The alarm clock rings and you jump straight out of bed, rattling downstairs to start the day. Or maybe you creak downstairs, each step a struggle because of stiffness and pain in your knees and other joints. If the second description fits the start of your day, then maybe, like 70 million Americans, you have arthritis, one of the most prevalent chronic health problems in the United States.Arthritis is an umbrella term for more than 100 medical conditions. What all forms of arthritis have in common is that they affect our joints-places where two or more bones meet. In healthy joints, the ends of the bones are covered with cartilage, a tough but smooth tissue that, like the oil in a car engine, reduces friction between the moving parts. In the most common form of arthritis-osteoarthritis-breakdown of this cartilage, which is called articular cartilage, means the bones rub together, causing pain and loss of movement. Risk factors for osteoarthritis include age and family history.If we could understand the molecular mechanisms that create and maintain articular cartilage, it might be possible to discover what goes wrong in our joints as we age and to find better treatments for arthritis. Embryologists have already discovered quite a bit about the earliest stages of joint formation. It is known, for example, that stripes of cells that form between developing bones subsequently develop into the permanent cartilage found in joints. Several members of a family of secreted proteins known as bone morphogenetic proteins (BMPs) are expressed in these stripes of cells, implicating BMP signaling (the transmission of messages produced by BMPs binding to cell-surface receptors) in early joint development.loxP sites on either side of Bmpr1a, a gene that encodes one of the BMP receptors. The loxP sites have no effect until an enzyme known as Cre is expressed, and then the DNA between the loxP sites is cut out and discarded. Because Kingsley's team knew that global inactivation of Bmpr1a early in development causes embryonic death, they linked the gene for Cre to DNA sequences that limit its expression to those regions of the embryo where joints eventually develop. The result: a mouse strain in which Bmpr1a receptor function is specifically lost only in tissues destined to become joints.David Kingsley's team has been investigating whether BMPs are also involved in the later development and maintenance of joint cartilage. To do this, the researchers designed a genetic system that inactivates BMP signaling late in mouse embryonic development. They inserted special DNA sequences called Most of the joints in this mouse strain formed normally. However, the mice rapidly developed severe arthritis after birth. By 7 days old, the expression of proteins normally found in cartilage was reduced, although at this stage the knee, for example, looked normal. By 7 weeks old (adulthood for mice), there were clear structural changes in the knee joints, and the articular cartilage was thinner and showed signs of wearing away. By 9 months old, the knees of the mutant mice largely lacked articular cartilage and the unprotected leg bones seemed to rub directly against each other.All told, the joints in these mutant mice closely resembled those in people with osteoarthritis, suggesting that BMP signaling is necessary for the maintenance of healthy articular cartilage. This raises the possibility that mutations in BMP signaling components may underlie some of the genetic variation in human osteoarthritis risk and suggests that treatments designed to mimic or augment BMP signaling might help to maintain healthy joints. Finally, the genetic system described by Kingsley and coworkers should be useful for future investigations into joint formation and maintenance.


A large number of microsatellites have been extensively used to comprehend the genetic diversity of different global groups. This paper entails polymorphism at 15 STR in four predominant and endogamous populations representing Karnataka, located on the southwest coast of India. The populations residing in this region are believed to have received gene flow from south Indian populations and world migrants, hence, we carried out a detailed study on populations inhabiting this region to understand their genetic structure, diversity related to geography and linguistic affiliation and relatedness to other Indian and global migrant populations.ST value. Iyengar and Lyngayat were placed above the regression line in the R-matrix analysis as opposed to the Gowda and Muslim. AMOVA indicated that majority of variation was confined to individuals within a population, with geographic grouping demonstrating lesser genetic differentiation as compared to linguistic clustering. DA distances show the genetic affinity among the southern populations, with Iyengar, Lyngayat and Vanniyar displaying some affinity with northern Brahmins and global migrant groups from East Asia and Europe.Various statistical analyses were performed on the microsatellite data to accomplish the objectives of the paper. The heretozygosity was moderately high and similar across the loci, with low average GThe microsatellite study divulges a common ancestry for the four diverse populations of Karnataka, with the overall genetic differentiation among them being largely confined to intra-population variation. The practice of consanguineous marriages might have attributed to the relatively lower gene flow displayed by Gowda and Muslim as compared to Iyengar and Lyngayat. The various statistical analyses strongly suggest that the studied populations could not be differentiated on the basis of caste or spatial location, although, linguistic affinity was reflected among the southern populations, distinguishing them from the northern groups. Our study also indicates a heterogeneous origin for Lyngayat and Iyengar owing to their genetic proximity with southern populations and northern Brahmins. The high-ranking communities, in particular, Iyengar, Lyngayat, Vanniyar and northern Brahmins might have experienced genetic admixture from East Asian and European ethnic groups. The Indian subcontinent is regarded as a natural genetic laboratory, owing to the co-existence and interaction of socio-culturally, linguistically, ethnically and genetically diversified endogamous populations in a geographical terrain. It is believed that the earliest humans leaving Africa for Eurasia might have taken a coastal route across Saudi Arabia, through Iraq, Iran, to Pakistan and finally entered India along the coastlines . A seconAnthropologically, southern and northern populations are distinct and these differences are further substantiated by (i) the presence of Neolithic sites in this region suggests that Neolithic people of southern India came from north by land and the west-coast by sea , (ii) thThe present study was carried out on one of the largest southern states, Karnataka, positioned on the southwest coast of India, with a dwelling of about 50 million people. This expanse has been a rich source of prehistoric discoveries dating back to the Paleolithic era that are akin to those seen in Europe . KarnataA few studies utilizing classical markers have been carried out on southern populations ,11,12, iAmong the different caste and tribal groups inhabiting the southwest coast of India, we have selected four predominant Dravidian-speaking communities from Karnataka: Iyengar Brahmin, Lyngayat, Gowda and Muslim, they not only belong to dissimilar groups of the Indian caste hierarchy but also have varied migration histories, conferring them uniqueness and significance from a genetic perspective. The present microsatellite study primarily attempts to understand the genetic structure of the four selected populations and to determine their genetic relationship with other linguistically and ethnically similar groups of southern India and Brahmin groups of northern India. It has been suggested that that despite the linguistic homogeneity in southern India, these populations have remained genetically diversified . Hence, ST value of 0.009 elucidates the low degree of genetic differentiation in them. However, the GST value for the pooled Indian and global populations demonstrated a high value at 2.3% (data not shown). Genetic relationship of studied populations with other similar southern groups; Vanniyar, Gounder, Pallar and Tanjore Kallar [A distances indicate that among these groups, Iyengar, Lyngayat and Vanniyar are more similar to the northern Brahmins (0.030). Furthermore, genetic distances emphasize the affinity of Lyngayat with Tanjore Kallar (0.029), Iyengar (0.026) and Vanniyar (0.028). Estimation of relatedness between the southern and global populations shows that all the southern communities formed a separate cluster, nevertheless, genetic distances disclose the affinity of upper caste Indian communities; Iyengar, Lyngayat, Vanniyar, Bihar and Oriya Brahmin with Europeans and East Asians. The Indian populations were most distant to Africans.Allele frequency at 15 STR was used to compute the heterozygosity (observed) for the four studied populations, which varied for each locus, and population but reflected similar values, ranging between 0.724 and 0.797 , irrespective of their geographic or linguistic grouping Table . The geoIn recent years, population genetics has witnessed extensive use of microsatellite markers to understand and evolutionary histories of contemporary human populations ,32-34. TThe most distinctive feature revealed by the fifteen microsatellites was the considerable genetic homogeneity amongst the four diverse caste groups residing in southwest India. The presence of an almost similar allele frequency pattern , suggestAMOVA test strongly suggests that genetic diversity among the southern populations was mainly confined to intra-population variation, further emphasizing the genetic homogeneity in them. Analysis using different genetic markers corroborate with our finding that the genetic diversity in human populations can be mainly attributed to variation within populations ,34,36,37An exploration of the genetic differentiation based on geographical grouping of populations discloses the genetic similarity among populations residing in a region. Nevertheless, the geographic affinity was comparably lesser to that observed within the two linguistic families, viz., Dravidian and Indo-European. Our finding provides evidence to the strong linguistic affinity prevailing amongst the Dravidian speaking populations and imparts them genetic distinctness from the Indo-European linguistic group. Even though prior studies have indicated that genetic clusters often correspond closely to predefined regional and linguistic groups , AMOVA sThe genetic differentiation of the studied populations with relevant global migrant groups was estimated to be 2.3%, relatively lower than the 9% observed in another similar study , which hThe populations selected in this study comprise of three major Hindu castes-Iyengar, Lyngayat, Gowda and a Muslim community, inhabiting the southwest coastal terrain of Karnataka (11.3 – 18.45°N latitudes and 74.12 – 78.40°E longitudes). All the populations belong to the Dravidian linguistic family and are speakers of the local dialect, Kannada, but differ in caste hierarchy and socio-religious practices. Consanguineous marriages have been reported in Karnataka, with inbreeding levels of the order 0.020 to 0.033, in general .Iyengar hold a high position in the Indian caste hierarchy and sporadic accounts on Brahmin, suggests that they primarily migrated from the upper Gangetic plains to southern India. Nonetheless, few bioanthropological studies have revealed that morphologically Brahmins of a geographical region are similar to the local groups.Lyngayat community was initially formed, as a religious cult by the amalgamation of people from different castes and geographical regions but later developed into a distinct community practicing strict marriage endogamy with social sub-divisions such as clans, sub-castes and sects .Gowda is a low ranking agriculturist caste group that typically exhibits the Dravidian socio-cultural characteristic of consanguineous marriage. It is believed to have moved in from the adjoining area of Tamil Nadu.th century, introduced new genes only in northern India, suggesting that Muslims from Southern India are mainly local converts [Muslim is a linguistically heterogeneous, complex religio-ethnic group, . It is bconverts .The 15 STR marker set analyzed in this study consists of thirteen tetra nucleotide repeat loci: D3S1358, THO1, D21S11, D18S51, D5S818, D13S317, D7S820, D16S539, CSF1PO, vWA, D8S179, TPOX, FGA and two penta nucleotide repeat loci: Penta D, Penta E. Their repeat size makes them less prone to slippage of polymerase during enzymatic amplification compared to the dinucleotide repeats, allowing unambiguous typing [The blood samples were collected from unrelated individuals belonging to – Iyengar (65), Lyngayat (98), Gowda (59) and Muslim (45) communities, residing in different districts of Karnataka. DNA was extracted from blood by the phenol-chloroform method , followeST), observed heterozygosity and pairwise genetic distances (DA) were computed using allele frequencies [Allele frequencies of the 15 STR loci were calculated using the gene counting method . The genquencies . The DA quencies . Neighboquencies , and itsAnalysis of molecular variance (AMOVA) was performed using the Arlequin Ver. 2.00 package . Two levTo assess the gene flow experienced by these populations, the rii value, i.e., the genetic distance of a population from the centroid was calculated using the regression model . This moii is the distance from the centroid, pi is the frequency of the allele in ith population and  is the mean allelic frequency.where, rSTR – Short Tandem RepeatAMOVA – Analysis of Molecular VarianceNJ tree – Neighbor-Joining treeRR carried out the molecular studies, analyzed the genetic data and drafted the manuscript. VKK participated in the design, conceiving and preparation of manuscript. Both authors read and approved the final manuscript.


