Downregulation of hsp22 gene expression in Drosophila melanogaster from sites located near chemical plants.

A common physiological response of organisms to environmental conditions is variation in gene expression, especially true for genes encoding for heat shock proteins. In insects, this process has been examined for induced heat or cold stress. The putative long-term imprinted/acquired heat shock protein response due to unfriendly environmental conditions has been far less studied. The Drosophila melanogaster hsp22 gene, which has been extensively reviewed as being sensitive to different changing life conditions, was examined by qRT-PCR, using carboxy-X-rhodamine. In the present study, we focused on the detection of hsp22 level of transcription in three D. melanogaster isolates, collected from sites located near different chemical plants in Romania and subjected to one-year adaptation to laboratory conditions. In all isolates, the hsp22 gene expression was determined using the housekeeping genes Gapdh1 and UbcD10 as internal controls. According to our experimental results, the D. melanogaster hsp22 gene was significantly downregulated compared to the same gene in w(1118)iso, used as a calibrator. We showed that hsp22 could play an important role in relation to stress resistance and adaptation. This study highlights the importance of in vivo studies to demonstrate genome plasticity to overcome different damages induced by any presumed source of stress.

Expression, phosphorylation, and glycosylation of CNS proteins in aversive operant conditioning associated memory in Lymnaea stagnalis.

Long-term memory formation requires "de novo" expression and post-translational modification of many proteins. Understanding the temporal and spatial regulatory pattern of these proteins is fundamental to decoding the molecular basis of learning and memory. We characterized changes in expression, phosphorylation, and glycosylation of CNS proteins after operant conditioning in pond snail Lymnaea stagnalis. The phosphorylation and the glycosylation levels of proteins, measured by the ratio of Pro-Q Diamond (phosphoproteins) or Pro-Q Emerald (glycoproteins) vs. SYPRO-Ruby (total proteins) signals, increased during memory formation. Proteins whose modulation of phosphorylation might be involved in learning and memory were identified by mass spectrometry (MS) and are associated with cytoskeleton, glutamine cycle, energy metabolism, G-protein signaling, neurotransmitter release regulation, iron transport, protein synthesis, and cell division. Phosphorylation of actin increased during memory formation. To identify proteins whose expression levels changed in long-term memory formation we used two-dimensional difference gel electrophoresis followed by MS. The up-regulated proteins are mostly associated with lipoprotein and cholesterol metabolism, protein synthesis and degradation, cytoskeleton, nucleic acid synthesis, and energy supply. The down-regulated proteins are enzymes of aspartic acid metabolism involved in regulation of protein synthesis. Our proteomic analyses have revealed a number of candidate proteins associated with memory formation. These findings provide new directions for further investigation into the signaling networks required for memory formation and consolidation.

Detection of possible restriction sites for type II restriction enzymes in DNA sequences.

In order to make a step forward in the knowledge of the mechanism operating in complex polygenic disorders such as diabetes and obesity, this paper proposes a new algorithm (PRSD -possible restriction site detection) and its implementation in Applied Genetics software. This software can be used for in silico detection of potential (hidden) recognition sites for endonucleases and for nucleotide repeats identification. The recognition sites for endonucleases may result from hidden sequences through deletion or insertion of a specific number of nucleotides. Tests were conducted on DNA sequences downloaded from NCBI servers using specific recognition sites for common type II restriction enzymes introduced in the software database (n = 126). Each possible recognition site indicated by the PRSD algorithm implemented in Applied Genetics was checked and confirmed by NEBcutter V2.0 and Webcutter 2.0 software. In the sequence NG_008724.1 (which includes 63632 nucleotides) we found a high number of potential restriction sites for ECO R1 that may be produced by deletion (n = 43 sites) or insertion (n = 591 sites) of one nucleotide. The second module of Applied Genetics has been designed to find simple repeats sizes with a real future in understanding the role of SNPs (Single Nucleotide Polymorphisms) in the pathogenesis of the complex metabolic disorders. We have tested the presence of simple repetitive sequences in five DNA sequence. The software indicated exact position of each repeats detected in the tested sequences. Future development of Applied Genetics can provide an alternative for powerful tools used to search for restriction sites or repetitive sequences or to improve genotyping methods.

