📋 Browse Articles

Tobacco smoking, counted as one of the alternating external cardiovascular risk factors, can cause disadvantageous changes of concentration of compounds considered to be the new internal risk factors. The aim of the study was to determine the influence of tobacco smoke on concentration levels of homocysteine, dimethylarginine (ADMA), fibrinogen and C-reactive protein (CRP) in healthy smoking male's blood plasma. 71 healthy male volunteers, aged 30-59, were chosen for the study. Based on a questionnaire they were divided into non-smoking group (37 individuals) and active smokers group (34 individuals). The results obtained indicate the most significant increasing effect of tobacco smoke on blood plasma homocysteine levels (by 11.9%) and then, successively, on fibrinogen (by 10.1%) and ADMA (by 9.1%). Changes of CRP concentration, although differentiated similarly to other factors, were insignificant. Since the coexistence of elevated risk factors shows a synergistic effect on cardiovascular risk level in general, it is advisable to perform tests of new risk factors among people exposed to tobacco smoke. The tests will appoint the ones at risk of disease and help to cover them with a medical care. Show less

The first paper concerning hair nicotine determination was published in 1983 by Ishiyama et al. Since then, examining hair nicotine content has become a valuable tool facilitating assessment of exposure to tobacco smoke in various groups of people (active and passive smokers or even neonates and fetus). The present article describes briefly recent research studies on the use of hair nicotine as a biomarker. Furthermore, the paper describes various determination techniques related to nicotine in hair, as well as advantages and disadvantages of hair nicotine as a biomarker. Show less

In Poland approximately 33% adults are active smokers. Both active smoking and passive exposition to tobacco smoke leads to serious diseases and causes many other problems. This is why the decrease of smoking prevalence seems to be one of the most important targets of contemporary medicine. In this task the attitude of medical staff plays a great role (prevention, education, behaviour of a medical employee as a positive example). The research was carried out among 48 employees of Regional Station of Ambulance Service in Poznan (16 physicians, 19 medical rescuers and 13 nurses). The group comprised 21 women and 27 men. The investigative tool was a questionnaire concerning smoking, passive exposition to tobacco smoke and attitude of the examined persons towards smoking. We found that smoking prevalence in the investigated group considerably exceeds the average values for Polish population (particularly the number of smoking women) whereas passive exposition to tobacco smoke affects everyone in this group. In the opinion of the examined people the main factors inducing and maintaining tobacco smoking are: addiction, pleasure connected with smoking or influence of someone's company. In a case of medical personnel tobacco smoking (similarly to other behaviours which are harmful for someone's health) weakens the authority of a health care provider as an example of health promotion. Show less

The aim of this study was analysis of kind of interactions between Fe and the other metals: Al, Ba, Ca, Cr, Cu, Mg, Mn, Zn, K i Na in pharyngeal tonsils from girls and boys in several group of age (2-5 years, 6-9 years and 10-15 years) exposed and no exposed to tobacco smoking. This study showed that the kind of interaction dependent on sex, age and exposed to ETS. Show less

The objective of this study was qualification of content cadmium and lead in selected elements of the hip joint in aspect of tobacco smoking. The material for the research were 5 elements of hip joint (articular cartilage, trabecular bone and cortical bone femur head, fragment articular capsule and fragment trabecular bone from region intertrochanteric femoral bone), obtained intraoperatively during endoprothesoplastic surgeries. The samples come from habitants of Upper Silesian Region. Determination of trace elements contents were performed by ASA method (Pye Unicam SP-9) in acetylene-oxygen flame. Higher contents of lead were observed for smoking people, however in case of cadmium the differences of this element were not statistical essential between smokers and non-smokers. Show less

Most high throughput structural proteomics centers use the sitting-drop method to obtain diffracting crystals for three-dimensional (3D) structure determination of biological macromolecules by x-ray crystallography. Although several robotic systems are available for dispensing the initial sitting-drop screening conditions, generally they are not used for optimization of crystallization conditions. This chapter describes a protocol for such automated systems, which permits easy construction of pH optimization grids using any desired fixed buffer set with varying ionic strengths directly dispensed into the crystallization plate. Show less