Increased hepatic iron is assumed to potentiate progression towards liver fibrosis in chronic hepatitis C virus (HCV) infection. In this study we have evaluated the potentiating effect of marked hepatic iron overload and chronic HCV infection on hepatic fibrosis in thalassemic patients.Liver biopsies of one group of patients with beta thalassemia major and chronic HCV infection (group 1) was compared with two groups of patients (groups 2&3) with either chronic HCV infection or thalassemia major, respectively (20 patients in each group). Necroinflammation, fibrosis, and iron overload were graded and compared.Stage of fibrosis in group 1 patients was significantly higher than the other two groups (p < 0.05). Necroinflammatory grade was significantly lower, but iron score was significantly higher in thalassemic patients (group 3) in comparison to groups 1 and 2 (p < 0.05).Our results indicate that marked liver iron overload and HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis. Thalassemia major is an inherited disorder particularly common in people of Mediterranean, African, and South-east Asian ancestry. It is characterized by decreased production of beta chain of hemoglobin. Clinical features result from anemia, markedly expanded marrow space, and transfusional and absorptive iron overload. In these patients iron overload is often inevitable, especially when iron chelating agents are not used properly . HepaticHCV is responsible for 80–90% of post-transfusional cases of hepatitis in patients who have received blood transfusion(s) prior to the introduction of routine blood products screening in 1990 . More thIncreased hepatic iron may potentiate progression towards liver fibrosis in chronic HCV infection, and may contribute to poor response to interferon therapy ,6,7. AccA retrospective cross-sectional study was performed on sixty patients in three different groups as outlined below:Group 1: Twenty patients  with the diagnosis of thalassemia major and chronic HCV infection (BTM/CHI). The only risk factor of HCV infection in these patients was blood transfusion before 1990.Group 2: Twenty patients  with chronic HCV infection (CHI). The route of infection and the duration of disease were not known in most of the patients.Group 3: Twenty patients  with thalassemia major.Thalassemia major was diagnosed by appropriate clinical and laboratory findings, and confirmed by hemoglobin electrophoresis. All the thalassemic patients had received multiple blood transfusions since childhood, and only those with at least moderate hepatic iron overload (2/4 based on Marx and Sindram hepatic iron scoring ) were inCHI was confirmed by positive anti-HCV (enzyme-linked immunosorbant assay), a positive HCV RNA by polymerase chain reaction and appropriate findings on liver biopsy.Patients with history of alcohol intake, smoking, positive HIV serology, positive HBs antigen or HBc antibody, and other liver diseases  were excluded from all the three groups.All of the patients whose liver biopsies were submitted to the Central Pathology Department of Imam Khomeini Hospital  between April 2001 and January 2004 were retrieved. During this period, there were only 20 thalassemic patients with moderate to marked hepatic iron overload, so all of them were included in our study. Since the sex and age distribution of patients with thalassemia major and BTM/CHI were relatively the same, 20 patients with BTM/CHI and at least moderate hepatic iron overload were randomly selected from all the patients with this diagnosis. On the other hand, patients with CHI had a considerably higher age, so to match the age of the patients, we selected 20 of the youngest patients.For all the sixty patients, we had hematoxylin and eosin, Masson's trichrome and Perls' Prussian blue stains. All the slides were reviewed by a single pathologist (FAA) who was blind to the diagnoses. The necroinflammation, fibrosis, and iron deposition were scored using modified Hepatitis Activity Index (HAI) grading, modified HAI staging, and Marx/Sindram hepatic iron scoring systems, respectively ,10. Pres2 tests for other variables with p < 0.05 considered to be significant.The results are presented as mean value +/- standard deviation (for age), median and range values (for modified HAI grade, stage, and iron score) and percentages (for steatosis). Group comparisons were made using the two-tailed independent Student's t-test for age and two-sided Fisher's exact or ChiFor each patient with BTM/CHI (doubly exposed cases), there were two different unexposed cases, i.e. thalassemic cases (unexposed to HCV) and CHI cases (unexposed to iron overload). Considering "disease" as higher stages of fibrosis (modified HAI stage ≥ 3), our guesstimate of the expected frequency of disease in unexposed and exposed patients were about 25% and 75%, respectively. So the sample size was calculated as about 20 patients for each group with confidence level of 95% and power of 80%.HFE mutations.The results of the study are summarized in table Angelucci et al showed for the first time the role of iron overload and HCV positivity as independent risk factors for hepatic fibrosis progression in thalassemic patients following successful bone marrow transplantation . The AngThe potentiating effect of iron overload and HCV infection can be explained by the fact that both these agents produce oxidative stress in the liver ,13. In oLimitations of our study were that it was a retrospective study with limited number of patients in each group. The duration of HCV infection and genotype of virus were not known in our patients. The routes of infection in most of the CHI patients were not known and where probably different from our thalassemic patients. The age of our CHI group was significantly higher than the other two groups. Since older age at the time of infection is considered to be a risk factor for progression of chronic hepatitis C, the stage of fibrosis should have been higher in our CHI patients than BTM/CHI patients if iron had not had any potentiating effect on liver fibrosis. Our study was performed on thalassemic patients with moderate to severe liver iron deposition. At these stages of iron overload, the pattern of hepatic iron deposition in HH and thalassemic patients are nearly the same. So this study does not reveal the effect of only sinusoidal iron deposition on progression of fibrosis in HCV infected thalassemic patients.Our results show that moderate to severe liver iron overload and chronic HCV infection in thalassemic patients have potentiating effect on hepatic fibrogenesis. So the proper use of chelating agents in HCV-infected thalassemic patients seems to be of great importance in delaying progression of the liver disease. The presence of liver siderosis has been shown to be related to poor response to interferon alpha (IFN) in non-thalassemic patients ,12,14,15None declared.FAA reviewed all of the slides, participated in study design and statistical analysis of results, and prepared the manuscript. MRFO assisted in collection and entering of the data, cooperated in reviewing slides, statistical analysis of results, and preparing the manuscript. MN was the clinical consultant. GI reviewed some of the problematic slides. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


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Upon hyperoxic exposure, the inducible nitric oxide synthase (iNOS) is activated by a variety of proinflammatory cytokines both in vitro and in vivo. In the present study, we used a murine hyperoxic model to evaluate the effects of iNOS deficiency on the inflammatory response.Hyperoxic exposures are often found in clinical settings of respiratory insufficient patients, although oxygen therapy (>50% O2 or >95% O2 for 72 h.Wild-type and iNOS-deficient mice were exposed to normoxia, 60% O2 resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. No significant effects of iNOS deficiency on cell differential in bronchoalveolar lavage fluid were observed. However, hyperoxia induced a significant increase in total cell count, protein concentration, LDH activity, lipid peroxidation, and TNF-α concentration in the bronchoalveolar lavage fluid compared to iNOS knockout mice. Moreover, binding activity of NF-κB and AP-1 appeared to be higher in wild-type than in iNOS-deficient mice.Exposure to >95% OTaken together, our results provide evidence to suggest that iNOS plays a proinflammatory role in acute hyperoxic lung injury. The pat-13. HoweReactive oxygen species can also react with other free radicals such as nitric oxide (NO) to yield more cytotoxic species including peroxynitrite anion ,15. PeroNO is synthesized from L-arginine by two main isoforms of the NO synthase: the constitutive and the inducible isoform . The NOSInappropriate regulation of nuclear factor-κB (NF-κB)-and activator protein-1 (AP-1)-mediated transcription has also been associated with pathological conditions, including acute inflammation such as hyperoxic exposure . Intrace concentration were analyzed. Binding activity of NF-κB and AP-1 was investigated in order to elucidate transcriptional mechanisms for iNOS and TNF-α expression.The objective of this study was to investigate the effect of iNOS deficiency on acute hyperoxic lung injury. As indicators of lung hyperpermeability, lavageable lung protein and LDH activity were measured; lung lipid oxidation was assessed based on the levels of thiobarbituric acid reactive substances. To characterize the inflammatory process, lavageable cell counts, cell differential, and TNF-Inducible NOS-deficient mice (C57BL6/J × 129SvEv) were originally provided by Dr. J. Mudgett (Merck & Co., Rathway, New Jersey, USA), Dr. J. MacMicking, and Dr. C. Nathan  . As cont3). Animals were randomized and exposed to 60% O2 and >95% O2 with a gas flow rate of 6 l/min at atmospheric pressure for 72 h. Mice exposed to room air in the same chamber served as controls. O2 levels were monitored twice daily with an oxygen analyzer (Drägerwerk AG, Lübeck, Germany). The environmental temperature was maintained at 24°C ± 1, relative humidity was 73% ± 13, and air pressure was 947 mbar ± 5. Oxygen was humidified by bubbling through a water chamber. The Plexiglas chamber bottom was lined with soda lime for CO2 absorption . Exposures were continuous for the time indicated except for 5 – 10 min daily when the chamber was opened for housekeeping purposes. Animals were kept on a 12 h light/dark cycle. Standard rodent food and water were available ad libitum.Male mice (12 – 16 weeks old, body weight between 26.1 g and 27.3 g) were kept in groups