MTRR polymorphism and the risk for colorectal and breast cancer in Romanian patients--a preliminary study.

BACKGROUND: The risk of colorectal cancer (CRC) and breast cancer (BC) is influenced by polymorphisms located in the genes encoding enzymes of the folate pathway. The aim of this study was to evaluate if A66G MTRR (rs1801394) polymorphism is involved in predisposition for colorectal and breast carcinogenesis in Romanian patients. MATERIALS AND METHODS: In the present case-control study, 300 individuals divide in four groups: sporadic CRC patients (n = 120), control CRC (n = 60), BC patients (n = 60) and control BC (n = 60), were genotyped by PCR-RFLP method. RESULTS: Frequency of genotype AA was 11.7% in CRC control and 5% respectively in BC control. For cancer groups the frequency of genotype AA was 9.2% in CRC and 0% in BC. CONCLUSIONS: Study results do not demonstrate an association between A66G MTRR polymorphism and CRC or BC in Romanian patients.

Spinal muscular atrophy disease: a literature review for therapeutic strategies.

Currently, there is no cure for the treatment of spinal muscular atrophy (SMA). Based on the available clinical and molecular findings, different therapeutic strategies were tested in vitro and in vivo and clinical trials are ongoing. The main therapeutic direction is focused on the enhancement of SMN expression by increasing the full-length (fl) SMN2 transcript levels, preventing the SMN exon 7 from skipping or from protein stabilizing. In addition, the action of neurotrophic, neuroprotective or anabolic agents is tested and stem cell and gene therapy approaches are in a promising development.

Lack of association between ACE ID polymorphism and colorectal cancer in Romanian patients.

BACKGROUND: The insertion/deletion polymorphism of the angiotensin I-converting enzyme (ACE) gene has recently been linked to the pathogenesis of human cancers. The goal of this study was to analyze the possible association between ACE gene I/D polymorphism and colorectal cancer in Romanian patients. METHODS: Blood samples were obtained, after informed consent, from individuals with colorectal cancer (n=108, M:W = 64:44), and healthy persons (n=150, M:W = 84:66). Genomic DNA was extracted from peripheral blood leucocytes using commercial kits and the insertion (I) / deletion (D) polymorphism was assessed by PCR. Statistical analysis was done using the chi2 test. We determined the odds ratio using the genotype II as risk factor. A p value < 0.05 was considered statistically significant. RESULTS: The distribution of ACE II: ID: DD genotypes was 23.1%: 46.3%: 30.6% in patients and respectively 20%: 48.7%: 31.3% in controls. The distribution of genotype (chi2 0.37, p = 0.54) and alleles (chi2 0.19, p = 0.65) did not differ significantly between cancer patients and control. CONCLUSIONS: Study results do not demonstrate an association between ACE ID polymorphism and colorectal cancer in our patients.

Neural phosphoproteomics of a chronic hypoxia model--Lymnaea stagnalis.

Chronic hypoxia is a common clinical event that induces adaptive responses and can result in behavioral deterioration. The reduction of metabolic rate during hypoxia may limit overall protein phosphorylation owing to the lack of high energy phosphate. However, the hypoxia-induced regulation of phosphoproteins is poorly understood. Here, we characterized the CNS phosphoproteome of Lymnaea stagnalis, a freshwater snail that has been used as a model to study chronic hypoxia-induced neural depression. After hypoxia treatment for 4 days, the motor behavior of the snail was suppressed. Electrophysiological measurements from Pedal A (PeA) interneurons showed that hypoxia increased the frequency of spontaneous postsynaptic excitatory potentials (sEPSPs), but reduced the firing frequency, the amplitude, and the half-width duration (APD(50)) of spontaneous action potentials. Imaging with a fluorescent phosphate label, Pro-Q Diamond, revealed that the neuronal phosphoprotein level was reduced after the hypoxia treatment. The hypoxia-induced changes in the phosphoproteome of the central ganglia were quantified using one-dimensional gel-electrophoresis by comparing the fluorescence intensity ratio of phospholabeled phosphoproteins versus total proteins between the hypoxia and control groups. We analyzed 16 protein bands: eight showed decreased phosphorylation levels after hypoxia treatment, and eight did not change. Using mass spectrometry analysis and protein database matching we found three phosphoproteins that may be associated with chronic hypoxia-induced neuronal adaptive response of the snail. This is the first proteomic screening for neural phosphoproteins in chronic hypoxia.

