Biomedical subjects

S Ghosh

Publications and source records attributed to S Ghosh.

At least 271 records · Page 15Linked to original sources

PubMed2000

Cloning and characterization of the gene encoding mouse IkappaBbeta.

IkappaBbeta is a member of the IkappaB family of structurally related proteins that are important regulators of the inducible transcription factor NF-kappaB. In the present study, the mouse IkappaBbeta gene was cloned and sequenced, and its structure determined. The mouse IkappaBbeta gene contains six exons and five introns that span 7435 nucleotides. A single major transcription initiation site is located 59 nucleotides upstream of the initiating methionine. A translation termination codon and a single polyadenylation signal are found within exon 6. The exon/intron structure of IkappaBbeta and IkappaBalpha genes were compared and found to be very similar, with individual ankyrin repeats being maintained on corresponding exons. This suggests a close evolutionary relationship between these two IkappaB isoforms. The significance of this evolutionary relationship is discussed.

Amino Acid Sequence↗

PubMed2000

Transcriptional activation of the cyclooxygenase-2 gene in endotoxin-treated RAW 264.7 macrophages.

Cyclooxygenase-2 (COX-2), the enzyme primarily responsible for induced prostaglandin synthesis, is an immediate early gene induced by endotoxin in macrophages. We investigated the cis-acting elements of the COX-2 5'-flanking sequence, the transcription factors and signaling pathways responsible for transcriptional activation of the COX-2 gene in endotoxin-treated murine RAW 264.7 macrophages. Luciferase reporter constructs with alterations in presumptive cis-acting transcriptional regulatory elements demonstrate that the cyclic AMP-response element and two nuclear factor interleukin-6 (CCAAT/enhancer-binding protein (C/EBP)) sites of the COX-2 promoter are required for optimal endotoxin-dependent induction. In contrast, the E-box and NF-kappaB sites are not required for endotoxin-dependent induction. Inhibition of endotoxin-induced NF-kappaB activation by expression of an inhibitor-kappaB alpha mutant does not block endotoxin-dependent COX-2 reporter activity. Overexpression of c-Jun, C/EBPbeta, and C/EBPdelta enhances induction of the COX-2 reporter, while overexpression of cyclic AMP-response element-binding protein or "dominant negative" C/EBPbeta represses COX-2 induction. In addition, endotoxin rapidly and transiently elicits c-Jun phosphorylation in RAW 264.7 macrophages. Cotransfection of the COX-2 reporter with dominant negative expression vectors shows that endotoxin-induced COX-2 gene expression requires signaling through a Ras-independent pathway involving the adapter protein ECSIT and the signaling kinases MEKK1 and JNK. In contrast, endotoxin-induced COX-2 reporter activity is not blocked by overexpression of dominant-negative forms of Raf-1, ERK1, or ERK2.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Plasmid curing from an acidophilic bacterium of the genus Acidocella.

Preservation of the acidophilic heterotroph, Acidocella sp. strain GS19h, at 4 degrees C in stab culture eliminated all indigenous plasmids from this bacterium. Growth at 42 degrees C initially caused changes in the plasmid profile followed by total elimination of plasmids after 10 cycles of growth. Concomitant to this loss of all plasmids, the cured derivatives became sensitive to CdSO(4) and ZnSO(4), and the MIC value of the salts dropped from 1 M for each in the case of parental strain to 2 mM and 5 mM, respectively, suggesting plasmid-mediated inheritance of metal resistance in this bacterium. The cured derivatives could not utilise lactose, indicating this metabolic activity to be plasmid-associated in this strain.

Bacteria↗

PubMed2000

A subclass of Ras proteins that regulate the degradation of IkappaB.

Small guanosine triphosphatases, typified by the mammalian Ras proteins, play major roles in the regulation of numerous cellular pathways. A subclass of evolutionarily conserved Ras-like proteins was identified, members of which differ from other Ras proteins in containing amino acids at positions 12 and 61 that are similar to those present in the oncogenic forms of Ras. These proteins, kappaB-Ras1 and kappaB-Ras2, interact with the PEST domains of IkappaBalpha and IkappaBbeta [inhibitors of the transcription factor nuclear factor kappa B (NF-kappaB)] and decrease their rate of degradation. In cells, kappaB-Ras proteins are associated only with NF-kappaB:IkappaBbeta complexes and therefore may provide an explanation for the slower rate of degradation of IkappaBbeta compared with IkappaBalpha.