of seven in a sealed Plexiglas chamber . Tracheotomy was performed and a 20 G × 32 mm needle (Abbocath®-T, Venisystems, Sligo, Ireland) was inserted and secured. Bronchoalveolar lavage (BAL) was performed five times with 1 ml of sterile non-pyrogenic phosphate-buffered saline solution  in each animal. After centrifugation at 300 × g for 10 min, the supernatant was collected and stored at -20°C and -80°C for later protein assays. The BAL cell pellet was resuspended in PBS and washed by centrifugation. Cells were stained with May-Grunwald-Giemsa (Varistain 4, Shandon Labortechnik GmbH, Frankfurt, Germany) to identify cellular populations. Total cell counts were assessed with a hemacytometer (Coulter Ac T 8, Coulter Electronics, Krefeld, Germany).Immediately following exposure, mice were anaesthetized by intraperitoneal injection of sodium pentobarbital  based on a method of Smith et al. .To evaluate lactate dehydrogenase (LDH) activity in cell free BAL fluids, a commercially available kit was used (LDH Optimiert, Roche Diagnostics, Mannheim, Germany).Concentration of TNF-α in cell free BAL fluid was measured by an enzyme linked immunosorbent assay using a commercially available kit (EM-TNFA, Endogen, Woburn, Massachusetts, USA). Briefly, 50 ml biotinylated antibody reagent were added to 50 ml-samples in an anti-mouse TNF-α pre-coated strip well plate. After incubation for 2 h at room temperature, the plate was washed, a streptavidin horseradish peroxidase solution and the 3, 3',5, 5'-tetramethylbenzidine substrate solution were added and incubated in the dark. The absorbance was detected at 450 nm in a microplate reader . A standard curve was used to determine the amount of TNF-α concentration in the samples.Total RNA was isolated from non-lavaged lung homogenate of each mouse (RNeasy Mini Kit, Quiagen, Hilden, Germany), reverse transcribed into cDNA in a volume of 20 ml, containing 2 μg RNA, 1.5 μM Oligo-p(dT)15-primer, 5 × PCR-buffer, 0.1 M DTT, 10 nM dNTP-mix and 200 U/μl of Moloney murine leukemia virus reverse transcriptase. Reverse transcriptase-polymerase chain reaction (RT-PCR) amplifications were performed with aliquots of cDNA (3 μl) in total volume of 50 μl (5 μl 10 × PCR reaction-buffer, 1 μl dNTP-mix, 1 μl each of forward and reverse single strand DNA primers specific for mouse iNOS, 38.8 μl sterile deionized water, 0.2 μl Taq DNA-polymerase 1 U/ml). Oligonucleotide primers for iNOS were 5'-CAC AAG GCC ACA TCG GAT TTC-3' (sense) and 5'-TGC ATA CCA CTT CAA CCC AG-3' (antisense). Co-amplification of the housekeeping gene β-actin served as an internal control, using the following primers, 5'-GGA CTC CTA TGT GGG TGA CGA GG-3' (sense), 5'-GGG AGA GCA TAG CCC TCG TAG AT-3' (antisense). RT-PCR was started with 1 min incubation at 95°C followed by the steps of denaturation at 95°C for 45 sec, annealing at 55 – 64°C for 45 sec, elongation at 72°C for 1 min. The number of cycles (30 – 35 each) was chosen to ensure that the amplification product did not reach the level of saturation. Reactions were electrophoresed in 1% agarose gel and stained with ethidium bromide. The densitometry of each cDNA band was quantified using BIO-1D.V96 software and the ratio of iNOS cDNA to β-actin cDNA was determined.32P]-dATP end-labeled double-stranded oligonucleotides containing an NF-κB-consensus sequence (5'-AGT TGA GGG GAC TTT CCC AGG C-3') or AP-1-consensus sequence (5'-CGC TTG ATG AGT CAG CCG GAA-3') were added to the reaction followed by an incubation for 1 h at 37°C. The mixture was subjected to electrophoresis on a 6% PAA-Gel (75% H2O, 45 mM Tris, 45 mM bore acid, 1 mM EDTA, pH 8. 6% APS, 60 μl TEMED) for 2 h at 250 V.Nuclear protein extracts were prepared from pooled lung tissue as previously described . BrieflyConcentration of thiobarbituric acid reactive substances (TBARS) was evaluated with an assay according to Thiery et al. . BAL fluResults are presented as the group mean ± standard error of the mean (SEM). Statistical comparison between values of the three oxygen concentrations was performed by using analysis of variance on ranks and Mann-Whitney rank sum test followed by Bonferroni's correction. Statistical comparison between wild-type and iNOS knockout mice was analyzed by using Mann-Whitney rank sum test. Significance was accepted at p < 0.05.2, animals showed signs of reduced general conditions and reactions. Hyperoxic exposure >95% O2 also caused a significant reduction in body weight of wild-type mice compared to normoxic conditions and 60% oxygen exposure within the 72 h experimental period. In contrast, there was no significant change in body weight of iNOS knockout mice before and after normoxia and hyperoxia, respectively . Results presented in table 2, there was a significant decrease in the percentage of alveolar macrophages as well as a significant increase in the percentage of neutrophils and lymphocytes in both wild-type and iNOS knockout mice compared to normoxic conditions. No significant differences between wild-type- and iNOS knockout mice were found. However, hyperoxic exposure (>95% O2) resulted in a significant increase in total BAL cell counts after 72 h in wild-type (0.54 ± 0.05 × 106/ml) and in iNOS knockout mice (0.38 ± 0.04 × 106/ml) compared to normoxia (0.20 ± 0.03 × 106/ml and 0.16 ± 0.02 × 106/ml, respectively) and 60% O2 exposure (0.24 ± 0.04 × 106/ml and 0.19 ± 0.04 × 106/ml, respectively). This increase in BAL total cell counts under >95% O2 was significantly higher in wild-type than in iNOS knockout mice  and knockout mice . Exposure to >95% O2 resulted in a significant increase in protein concentration in wild-type mice (973.8 ± 95.7 μg/ml) and only in a modest increase in iNOS knockout mice (326.8 ± 90.4 μg/ml) that did not reach statistical significance.Total protein concentration in the BAL fluid was determined as an indicator of lung hyperpermeability induced by hyperoxic exposure. Under normoxia and 60% O2, LDH activity was comparable between wild-type  and iNOS knockout mice . Exposure to >95% O2 resulted in a significant enhancement of LDH activity in wild-type mice (41.8 ± 10.8 U/min/ml) compared to iNOS knockout mice (9.6 ± 3.0 U/min/ml) and to normoxia (figure As an indicator of cellular damage, LDH activity was measured in BAL fluid. Under normoxic conditions and 60% O2 exposure (29.2 ± 2.6 pg/ml and 25.4 ± 6.0 pg/ml, respectively). However, there was a significantly enhanced TNF-α release measured upon >95% O2 exposure in wild-type (83.0 ± 9.8 pg/ml) and iNOS knockout mice (54.9 ± 9.0 pg/ml) compared to normoxic conditions. TNF-α concentration was significantly higher in wild-type than in iNOS knockout animals (figure TNF-α concentrations were determined in BAL fluid to investigate inflammatory cytokine release. Under normoxic conditions, TNF-α release did not differ between wild-type (28.5 ± 3.8 pg/ml) and iNOS knockout mice 35.0 ± 5.4 pg/ml), the same as upon 60% O pg/ml, t2 exhibited a pronounced increase in concentration of TBARS (146.0 ± 62.0 nmol/ml) compared to normoxia (35.0 ± 14.0 nmol/ml) and 60% O2 (31.0 ± 17.0 nmol/ml). In iNOS knockout mice, concentrations of TBARS after >95% O2 (52.0 ± 18.0 nmol/ml) did not differ from those after normoxic conditions (26.0 ± 0.0 nmol/ml) and 60% O2 exposure (35.0 ± 26.0 nmol/ml), respectively.Lung lipid peroxidation was assessed based on the levels of thiobarbituric acid reactive substances in BAL figure . Wild-ty2 compared to normoxia and 60% O2. This enhancement of binding activity under hyperoxia appeared to be more prominent in the group of wild-type mice in comparison to iNOS knockout animals.In an effort to elucidate transcriptional mechanisms for increased iNOS and TNF-α expression after hyperoxic exposure, electrophoretic mobility shift assays for NF-κB and AP-1 were performed figure . NF-κB a2) induced an increased expression of iNOS mRNA in lung samples compared to normoxic situation. Densitometric analysis was performed by determining the ratio of iNOS cDNA to β-actin cDNA. Results demonstrated a significant increase in iNOS mRNA expression upon >95% O2 (1.2 ± 0.1) compared to 60% O2 (0.8 ± 0.1).To investigate the induction of the iNOS gene in lung tissue, expression of iNOS mRNA was analyzed figure . As expe2) during an intensive care setting to maintain arterial pO2 can lead to progressive lung injury. Several cellular systems including alveolar macrophages and leukocytes are involved in this process. Activation of inflammatory cells causes the release of reactive oxygen species and proinflammatory cytokines, resulting in endothelial dysfunction, tissue and alveolar edema formation, and surfactant inactivation. Furthermore, high levels of NO produced by inducible NO synthase may contribute to tissue damage. NO is directly cytotoxic or can combine with superoxide anions to form the more reactive oxidant peroxynitrite. Although a large amount of literature exists concerning the pulmonary response to oxidant exposure, some issues remain unresolved.Prolonged exposure to high concentrations of oxygen (>50% O2 induced a significant increase in iNOS mRNA expression. This enhanced iNOS mRNA expression during hyperoxic exposure seems to contradict findings reported in a study published by Arkovitz and colleagues, in which hyperoxia did not induce iNOS expression in lungs of mice [2 induced a prominent expression of iNOS protein in the lungs from wild-type mice (data not shown).Our findings confirm previous results showing that hyperoxia is able to upregulate iNOS expression in lung tissue ,34. As e of mice . This ma of mice -30, we fin vivo oxygen exposure significantly elevated total BAL cell count after 72 h >95% O2 both in wild-type and in iNOS knockout mice. According to this, oxygen exposure resulted in a significant enhancement in the number of neutrophils and lymphocytes in BAL fluid, combined with a significant reduction in the number of alveolar macrophages both in wild-type and iNOS knockout mice. Dedhia et al. also found elevated numbers of neutrophils and lymphocytes combined with decreased numbers of alveolar macrophages in rat lungs [The data from the present study demonstrate that at lungs . Recent at lungs , iNOS-deat lungs . These rat lungs . In our at lungs .2 was attenuated in iNOS-deficient mice (data not shown). Formation of nitrotyrosine was proposed as a relatively specific marker for detecting endogenous generation of peroxynitrite. However, recent evidence indicates that alternate reactions are capable of inducing nitration of tyrosine in proteins, for example the reaction of myeloperoxidase with hydrogen peroxide. Therefore, increased