The Cyt family of proteins consists of delta-endotoxins expressed during sporulation of several subspecies of Bacillus thuringiensis. Its members possess insecticidal, hemolytic, and cytolytic activities through pore formation and attract attention due to their potential use as vehicles for targeted membrane destruction. The delta-endotoxins of subsp. israelensis include three Cyt species: a major Cyt1Aa and two minor proteins, Cyt2Ba and Cyt1Ca. A cleaved Cyt protein that lacks the N- and C-terminal segments forms a toxic monomer. Here, we describe the crystal structure of Cyt2Ba, cleaved at its amino and carboxy termini by bacterial endogenous protease(s). Overall, its fold resembles that of the previously described volvatoxin A2 and the nontoxic form of Cyt2Aa. The structural similarity between these three proteins may provide information regarding the mechanism(s) of membrane-perforating toxins. Show less

The increased plasma level of homocysteine have been shown to be the sensitive marker for the folate, vitamin B6 and cobalamins deficiency and an independent risk factor for the cardiovascular disease, neutral tube defects and a potential causal risk factor for neuropsychiatric disorders. The blood and plasma homocysteine levels except for genetic defects are influenced by age, gender, efficiency of detoxication systems, one or more unhealthy lifestyle factors, such as high alcohol consumption, low nutritional intake of vitamins, high coffee consumption, acquired disorders and lack of physical exercise. Many studies confirm that active tobacco smoking and environmental tobacco smoke (ETS) have been significantly associated with hiperhomocysteinemia. In metabolic pathway of homocysteine the important role played folic acid, as a donor of methyl group in re-methylated reaction to methionine and vitamin B6. It acts as the cofactor in transsuphuration reactions of homocysteine to cystathionine and cysteine. Hence, the aim of this work was to compare the plasma folate and vitamin B6 concentrations in smokers and passive smokers with a hiperhomocysteinemia (> 15 micromol/L). It was observed that the plasma folate levels in active (n = 30) and passive smokers (n = 29) groups decrease statistically significant (P < 0.001) in comparison to non-smokers (n = 37). The calculated Spaermann's correlations coefficient of total plasma homocysteine level and plasma folate concentrations in the non-smoker group indicated a weak, statistically insignificant correlation (r = -0.103, P = 0.542). However, the above relationship in passive and active smokers were statistically significant (r = -0.495, P 0 0.01; r = -0.672, P < 0.001, respectively). The decrease of vitamin in B6 plasma was observed in all active smokers group (P < 0.01) and men smokers comparing to non-smokers (P < 0.001). There was no observed significant correlations between hiperhomocysteinemia and vitamin B6 in all studied groups. The results indicated that hiperhomocysteinemia have strong negative impact on folate levels in active and passive smokers. The tobacco smoke exposure have negative influence on the status of vitamin B6. The lack of significant correlation between increased homocysteine levels and vitamin B6 status confirmed hypothesis that hiperhomocysteinemia is not depended on vitamin B6 concentrations in plasma. Show less

It has been already proved in many experimental studies that tobacco smoke has multiple toxic effects on respiratory tract cells. Alterations in cilliary epithelium of rats trachea after short exposition to high tobacco smoke concentrations in inhaled air were been determined in current study. Morphological evaluation revealed in lining epithelium decrease in number of cilliary cells and increase in number of goblet cells. The mucous membrane was thickened and infiltrated with inflammatory cells. Show less