Adenosine Triphosphate↗

PubMed2000

Stereoselective preparation of enantiomerically pure annulated carbohydrates using ring-closing metathesis

Ring-closing metathesis has been applied to a series of glucose derivatives to produce cyclopentene derivatives 5a and 5b, cyclohexene derivatives 8 and 9, cycloheptene 12, and cyclooctene 14. Spirocyclic dihydrofurans 19, 26a, and 26b, along with dihydropyran 22, were also produced. A range of fused oxepine derivatives 29a-c and one oxo-cyclononene 31 were also prepared. Cyclopentene 5b was subjected to a sequence of hydrogenation, NBS bromination, and treatment with powdered zinc to furnish the ring-expanded product 35. No such ring expansion occurred when the cyclohexaannulated compound 8 was treated with NBS followed by powdered zinc, leading to aldehyde 39. The spiro dihydrofuran derivative 19 was converted to the aldehyde 42 via the same reaction sequence used to fragment cyclopentene derivative 5b.

Journal Article↗

PubMed2000

Cholesteryl ester hydrolase in human monocyte/macrophage: cloning, sequencing, and expression of full-length cDNA.

The sensitive technique of RT-PCR was used to identify cholesteryl ester hydrolase (CEH) expressed in human macrophages. This enzyme is thought to regulate the availability of intracellular free cholesterol for efflux. The expected 667-bp product was obtained starting with RNA from human peripheral blood and THP-1 monocytes and macrophages. The cDNA for human macrophage CEH was then cloned by PCR-based screening of a lambda-gt11 cDNA library. The full-length cDNA was sequenced and found to exhibit 76% homology (at the nucleotide and conceptually translated protein level) to hepatic CEH, an enzyme shown to be involved in hepatic cholesterol homeostasis and regulated by cholesterol at the transcription level via sterol response elements in the proximal promoter. Identification of the conserved catalytic triad (Ser(221 , His(468), and Glu354)) and the SEDCLY motif places human macrophage CEH in the family of carboxylesterases. A greater than 20-fold increase in CEH activity was observed when COS-1 and COS-7 cells were transiently transfected with an eukaryotic expression vector, pcDNA3.1/V5/His-TOPO, containing the cDNA for human macrophage CEH. Using this full-length cDNA as a probe, a 2.2-kb transcript was identified by Northern blot analysis of total RNA from human peripheral blood and THP -1 macrophages. Overexpression of human macrophage CEH resulted in an impairment of upregulation of low-density lipoprotein (LDL) receptor mRNA in Chinese hamster ovary (CHO-K1) cells grown in cholesterol-deficient environment. These data identify the human macrophage CEH, demonstrate its expression in human peripheral blood macrophage and human macrophage cell line, THP-1, and suggest its role in the intracellular cholesteryl ester metabolism.

Amino Acid Sequence↗

PubMed2000

Preconditioning the human myocardium by simulated ischemia: studies on the early and delayed protection.