nitrotyrosine staining is considered as an indicator of "increased nitrative stress" rather than a "footprint" for the formation of peroxynitrite [Recent studies indicate that iNOS also plays a proinflammatory role in the development of asbestosis-related pulmonary disorders, measured as a significantly decreased total protein count, LDH activity, and nitrotyrosine staining in iNOS-deficient mice . In contynitrite ,50. Amplynitrite . In our in vivo resulting in inflammatory liver injury in mice by stimulation of TNF-α production [Cytokines may also play a role in oxygen toxicity. Several studies point out that TNF-α is produced during hyperoxic exposure ,52. Furtoduction . To inveoduction . Moreoveoduction . Data froduction  also indoduction .2, proinflammatory effects of iNOS may be predominant, thereby contributing to the extent of acute hyperoxic lung injury.Taken together, our data show that the absence of the iNOS gene does attenuate, but not fully abolish, oxidation, nitration, and cytotoxicity in response to acute hyperoxic exposure. The degree of transcriptional activation, inflammation, and oxidative lung injury caused by hyperoxia is significantly reduced in iNOS knockout mice compared to wild-type animals. In conclusion, these findings provide evidence to suggest that, upon hyperoxic exposure to >95% OAKH carried out the hyperoxic model, subsequent cytological and biochemical analyses, and writing and preparation of the manuscript. MD and FK participated in the direction of the study as well as in writing and preparation of the manuscript. CK carried out the electrophoretic mobility shift assays. The data presented in this paper are part of the doctoral thesis of AKH. All authors read and approved the final manuscript.AP-1 activator protein-1BAL bronchoalveolar lavageiNOS inducible nitric oxide synthaseLDH lactate dehydrogenaseLPS lipopolysaccharideNF-κB nuclear factor-kappa BNO nitric oxideTBARS thiobarbituric acid reactive substancesTNF-α tumor necrosis factor-alpha


Rhodobacter capsulatus.The enzyme porphobilinogen synthase (PBGS), which is central to the biosynthesis of heme, chlorophyll and cobalamins, has long been known to use a variety of metal ions and has recently been shown able to exist in two very different quaternary forms that are related to metal ion usage. This paper reports new information on the metal ion independence and quaternary structure of PBGS from the photosynthetic bacterium R. capsulatus PBGS was amplified from genomic DNA and sequencing revealed errors in the sequence database. R. capsulatus PBGS was heterologously expressed in E. coli and purified to homogeneity. Analysis of an unusual phylogenetic variation in metal ion usage by PBGS enzymes predicts that R. capsulatus PBGS does not utilize metal ions such as Zn2+, or Mg2+, which have been shown to act in other PBGS at either catalytic or allosteric sites. Studies with these ions and chelators confirm the predictions. A broad pH optimum was determined to be independent of monovalent cations, approximately 8.5, and the Km value shows an acidic pKa of ~6. Because the metal ions of other PBGS affect the quaternary structure, gel permeation chromatography and analytical ultracentrifugation experiments were performed to examine the quaternary structure of metal ion independent R. capsulatus PBGS. The enzyme was found to be predominantly hexameric, in contrast with most other PBGS, which are octameric. A protein concentration dependence to the specific activity suggests that the hexameric R. capsulatus PBGS is very active and can dissociate to smaller, less active, species. A homology model of hexameric R. capsulatus PBGS is presented and discussed.The gene for R. capsulatus PBGS as not requiring any metal ions for function. Unlike other wild-type PBGS, the R. capsulatus protein is a hexamer with an unusually high specific activity when compared to other octameric PBGS proteins.The evidence presented in this paper supports the unusual position of the  The ∝-proteobacteria synthesize ALA from succinyl-CoA and glycine while other photosynthetic organisms use glutamic acid to make ALA [The enzyme porphobilinogen synthase PBGS, EC 4.2.1.24) catalyzes the first common step in the biosynthesis of the tetrapyrrole pigments such as heme, chlorophyll, and cobalamin . As of 2.2.1.24 crganisms . Howevermake ALA .Rhodobacter capsulatus with emphasis on understanding the enzyme's unique characteristics. Since other PBGS have been shown to absolutely require divalent cations for catalytic activity, and in light of the enhanced purity of modern reagents, it is important to revisit the metal ion requirements of R. capsulatus PBGS to test the predictions of the sequence analysis that suggests the absence of any metal binding determinants. Herein we present evidence that there is absolutely no effect of Zn2+ or Mg2+ on the activity of the enzyme and no other metal ions appear to be required for enzyme function. Prior studies have also shown that some PBGS enzymes exhibit a pH rate profile whose pKa value is altered by the presence of monovalent cations [In light of the vast information now available on phylogenetic variations in tetrapyrrole biosynthesis and on the PBGS that require a catalytic zinc and/or that utilize an allosteric magnesium, the current study revisits the PBGS of  cations ,8. HenceE. coli, and Pseudomonas aeruginosa, and noncrystallographic cross-linking data on PBGS from the green plant pea [R. capsulatus PBGS, and results suggest that the protein is a homo-hexamer. We present a homology model of the hexameric R. capsulatus PBGS structure.The native holoenzyme quaternary structure for PBGS from most species is a homo-octamer as supported by 18 deposited PBGS crystal structures from yeast, human, lant pea -14. Howelant pea . The hexlant pea . In the hemB gene (which encodes PBGS) from R. capsulatus was accomplished by PCR using primers based upon the sequence of Indest and Biel [[R. capsulatus and R. sphaeroides sequences (GenBank accession number AAL 26883) as well as the sequence determined from our PCR product (GenBank accession number AY618996). The changes in the predicted polypeptides are due to three differences that we observe when comparing the nucleotide sequence determined herein with the published sequence: a deletion of G at position 425, an insertion of an A at position 468, and reversal of the AC at positions 643–644 to CA. The first two sequence differences result in alteration of the predicted amino acid sequence from amino acids 72 to 86. The third sequence difference alters the amino acid at position 145 from leucine to isoleucine. Based on the aligned polypeptide sequence, the newly determined sequence appears more homologous to R. sphaeroides PBGS, and hence is deduced to be the correct sequence.The cloning of the nd Biel [, GenBankR. capsulatus PBGS. The specific activities of the ultracentrifuge supernatant, redissolved ammonium sulfate pellet, phenyl-sepharose pool, DEAE pool, and concentrated S-300 pool were 130, 250, 212, 176, and 364, μmol h-1 mg -1 respectively. We suspect that some of this variation is due to a protein concentration dependence to the specific activity (see below). The enzyme has an apparent molecular weight that is in agreement with the predicted mass of 35.8 kDa  suggest that there is little if any effect of the monovalent cations K+, Na+ or NH4+ over a wide range of concentrations (0–100 mM). Figure -1 protein concentration.Previous experiments with a wide variety of PBGS enzymes suggest that monovalent cations can affect the activity of the enzyme. Such cations were clearly shown to shift the pH rate profile for some PBGS ,8. ResulVmax and Km values at ~1 μg ml-1 protein at a variety of pH values as presented in Figure Km value drastically increases at lower pH values, which is similar to what has been observed for other PBGS enzymes e.g., human PBGS [E. coli PBGS [B. japonicum PBGS [Km at low pH appears to be independent of the metal ion requirements for PBGS.To determine the optimal pH for the enzyme we determined the man PBGS , E. colioli PBGS , or B. jcum PBGS . Based oR. capsulatus PBGS, a native gel analysis was performed and the gel was stained for enzyme activity followed by Coomassie staining for protein . Regions of highest uncertainty are places where alignment of R. capsulatus PBGS and P. aeruginosa PBGS contain insertions and deletions. These regions are illustrated in the Figure; they are all on the solvent exposed surface of the oligomer and thus unlikely to affect subunit interactions. In an attempt to understand why, unlike other PBGS, R. capsulatus PBGS does not predominate as an octamer, we analyzed points of subunit contact in the octamer of the highly homologous P. aeruginosa PBGS. Each P. aeruginosa PBGS monomer contains thirty-six residues that are within 3.2 Å of an adjacent subunit of the octamer. Twenty-five of these residues are identical in R. capsulatus PBGS; eleven are different. The majority of these differences result in a reduced ability in R capsulatus PBGS to form an ion pair or hydrogen bonding interaction at the subunit interface. The differences are tyrosine to phenylalanine or isoleucine, arginine to glutamine or glycine, valine to isoleucine, aspartic acid to asparagine or alanine, histidine to arginine or leucine, and glutamic acid to alanine or asparagine. This analysis is consistent with the observation that the interactions between PBGS subunits are largely hydrophilic in character with ordered water molecules at the subunit interfaces (see Discussion).Figure R. capsulatus and R. sphaeroides PBGS. The revised sequence for R. capsulatus PBGS presented shows close homology between the PBGS of the two Rhodobacter species. This is interesting in light of the apparent differences between these enzymes regarding monovalent cation usage. The R. sphaeroides enzyme is reported to be stimulated by monovalent cations [R. capsulatus is not affected by monovalent cations. While it was previously tempting to ascribe the difference in response between the enzymes to this region, clearly this is not the case.Prior to the current work, it appeared that there was a considerable sequence diversion in a 13 amino acid segment between  cations , while tR. capsulatus PBGS, the enzyme was purified after being heterologously expressed in E. coli. The purification has allowed us to carry out definitive experiments on the requirements for R. capsulatus PBGS function.To further analyze the enzymatic characteristics of the Rhodobacter PBGS appear to constitute a unique form of the enzyme that does not require