Agrobacterium tumefaciens infects its plant hosts by a mechanism of horizontal gene transfer. This capability has led to its widespread use in artificial genetic transformation. In addition to DNA, the bacterium delivers an abundant ssDNA binding protein, VirE2, whose roles in the host include protection from cytoplasmic nucleases and adaptation for nuclear import. In Agrobacterium, VirE2 is bound to its acidic chaperone VirE1. When expressed in vitro in the absence of VirE1, VirE2 is prone to oligomerization and forms disordered filamentous aggregates. These filaments adopt an ordered solenoidal form in the presence of ssDNA, which was characterized previously by electron microscopy and three-dimensional image processing. VirE2 coexpressed in vitro with VirE1 forms a soluble heterodimer. VirE1 thus prevents VirE2 oligomerization and competes with its binding to ssDNA. We present here a crystal structure of VirE2 in complex with VirE1, showing that VirE2 is composed of two independent domains presenting a novel fold, joined by a flexible linker. Electrostatic interactions with VirE1 cement the two domains of VirE2 into a locked form. Comparison with the electron microscopy structure indicates that the VirE2 domains adopt different relative orientations. We suggest that the flexible linker between the domains enables VirE2 to accommodate its different binding partners. Show less

The asymmetric unit of the title compound, {[Cu(CO(3))(C(14)H(14)N(4))(1.5)] x 0.5 C(14)H(14)N(4) x 5 H(2)O}(n), contains one Cu(II) cation in a slightly distorted square-pyramidal coordination environment, one CO(3)(2-) anion, one full and two half 1,4-bis(imidazol-1-ylmethyl)benzene (bix) ligands, one half-molecule of which is uncoordinated, and five uncoordinated water molecules. One of the coordinated bix ligands and the uncoordinated bix molecule are situated about centers of symmetry, located at the centers of the benzene rings. The coordinated bix ligands link the copper(II) ions into a [Cu(bix)(1.5)](n) molecular ladder. These molecular ladders do not form interpenetrated ladders but are arranged in an ABAB parallel terrace, i.e. with the ladders arranged one above another, with sequence A translated with respect to B by 8 A. To best of our knowledge, this arrangement has not been observed in any of the molecular ladder frameworks synthesized to date. The coordination environment of the Cu(II) atom is completed by two O atoms of the CO(3)(2-) anion. The framework is further strengthened by extensive O-H...O and O-H...N hydrogen bonds involving the water molecules, the O atoms of the CO(3)(2-) anion and the N atoms of the bix ligands. This study describes the first example of a molecular ladder coordination polymer based on bix and therefore demonstrates further the usefulness of bix as a versatile multidentate ligand for constructing coordination polymers with interesting architectures. Show less

Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia are recessive spondyloepimetaphyseal dysplasias caused by loss-of-function mutations in dymeclin (Dym), a gene with previously unknown function. Here we report that Dym-deficient mice display defects in endochondral bone formation similar to that of Dyggve-Melchior-Clausen syndrome and Smith-McCort dysplasia, demonstrating functional conservation between the two species. Dym-mutant cells display multiple defects in vesicle traffic, as evidenced by enhanced dispersal of Golgi markers in interphase cells, delayed Golgi reassembly after brefeldin A treatment, delayed retrograde traffic of an endoplasmic reticulum-targeted Shiga toxin B subunit, and altered furin trafficking; and the Dym protein associates with multiple cellular proteins involved in vesicular traffic. These results establish dymeclin as a novel protein involved in Golgi organization and intracellular vesicle traffic and clarify the molecular basis for chondrodysplasia in mice and men. Show less

Analysis of the three-dimensional structures of three closely related mesophilic, thermophilic, and hyperthermophilic alcohol dehydrogenases (ADHs) from the respective microorganisms Clostridium beijerinckii (CbADH), Entamoeba histolytica (EhADH1), and Thermoanaerobacter brockii (TbADH) suggested that a unique, strategically located proline residue (Pro100) might be crucial for maintaining the thermal stability of EhADH1. To determine whether proline substitution at this position in TbADH and CbADH would affect thermal stability, we used site-directed mutagenesis to replace the complementary residues in both enzymes with proline. The results showed that replacing Gln100 with proline significantly enhanced the thermal stability of the mesophilic ADH: DeltaT(1/2) (60 min) = + 8 degrees C (temperature of 50% inactivation after incubation for 60 min), DeltaT(1/2) (CD) = +11.5 degrees C (temperature at which 50% of the original CD signal at 218 nm is lost upon heating between 30 degrees and 98 degrees C). A His100 --> Pro substitution in the thermophilic TbADH had no effect on its thermostability. An analysis of the three-dimensional structure of the crystallized thermostable mutant Q100P-CbADH suggested that the proline residue at position 100 stabilized the enzyme by reinforcing hydrophobic interactions and by reducing the flexibility of a loop at this strategic region. Show less