Prenatal molecular diagnosis of beta-thalassemia: report on the first two cases in Romania.

Thalassaemia major is a classical example of a disease that can be prevented by prenatal diagnosis. In Romania there are currently 300 patients with thalassaemia major under the management of specialized institutions. Prenatal diagnoses of thalassemia have offered a new dimension to the prevention of this disease, but in order to implement prenatal diagnosis, knowledge of mutations and of their incidence is essential. Molecular testing using Denaturing Gradient Gel Electrophoresis (DGGE) scanning and direct mutation detection with Amplificaton Refractory Mutation System-PCR (ARMS-PCR) and Restriction endonuclease Analysis of PCR fragments (PCR-RFLP) was performed by using amplified DNA from amniotic cells samples, while mutations in the parents were determined in advance. Using our experience in molecular diagnosis, we were able to perform the first prenatal diagnosis for two young couples at risk for thalassaemia major. Foetal samplings were collected by amniocentesis and chorionic villus sampling in the second trimester of the pregnancies. Maternal contamination of the foetal DNA was ruled out by STR genotyping. The prenatal diagnosis revealed affected foetuses with homozygous status of beta-thalassemia major. The IVSI-110 (G-A)/IVS II-745 (C-G) genotype in the first case foetus and ed 8 (-AA)/cd 8 (-AA) in the second case foetus were reported. The results of this study point to a successful future prenatal diagnosis of beta-thalassnemia in Romania, using a rapid and accurate molecular method. Together with the implementation of proper preventive health measures and the education of parents regarding their carrier status, we are hoping that this method will be used as the common application approach to decrease the incidence of thalassacmia major.

Molecular analysis of mutations for the adenomatous polyposis coli (APC) gene in Romanian patients with colorectal cancer.

Mutations in adenomatous polyposis coli (APC) gene have not been previously characterized among Romanian patients with colorectal cancer (CRC). We initiate this study to detect the mutations in APC gene in blood and tumor samples collected from 16 patients (10 men and 6 women) and blood samples from 21 first and second degree relatives of the patients. For this the presence of mutations in exons 6, 7, 12, 13, 14 as well as in regions B, L and W of exon 15 was investigated using PCR multiplex. In the same time, we have searched for 5 bp deletions at codon 1061 of APC gene by PAGE and SSCP methods. These methods allowed us to evidence identification of the presence of mutations in samples from 7 individuals. In one patient, was detected a deletion of exon 13th of APC gene both in DNA extracted from blood and tumor samples. Multiple deletions (e.g. in exon 6, 12, and in 15L and 15W regions) in DNA extracted from the tumor sample were detected, but not in DNA probe obtained from blood cells. We can speculate that these mutations are an example of genomic instability accompanying the malignancy. Till now, no mutation affecting 1061 codon of APC gene was identified in the patients investigated in our study.

NIDDM: new once-daily intervention for type 2 diabetes mellitus: Diaprel MR.