metal ions for structure, activity, or allosteric regulation [Rhodobacter PBGS enzymes [R. capsulatus PBGS. R. capsulatus PBGS activity is independent of all metal ions tested.Based on sequence comparisons and known crystal structures for some PBGS, the gulation . Althoug enzymes ,5 did noRhodobacter capsulatus makes a similar pigment, bacteriochlorophyll, the PBGS enzyme from this organism does not exhibit any regulation by divalent cations. In the absence of the allosteric magnesium, Rhodobacter must use alternative mechanisms to prevent the accumulation of photoreactive intermediates in the biosynthesis of its physiologically relevant tetrapyrroles.It has been proposed that chloroplast containing photosynthetic organisms use the allosteric regulation of PBGS by magnesium as part of a complex control of the biosynthesis of chlorophyll . AlthougVmax and Km in a systematic fashion. Based upon these analyses, the apparent pH optimum is approximately pH 8.0, but the enzyme demonstrates significant activity from pH 7–9. The Km value at optimal pH is still high (0.5 mM) relative to other PBGS at their optimal pH in the presence of their required metal ions (~150 μM). This suggests that an unknown factor may be required in vivo. Because there have been reports for stimulation of PBGS enzymes from other organisms by the addition of monovalent cations, several monovalent cations were tested for their ability to stimulate enzyme function at the pH optimum. At the pH optimum no stimulation by monovalent cations was observed  directed to the 5' and 3' ends of the coding region based on published sequence [CATATGACCCTGATCACCCCCCCC-3') introduced an NdeI site and the reverse primer PBGS 3' (5'-CGGATCCGCGGTCAGGCGCCGATCAGC-3') introduced a BamHI site. The PCR reaction was performed using a thermocycler from MWG Scientific and Pfu polymerase (Stratagene) with the following program: 45 sec at 95°C, 45 sec at 48°C, 1 minute at 72°C. The resulting PCR fragment was cloned into vector pPCRScript Amp (Stratagene) creating plasmid pPBGS1. The PCR fragment was removed from the vector by digestion with NdeI and BamHI and ligated into the vector pET11a (Novagen) digested with the same restriction enzymes. The resultant plasmid pPBGS4 was sequenced in the FCCC DNA Sequencing Facility using external and internal primers to confirm the sequence in both directions. For expression, the recombinant plasmid pPBGS4 was transformed into strain BLR (DE3).The DNA encoding PBGS was amplified from sequence . The forg) and resuspended in fresh LB medium containing no glucose, but with 100 μM isopropyl-1-thio-β-D-galactopyranoside (Research Organics) and grown for 45 hr at 15°C. From this point all steps were performed on ice or at 4°C. Cells were harvested by centrifugation for 10 min at 10,800 × g with a yield of 5.74 g wet weight. The cells were washed with 0.1 M BisTris Propane (BTP, Research Organics) pH 8.5 and then resuspended in 15 ml of 0.1 M BTP pH 8.5 and lysed by two passes through a French Press in the presence of Benzonase™ (Novagen) nuclease and 1 mM AEBSF . Unbroken cells, inclusion bodies, and debris were removed by centrifugation for 15 min at 21,500 × g. The sample was then ultracentrifuged for 1 hour at 141,000 × g to remove membranes. The enzyme was precipitated from solution by treatment with 25% ammonium sulfate and collected by centrifugation for 20 min at 31,000 × g. The pellet was resuspended in 0.1 M BTP pH 7.0 and loaded onto a 100 ml Phenyl-Sepharose (Amersham Biosciences) column pre-equilibrated in 20% ammonium sulfate, 0.1 M BTP pH 7.0 buffer. The enzyme was eluted from the column with a two column volume gradient running from 20% to 0% ammonium sulfate in 30 mM BTP pH 7.0. The PBGS eluted from the column very close to the end of the gradient. Fractions with specific activity greater than 100 μmol h-1 mg-1 were then pooled and loaded to a 100 mL DEAE-Sepharose column equilibrated in 30 mM BTP pH 7.5 and eluted with a two column volume gradient from 0 to 0.4 M KCl in 30 mM BTP pH 7.0. The fractions with a specific activity greater than 300 μmol h-1 mg-1 eluted near the end of the gradient and were pooled and concentrated using centrifugal concentrators. The concentrated DEAE fraction (0.9 mg ml-1) was then loaded on to a 300 mL S-300 column (2.6 cm × 60 cm) (Amersham Biosciences) pre-equilibrated with 0.1 M BTP pH 7.0, and eluted with the same buffer. The fractions with specific activity greater than 185 μmol h-1 mg-1 were pooled and concentrated. The enzyme appeared to be greater than 95% pure based on SDS-PAGE analysis.A 1 L culture of LB broth with 0.4% glucose was inoculated with a single colony from a fresh transformation and grown for 16 hours at 37°C. The cells were harvested by centrifugation  to a final concentration of 10 mM and were allowed to run for 5 minutes in a final volume of 1 ml. Assays were terminated by the addition of 0.5 ml of 20% TCA. The product was then quantified by reaction of the stopped assay mixture with an equal volume of modified Ehrlich's reagent and measurement of absorbance at 555 nm approximately 8–10 minutes later. All assays were performed in duplicate or triplicate. If the amount of product resulted in an absorbance above 1.0 OD, the stopped assay mixture was diluted prior to the addition of Ehrlich's reagent. Inhibition by 1,10-phenanthroline was carried out as described previously . InhibitKm and Vmax as a function of pH, the enzyme was at 0.13 μg ml-1. Since the substrate is an acid, the actual pH values for assays were determined by running a mock assay without enzyme and measuring the actual pH of the combined reaction. When ALA concentration was varied for determination of Vmax, the pH of the assay mixture was adjusted with 0.5 M HCl to control the final pH. For determination of the pH rate profile with and without 0.1 M KC1, the enzyme was at 15 μg ml-1.For assays performed as a function of pH, the buffer was 0.1 M BTP . For determination of -1 for 10 minutes in 0.3 M Tris pH 6.8 with additions of 100 μM Zn, 13 mM ALA or both prior to loading. 15 μl of the preincubated sample was then loaded to each well.SDS-PAGE was performed using the Laemmli system and precast 4–20% gradient gels from Cambrex (Rockland, ME). Gels were stained using a silver stain kit from Pierce Chemical (Rockford, IL). Native gels were performed using the same gel system but omitting SDS from all buffers. Activity staining of the gel was performed as described previously . Followi-1 solution. The column was calibrated with standards obtained from the manufacturer. For AU experiments, protein samples were dialyzed against 10 mM Tris-HCl pH 7.7 with either 0.1 mM DTT or 1 mM βME as reducing agent. Although TCEP (Tri(2-carboxyethyl)phosphine) is the preferred reducing agent for such biophysical experiments, we discovered that the oligomeric state was destabilized in the presence of this reagent with other PBGS; weak chelation of divalent cations has been observed for this reagent suggesting a mechanism for this destabilization. Protein loading concentrations were 12.3 μM in monomer (440 μg ml-1). Concentrations were determined by UV absorption at 280 nm. The extinction coefficient used for the protein was 29,870 L mol-1 cm-1 and represents the sum of individual tyrosine and tryptophan absorbance coefficients. Sedimentation equilibrium (SE) experiments were carried out at 4°C using a Beckman Optima XL-A analytical ultracentrifuge equipped with an An60 Ti four-hole rotor. Samples were loaded into six-channel charcoal-filled Epon centerpieces. Temperature-corrected partial specific volumes and solution densities were calculated using the Sednterp program [-1 and partial specific volume was 0.7304 ml g-1. Data analysis was performed using the WinNonlin V.1.060 nonlinear least squares fitting program obtained from the National Analytical Ultracentrifugation Facility at the University of Connecticut.The size of the enzyme was determined both by size exclusion chromatography and by analytical ultracentrifugation (AU). Size exclusion chromatography was performed using a Waters 600 system equipped with a Waters 996 photodiode array for detection of protein elution at 280 nm. The column used was a Superose 6 (10 × 300 mm, Amersham Pharmacia) and was run at 0.5 ml/min with 100 mM BTP pH 8.6 with 100 μL of a 200 μg ml program ; the solR capsulatus PBGS is that of hexameric human PBGS clinical variant F12L, PDB code 1PV8 [R. capsulatus PBGS. The chosen structure is PDB code 1GZG [Pseudomonas aeruginosa PBGS, and 56% sequence identical to R. capsulatus PBGS. The model building procedure was a two step process. The first step was construction of a model of a hexameric form of P. aeruginosa PBGS; the second step was to use that hexamer to build the R. capsulatus PBGS hexamer.The only existing crystal structure on which one can base a model of hexameric ode 1PV8 . Unfortuode 1PV8 . Hence, ode 1GZG , which iP. aeruginosa PBGS hexamer preparation used various capacities of the program Swiss-PDB Viewer [P. aeruginosa PBGS αβ-barrels. There is no significant sequence identity between the N-terminal arms of human and P. aeruginosa PBGS, hence there is an alignment ambiguity when trying to build the outstretched arms of the P. aeruginosa PBGS hexamer. However, there is a region of the arm that is α-helical in both the human octamer and the human hexamer. Hence, a structure alignment of octameric forms of human PBGS and P. aeruginosa PBGS was used to determine the proper sequence alignment for this α-helical segment. This information was used to spatially position the amino acids 22 – 29 of P. aeruginosa PBGS in the hexamer according to the position of this helix in the hexamer of human PBGS. Loop and side-chain prediction was performed in a graphical user environment, developed in the FCCC Molecular Modeling Facility, that integrates the functions of the programs Loopy [P. aeruginosa PBGS, which could then be used for preparing hexameric models of other PBGS as we have done before for the octameric forms of pea and D. melanogaster PBGS [R. capsulatus PBGS was built using the same integrated graphical environment developed in house. This software integrates sequence alignment, threading, loop model building to accommodate insertions and deletions, and side chain optimization similar to that used for our previously published models [B Viewer  and somems Loopy , and SCWms Loopy . Within ms Loopy  was usedms Loopy  was usedter PBGS ,9. The md models ,9.DWB was responsible for the initial cloning by PCR, purification of the enzyme, initial assays of the enzyme, and preparation of Figs.


