AIM: To characterize oxidase- and urease-producing bacterial isolates, grown aerobically, that originated from antral biopsies of patients suffering from acid peptic diseases.
METHODS: A total of 258 antral biopsy specimens were subjected to isolation of bacteria followed by tests for oxidase and urease production, acid tolerance and aerobic growth. The selected isolates were further characterized by molecular techniques viz. amplifications for 16S rRNA using universal eubacterial and HSP60 gene specific primers. The amplicons were subjected to restriction analysis and partial sequencing. A phylogenetic tree was generated using unweighted pair group method with arithmetic mean (UPGMA) from evolutionary distance computed with bootstrap test of phylogeny. Assessment of acidity tolerance of bacteria isolated from antrum was performed using hydrochloric acid from 10-7 mol/L to 10-1 mol/L.
RESULTS: Of the 258 antral biopsy specimens collected from patients, 179 (69.4%) were positive for urease production by rapid urease test and 31% (80/258) yielded typical Helicobacter pylori (H. pylori) after 5-7 d of incubation under a microaerophilic environment. A total of 240 (93%) antral biopsies yielded homogeneous semi-translucent and small colonies after overnight incubation. The partial 16S rRNA sequences revealed that the isolates had 99% similarity with Pseudomonas species. A phylogenetic tree on the basis of 16S rRNA sequences denoted that JQ927226 and JQ927227 were likely to be related to Pseudomonas fluorescens (P. fluorescens). On the basis of HSP60 sequences applied to the UPGMA phylogenetic tree, it was observed that isolated strains in an aerobic environment were likely to be P. fluorescens, and HSP60 sequences had more discriminatory potential rather than 16S rRNA sequences. Interestingly, this bacterium was acid tolerant for hours at low pH. Further, a total of 250 (96.9%) genomic DNA samples of 258 biopsy specimens and DNA from 240 bacterial isolates were positive for the 613 bp amplicons by targeting P. fluorescens-specific conserved putative outer membrane protein gene sequences.
CONCLUSION: This study indicates that bacterial isolates from antral biopsies grown aerobically were P. fluorescens, and thus acid-tolerant bacteria other than H. pylori can also colonize the stomach and may be implicated in pathogenesis/protection.
Antral biopsy; Helicobacter pylori; Pseudomonas fluorescens; HSP60; Nested polymerase chain reaction; Acid-tolerant bacteria
Kartagener's syndrome is a rare, autosomal recessive genetic ciliary disorder comprising the triad of situs inversus, chronic sinusitis, and bronchiectasis. The basic problem lies in the defective movement of cilia, leading to recurrent chest infections, ear/nose/throat symptoms, and infertility. We hereby report three unusual cases of this rare entity – an infertile male with azoospermia in whom Bochdalek's diaphragmatic hernia coexisted, another case of an infertile female, and a third of an infertile male with oligospermia. The need for a high index of suspicion to make an early diagnosis cannot be overemphasized in such patients so that wherever possible, options for timely treatment of infertility may be offered and unnecessary evaluation of symptoms is avoided.
Bronchiectasis; Kartagener's syndrome; sinusitis; situs inversus
Two uncommon cases of locally invasive pulmonary inflammatory myofibroblastic tumor are reported. Diagnosis was established by a prior thoracotomy and incisional biopsy. Complete excision was curative and both children remain asymptomatic at last follow up.
Inflammatory myofibroblastic tumors; inflammatory pseudotumors; pulmonary tumors
Traditional analyses of calcium homeostasis have separately quantified either calcium accumulation or release mechanisms. To define the system as a whole, however, requires multiple experimental techniques to examine both accumulation and release. Here we describe a technique that couples the simultaneous quantification of radio-labeled calcium accumulation in endoplasmic reticulum (ER) microsomes with the release of inorganic phosphate (Pi) by the hydrolytic activity of sarco-endoplasmic reticulum calcium ATPase (SERCA) all in the convenience of a 96-well format.