The aim of the study was to evaluate the reliability of self-reported smoking status and environmental tobacco smoke exposure (ETS) during pregnancy, assessing serum cotinine level. The prospective cohort study was conducted in 2 antenatal care units in Lodz, Poland. Study population consisted of 183 pregnant women between 20-24 weeks of pregnancy. All of the women who agreed to participate in the study were interviewed to investigate certain socio-demographic, lifestyles, behavioural characteristics and obstetric background. Self-reported smoking status and ETS exposure were verified with the help of serum cotinine level. Cotinine level was analyzed by means of gas chromatography with mass spectroscopy (GC-MS). We choose more than 15 ng/ml as serum cotinine level for smokers, 2-15 ng/ml for ETS exposure and less than 2 ng/ml for non-smokers not exposed to ETS. Among non-smoking and not ETS-exposed women, 17% had cotinine level indicating active smoking and 74% ETS exposure. About 4% of the women who indicated ETS exposure during pregnancy had serum cotinine level higher than 15 ng/ml indicating active smoking. The information about active and passive smoking during pregnancy obtained from mothers and based on the questionnaire does not indicate objective maternal exposure to tobacco smoke. Show less

Spermatogenesis is the process by which spermatogonial stem cells divide and differentiate into sperm. The role of growth factor receptors in regulating self-renewal and differentiation of spermatogonial stem cells remains largely unclear. This study was designed to examine Gfra1 receptor expression in immature and adult mouse testes and determine the effects of Gfra1 knockdown on the proliferation and differentiation of type A spermatogonia. We demonstrated that GFRA1 was expressed in a subpopulation of spermatogonia in immature and adult mice. Neither Gfra1 mRNA nor GFRA1 protein was detected in pachytene spermatocytes and round spermatids. GFRA1 and POU5F1 (also known as OCT4), a marker for spermatogonial stem cells, were co-expressed in a subpopulation of type A spermatogonia from 6-day-old mice. In addition, the spermatogonia expressing GFRA1 exhibited a potential for proliferation and the ability to form colonies in culture, which is a characteristic of stem cells. RNA interference assays showed that Gfra1 small interfering RNAs (siRNAs) knocked down the expression of Gfra1 mRNA and GFRA1 protein in type A spermatogonia. Notably, the reduction of Gfra1 expression by Gfra1 siRNAs induced a phenotypic differentiation, as evidenced by the elevated expression of KIT, as well as the decreased expression of POU5F1 and proliferating cell nuclear antigen (PCNA). Furthermore, Gfra1 silencing resulted in a decrease in RET phosphorylation. Taken together, these data indicate that Gfra1 is expressed dominantly in mouse spermatogonial stem cells and that Gfra1 knockdown leads to their differentiation via the inactivation of RET tyrosine kinase, suggesting an essential role for Gfra1 in spermatogonial stem cell regulation. Show less

Cigarette smoking is associated with oxidative stress and increased risk of many chronic diseased. Smoking inducts depletion of cellular antioxidant and is also known to be associated with an increased homocysteine level. Exposure to tobacco smoke has negative impact on the folic acid level. Folic acid is cofactor by demethyla-tion of homocysteine to non toxic methionine. The aim of this study was to determine the concentration of total plasma homocysteine and 5-methyltetrahydrofolic acid in active and non smokers and to evaluate the influence of the tobacco smoke exposure on urinary cotinine levels. The results indicated significant increase of total plasma homocysteine in smokers, than non smokers. We also investigated the negative influence of tobacco smoke on the level of folic acid in plasma and it suggests, that additional supplementation of folic acid in smokers is necessary. Show less