UNLABELLED: Pharmacological treatment of hyperglycemia should address to both abnormalities in T2DM treatment, that is reduction of insulin resistance and restoration of normal insulin secretion. Gliclazide is a sulfonylurea compound oral hypoglycemic drug that has a unique feature of restoring the first-phase insulin secretion, which is lost in T2DM being one of the early features of disease. MATERIAL AND METHOD: Laboratoires Servier conducted in Romania a an open non randomized surveillance on the efficacy and safety of Diaprel MR in type 2 diabetic patients. 199 patients in 14 treatment centers were enrolled. Eligibility criteria were as it follows: men and women with diabetes, previously on diet alone and not treated with other OAD, over 35 years old with FPG (mg/dl) at enrollment between 126 and 180. The clinical trial lasted for 16 weeks. During this period the doctors examined the patients 6 times. First visits were at a 2 weeks interval and the last two visits at a 4 weeks interval. At each visit the doctor renewed the prescription for the subsequent period according to the following protocol: the starting dose was 30 mg Diaprel MR/day, if the FPG (mg/dl) was over 140 (at the next visit) the dosage was increased with 30 mg Diaprel MR/day, if the FPG (mg/dl) was under 140 the dosage remained the same as the previous dosage. The maximum dosage was 120 mg Diaprel MR/day. The following parameters were measured on first and last (seventh) visit: blood pressure (systolic and diastolic), heart rate (bpm), body mass index-BMI (kg/m2), fasting plasma glucose FPG (mg/dl and mmol/l), glycated hemoglobin HbA1c (%) and Hb-Hct (mg/dl-%), creatinine (mg/dl), SGPT (UI/I), cholesterol (mg/dl) and triglycerides (mg/dl). Blood pressure (systolic and diastolic), heart rate, BMI and FPG were measured from the second to sixth visit also. On each visit there was registered other data such as: associated illnesses, concomitant medication and adverse events. RESULTS: Primary end points. The average values of end points HbA1c (%) and PFG (mg/dl) registered a significant decrease during the 16 weeks of medication, from the enrollment moment (S0) to the last week (S16). The decrease was significant on the total sample of the main analysis group but also on subsamples of age, gender and BMI. HbA1c (%) average values decreased in the main analysis group (S16 compared to S0): with 22% on the total sample (from 7.7 to 6.0); p < 0.05. FPG (mg/dl) average values decreased in the main analysis group (S16 compared to S0): with 21% on the total sample (from 159 to 126); p < 0.05. Secondary end points. There were no significant changes registered in the average level of cholesterol and triglycerides, BMI, diastolic blood pressure, heart rate, creatinine, SGPT. A significant decline of the average systolic blood pressure was registered. CONCLUSION: Diaprel MR can be used safely in diabetic patients newly diagnosed, uncontrolled on diet or other oral antidiabetic drugs, overweight, safely in those with cardio-vascular disease, or in patients with creatinine clearance 50-80 ml/min.

Y chromosomal haplogroup J as a signature of the post-neolithic colonization of Europe.

In order to attain a finer reconstruction of the peopling of southern and central-eastern Europe from the Levant, we determined the frequencies of eight lineages internal to the Y chromosomal haplogroup J, defined by biallelic markers, in 22 population samples obtained with a fine-grained sampling scheme. Our results partially resolve a major multifurcation of lineages within the haplogroup. Analyses of molecular variance show that the area covered by haplogroup J dispersal is characterized by a significant degree of molecular radiation for unique event polymorphisms within the haplogroup, with a higher incidence of the most derived sub-haplogroups on the northern Mediterranean coast, from Turkey westward; here, J diversity is not simply a subset of that present in the area in which this haplogroup first originated. Dating estimates, based on simple tandem repeat loci (STR) diversity within each lineage, confirmed the presence of a major population structuring at the time of spread of haplogroup J in Europe and a punctuation in the peopling of this continent in the post-Neolithic, compatible with the expansion of the Greek world. We also present here, for the first time, a novel method for comparative dating of lineages, free of assumptions of STR mutation rates.

Scanning of beta-globin gene for identification of beta-thalassemia mutation in Romanian population.

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.

Screening for microdeletions in human Y chromosome--AZF candidate genes and male infertility.