Plasmodium vivax, although causing a less serious disease than Plasmodium falciparum, is the most widespread of the four human malarial species. Further to the recent recrudescence of P. vivax cases in the Newly Independent States (NIS) of central Asia, a survey on the genetic diversity and dissemination in Azerbaijan was undertaken. Azerbaijan is at the crossroads of Asia and, as such, could see a rise in the number of cases, although an effective malaria control programme has been established in the country.P. vivax isolates from Central Azerbaijan were characterized by analysing the genetic polymorphism of the circumsporozoite protein (CSP) and the merozoite surface protein 1 (MSP-1) genes, using PCR amplifications and amplicons sequencing.Thirty-six Analysis of CSP sequences showed that all the processed isolates belong to the VK 210 type, with variations in the alternation of alanine residue (A) or aspartic acid residue (D) in the repeat motif GDRA(A/D)GQPA along the sequence. As far as MSP-1 genotyping is concerned, it was found that the majority of isolates analysed belong to Belem and Sal I types. Five recombinant isolates were also identified. Combined analysis with the two genetic markers allowed the identification of 19 plasmodial sub-types.P. vivax clones circulating in Azerbaijan and, consequently, a careful malaria surveillance could be of paramount importance to identify, at early stage, the occurrence of possible P. vivax malaria outbreaks.The results obtained in the present study indicate that there are several  Plasmodium vivax is the most widely distributed human parasite, with an estimated burden of 70–80 million cases annually [Plasmodium falciparum, being rarely lethal, P. vivax affects the working capacity of the population and the lack of efficient drug distribution favors the onset of drug resistant strains [P. falciparum infections account for the majority of cases (64%), P. vivax is responsible for an additional 23% [P. vivax infection represents a permanent risk for the occurrence of P. vivax indigenous malaria cases, as recently occurred in central Italy [annually . In some strains ,3. Impor strains . Althougf cases 6%, P. vival Italy ,6. SinceP. falciparum, molecular studies of the genetic diversity and dissemination of P. vivax are scanty. Recently, 33 polymorphic tandem repeats (TRs) of P. vivax and a P. vivax polymorphic microsatellite have been identified and shown to be useful in population studies [P. vivax populations, since PCR-RFLP products indicate the presence of up to 13 alleles [P. vivax endemic areas.In comparison with  studies ,10. The  alleles ,12. HoweP. vivax natural populations present in this endemic country. For this purpose the extent of polymorphism of CSP and MSP-1 genes were analysed in parasite isolates from five localities of central Azerbaijan by using PCR amplification and sequencing.In the frame of a malaria research project funded by the European Commission, a molecular study was undertaken in Azerbaijan, aimed at collecting information on the genetic make-up of 2, with a populations of approximatively 2,5 millions. The climate is typically continental with an average temperature between 12 and 15°C and a rainfall between 200 and 600 mm per year. Climatic and agro-ecological conditions of this area make the environment favourable to mosquito vectors breeding. The major malaria vector is Anopheles sacharovi that breeds preferably in lakes, swamps, irrigation canals and pools. Although it prefers well-oxygenated water, it is known to tolerate moderate salty water. Other anopheline species found in this area are A. maculipennis, A. subalpinus, A. superpictus and A. hyrcanus, all of which are considered secondary vectors of malaria transmission [Azerbaijan covers an area of 29.540 Kmsmission . MalariaDuring summer 2002, a malaria epidemiological survey was performed in central Azerbaijan, in the frame of a Malaria Surveillance Programme launched in year 2001 by the Ministry of Health in collaboration with WHO. Active case detection was carried out in five districts included in previously identified sentinel sites, namely Mingaçevir, Beylagan, Imisli, Saatli and Sabirabad. A map of Azerbaijan with the study area is shown in Figure All individuals who visited the district health centres or were found in villages with a history of recent fever and no history of travel in the past few months were considered. In this context, a total of 36 infected individuals with positives blood smears at the microscopic examination, collected between August and September 2002, were selected for the genetic study. The age of patients ranged from 6 to 78 years and parasitaemia varied from 288 to 12,800 parasites/μl. For the molecular analysis, a blood sample of about 1 ml was taken from by venipuncture before drug treatment was given. Patients or the guardians of children were informed about the study. According to the international rules for research involving human subjects, any information which would identify a participant was removed in order to keep each sample processed anonymous. Number of samples for each district is shown in Table Plasmodial DNA was extracted from 200 μl of each infected blood sample using QIAamp DNA blood kit following the manufacturer's instructions (Qiagen, CA).2, 80 μM of each deoxynucleotide triphosphate, 6 pmol of each primer and 1.3 U of Taq polymerase (Promega, Madison, USA). The PCR programme was: denaturation at 94°C for five minutes; 34 cycles of one minute at 94°C, one minute at 54°C and two minutes at 72°C. The PCR products were separated using electrophoresis on a 1.5 % NuSieve gel and the band of interest was cut out and purified using the QIAquick PCR purification Kit (Qiagen). The purified product was sequenced in both directions using an ABI-PRISM 373 sequencer. Nucleotide or amino acid sequences were aligned first using the CLUSTAL X programme [ was used to convert nucleotide sequences into amino acid sequences. The GenBank accession numbers of the eight sub-types of VK210 type are from AY792359 to AY 792366.The CSP gene was amplified for the most part of samples using PV5 and PV6 primers . Samplesrogramme  with manrogramme . The ExP2, 200 μM of each deoxynucleotide triphosphate, 30 pmol of each primer and 2.5 U of Taq polymerase (Promega, Madison, USA). For the second round, 10 μl of the first amplification product was added to a fresh PCR mixture with 30 pmoles of each inner primer. The thermal profile was: denaturation at 94°C for five minutes; 35 cycles of 94°C for one minute, 60°C for one minute and 75°C for three minutes. All nested-PCR products were purified by Microcon-PCR (Millipore), following the manufacturer's instructions and sequenced in both directions at the MWG Biotech. The results were analysed by means of Omiga 2.0 (ACCELRYS, Cambrige) and Mega 2 (S. Kumar, K. Tamura, M. Nei and Pennsylvania State University) computer programmes. The GenBank accession numbers of the 36 nucleotide sequences from P. vivax isolates are from AY789657 to AY789692.A portion of the MSP-1 gene (the region encompassing the interspecies conserved blocks ICB5 and ICB6) was amplified using a nested PCR with, respectively, the two outer primers A5 and A6  and the The aligned nucleotide sequences of CSP were converted to a distance matrix (% of differences) using the Net algorithm of the MUST package . The denMSP-1 and CSP data were analysed using the Cavalli-Sforza distance  from GenP. vivax isolates were found to belong to the VK210 type [CSP sequences obtained from 36 Azerbaijan 210 type . The iso210 type . In summP. vivax isolates were identified as recombinant types. Isolate Satl1 (sequence named in the present study as sub-type T) could be ascribed to the type 3a GQPAA and ANGAGNQPG, respectively, with some variant positions within the sequence ,23. ThesP. vivax malaria as well, where the authors detected the two types Belem and Sal I, together with several recombinant types. In particular, in the study carried out in Iran by Zakeri et al., the analysis of MSP1 genetic diversity on a total of 16 plasmodial isolates leaded to the identification of 14 genetic sub-types. It is worth noting that such a high level of diversity is probably due to the small sample size.Our results confirmed that MSP-1 is a good polymorphic marker. In particular, the region of the gene known to be highly polymorphic and discriminative between the Belem and Sal I types was analysed . A variaet al., it is possible to speculate that the observed genetic diversity could be also explained considering the studied area, i.e. central Azerbaijan, as transit road of the country and also of neighboring Asian country, where the circulating P. vivax populations show considerable MSP-1 genetic diversity. However, further studies aimed at collecting more information about people moving within the whole country and to closer countries are needed to verify the above hypothesis. The combined analysis of CSP and MSP1 sequence polymorphism has led to the identification of a total of 19 P. vivax sub-types, confirming that the simultaneous use of more than one genetic marker in this kind of study enhances the knowledge of genetic diversity existing in the parasite populations. The results of the current study show the circulation of multiple plasmodial clones in the studied area thus leading to the conclusion that malaria surveillance activities must be maintained in Azerbaijan in order to avoid serious disease outbreaks in the future.Our results show 17 genetic sub-types on a total of 36 isolates analysed and a quite high MSP-1 polymorphism also in Azerbaijan. As suggested in other studies  and alsoP. vivax field populations could help in implementing malaria control activities being a crucial step for the development of a malaria vaccine.The understanding of the polymorphism extent in surface antigens as CSP and MSP-1 and the resulting genetic diversity in S. Mammadov, N. Aliyev, E. Gasimov were involved in field collection of blood samples and microscopy examinations. M.C. Leclerc and A. Cligny did the CSP sequence analysis and M. Menegon did the MSP-1 sequence analysis. M.C. Leclerc and J.L. Noyer did the distance analyses. M.C. Leclerc wrote the report with major contributions of C Severini, M Menegon and G. Majori. G. Majori coordinated the field activities carried out in Azerbaijan. C. Severini, as scientific coordinator of the VIVAXNIS project mentioned below, got the financial support.