Purpose of this work was to judge progress of diagnosed bronchial asthma among children and youth that are exposed on tobacco smoke in aspect of number of exacerbation of disease, therapy (doses of corticosteroids) and number of hospitalizations. There were two groups of patients with bronchial asthma under examination, first one were children and youth that are exposed on tobacco smoke, and the other group of children and youth that are not exposed on tobacco smoke. We discovered statistically significant difference in number of exacerbation of disease among those two groups, also bigger therapeutic and prophylactic doses of inhaled steroids during treatment, and much more often hospitalizations among children and youth that are exposed on tobacco smoke. Show less

Prevalence of tobacco smoking among young people is one of the most important aspects in prevention of smoking and smoking-related diseases. The aim of this study was assessment of pharmacy students' exposition to tobacco smoke (active and passive smoking) and searching for circumstances promoting tobacco smoking and exposition to ETS (environmental tobacco smoke). In the examined cohort of 161 students we found 37 smokers (23%; 21.2% women and 31% men), among them 14 (8.7%) everyday smokers. ETS affected 155 persons (96.3%) and usually took place in a circle of friends. The most common factors promoting tobacco smoking were: pleasure connected with smoking, smoking as a support in stress situation or as a facilitation of society functioning and getting to know new friends. We must emphasize again the leading role of prevention of smoking initiation in young people in all preventive activities. Show less

Toxic effects of tobacco smoke may manifest by the increase of concentration of aterogenic compounds in the human body. One of them is asymmetric dimetyloarginine (ADMA) which is perceived not only as a significant risk factor of endothelium dysfunction and cardiovascular disease but also predicts all-cause and cardiovascular mortality independently of established and emerging cardiovascular risk factor. The aim of this work was to check to what degree ADMA concentration in plasma changes for people exposed to tobacco smoke in various intensity. The study population consisted of 168 healthy males between the age of 18 and 60. The exposure to tobacco smoke in examined groups was determined on the basis of questionnaires and cotinine concentration in plasma. Three groups were selected: non-smokers (53 people), passive smokers (48 people) and active smokers (67 people). Insignificant differences of ADMA concentration between passive and active smokers in relation to non-smokers were determined, 15.4% (0.45 vs. 0.39 mmol/l,) and 7.7% (0.42 vs. 0.39 mmol/l) respectively. There was no significant relationship between concentration of ADMA and cotinine in plasma (r=0.173; p=0.239 for passive smokers and r=0.218; p=0.076 for active smokers). The results obtained indicate that tobacco smoke has small influence on ADMA level in healthy males' plasma. Show less

To investigate the effects of 2-methoxyestradiol (2-ME) on 2 myeloid leukemia cell lines HL-60 and U937, and to explore its mechanisms. Human myeloid leukemia cells HL-60 and U937 were used. Measurement of mitochondrial membrane potential (Dym) was performed using 5,5',6,6'-Tetrachloro-1,1',3,3'- tetraethylbenzimidazolylcarbocyanine iodide ( JC-1). Apoptosis and cellular nitric oxide (NO) were detected by flow cytometry using Annexin V and NO sensor dye. Superoxide anion was measured with a fluorescent plate reader by dihydroethidium (DHE). Cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. 2-ME resulted in viability decrease in a dose-dependent manner. 2-ME treatment also generated reactive oxygen species (ROS), including NO and superoxide anions, which resulted in mitochondria damage. 2-ME-induced apoptosis was correlated with an increase in ROS. The quenching of ROS with N-acetyl-L-cysteine protected leukemia cells from 2-ME cytotoxicity and prevented apoptosis induction by 2-ME. Furthermore, the addition of manumycin, a farnesyltransferase inhibitor, significantly enhanced apoptosis induced by 2-ME. Cellular ROS generation plays an important role in the cytotoxic effect of 2-ME. It is possible to use ROS generation agents, such as manumycin, to enhance the antileukemic effect. The combination strategy needs further in vivo justification and may have potential clinical application. Show less