BACKGROUND: There are data supporting the existence of ischemic preconditioning in man. This study investigated the most effective preconditioning protocol for the human myocardium and whether the second window of ischemic preconditioning (24 h) is as protective as the first window (< or = 2 h). METHODS AND RESULTS: Right atrial appendages (n = 6/group) obtained during coronary bypass surgery were prepared and superfused with normoxic and normothermic Krebs-Henseleit solution. After 30 min stabilisation, muscles were subjected to various preconditioning protocols followed by 90 min ischemia and 120 min reperfusion. At the end of each protocol, the leakage of creatinine kinase (CK, U/g wet wt) and the reduction of MTT to insoluble formazan dye (OD/mg wet wt), an index of cell viability, were measured. In study 1, preconditioning was induced by 2, 3, 5 and 10 min of ischemia followed by 5 min reperfusion. In study 2, 1-4 cycles of 2 or 5 min ischemia-5 min reperfusion were applied. In study 3, preconditioning was induced by 5 min ischemia-5 min reperfusion followed by 1, 2, 3 or 4 h reperfusion before the subsequent 90 min ischemia. In study 4, preconditioning with 5 min ischemia followed by 5 min reperfusion either immediately preceded 30 or 90 min ischemia/120 min reperfusion or was applied 24 h before. In study 1 and 2, optimal protection was achieved with 5 min or two cycles of 2 min preconditioning ischemia (CK = 3.06 +/- 0.31 and 2.89 +/- 0.02; MTT = 0.56 +/- 0.05 and 0.47 +/- 0.09, respectively vs. CK = 5.56 +/- 0.52 and MTT = 0.18 +/- 0.04 in ischemia alone group; P < 0.05). In study 3, protection was observed 2 h after preconditioning (CK = 3.43 +/- 0.22 and MTT = 0.46 +/- 0.09; P < 0.01 vs. ischemia alone group) but it was lost beyond 2 h (CK = 6.30 +/- 0.56 and MTT = 0.16 +/- 0.02 after 3 h; P = NS vs. ischemia alone group). In study 4, protection was observed 24 h following preconditioning when the atrial specimens were exposed to 30 min ischemia (CK = 2.96 +/- 0.38 and MTT = 0.61 +/- 0.01 vs. CK = 4.56 +/- 0.26 and MTT = 0.43 +/- 0.02 in ischemia alone group, P < 0.05); however, when the period of ischemia was extended to 90 min the beneficial effect of preconditioning was lost (CK = 10.28 +/- 0.05 and MTT = 0.11 +/- 0.05 vs. CK = 9.56 +/- 0.62 and MTT = 0.104 +/- 0.05 in ischemia alone group, P = NS). CONCLUSIONS: In the isolated human myocardium maximal protection induced by preconditioning is achieved by a total 4-5 min ischemic stimulus, an effect that is lost beyond 2 h of its application. Two windows of protection were identified, the first (< or = 2 h) being more potent than the second (24 h).

Analysis of Variance↗

PubMed2000

A development-specific histone H3 localizes to the developing macronucleus of Euplotes.

During the process of macronuclear development, the ciliate Euplotes crassus undergoes extensive programmed DNA rearrangement. Previous studies have identified a gene, H3(P), that is expressed only during sexual reproduction and is predicted to encode a variant histone H3 protein. In the current study, an antiserum to the H3(P) protein has been generated. The antiserum has been used to demonstrate that H3(P) is maximally expressed during the polytene chromosome stage of macronuclear development. Moreover, H3(P) is localized to the developing macronucleus, but not other nuclei present within the cell. Additional studies indicate that at least one additional variant histone is also present within the developing macronucleus. The results indicate that there are significant changes in nucleosome composition within the developing macronucleus, and provide additional support for the notion that changes in chromatin structure play a role in the DNA rearrangement processes of macronuclear development. genesis 26:179-188, 2000.

Amino Acid Sequence↗

PubMed2000

Further evidence of the absence of measles virus genome sequence in full thickness intestinal specimens from patients with Crohn's disease.

Specimens of macroscopically inflamed and normal intestine along with mesenteric lymph nodes were obtained at resection from patients with Crohn's disease. The samples were systematically examined by RT-PCR-nested PCR targeting N, M and H gene regions of the measles virus genome. None of the samples examined gave any evidence of the persistence of measles virus in the intestine of Crohn's disease patients. The study supports previous findings produced by this laboratory and others using highly sensitive measles virus specific PCR diagnostic technology.

Adult↗

PubMed2000

Diverse patterns of expression of the 67-kD laminin receptor in human small intestinal mucosa: potential binding sites for prion proteins?