Calcium; SERCA activity; Microsomes; Inorganic phosphate; Malachite green
Brain cells expend large amounts of energy sequestering calcium (Ca2+), while loss of Ca2+ compartmentalization leads to cell damage or death. Upon cell entry, glucose is converted to glucose-6-phosphate (G6P), a parent substrate to several metabolic major pathways, including glycolysis. In several tissues, G6P alters the ability of the endoplasmic reticulum (ER) to sequester Ca2+. This led to the hypothesis that G6P regulates Ca2+ accumulation by acting as an endogenous ligand for sarco-endoplasmic reticulum calcium ATPase (SERCA). Whole brain ER microsomes were pooled from adult male Sprague-Dawley rats. Using radio-isotopic assays, 45Ca2+ accumulation was quantified following incubation with increasing amounts of G6P, in the presence or absence of thapsigargin, a potent SERCA inhibitor. To qualitatively assess SERCA activity, the simultaneous release of inorganic phosphate (Pi) coupled with Ca2+ accumulation was quantified. Addition of G6P significantly and decreased Ca2+ accumulation in a dose-dependent fashion (1–10 mM). The reduction in Ca2+ accumulation was not significantly different that seen with addition of thapsigargin. Addition of glucose-1-phosphate or fructose-6-phosphate, or other glucose metabolic pathway intermediates, had no effect on Ca2+ accumulation. Further, the release of Pi was markedly decreased, indicating G6P-mediated SERCA inhibition as the responsible mechanism for reduced Ca2+ uptake. Simultaneous addition of thapsigargin and G6P did decrease inorganic phosphate in comparison to either treatment alone, which suggests that the two treatments have different mechanisms of action. Therefore, G6P may be a novel, endogenous regulator of SERCA activity. Additionally, pathological conditions observed during disease states that disrupt glucose homeostasis, may be attributable to Ca2+ dystasis caused by altered G6P regulation of SERCA activity.
glucose-6-phosphate; G6P; calcium; SERCA; microsome; endoplasmic reticulum; thapsigargin; brain
In vitro experiments have shown that protoporphyrin IX (PPIX) binds to the translocator protein 18 kD (TSPO), which transports cholesterol across the outer mitochondrial membrane. The purpose of this study was to examine whether binding of PPIX to TSPO can also be detected in vivo using positron emission tomography (PET) and [11C]PBR28, a radioligand that binds with high affinity and selectivity to TSPO. Rats were injected with a high dose of 5-aminolevulinic acid (ALA, 200 mg/kg i.v.), which is a precursor for PPIX. ALA-pretreatment significantly decreased the uptake of [11C]PBR28 in TSPO-rich organs such as heart, kidneys, lungs, parotid glands, and spleen by 57% to 80%. As a control experiment, injection of a receptor-saturating does of PK 11195, which is selective for TSPO, produced a pattern of displacement similar to that after ALA but with greater magnitude (88% to 97%). This study provides the first evidence that PPIX binds in vivo to TSPO. Although PPIX at physiological concentrations would likely occupy an insignificant percentage of TSPOs, it does reach high enough concentrations in porphyria to occupy and have pharmacological effects via this target.
Peripheral benzodiazepine receptor; 5-Aminolevulinic acid; porphyria
Glucose metabolism in nervous tissue has been proposed to occur in a compartmentalized manner with astrocytes contributing largely to glycolysis and neurons being the primary site of glucose oxidation. However, mammalian astrocytes and neurons both contain mitochondria and it remains unclear why in culture neurons oxidize glucose, lactate, and pyruvate to a much larger extent than astrocytes. The objective of this study was to determine whether pyruvate metabolism is differentially regulated in cultured neurons vs. astrocytes. Expression of all components of the pyruvate dehydrogenase complex (PDC), the rate-limiting step for pyruvate entry into the Krebs cycle, was determined in cultured astrocytes and neurons. In addition, regulation of PDC enzymatic activity in the two cell types via protein phosphorylation was examined. We show that all components of the PDC are expressed in both cell types in culture but that PDC activity is kept strongly inhibited in astrocytes through phosphorylation of the pyruvate dehydrogenase alpha subunit (PDHα). In contrast, neuronal PDC operates close to maximal levels with much lower levels of phosphorlyated PDHα. Dephosphorylation of astrocytic PDHα restores PDC activity and lowers lactate production. Our findings suggest that the glucose metabolism of astrocytes and neurons may be far more flexible than previously believed.