The aim of the study was to analyze the active smoking by pregnant women in different weeks of pregnancy. The study population consisted of 136 women before 22 weeks of pregnancy. The women were interviewed three times during pregnancy. From all women included into the study the saliva sample was collected three times to verified smoking status in pregnancy. Cotinine level in saliva was analyzes using Liquid Chromatography with Tandem Mass Spectrometry. Based on interview about 25-30% of pregnant women smoked cigarettes during pregnancy. This percentage was higher when the smoking status was verified using cotinine level in saliva (33-41%). In the group of pregnant women who smoked cigarettes there was no decreasing trend in cotinine level during pregnancy whereas in nonsmokers the decreasing trend was observed. Unmarried women, those with lover educational level, and unemployed had the highest risk of smoking during pregnancy. Show less

Gaucher's disease, a lysosomal storage disorder caused by mutations in the gene encoding glucocerebrosidase (GCD), is currently treated by enzyme replacement therapy using recombinant GCD (Cerezyme) expressed in Chinese hamster ovary (CHO) cells. As complex glycans in mammalian cells do not terminate in mannose residues, which are essential for the biological uptake of GCD via macrophage mannose receptors in human patients with Gaucher's disease, an in vitro glycan modification is required in order to expose the mannose residues on the glycans of Cerezyme. In this report, the production of a recombinant human GCD in a carrot cell suspension culture is described. The recombinant plant-derived GCD (prGCD) is targeted to the storage vacuoles, using a plant-specific C-terminal sorting signal. Notably, the recombinant human GCD expressed in the carrot cells naturally contains terminal mannose residues on its complex glycans, apparently as a result of the activity of a special vacuolar enzyme that modifies complex glycans. Hence, the plant-produced recombinant human GCD does not require exposure of mannose residues in vitro, which is a requirement for the production of Cerezyme. prGCD also displays a level of biological activity similar to that of Cerezyme produced in CHO cells, as well as a highly homologous high-resolution three-dimensional structure, determined by X-ray crystallography. A single-dose toxicity study with prGCD in mice demonstrated the absence of treatment-related adverse reactions or clinical findings, indicating the potential safety of prGCD. prGCD is currently undergoing clinical studies, and may offer a new and alternative therapeutic option for Gaucher's disease. Show less

Genetic variation across inbred and outbred mouse strains have been observed for intake of sweet solutions, salts, bitter tastants and a high-fat diet. Our laboratory recently reported marked strain differences in the amounts and/or percentages of kilocalories of sucrose consumed among 11 inbred and one outbred mouse strains exposed to a wide range of nine sucrose concentrations (0.0001-5%) in two-bottle 24-h preference tests. To assess whether differences in fat intake were similarly associated with genetic variation, the present study examined intake of chow, water and an emulsified fat source (Intralipid) across nine different concentrations (0.00001-5%) in the same 11 inbred and 1 outbred mouse strains using two-bottle 24-h preference tests, which controlled for Intralipid concentration presentation effects, Intralipid and water bottle positions, and measurement of kilocalorie intake consumed as Intralipid or chow. Strains displayed differential increases in Intralipid intake relative to corresponding water with significant effects observed at the seven (BALB/cJ: 0.001% threshold sensitivity), four (AKR/J, C57BL/6J, DBA/2J, SWR/J: 0.5% threshold sensitivity), three (CD-1, C57BL/10J, SJL/J: 1% threshold sensitivity) and two (A/J, CBA/J, C3H/HeJ, 129P3/J: 2% threshold sensitivity) highest concentrations. In assessing the percentage of kilocalories consumed as Intralipid, SWR/J mice consumed significantly more at the three highest concentrations to a greater degree than BALB/cJ, C57BL/6J, CD-1, C3H/HeJ, DBA/J and 129P3/J strains which in turn consumed more than A/J, AKR/J, CBA/J, C57BL/10J and SJL/J mice. Relatively strong (h2 = 0.73-0.79) heritability estimates were obtained for weight-adjusted Intralipid intake at those concentrations (0.001-1%) that displayed the largest strain-specific effects in sensitivity to Intralipid. The identification of strains with diverging abilities to regulate kilocalorie intake when presented with high Intralipid concentrations may lead to the successful mapping of genes related to hedonics and obesity. Show less