It has been shown that the 67-kD laminin receptor (LR) may function as a receptor for Sindbis and tick-born encephalitis viruses. Recent data indicate that the 37-kD precursor (LRP) for this molecule acts as a receptor for prion proteins (PrP), self-proteins implicated in the pathogenesis of transmissible spongiform encephalopathies including new variant Creutzfeldt-Jakob disease (nvCJD). Laminin and PrP share the same binding site on LRP, which is incorporated into the mature LR as a functional binding domain. To localize PrP binding sites potentially relevant to oral infection, the expression of the LR in human small intestinal mucosa was studied. Expression of the LR was determined by immunohistochemistry in duodenal and jejunal biopsies using a monoclonal antibody (MLuC5) which specifically recognizes the 67-kD LR. Biopsy material was obtained from 39 control patients, 15 patients with ulcerative colitis, 15 patients with Crohn's disease and uninvolved small bowel, and 28 patients with active coeliac disease. Two distinctive patterns of LR expression were found within each group of patients. One pattern was characterized by LR expression in the brush border and Golgi apparatus region of villus and crypt enterocytes. Paneth cell secretory granules were positive for LR in these samples. Brush border expression of LR was found in approximately 40% of samples, with the exception of Crohn's disease (6.7% of samples were positive). Another pattern of LR expression was characterized by positively stained endothelium, while the epithelium was generally negative (45 of 97). The use of two polyclonal antibodies which recognize both the LRP and the LR confirmed brush border and paranuclear expression of the LR, but also showed varying cytoplasmic and apical surface immunoreactivity in MLuC5-negative epithelium, reflecting the distribution of LRP as opposed to the mature receptor. In conclusion, expression of the LR in the brush border and in Paneth cell secretory granules suggests that this molecule might be involved in both secretory and endocytotic functions. The major implication of intestinal epithelial/brush border expression of the LR may be an increased susceptibility to oral infection with prion proteins.

Adult↗

PubMed2000

Mapping a quantitative trait locus via the EM algorithm and Bayesian classification.

Mapping a locus controlling a quantitative genetic trait (e.g., blood pressure) to a specific genomic region is of considerable interest. Data on the quantitative trait under consideration and several codominant genetic markers with known genomic locations are collected from members of families and statistically analyzed to draw inferences on the genomic position of the trait locus. The vector of parameters of interest comprises the pairwise recombination fractions, theta, between the putative quantitative trait locus and the marker loci. One of the major complications in estimating theta for a quantitative trait in humans is the lack of haplotype information on members of families. The purpose of this study was to devise a computationally simple and efficient method of estimation of theta in the absence of haplotype information. We have proposed a two-stage estimation procedure using the expectation-maximization (EM) algorithm. In the first stage, parameters of the QTL are estimated based on data of a sample of unrelated individuals. From estimates thus obtained, we have used a Bayes' rule to infer QTL genotypes of parents in families. Finally, in the second stage of the procedure, we have proposed an EM algorithm for obtaining the maximum likelihood estimate of theta based on data of informative families (which are identified upon inferring parental QTL genotypes performed in the first stage). We have shown, using simulated data, that the proposed procedure is cost-effective, computationally simple, and statistically efficient. As expected, analysis of data on multiple markers jointly is more efficient than the analysis based on single markers.

Algorithms↗

PubMed2000

PCR detection of Giardia lamblia in stool: targeting intergenic spacer region of multicopy rRNA gene.

A PCR based detection that amplifies the 552-bp intergenic spacer (IGS) region of multicopy rRNA gene of Giardia lamblia and 320-bp internal sequences to first PCR product has been used in diagnosis of giardiasis in stool sample. The primers were found highly specific to Giardia spp. only, because no amplification was observed with DNAs from other enteric pathogens like Escherichia coli, Shigella dysenteriae and Entamoeba histolytica. The test could detect even less than 2 pg of genomic DNA from Giardia trophozoites. In direct diagnosis of Giardia lamblia in stool samples, it was observed that PCR amplification of IGS followed by nested PCR could enhance the sensitivity and specificity of the tests manifold and the system was able to detect as low as 10 parasites in 100 microl of stool. The comparative evaluation of the present system with conventional microscopy, CIEP and ELISA in the diagnosis of giardiasis from diarrhoeic stool samples and control subjects demonstrated a 100% correlation among nested PCR, microscopic examination and ELISA in patients suggestive of giardiasis (Group I) and control subjects (Group II). In Group I cases (patients suffering from other than giardiasis), CIEP, ELISA and nested PCR showed better results than microscopic examination. However, among them, PCR was found most sensitive and specific because 20% positivity was noticed by PCR whereas CIEP and ELISA showed only 7.14% and 12.85%, respectively. Break-up results showed that all the samples which were positive by CIEP or ELISA, also found positive by PCR. The present observation clearly suggests the use of PCR that amplifies the intergenic spacer region of multicopy rRNA gene of Giardia lamblia followed by nested PCR for routine, quick and reliable detection of Giardia lamblia in stool samples.