Glycolysis; oxidation; lactate; dichloroacetate
Costochondral graft (CCG) replacement of the mandibular condyle was first described by Gilles in 1920. Since then CCGs have gained increasing popularity in reconstruction of the TMJ and condyle in children. The influence of CCGs on mandibular growth and function is not known in detail. Adaptation of the graft has been observed to be better in children, but CCGs have also been shown to grow in adult patients. One of the major disadvantages of the CCGs is its growth pattern, which is extremely unpredictable and may manifest as excessive growth or no growth at all. A mandibular overgrowth on the grafted site can actually be more troublesome than lack of growth. Furthermore, maxillary growth is proportionality influenced by vertical mandibular growth of the graft. This is a report of such a case in which a bizarre overgrowth of the graft was seen following a reconstruction of TMJ by CCG and the devastating outcomes of the treatment. He required one further resection because the grafted tissue had overgrown five years later.
Costochondral graft; overgrowth; TMJ ankylosis
Survey on the prevalence of various intestinal parasitic infestations in different geographic regions is a prerequisite to obtain an accurate understanding of the burden and cause of intestinal parasitic infestations in a particular area. The aim of the present study was to determine the intestinal parasitic infestation among children in a semi-urban area.
Materials and Methods:
A total of 335 stool samples were collected, processed, and microscopically examined for intestinal parasites.
One hundred twenty-eight (38%) stool samples showed presence of ova/cysts. Multiple parasites were seen in 42 (32.8%) samples. Among the protozoans, Entamoeba histolytica (55.3%) was the most common followed by Giardia lamblia (40.4%). Ascaris lumbricoides and Hymenolepis nana (24.2%) were the most common helminths detected.
In most of the cases, intestinal parasitic infestation spreads due to low standards of personal hygiene, poor sanitation, non-usage of toilets and an illiterate population, thus suggesting regular surveys to help in devising optimum methods of control.
Diarrhea; intestinal parasites; semi-urban population
The rapidly growing availability of diverse full genome sequences from across the world is increasing the feasibility of studying the large-scale population processes that underly observable pattern of virus diversity. In particular, characterizing the genetic structure of virus populations could potentially reveal much about how factors such as geographical distributions, host ranges and gene flow between populations combine to produce the discontinuous patterns of genetic diversity that we perceive as distinct virus species. Among the richest and most diverse full genome datasets that are available is that for the dicotyledonous plant infecting genus, Begomovirus, in the Family Geminiviridae. The begomoviruses all share the same whitefly vector, are highly recombinogenic and are distributed throughout tropical and subtropical regions where they seriously threaten the food security of the world's poorest people.
We focus here on using a model-based population genetic approach to identify the genetically distinct sub-populations within the global begomovirus meta-population. We demonstrate the existence of at least seven major sub-populations that can further be sub-divided into as many as thirty four significantly differentiated and genetically cohesive minor sub-populations. Using the population structure framework revealed in the present study, we further explored the extent of gene flow and recombination between genetic populations.
Although geographical barriers are apparently the most significant underlying cause of the seven major population sub-divisions, within the framework of these sub-divisions, we explore patterns of gene flow to reveal that both host range differences and genetic barriers to recombination have probably been major contributors to the minor population sub-divisions that we have identified. We believe that the global Begomovirus population structure revealed here could facilitate population genetics studies into how central parameters of population genetics namely selection, recombination, mutation, gene flow, and genetic drift shape the global begomovirus diversity.