After fertilization, embryo development involves differentiation, as well as development of the fetal body and extra-embryonic tissues until the moment of implantation. During this period various cellular and molecular changes take place with a genetic origin, e.g. the elongation of embryonic tissues, cell-cell contact between the mother and the embryo and placentation. To identify genetic profiles and search for new candidate molecules involved during this period, embryonic gene expression was analyzed with a custom designed utero-placental complementary DNA (cDNA) microarray.k-means and hierarchical clustering methods identified the expression patterns of differentially expressed genes across pre-implantation period. Novel candidate genes were confirmed by real-time RT-PCR.Bovine embryos on days 7, 14 and 21, extra-embryonic membranes on day 28 and fetuses on days 28 were collected to represent early embryo, elongating embryo, pre-implantation embryo, post-implantation extra-embryonic membrane and fetus, respectively. Gene expression at these different time points was analyzed using our cDNA microarray. Two clustering algorithms such as k-means clustering. Comparison of day 7 and day 14 revealed most genes increased during this period, and a small number of genes exhibiting altered expression decreased as gestation progressed. Clustering analysis demonstrated that trophoblast-cell-specific molecules such as placental lactogens (PLs), prolactin-related proteins (PRPs), interferon-tau, and adhesion molecules apparently all play pivotal roles in the preparation needed for implantation, since their expression was remarkably enhanced during the pre-implantation period. The hierarchical clustering analysis and RT-PCR data revealed new functional roles for certain known genes (dickkopf-1, NPM, etc) as well as novel candidate genes (AW464053, AW465434, AW462349, AW485575) related to already established trophoblast-specific genes such as PLs and PRPs.In total, 1,773 individual genes were analyzed by complete A large number of genes in extra-embryonic membrane increased up to implantation and these profiles provide information fundamental to an understanding of extra-embryonic membrane differentiation and development. Genes in significant expression suggest novel molecules in trophoblast differentiation. Embryogenesis in the period from fertilization to implantation involves various morphological, cellular, and biochemical changes related to genomic activity . These cWe have developed a bovine utero-placental specific cDNA microarray that contains various genes known to play key roles in extra-embryonic membrane development in the bovine . To our k-means [k-means and hierarchical clustering methods to identify genes involved in trophoblastic differentiation.Although there is no defined standard method that has been developed for the analysis of microarray data, application of k-means ,17 and hk-means  methods k-means -22 and mk-means ,23,24. W2α  on three days after the initial injection of FSH. Artificial insemination was performed both on the day of estrus (Day 0 of gestation) and the next morning, using frozen semen from Japanese black bulls. Twelve blastocysts collected on Day 7 were pooled to represent the Day 7 embryo (abbreviated as Day 7E), two expanded and elongated embryos collected on Day 14 were pooled to represent Day 14E, two embryos collected on Days 19 and 21 were used individually but designated as Day 21E (n = 2). Extra-embryonic membranes collected on Days 27 (two samples) and 28 (one sample) were used as Day 28 extra-embryonic membranes (28EEM) (n = 3), and two fetuses, excluding the extra-embryonic membranes, collected on Days 27 and 28 were used as the Day 28 fetus (28F) (n = 2). All samples were snap frozen in liquid nitrogen immediately after collection and stored at -80°C until RNA extraction. The days of collection were selected depending on the macro morphological changes of the embryos during the peri-implantation periods; bovine embryo passes into the uterus around a morula stage and develop to a blastocyst stage by Day 7. Embryo begins to elongate on Day 14 and form a long embryo of 20 to 30 cm in length on Day 20 just before implantation occurs. The embryo is easily identifiable around Day 30 around which placentome formation is visible [Totally five Japanese black cows were slaughtered on Days 19, 21, 27 (two) and 28 of gestation , and collected embryonic membrane and fetus. Embryos were collected non-surgically on Days 7 and 14 of gestation from superovulated Japanese black cows. Namely, a follicle-stimulating hormone  was injected twice daily at doses of 5 mg, 3 mg, and 2 mg on the first, second, and third day of treatment, respectively. Luteolysis was induced by injecting prostaglandin FThe total RNA was isolated from the embryos using ISOGEN (NipponGene, Toyama, Japan) according to the manufacturer's instructions. Briefly, Samples were mixed and homogenized with ISOGEN. The aqueous phase was collected after the centrifugation. This phase was washed by chloroform for the RNA purification. The aqueous phase was collected after the centrifugation again. The total RNA pellet in the aqueous phase was obtained using the isopropanol sedimentation. Total RNA was directly used for the T7-based linear amplification for the cDNA microarray analysis. Messenger RNA (mRNA) were reverse transcribed by using the T7-oligo dT primer in the T7-based linear amplification system.Linear-amplified antisense RNA (aRNA) was used for the cDNA microarray experiment. Because the amount of total RNA extracted was small from Day 7 blastocysts for the microarray analysis, mRNA from Day 7 blastocysts was amplified along with all other mRNA samples that were collected from Days 14 to 28 of gestation to eliminate technical variations and to efficiently compare the array data.We used a MessageAmp aRNA kit (Ambion, Austin, TX, USA) for the T7-based linear amplification of mRNA. Synthesis and purification of cDNA and aRNA were carried out according to the manufacturer's instructions. The aRNA amplification procedure consisted of (i) first-strand cDNA synthesis (reverse transcription of mRNA), (ii) second-strand cDNA synthesis, (iii) cDNA purification, (iv) in vitro transcription, and (v) aRNA purification. The aRNA amplification for Day 7 blastocysts was done with two steps purification. The comparison between first and second round amplification was r = 0.96 (data not shown).For the series of present experiments we used a utero-placental custom cDNA microarray developed in our laboratory, as described previously ,15. BrieWe have previously reported our microarray hybridization procedures . Briefly) compliance was met by depositing all the data in the Gene Expression Omnibus (GEO) repository . The GEO accession numbers are as follows. Platform: GPL1221; Samples: GSM23324, GSM26510, GSM26511, GSM23327, GSM23328, GSM23329, GSM23330, GSM23331, and GSM23332; Series: GSE1414.Data normalization was performed by following the procedures described previously ,31. The 2 transformed and used for cluster analysis. The TIGR (The Institute for Genome Research) MultiExperiment Viewer (MeV) program  was used to derive the k-means and for hierarchical tree clustering analysis [k-means algorithm. Data for each gene were represented by an eight-dimensional vector. K-means clustering was performed by division into 12 centroid centers. The distance between gene vectors was calculated by the cosine coefficient (vector angle). Hierarchical tree clustering was performed on the annotated and EST genes that demonstrated up-regulation when the comparison was made between Day 28EEM vs. Day 28F. Genes that displayed two-fold (and greater) differences were selected and the normalized expression of each sample was utilized for the cluster analysis. The distance between gene vectors was also calculated by the cosine coefficient. We adopted average linkage clustering in the hierarchical tree clustering method. Data used for the cluster analyses were as follows: Day 21E ; Day 28EEM ; and Day 28F . Other samples were used independently for cluster analysis.Data for individual genes were obtained by averaging the intensity values of analogous spots on the microarray. Data were loganalysis . The genGene expression profiles derived from microarray analyses were confirmed quantitatively by real-time RT-PCR analysis. The selected genes for RT-PCR included two key known genes  and prolactin-related protein-1 (PRP-I)) and four apparently up-regulated ESTs, and compared their expression from Day 7 through Day 28. The ESTs selected have the GenBank accession numbers AW464053, AW465434, AW462349, and AW485575.2 and an RNase inhibitor. Primer pairs and oligonucleotide probes labeled with a reporter fluorescence dye at the 5' end and a quencher fluorescence dye at the 3' end were designed using Primer Express computer software (Applied Biosystems, Foster City, CA, USA). The primers and probes for the selected genes are listed in Table T) is related to the initial quantity of the target gene in each sample determined in real time using an ABI Prism 7700 sequence detector (Applied Biosystems). The relative difference in the initial amount of each mRNA species (or cDNA) was determined by comparing the CT values between the samples at different stages. Bovine GAPDH was used as endogenous control. The standard curve for each gene was generated by serial dilution of plasmid containing PL-Ala, PRP-I, four ESTs, or GAPDH cDNA to quantify mRNA concentrations. A ratio of PL-Ala, PRP-I and four ESTs mRNA to GAPDH mRNA was calculated to adjust for any variation in the RT-PCR reaction. All values are presented as mean ± SD. Data were analyzed initially by ANOVA and followed by either Tukey-Kramer multiple comparison test. P-values of <0.05 were considered significant.The details of real-time RT-PCR procedures have been described in previous reports ,33. BrieP < 0.05; data not shown) between the unamplified mRNA and amplified aRNA. Confirmation experiments were conducted twice with reverse labeling . Previous reports have also described substantial cDNA microarray population correlation between unamplified mRNA and amplified aRNA [We confirmed the reliability of this amplification method by comparing unamplified mRNA vs. amplified mRNA (aRNA) derived from the same bovine placental tissue after hybridization into the utero-placental cDNA microarray. We observed a significant correlation  increased only from Day 7 to Day 14 and thereafter either remained constant or decreased slightly (clusters 2, 3, 12); (ii) Continued to increase until Day 21 (clusters 1, 6, 7, 8, 9, and 10); and (iii) progressively increased until Day 21 (clusters 4, 5, and 11). Clusters 1 and 7 contained more than 200 genes that included annotated and ESTs, whereas clusters 4, 5, and 11 contained fewer than 100 genes. Clusters 5 and 11 contained mostly members of the cytokine family, and clusters 4 and 5 contained ECM-related genes. In particular, trophoblast-specific genes, such as placental lactogens (PLs), prolactin-related proteins (PRPs), and pregnancy-associated glycoproteins (PAGs), were found in cluster 11.Sixty-six genes out of a total of 1,773 were exempted from cluster analysis due to low expression intensity values. Consequently, 1,707 genes, including 833 previously annotated, were classified into 12 categories by  as supplemental tables .Pair-wise comparisons were made to identify genes displaying differential expression; the results are given in Fig.  were also strongly expressed. Other cytokine molecules, such as HDGF (NM_00494), had their expression accented. Cell-function-related genes such as integrin, mucin, ADAMTS, keratin, and cytokeratin were found to be significantly increased in their expression.A comparison between Day 7E and Day 14E clearly indicates that only 26 genes were down-regulated (14 annotated and 12 ESTs), with many genes (680) being up-regulated from Day 7 through Day 14E. The most significant genes during this period are listed in Table A total of 452 genes were significantly up-regulated from Day 14 through Day 21, and half of these (226 genes) were annotated by BLASTn  to interpret comprehensive genes expression, because they were frequently found in the list of significant genes and previous data had revealed their importance in early embryo development . Ten HSP which inhibits luteolysis and plays a role in embryonic survival by mediating embryo-maternal crosstalk [Intensive IFN-τ expression was found during the implantation period (Days 14E to 21E). The trophoblast elongates and grows into a thread-like structure during this period and produces IFN-rosstalk ,43,53-56rosstalk . It has rosstalk  and alsorosstalk . It is iCell-adhesion molecules and cytokines, such as the integrin family, have been emphasized their importance for initiation of implantation -62. Recek-means cluster 11 in the present study, whereas PAG-8 was distributed to k-means cluster 3  in Sus scrofa clone RP44-519O7 (AC096884). The AW485575 gene has 436 bp sequences and was found to be common for a slight 157/181 bp  in Homo sapiens hypothetical protein MGC39389 (BC003531). The AW464053 gene expression patterns were similar to the expression pattern of PRP-I, and the gene sequence has a portion in common with an e-value = 1 × 10-102 and 4 × 10-31 (common sequence = 326/371, 87% and 105/116, 90%) for PRP-VI (X59504), or an e-value = 1 × 10-95 (common sequence = 351/408, 86%) for PRP-III (M27240 or NM_174160). We suggest this EST was new member of bPRP.The most straightforward explanation for present study is that EST genes that display similar expression patterns are functionally related . AW46405This study provides developmental expression changes of a large number of genes in the bovine embryo during the peri-implantation period, with particular focus on genes that express in extra-embryonic membranes. The new gene candidates that were discussed here may address a new set of annotated genes and ESTs for embryonic differentiation and development. Participation of several known genes like Ezrin, CIB, Dickkopf-1, Grancalcin and EST (AW464053, AW465434 etc) in extra-embryonic membrane differentiation and development is elucidated by this microarray analysis. Fundamental investigations of this sort contribute significantly to a better understanding of the effects of various cultural conditions and cellular and genetic manipulation of embryos, including in vitro fertilization, in vitro maturation, and embryo cloning technology.Supplement tables list the whole genes which produced the expression difference in Fig. Click here for file