Animals↗

PubMed2000

Depression of neuronal firing rates in somatosensory and posterior parietal cortex during object acquisition in a prehension task.

Prehension is an object-oriented behavior consisting of four components: reach, grasp, manipulation, and release. To determine how such actions are represented in primary somatosensory (S-I) and posterior parietal cortex (PPC), we used digital video to synchronize spike trains of neurons recorded in Brodmann's areas 3b, 1, 2, 5, and 7 with the hand kinematics as monkeys performed a prehension task. Statistical analyses indicated that one-third of task-modulated neurons showed significantly depressed firing rates during object acquisition and/or manipulation. This population was dominated by neurons innervated by deep receptors that sensed extension movements of the fingers, or by tactile receptors in hairy skin sensing stretch. Grasp-inhibited responses were the most common type. Tonic firing rates of these cells dropped significantly during approach as the hand was preshaped for grasping, or at contact when grasp was initiated, and persisted until hand motion ceased or as the grip relaxed. Maximum suppression of firing occurred at grasp completion. Their lack of specificity for particular hand behaviors formed the inhibitory counterpart of broadly tuned cells that fired prolonged bursts during grasp and manipulatory stages of prehension. The remainder of the task-inhibited population showed biphasic responses. Firing rates were significantly depressed during grasping and manipulation when the hand interacted directly with the object, but were enhanced prior to contact, when the hand was preshaped (approach-tuned), or upon relaxation of grasp and release of the object from the hand (loweror relax-tuned). Grasp-inhibited responses occurred primarily in S-I, whereas biphasic inhibitory activity was recorded mainly in PPC. Suppression of activity within these populations may thereby increase the saliency of excitatory responses to acquisition and manipulation of objects. Reduction of firing during prehension might also signal the flexed postures used to retain objects in the hand, rather than a generalized gating of sensory information. The similarity of responses to active and passive extension movements suggests that the inhibitory responses may provide important postural and motor information about the hand kinematics when performing skilled tasks.

Action Potentials↗

PubMed2000

Cardiotrophin-1 protects the human myocardium from ischemic injury. Comparison with the first and second window of protection by ischemic preconditioning.

BACKGROUND: There are reports suggesting that cardiotrophin 1 (CT-1) is cytoprotective. We investigated the cardioprotective effects of CT-1 on the human myocardium and compared this benefit with the early and delayed protection afforded by ischemic preconditioning (PC). METHODS: Right atrium specimens were prepared and incubated in buffer solution at 37 degrees C for 30 min stabilisation, before entering one of the three following studies. In study 1, muscles (n=6/group) were allocated to one of four groups: (i) aerobic control - incubated in oxygenated media for 210 min, (ii) ischemia alone - 90 min ischemia followed by 120 min reoxygenation, (iii) PC by 5 min ischemia-5 min reoxygenation before 90 min ischemia-120 min reoxygenation and (iv) CT-1 (1 nM) - 90 min ischemia-120 min reoxygenation with exposure to CT-1 throughout the protocol. In study 2, muscles (n=6/group) were allocated to one of four protocols as in study 1with the exception that were incubated for 24 h followed by 30 or 90 min ischemia-120 min reoxygenation on day 2. In study 3, the same groups were employed as in study 2 with the exception that only a 30-min period of ischemia was used and that CT-1 antibody (5 microg/ml) was added to all groups throughout the experimental protocol. Creatine kinase (CK, U/g wet wt.) leakage into the medium and MTT reduction (OD/mg wet wt.), an index of cell viability, were assessed at the end of the experiment. RESULTS: In study 1, a first window of cardioprotection was observed with PC (CK=4.39+/-0.34; MTT=0.58+/-0.03 vs. CK=7.11+/-0.4;MTT=0.32+/-0.02 in the ischemic alone group; P<0.001) but not with CT-1(CK=6.65+/-0. 67; MTT=0.31+/-0.03, P=NS vs. ischemia alone). In study 2, PC applied on day 1 was protective against 30-min ischemia (CK=3.28+/-0. 43; MTT=0.68+/-0.046, P<0.001 vs. ischemia alone) but not against 90-min ischemia (CK=7.13+/-0.66; MTT=0.24+/-0.03, P=NS vs. ischemia alone) induced on day 2 (second window). However, when the tissue was exposed to CT-1 for 24 h, protection was similar to that of PC when subjected to 30 min of ischemia (CK=2.95+/-0.71; MTT=0.77+/-0. 05, P=NS vs. PC) and greater than PC when subjected to 90 min of ischemia (CK=4.56+/-0.51; MTT=0.39+/-0.03, P=0.002 vs. PC). In study 3, the CT-1 antibody did not affect the protection induced by PC (CK=3.36+/-0.6; MTT=0.69+/-0.06) but it abolished the protection obtained with CT-1(CK=5.15+/-0.81; MTT=0.42+/-0.06, P=NS vs. ischemia alone group). CONCLUSIONS: CT-1 exhibits a significant protection of the human myocardium against ischemic injury when tissue is exposed to this factor for a long period (e.g. 24 h) but not when exposed for a short period (e.g. 2 h). In addition, the protection afforded by long exposure to CT-1 is as potent or even greater than the one obtained by the second window of PC. The protection induced by CT-1 but not that induced by PC can be abolished by CT-1 antibody suggesting that their beneficial action is attained by different mechanisms.