About 30% of couple infertilities are of male origin, some of them caused by genetic abnormalities of the Y chromosome. Deletions in AZF region can cause severe spermatogenic defects ranging from non-obstructive azoospermia to oligospermia. The intracytoplasmatic sperm injection technique (ICSI) is rapidly becoming a versatile procedure for human assisted reproduction in case of male infertility. The use of ICSI allows Y chromosome defects to be passed from father. The goal of our study is to evaluate the frequency of microdeletions in the long arm of Y chromosome, within the AZF regions, in these cases of infertilities, using molecular genetics techniques. Thirty infertile men with azoospermia or oligozoospermia, determined by spermogram, were studied after exclusion of patients with endocrine or obstructive causes of infertility. Peripheral blood DNA was extracted from each patient, then amplified by multiplex PCR with STS genomic markers from the Y chromosome AZF zones. Each case was checked by multiplex PCR through coamplification with the SRY marker. Three men with microdeletions of the long arm of the Y chromosome were diagnosed among the 30 patients, corresponding to a proportion of 10%. The relatively high proportion of microdeletions found in our population suggest the need for strict patient selection to avoid unnecessary screening for long arm Y chromosome microdeletions. The molecular diagnostics was performed according to the current European Academy of Andrology laboratory guidelines for molecular diagnosis of Y chromosomal microdeletions.

A multistep process for the dispersal of a Y chromosomal lineage in the Mediterranean area.

In this work we focus on a microsatellite-defined Y-chromosomal lineage (network 1.2) identified by us and reported in previous studies, whose geographic distribution and antiquity appear to be compatible with the Neolithic spread of farmers. Here, we set network 1.2 in the Y-chromosomal phylogenetic tree, date it with respect to other lineages associated with the same movements by other authors, examine its diversity by means of tri- and tetranucleotide loci and discuss the implications in reconstructing the spread of this group of chromosomes in the Mediterranean area. Our results define a tripartite phylogeny within HG 9 (Rosser et al. 2000), with the deepest branching defined by alleles T (Haplogroup Eu10) or G (Haplogroup Eu9) at M172 (Semino et al. 2000), and a subsequent branching within Eu9 defined by network 1.2. Population distributions of HG 9 and network 1.2 show that their occurrence in the surveyed area is not due to the spread of people from a single parental population but, rather, to a process punctuated by at least two phases. Our data identify the wide area of the Balkans, Aegean and Anatolia as the possible homeland harbouring the largest variation within network 1.2. The use of recently proposed tests based on the stepwise mutation model suggests that its spread was associated to a population expansion, with a high rate of male gene flow in the Turkish-Greek area.

Chromatin and chromosomal fine structure in spermatogenesis of some species of amphibians.

Spermatogenesis is a complex differentiation process which is characterised, among other features, by conspicuous stage-specific nuclear events such as the pairing of homologous chromosomes coupled with the formation of synaptonemal complexes, the replacement of histones with sperm-specific proteins during spermiogenesis and, as a result, chromatin condensation and its inactivation in sperm cells. The chromatin of spermatogenic cells undergoes dramatic conformational changes upon differentiation from spermatogonia to mature spermatozoa. During the haploid stage of spermatogenesis, histones are gradually replaced, firstly by transition proteins and later by sperm-specific proteins. As a result of the high degree of condensation and inactivation of spermatid and sperm chromatin, Sertoli cells are responsible for the nourishment of germ cells with ribosomal RNA and nutritive substances.

Regulation of the tip-high [Ca2+] gradient in growing hyphae of the fungus Neurospora crassa.