In the treatment of tuberculosis there are special therapeutic problems related to adverse effects of drugs, compliance to treatment, and microbial resistance. Thrombocytopenia is an uncommon but potentially fatal adverse effect of certain anti-tubercular drugs when the incriminating drug is taken by a susceptible individual. We report a case of rifampicin-induced thrombocytopenia, which although rare, needs attention.
Rifampicin; thrombocytopenia; tuberculosis
Mitochondrial mutations have been identified in head and neck squamous cell carcinoma (HNSCC), but the pathways by which phenotypic effects of these mutations are exerted remain unclear. Previously, we found that mitochondrial ND2 mutations in primary HNSCC increased reactive oxygen species (ROS) and conferred an aerobic, glycolytic phenotype with HIF1α accumulation and increased cell growth. The purpose of present study was to examine the pathways relating these alterations.
Mitochondrial mutant and wild-type ND2 constructs were transfected into oral keratinocyte immortal cell line OKF6 and head and neck cancer cell line JHU-O19 and established transfectants. The protein levels of HIF1α, pyruvate dehydrogenease (PDH), phospho-PDH, and pyruvate dehydrogenease kinase (PDK) 2, together with ROS generation, were compared between the mutant and wild type. Meanwhile, the effects of small molecule inhibitors targeting PDK2, and mitochondrial targeted catalase, were evaluated on the ND2 mutant transfectants.
We determined that ND2 mutant downregulated PDH expression via upregulated PDK2, with an increase in phospho-PDH. Inhibition of PDK2 with dichloroacetate decreased HIF1α accumulation and reduced cell growth. Extracellular treatment with hydrogen peroxide, a ROS mimic, increased PDK2 expression and HIF1α expression, and introduction of mitochondrial targeted catalase decreased mitochondrial mutation mediated PDK2 and HIF1α expression and suppressed cell growth.
Our findings suggest that mitochondrial ND2 mutation contributes to HIF1α accumulation via increased ROS production, upregulation of PDK2, attenuating PDH activity, thereby increasing pyruvate, resulting in HIF1α stabilization. This may provide insight into a potential mechanism by which mitochondrial mutations contribute to HNSCC development.
Mitochondrial mutation; Head and neck cancer; HIF1α; Glycolysis; Pyruvate; Reactive oxygen species; PDK2
A number of postmortem studies have found decreased pH in brains of patients with schizophrenia. Insofar as lower pH has been associated with decreased mRNA expression in postmortem human brain, decreased pH in schizophrenia may represent an important potential confound in comparisons between patients and controls. We hypothesized that decreased pH may be related to increased concentration of lactic acid. However, in contrast to the previous notion that an increase in lactic acid represents evidence for primary metabolic abnormalities in schizophrenia, we hypothesized that this increase is secondary to prior antipsychotic treatment. We have tested this by first demonstrating that lactate levels in the cerebellum of patients with schizophrenia (n=35) are increased relative to control subjects (n=42) by 28%, p=0.001. Second, we have shown that there is an excellent correlation between lactate levels in the cerebellum and pH, and that this correlation is particularly strong in patients (r=− 0.78, p=3e-6). Third, we have shown in rats that chronic haloperidol (0.8 mg/kg/day) and clozapine (5 mg/kg/day) increase lactic acid concentration in the frontal cortex relative to vehicle (by 31% and 22% respectively, p<0.01). These data suggest that lactate increases in postmortem human brain of patients with schizophrenia are associated with decreased pH and that these changes are possibly related to antipsychotic treatment rather than a primary metabolic abnormality in the prefrontal cortex of patients with schizophrenia.
Life at altitude depends on adaptation to ambient hypoxia. In the Andes, susceptibility to chronic mountain sickness (CMS), a clinical condition that occurs to native highlanders or to sea level natives with prolonged residence at high altitude, remains poorly understood. We hypothesized that hypoxia-associated gene expression in children of men with CMS might identify markers that predict the development of CMS in adults. We assessed distinct patterns of gene expression of hypoxia-responsive genes in children of highland Andean men, with and without CMS.
