epithelial cells were polyhedral and grew as islands in a whorl-like wavy pattern around glandular fragments. High cell culture purity was confirmed with the positive immunohistochemical reaction against cytokeratin 7,8,18,19. Isolated human glands had a similar distribution pattern of estrogen receptor (ER) and progesterone receptor (PR) as observed in vivo, suggesting that glands have a functional hormone receptor system at the time of plating. Using a specific monoclonal antibody against glycodelin A (GdA), a characteristic cyclical expression was demonstrated during the menstrual cycle. The GdA reaction was weak in the proliferative phase, increasing significantly till the late secretory phase, suggesting a similar GdA concentration in vitro as observed in vivo glands. In conclusion, this method could be a model for studying endometrial glandular cells from different menstrual phases, endometrial cell interactions, implantation mechanisms, GdA regulation mechanisms, and pharmacological or other influences on ER and PR alteration.     

Molecular characterization of the Thermomonospora curvata aglA gene encoding a thermotolerant alpha-1,4-glucosidase

The cloning, sequencing and structural characterization of a gene encoding a thermostable alpha-1,4-glucosidase from Thermomonospora curvata is described. DNA sequence analysis revealed four open reading frames designated aglA, aglR, aglE and aglF. The aglA gene encodes a thermostable alpha-1,4-glucosidase from T. curvata and is situated between two genes, aglR and aglE. Genes aglA, aglE and aglF are transcribed in the same direction, while aglR is transcribed in the opposite direction. By comparing the amino acid sequence of the alpha-1,4-glucosidase from T. curvata with other alpha-glucanases, it appears that the enzyme is a member of the alpha-amylase family. The proteins of this family have an (alpha/beta)8 barrel super secondary structure. The topology of the alpha-1,4-glucosidase was predicted by computer-assisted analysis. The topology of the secondary structures of the alpha-1,4-glucosidase resembles the structure of barley alpha-amylase, but the primary structure resembles most closely the oligo-1,6-glucosidase from Bacillus cereus. Putative catalytic residues (D221, E281 and D343) and calcium binding residues (N116, E179, D191, H224 or G225) are proposed.     

The effect of suppressors on the resulting segregation ratios of some characters of barley in higher hybrid generations

1. 1.
   V reciprokých k?í?eních kombinací některých odr?d je?mene byly zji?těny potla?ující faktory pro projev recesivní i dominantní alternativy těchto znak?: ?adovost klasu, pluchatost zrna, charakter osin a utvá?ení báze klasu.     

Diagnosis of Bacteria In Vitro by Mass Spectrometric Fingerprinting:A Pilot Study

The identification of bacteria by using conventional microbiological techniques can be very time-consuming and circumstantial. In contrast, the headspace screening of bacterial cultures by analyzing their emitted volatile compounds using mass spectrometry might provide a novel approach in diagnostic microbiology. In the present study different strains of Escherichia coli, Klebsiella, Citrobacter, Pseudomonas aeruginosa, Staphylococcus aureus, and Helicobacter pylori were investigated. The volatile compounds emitted by these bacteria in vitro were analyzed using proton-transfer-reaction mass spectrometry, which allows rapid and sensitive measurement. The detected patterns of volatile compounds produced by the investigated bacteria were compared and substantial differences regarding both quantity and quality were observed. In conclusion, the present study is the first to describe headspace screening of bacterial cultures as a potential diagnostic approach in medical microbiology.     

Testing of the Biocan B inj. ad us. vet. vaccine and development of the new recombinant vaccine against canine borreliosis

Verification of the efficacy of Biocan B inj. ad us. vet. (Bioveta, a.s.) was done by challenge testing. Ticks collected in the nature were used as natural vectors of the infection. Six beagles and two control ones were used in the test. Formation of outer surface protein A specific antibodies (OspA antibodies) and borrelia specific immonoglobulins (IgG) was measured by Western blot and EIA in the sera samples. The tissue samples were used for detection of borreliae by cultivation method and dark field microscopy (DFM). Formation of IgG antibodies and OspA antibodies after vaccination was observed. The maximum titer level of antibodies was reached between 21. and 49. day after vaccination and then slowly decreased. Presence of borreliae was detected only in skin biopsies of non-vaccinated dogs. The post mortem tissue samples showed presence of borreliae in all of the samples of the non-vaccinated dogs. The tissues of the vaccinated dogs were not infected with borreliae, except for two samples of dog with low titer levels of OspA antibodies. The development of the new vaccine is based on preparation of recombinant outer surface proteins (e.g. rOspA and rOspC) of B. afzelii, B. burgdorferi and B. garinii origin. Chosen recombinant proteins were successfully expressed in E. coli. The obtained purified proteins are currently being tested on laboratory BALB/c mice. Formation of specific antibodies against some recombinant proteins has been confirmed. These proteins are suitable candidates for preparation of a vaccine prototype and they will be subsequently used in challenge tests.     

Increased level of advanced oxidation products (AOPP) as a marker of oxidative stress in patients with acute coronary syndrome

Oxidative stress impairs endothelial function and may play an important role in the pathogenesis of acute cardiovascular diseases. Advanced oxidation protein products (AOPP) were proposed as one of the possible markers of oxidative injury, which originates under oxidative and carbonyl stress and increase global inflammatory activity. The present study was undertaken to compare AOPP concentrations in a control group of healthy individuals without ICHS (I), patients with stable angina pectoris (II), patients with acute coronary syndrome over 48 hours without ST elevations (III), and patients with ST elevation myocardial infarction (IV). Coronaronary angiography, risk factors and anamnestic data were analyzed. We examined 73 probands with signs of myocardial ischemia, mean age of 61.5 years (64% males) subjected to coronarography and 21 healthy individuals. No significant difference was found between venous blood and coronary samples, or between infarction and non-infarction arteries in the group IV. AOPP concentrations in healthy individuals in the group I (82.9 +/- 29.3 mmol/l) did not differ significantly from patients in group II (89.6 +/- 26.7 mmol/l) and group III (112.3 +/- 54.6 mmol/l). A significant difference in AOPP values was found between the groups I and IV, and between the groups II and IV (82.9 +/- 29.3 mmol/l vs. 125.8 +/- 101 mmol/l, p = 0.02, and 89.6 +/- 26.7 mmol/l vs. 125.8 +/- 101 mmol/l, p = 0.02). No correlations were found between AOPP and body mass index (BMI), nicotinism, left ventricular ejection fraction, parameters of glucose and lipid metabolism. ROC analysis revealed that AOPP concentrations of 89 mmol/l had 64% sensitivity and 71% specificity for revealing an acute coronary syndrome (AUC 0.65, 95% CI 0.55-0.80). AOPP are significantly increased in patients with acute coronary syndromes with ST segment elevation, but also tend to increase in patients with non-ST elevation myocardial infarction. Our observations suggest that AOPP may be used as a marker of oxidative stress and as a prognostic factor for severe forms of cardiovascular disease. A cut-off value of 89 mmol/l can be used with 64% sensitivity and 71% specificity for revealing acute coronary syndrome.