Previous work has shown that hyphal elongation in the fungus Neurospora crassa requires a tip-high cytosolic Ca2+ gradient. The source of the Ca2+ appears to be intracellular stores as there is no net transplasma membrane Ca2+ flux at the elongating hyphal tip and modification of ion fluxes across the plasma membrane using voltage clamp is without effect on tip growth. To decode the internal mechanisms which generate and maintain the tip-high Ca2+ gradient we first identified calcium regulators which affect hyphal growth and morphology, then determined how they modify cytosolic [Ca2+] and the actin cytoskeleton using fluorescent dyes and confocal microscopy. Cyclopiazonic acid (a known inhibitor of the endoplasmic reticulum calcium ATPase) inhibits growth and increases cytoplasmic [Ca2+] in the basal region 10-25 microm behind the hyphal tip. 2-APB (2-aminoethoxydiphenyl borate, an inhibitor of IP3-induced Ca2+ release) inhibits hyphal elongation and dissipates the tip-high Ca2 gradient 0-10 microm from the tip. Microinjections of the IP3 receptor agonists adenophostin A and IP3 (but not control microinjections of the biologically inactive L-IP3) transiently inhibited growth and induced subapical branches. IP3 microinjections, but not L-IP3, lowered tip-localized [Ca2+] and increased basal [Ca2+]. Even though their effect on [Ca2+] gradients was different, both cyclopiazonic acid and 2-APB disrupted similarly the normal actin pattern at the hyphal apex. Conversely, disruption of actin with latrunculin B dissipated tip-localized Ca2+. We conclude that the tip-high Ca2+ gradient is generated internally by Ca2+ sequestration into endoplasmic reticulum behind the tip and Ca2+ release via an IP3 receptor from tip-localized vesicles whose location is maintained by the actin cytoskeleton.

Structure and function analysis of the calcium-related gene spray in Neurospora crassa.

The spray gene was cloned, and wildtype and mutant alleles were sequenced. Spray(+) has a 3452-bp open reading frame plus seven introns. The spray mutant had a T --> G transversion close to the carboxyl end, creating a stop codon (TGA). The sequence shows no match to genes of known function, but the carboxyl end shows seven transmembrane domains and matches putative membrane proteins of yeast. The most abundant transcript detected was 4.4 kb in size. Repeat-induced point mutagenesis produced the mutant spray phenotype. Electrophysiological analysis showed that ion fluxes in the spray plasma membrane are normal; furthermore, whereas the spray mutant was known to have no organelle-based calcium fluorescence, the cytosol shows a tip-high calcium gradient. The spray mutant is sensitive to calcineurin inhibitors. The results suggest that the SPRAY protein is located in an organellar membrane, regulating the distribution of Ca(2+) via calcineurin.

Y chromosome analysis reveals a sharp genetic boundary in the Carpathian region.

Nine single nucleotide (SNP) or indel binary polymorphisms were used to determine the frequencies and phylogenetic relationships of 12 Y chromosomal haplogroups in 289 males from Romania and the Republic of Moldova. Our data indicated a low but not null rate of the homoplasic appearance of the DYZ3 (-) allelic state. All other markers confirmed the previously proposed phylogeny. Based on the affinities between populations in terms of haplogroup frequencies, this work identified the geographical region of the Carpathians as a break point in the gene geography of Eastern Central Europe, providing a finer definition of one of the possible sharp genetic changes between Western and Eastern Europe.

Localizing genes in Drosophila melanogaster polytene chromosomes by fluorescence in situ hybridization.

This paper describes a method for the identification of single copy genes in Drosophila melanogaster polytene chromosomes, using fluorescence in situ hybridization (FISH). We demonstrate the detection of white (w), a gene previously mapped to 1-1.5 region of the linkage map, and to 3C2 region of the cytogenetic map of X chromosome. Squash preparations of polytene chromosomes from salivary glands dissected out from third instar larvae of Drosophila melanogaster were denatured and subjected to hybridization with a digoxigenin labeled probe, corresponding to mini-white gene. The preparations were then washed and incubated with anti-digoxigenin-fluorescein antibodies. After removal of the nonspecifically bound antibodies, the polytene chromosomes were counterstained with propidium iodide. Fluorescence microscopy revealed white locus in the X chromosome in a subterminal location, in agreement with the above mentioned maps. The protocol is efficient and adaptable for simultaneously multiple signal detection.