Dyggve-Melchior-Clausen syndrome (DMCS) is a rare autosomal recessive disorder produced by mutations in the Dymeclin gene recently identified. It is characterized by the association of a progressive spondylo-epi-metaphyseal dysplasia and mental retardation ranging from mild to severe. The clinical and radiological similarities at the onset of the condition with the Morquio disease may hinder its diagnosis and no biochemical abnormality that causes it has been described as of yet. An eight-year-old girl had progressive postnatal dwarfism. Platyspondyly and dysplasic epiphyses and metaphyses with biochemical studies that resembled those of Morquio's disease; however the presence of specific radiological features and mental retardation led to the diagnosis of DMCS. A missense Dym mutation in homozygosis was identified. This entity should be known as it may be easily confused with Morquio disease. Radiological appearance of the iliac crests are very pathognomonic of DMCS. Identification of Dym gene is an important step towards the prenatal diagnosis. Show less

The Dyggve-Melchior-Clausen syndrome is a progressive spondyloepimetaphyseal dysplasia characterized by a short trunk dwarfism, barrel chest, sternal protrusion, kyphoscoliosis, severe platyspondyly, with a central constriction, irregular iliac wings with a lacy appearance, rhizomelic shortening of the limbs, microcephaly, coarse face, and variable mental retardation. This condition is extremely rare and the diagnosis is difficult without any previous experience on it. It is inherited as an autosomal recessive condition, its gene (DYM) having been mapped in the 18q12-21.1 chromosomal region. At least 21 different mutations of this gene have been reported. We describe an affected Spanish child and include his molecular analysis. We also review the current knowledge on this syndrome. The diagnosis of this patient, based on his clinical and radiological features, was later confirmed by analysis of the DYM gene mutations. The patient had two different mutations, one inherited from the mother and the other inherited from the father. One of the mutations of this patient (exon 8) is extremely rare and has mostly been reported in patients with Spanish ancestors (from Chile, Argentina, Guam islands and a French patient with Spanish ancestors). These observations, together with that of the patient described here, led us to consider this mutation as having a possible Spanish/Portuguese origin. This condition may be more frequent in Spain than previously thought, especially due to misdiagnosis. This is important in order to undertake quaternary prevention, which is quite necessary for rare syndromes with polysystemic affectation. Show less

Spermatogonial stem cells are required for the initiation of spermatogenesis and the continuous production of sperm. In addition, they can acquire pluripotency and differentiate into derivatives of the three embryonic germ layers when cultured in the appropriate conditions. Therefore, understanding the signaling pathways that lead to self-renewal or differentiation of these cells is of paramount importance for the treatment of infertility, the development of male contraceptives, the treatment of testicular cancers, and ultimately for tissue regeneration. In this report, we studied some of the signaling pathways triggered by glial cell line-derived neurotrophic factor (GDNF), a component of the spermatogonial stem cell niche produced by the somatic Sertoli cells. As model systems, we used primary cultures of mouse spermatogonial stem cells, a mouse spermatogonial stem cell line and freshly isolated testicular tubules. We report here that GDNF promotes spermatogonial stem cell proliferation through activation of members of the Src kinase family, and that these kinases exert their action through a PI3K/Akt-dependent pathway to up-regulate N-myc expression. Thus, to proliferate, spermatogonial stem cells activate mechanisms that are similar to the processes observed in brain stem cells and lung progenitors. Show less