We compared molecular signatures in children of highland (HA) men with CMS (n = 10), without CMS (n = 10) and in sea level (SL) children (n = 20). Haemoglobin, haematocrit, and oxygen saturation were measured. Gene expression in white cells was assessed at HA and then, in the same subjects, within one hour of arrival at sea level.
HA children showed higher expression levels of genes regulated by HIF (hypoxia inducible factor) and lower levels of those involved in glycolysis and in the tricarboxilic acid (TCA) cycle. Pyruvate dehydrogenase kinase 1(PDK1) and HIF prolyl hydroxylase 3 (HPH3) mRNA expressions were lowest in children of CMS fathers at altitude. At sea level the pattern of gene expression in the 3 children's groups was indistinguishable.
The molecular signatures of children of CMS patients show impaired adaptation to hypoxia. At altitude children of CMS fathers had defective coupling between glycolysis and mitochondria TCA cycle, which may be a key mechanism/biomarker for adult CMS. Early biologic markers of disease susceptibility in Andeans might impact health services and social planning.
The study of the biology of evolution has been confined to laboratories and model organisms. However, controlled laboratory conditions are unlikely to model variations in environments that influence selection in wild populations. Thus, the study of “fitness” for survival and the genetics that influence this are best carried out in the field and in matching environments.
Therefore, we studied highland populations in their native environments, to learn how they cope with ambient hypoxia. The Andeans, African highlanders and Himalayans have adapted differently to their hostile environment.
Chronic mountain sickness (CMS), a loss of adaptation to altitude, is common in the Andes, occasionally found in the Himalayas; and absent from the East African altitude plateau.
We compared molecular signatures (distinct patterns of gene expression) of hypoxia-related genes, in white blood cells (WBC) from Andeans with (n = 10), without CMS (n = 10) and sea-level controls from Lima (n = 20) with those obtained from CMS (n = 8) and controls (n = 5) Ladakhi subjects from the Tibetan altitude plateau. We further analyzed the expression of a subset of these genes in Ethiopian highlanders (n = 8). In all subjects, we performed the studies at their native altitude and after they were rendered normoxic.
We identified a gene that predicted CMS in Andeans and Himalayans (PDP2). After achieving normoxia, WBC gene expression still distinguished Andean and Himalayan CMS subjects.
Remarkably, analysis of the small subset of genes (n = 8) studied in all 3 highland populations showed normoxia induced gene expression changes in Andeans, but not in Ethiopians nor Himalayan controls. This is consistent with physiologic studies in which Ethiopians and Himalayans show a lack of responsiveness to hypoxia of the cerebral circulation and of the hypoxic ventilatory drive, and with the absence of CMS on the East African altitude plateau.
Erythropoietin (Epo) is used for managing anemia in cancer patients. However, recent studies have raised concerns for this practice. We investigated the expression and function of Epo and the erythropoietin receptor (EpoR) in tumor biopsies and cell lines from human head and neck cancer. Epo responsiveness of the cell lines was assessed by Epoetin-α-induced tyrosine phosphorylation of the Janus kinase 2 (JAK2) protein kinase. Transmigration assays across Matrigel-coated filters were used to examine the effects of Epoetin-α on cell invasiveness. In 32 biopsies, we observed a significant association between disease progression and expression of Epo and its receptor, EpoR. Expression was highest in malignant cells, particularly within hypoxic and infiltrating tumor regions. Although both Epo and EpoR were expressed in human head and neck carcinoma cell lines, only EpoR was upregulated by hypoxia. Epoetin-α treatment induced prominent JAK2 phosphorylation and enhanced cell invasion. Inhibition of JAK2 phosphorylation reduced both basal and Epo-induced invasiveness. Our findings support a role for autocrine or paracrine Epo signaling in the malignant progression and local invasiveness of head and neck cancer. This mechanism may also be activated by recombinant Epo therapy and could potentially produce detrimental effects in rhEpo-treated cancer patients.
Erythropoietin; HIF; cancer; invasion; hypoxia