Adult↗

PubMed2000

Evidence for mitochondrial K ATP channels as effectors of human myocardial preconditioning.

BACKGROUND: Sublethal periods of ischemia preceding a prolonged interval of ischaemia protect the myocardium. This myocardial preconditioning (PC) appears to be effected by KATP channels. These channels occur both in the sarcolemma and the mitochondrial membrane. We investigated whether mitochondrial KATP channels are the end-effector of PC in the human myocardium. METHODS: Right atrium specimens obtained from patients undergoing cardiac surgery were prepared and incubated in buffer solution at 37 degrees C. After 30-min stabilisation, the muscles were made ischemic for 90 min and then reperfused for 120 min. The preparations were randomised into eight experimental groups (n = 6/group): (1) Aerobic control--incubated in oxygenated buffer for 210 min, (2) ischemia alone--90 min ischemia followed by 120 min reperfusion, (3) PC--preconditioned with 5 min ischemia/5 min reperfusion, (4) Glibenclamide (10 microM) in the incubation media for 10 min before PC, (5) 5-hydroxydecanoate (5-HD, MitoKATP blocker, 1 mM) in the incubation media for 10 min before PC, (6) HMR 1883 (SarcKATP blocker, 10 microM) in the incubation media for 10 min before PC, (7) Pinacidil (0.5 mM) in the incubation media for 10 min before ischemia, and (8) Diazoxide (MitoKATP opener, 0.1 mM) in the incubation media for 10 min before ischemia. Creatinine kinase leakage into the medium (CK, IU/g wet wt) and MTT reduction (OD/mg wet wt.), an index of cell viability, were assessed at the end of the experiment. RESULTS: Ischemia alone resulted in a significant increase in CK leakage (8.01 +/- 0.35) and decrease in MTT (0.15 +/- 0.01) from the values seen in the aerobic control (2.24 +/- 0.52 and 0.78 +/- 0.10 respectively, P < 0.05 in both instances). PC fully reversed the effect of ischemia (CK = 2.97 +/- 0.31 and MTT = 0.61 +/- 0.05; P < 0.05 vs. ischemia alone group but P = NS vs. aerobic control group). Both Glibenclamide and 5-HD abolished the protection induced by PC (CK = 6.23 +/- 0.5 and 7.84 +/- 0.64; MTT = 0.18 +/- 0.03 and 0.13 +/- 0.02, respectively, P < 0.05 vs. PC), but interestingly, the protective effect of PC was not abolished by HMR 1883 (CK = 2.85 +/- 0.24 and MTT = 0.58 +/- 0.05, P = NS vs. PC). Diazoxide mimicked the protective effect of PC (CK = 3.56 +/- 0.32 and MTT = 0.58 +/- 0.02, P = NS vs. PC), however pinacidil exhibited less protection than PC (CK = 4.02 +/- 0.16 and MTT = 0.30 +/- 0.02, P < 0.05 vs. PC). CONCLUSIONS: These studies demonstrate that KATP channels are the end-effectors of ischemic preconditioning and that protection is mediated by mitochondrial KATP channels in human right atrial myocardium.