The study of genetic variance in opioid receptor antagonism of sucrose and other forms of sweet intake has been limited to reductions in sweet intake in mice that are opioid receptor-deficient or lacking either pre-pro-enkephalin or beta-endorphin. Marked genetic variance in inbred mouse strains has been observed for sucrose intake across a wide array of concentrations in terms of sensitivity, magnitude, percentages of kilocalories consumed as sucrose and compensatory chow intake. The present study examined potential genetic variance in systemic naltrexone's dose-dependent (0.01-5 mg/kg) and time-dependent (5-120 min) ability to decrease sucrose (10%) intake in eleven inbred (A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL/6J, C57BL/10J, DBA/2J, SJL/J, SWR/J, 129P3/J) and one outbred (CD-1) mouse strains. A minimum criterion sucrose intake (1 ml) under vehicle treatment, designed to avoid "floor effects" of antagonist treatment was not achieved in three (A/J, AKR/J, CBA/J) inbred mouse strains. Marked genetic variance in naltrexone's ability to inhibit sucrose intake was observed in the remaining strains with the greatest sensitivity observed in the C57BL/10J and C57BL/6J strains, intermediate sensitivity in BALB/cJ, C3H/HeJ, CD-1 and DBA/2J mice, and the least sensitivity in 129P3/J, SWR/J and SJL/J strains with a 7.5-36.5 fold range of greater effects in the ID(50) of naltrexone-induced inhibition in C57BL/10J relative to the three less-sensitive strains across the time course. Naltrexone primarily affected the maintenance, rather than the initiation of intake in BALB/cJ, CD-1, C3H/HeJ, DBA/2J and SJL/J mice, but significantly reduced sucrose intake at higher doses across the time course in C57BL/6J, C57BL/10J and 129P3/J mice. Whereas SWR/J mice failed to display any significant reduction in sucrose intake at any time point following any of the naltrexone doses, naltrexone's maximal magnitude of inhibitory effects was small (35-40%) in 129P3/J and SJL/J mice, moderate ( approximately 50%) in BALB/cJ, C3H/HeJ, CD-1 and DBA2/J mice, and profound (70-80%) in C57BL/6J and C57BL/10J mice. Indeed, the latter two strains displayed significantly greater percentages of naltrexone-induced inhibition of sucrose intake than virtually all other strains. These data indicate the importance of genetic variability in opioid modulation of sucrose intake. Show less

Proteins bind one another in aqua's solution to form tight and specific complexes. Previously we have shown that this is achieved through the modular architecture of the interaction network formed by the interface residues, where tight cooperative interactions are found within modules but not between them. Here we extend this study to cover the entire interface of TEM1 beta-lactamase and its protein inhibitor BLIP using an improved method for deriving interaction maps based on REDUCE to add hydrogen atoms and then by evaluating the interactions using modifications of the programs PROBE, NCI and PARE. An extensive mutagenesis study of the interface residues indeed showed that each module is energetically independent on other modules, and that cooperativity is found only within a module. By solving the X-ray structure of two interface mutations affecting two different modules, we demonstrated that protein-protein binding occur via the structural reorganization of the binding modules, either by a "lock and key" or an induced fit mechanism. To explain the cooperativity within a module, we performed multiple-mutant cycle analysis of cluster 2 resulting in a high-resolution energy map of this module. Mutant studies are usually done in reference to alanine, which can be regarded as a deletion of a side-chain. However, from a biological perspective, there is a major interest to understand non-Ala substitutions, as they are most common. Using X-ray crystallography and multiple-mutant cycle analysis we demonstrated the added complexity in understanding non-Ala mutations. Here, a double mutation replacing the wild-type Glu,Tyr to Tyr,Asn on TEM1 (res id 104,105) caused a major backbone structural rearrangement of BLIP, changing the composition of two modules but not of other modules within the interface. This shows the robustness of the modular approach, yet demonstrates the complexity of in silico protein design. Show less

This paper reviews the developments in high-throughput and nanolitre-scale protein crystallography technologies within the remit of workpackage 4 of the Structural Proteomics In Europe (SPINE) project since the project's inception in October 2002. By surveying the uptake, use and experience of new technologies by SPINE partners across Europe, a picture emerges of highly successful adoption of novel working methods revolutionizing this area of structural biology. Finally, a forward view is taken of how crystallization methodologies may develop in the future. Show less