General dental practitioners provide the majority of endodontic treatment in Jordan. The aim of this study was to gather information on the methods, materials and attitudes employed in root canal treatment by dentists in North Jordan, in order to evaluate and improve the quality of current practice.A questionnaire was posted to all registered general dental practitioners working in private practice in Irbid Governate in North Jordan (n = 181). The questionnaire included information on methods, materials and techniques used in endodontic treatment.Reply rate was 72% (n = 131). The results demonstrated that only five dentists used rubber dam occasionally and not routinely. The majority used cotton rolls for isolation solely or in combination with a high volume saliva ejector (n = 116). The most widely used irrigants were sodium hypochlorite and hydrogen peroxide, which were used by 32.9% (n = 43) and 33.6% (n = 44) of the respondents, respectively. Forty eight percent of the respondents (n = 61) used the cold lateral condensation technique for canal obturation, 31.3% (n = 41) used single cone, 9.9% (n = 13) used vertical condensation and 12.2% (n = 16) used paste or cement only for the obturation. The majority used zinc oxide eugenol as a sealer (72.5%). All, but one, respondents used hand instruments for canal preparation and the technique of choice was step back (52.7%). More than 50% (n = 70) of the dentists took one radiograph for determining the working length, whilst 22.9% (n = 30) did not take any radiograph at all. Most practitioners performed treatment in three visits for teeth with two or more root canals, and in two visits for teeth with a single root canal.This study indicates that dentists practicing in North Jordan do not comply with international quality standards and do not use recently introduced techniques. Many clinicians never take a radiograph for determining the working length and never used rubber dam or intra-canal medicaments. Root canal treatment is considered an essential element in the dental services provided to the population in developed countries. Various investigations were, therefore, carried out to explore the standard of root canal treatment carried out by general dental practitioners in Europe -3.It is the responsibility of the academics and dental schools to prepare their students to adopt the guidelines and recommended standards in root canal debridement, shaping and obturation ,5. SeverThe majority of endodontic treatment in Jordan is provided by the general dental practitioners due to absence of specialists in endodontics and to the lack of postgraduate programs in Jordan. The purpose of the current study was, therefore, to investigate the attitude of dentists toward endodontic treatment and to explore the materials and methods employed by general dental practitioners in North Jordan and to compare these findings with well-acknowledged international academic standards.A postal survey of general dental practitioners in Jordan was carried out to investigate common materials and methods employed in root canal treatment. A questionnaire was developed and piloted by sending it to 20 general dental practitioners. According to the replies, the questionnaire was modified. Few questions were added and others were reworded. Additionally, the questionnaire was provided in Arabic and English language. The finally modified questionnaire was posted to all registered general dental practitioners (n = 181) listed in the records of the Jordanian Dental Association and working in private practices in Irbid Governate in North Jordan, 'Questionnaire see '. The qu2) test. The chosen level of significance was set at P < 0.05. Unanswered questions were treated as missing values.To investigate the influence of the years of practical experience on the materials and techniques employed, the sample was divided into groups based on the years of professional experience: group 1, up to 5 years; group 2, 6–10 years; group 3, 11–15 years; group 4, 16–20 years, and group 5, more than 20 years. The collected data were entered into a personal computer and analyzed using the statistical package SPSS. Simple descriptive statistics were used together with Chi-square .The distribution of the repondents according to the years of professional experience is shown in Table Table The majority of dentists instrumented the canal using the step back technique. The next most popular preparation technique was the filing (push-pull) technique followed by the step down technique Table .The vast majority used gutta-percha points as their priniciple root filling material (87.8%), whilst 12.2% reported using only paste or cement to obturate the canal. Cold lateral compaction was the most common obturation technique Table . The majIntracanal medication was used by 63% of the respondents. The most common material used was tricresol formalin followed by calcium hydroxide. Other formulations were also used Table .Sodium hypochlorite and hydrogen peroxide solutions were used equally as an irrigating solutions. The most popular concentration of sodium hypochlorite was 3% which was used by 14.5% (n = 19) of the repondents, with only 2.3% (n = 3) using a 0.5% concentration. The most commonly used concentration of hydrogen peroxide was 3%, which was used by 21.4% (n = 28) of the respondents. The remainder used either normal saline or local anesthetic solutions  or cotton rolls in combination with a high volume saliva ejector n = 116) to reduce contamination with saliva . All dentists reported using amalgam for posterior teeth and composite for anterior teeth as a permanent coronal restorative material. All practitioners completed the restorations themselves. Sixty four percent of the respondents preferred to wait from 1 to 2 weeks after obturation before placing the permanent coronal filling, whilst the remainder placed the restoration immediately after completion of the treatment.The response rate was 72%. It was higher than in many previous surveys conducted in Western countries with better communication infrastructure ,11.The vast majority of the respondents did not practice single visit root canal treatment. This finding was in agreement with the results of a previous study undertaken in another developing country, Sudan . HoweverAlthough the application of rubber dam is always recommended as a standard during root canal treatment procedure to provide isolation, protection and improve visual access, only five dentists reported using rubber dam very occasionally and not as a routine practice. Similar findings were found in Sudan (2%) and among Flemish dentists (3.4%) ,8. HowevIn the current survey, most general dental practitioners used hydrogen peroxide and sodium hypochlorite solutions as canal irrigants. The same result was demonstrated amongst dentists in Switzerland . Sodium The use of either sodium hypochlorite or hydrogen peroxide without isolating the field of operation tightly with a rubber dam presents an obviously hazardous practice in the use of potentially irritant irrigation solutions.Despite the fact that calcium hydroxide is recognized as the standard intracanal medicament for inter-appointment dressing , it was The step back technique was the most popular canal preparation technique among North Jordanian general dental practitioners. The filing (push-pull) technique, on the other hand, was used by 27.5% of the respondents. In another study, 60.4% of Flemish dentists used the standard filing technique . GeneralAlmost half of the general dental practioners in North Jordan used cold lateral compaction of gutta-percha to obturate the root canal space. This technique is acknowledged universally and is the most common obturation technique . HoweverThis study investigated the status of endodontic practice among general dental practitioners working in private offices in North Jordan. It demonstrated that dentists performed procedures which often deviated from well-acknowledged endodontic quality guidelines. Dentists did not use rubber dam for isolation and frequently use formaldehyde-containing materials for inter-appointments dressing. In addition, a significant proportion of dentists (n = 30) did not use radiographs at any stage of endodontic treatment. General practitioners did not seem to keep up with recently introduced techniques, but use more conventional methods.The North Jordanian general dental practitioners carried out endodontic treatment with few referals to specialists. However, the absence of postgraduate endodontic programs and continuing education courses in addition to economic restrictions could explain why dentists in Jordan do not carry out endodontic treatment in accordance with recognized international standards.None declared.The pre-publication history for this paper can be accessed here:Endodontic Survey Questionnaire text of the file contains questions related to endodontic practice among general dental practitioners.Click here for file


Thanks to the tools of molecular biology, our understanding of the 100-plus diseases known collectively as cancer has increased dramatically over the past decades. While each of these cancers exhibits unique characteristics reflecting the particular cell or tissue it springs from, the disease follows a similar arc in nearly all its forms. Cancer is a multistep disease that begins when genetic damage—initiated by a multitude of agents—unleashes a single cell from the normal constraints on cellular proliferation. This single transformed cell generates a colony of similarly abnormal progeny that can take decades to develop into malignancies.While events that stimulate uncontrolled cell division can promote cancer, mutations in tumor suppressor genes figure prominently in tumor progression. Disruptions in the pRb (retinoblastoma 1) tumor suppressor, for example, are often seen early in cancer development, sensitizing cells to tumorigenesis. pRb, along with other “pocket proteins”—so-called because they share an amino acid domain called the Rb pocket—regulate cell cycle progression, apoptosis (programmed cell death), and cellular differentiation. Some tumor suppressors, such as p53, can trigger apoptosis, ultimately sacrificing cells that have sustained DNA damage or other types of cellular stress.Mutations in both the pRb and p53 tumor suppressor pathways are commonly seen in human cancers, though their interactions appear to vary depending on the tissue. In mouse brain epithelial cells, for example, loss of p53 function coupled with loss of pRb results in reduced apoptosis and increased tumor growth, while p53 loss in mouse brain astrocytes (cells that support neurons) does not affect tumor growth. Building on this work, Terry Van Dyke and colleagues report that loss of the pRb tumor suppressor in mammary tissue has the same effect—predisposition to tumor formation—seen in these other cell types. Despite the different environment inherent in each cell type, the initial events following loss of the pRb pathway were the same: increased proliferation and apoptosis, followed by tumorigenesis. But, surprisingly, pRb and p53 interactions varied in different cell types.Like most cancers, mammary gland cancer has a long latency period, prompting the researchers to ask what events engineer tumor progression. To investigate the relative contribution of pRb and p53 in tumorigenesis, the researchers generated a novel mouse model with a dysfunctional pRb pathway and various levels of p53 function in several cell types. This is a significant achievement in itself, as many agents that inactivate the pRb pathway also disrupt the p53 pathway. pRb inactivation, they show, causes abnormalities in mammary cell proliferation, apoptosis, and tissue morphology. In these mammary-specific pRb-deficient mice, p53 was responsible for most of the apoptotic response—decreased levels of p53 resulted in reduced apoptosis and accelerated tumorigenesis, but had no effect on proliferation. Interestingly, in other mouse models where aberrant proliferation is caused by disabling other pathways, loss of p53 was associated with increased proliferation—rather than reduced apoptosis—and early tumor formation. And while p53 is the main effector of apoptosis in brain and mammary epithelial cells, this is not the case in all tissues: in astrocytes, for example, the tumor suppressor Pten regulates apoptosis in response to pRb inactivation. Together these results indicate that specific cellular responses to a cancer-causing stimulus vary depending on the nature of the initial genetic injury and the cell type and that pRb and p53 interact in different ways in different tissues. And p53, it appears, contributes to tumor suppression—and thus progression—through multiple mechanisms.BRCA1 and BRCA2 genes, to shed light on the complex series of events that ultimately cause breast cancer.By creating a mouse model that disentangles the pRb and p53 pathways, Van Dyke and colleagues have added a valuable resource for studying breast cancer. This model, they propose, will facilitate further investigations into the relative contributions of these overlapping pathways to cancer progression. What's more, the model offers a vehicle for examining how pRb interacts with other breast cancer mutations, like the inherited mutations in the human