Analysis of Variance↗

PubMed2000

PPARgamma: observations in the hematopoietic system.

Human Peroxisome Proliferator-Activated Receptor gamma (PPARgamma) was originally cloned from a human bone marrow library. What role does this ligand activated transcription factor play in hematopoiesis and the immune system? We note that: a) PPARgamma has potential to interact/interfere or synergize with retinoid biology, b) fatty acids and a prostaglandin have been identified as ligands, and c) lymphocytes, monocytes and neutrophils use fatty acids as a major source of energy production, d) PPARgamma has been shown to oppose TNFalpha and down regulate cytokine production in monocytes. Therefore, we undertook a review of the literature and an expression survey of PPARgamma in a number of major organs and cells involved in the hematopoietic system, for the purpose of building a database towards understanding the role and function of PPARgamma gene regulation in the developing blood and immune systems. PPARgamma is expressed before mesodermal induction in tissue in and around Speymann's organizer in the xenopus blastocyst, in erythroid precursors of blood islands and in the circulation of the day 10.0 murine embryo, in human 19 week fetal liver, in some but not all murine and human bone marrow erythroid, myeloid, and monocytoid progenitors, bone marrow stromal cells and adipocytes, osteoblasts, endothelial cells, some T, and B lymphocytes, monocytes, macrophages, and other monocytic derivatives. It can be found in the cells of Peyer's patches, lymphoid follicles, spleen, and thymus. It is not clear if it is ever or transiently expressed in megakaryocytes, mast cells, or neutrophils. Based on the above data and a review of the literature, PPARgamma seems to play a role during the elicitation of immune responses. We propose PPARgamma may be involved in changes in energy states required during activation and development of many cell types involved, and has additional immunologically relevant effects in erythroid, myeloid, monocytic, T and B lymphocytic, stromal, and endothelial cell function.

Amino Acid Sequence↗

PubMed2000

NF-kappa B activation by the pre-T cell receptor serves as a selective survival signal in T lymphocyte development.

Activation of the transcription factor NF-kappa B and pre-T cell receptor (pre-TCR) expression is tightly correlated during thymocyte development. Inhibition of NF-kappa B in isolated thymocytes in vitro results in spontaneous apoptosis of cells expressing the pre-TCR, whereas inhibition of NF-kappa B in transgenic mice through expression of a mutated, superrepressor form of I kappa B alpha leads to a loss of beta-selected thymocytes. In contrast, the forced activation of NF-kappa B through expression of a dominant-active I kappa B kinase allows differentiation to proceed to the CD4(+)CD8(+) stage in a Rag1(-/-) mouse that cannot assemble the pre-TCR. Therefore, signals emanating from the pre-TCR are mediated at least in part by NF-kappa B, which provides a selective survival signal for developing thymocytes with productive beta chain rearrangements.

Animals↗

PubMed2000

Coupled in vitro synthesis and splicing of RNA polymerase II transcripts.

Compelling in vivo studies suggest a tight functional linkage between RNA polymerase II transcription and premessenger RNA splicing. At present, the specific interactions involved in this coupling are poorly understood and deserve investigation. To this end, we developed an in vitro system that permits study of coupled transcription and splicing. Transcripts generated by RNA polymerase II were accurately and efficiently spliced under reaction conditions that permitted both transcription and splicing to occur simultaneously. The splicing of RNA-polymerase-II-driven transcripts was accelerated relative to that of the same transcripts driven by T7 RNA polymerase. Moreover, the product of exon ligation was found associated with the DNA template in reactions driven by RNA polymerase II. These two findings indicate that transcription and splicing were coupled in the in vitro system driven by RNA polymerase II, and suggest that this system will be useful for the biochemical study of this coupling.

Base Sequence↗