DEEP BIOSPHERE. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor.

Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~10(4) cells cm(-3). Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Nimesulide, a COX-2 inhibitor, does not reduce lesion size or number in a nude mouse model of endometriosis.

BACKGROUND: Women with endometriosis have elevated levels of cyclooxygenase-2 (COX-2) in peritoneal macrophages and endometriotic tissue. Inhibition of COX-2 has been shown to reduce inflammation, angiogenesis and cellular proliferation. It may also downregulate aromatase activity in ectopic endometrial lesions. Ectopic endometrial establishment and growth are therefore likely to be suppressed in the presence of COX-2 inhibitors. We hypothesized that COX-2 inhibition would reduce the size and number of ectopic human endometrial lesions in a nude mouse model of endometriosis. METHODS: The selective COX-2 inhibitor, nimesulide, was administered to estrogen-supplemented nude mice implanted with human endometrial tissue. Ten days after implantation, the number and size of ectopic endometrial lesions were evaluated and compared with lesions from a control group. Immunohistochemical assessment of vascular development and macrophage and myofibroblast infiltration in control and treated lesions was performed. RESULTS: There was no difference in the number or size of ectopic endometrial lesions in control and nimesulide-treated nude mice. Nimesulide did not induce a visually identifiable difference in blood vessel development or macrophage or myofibroblast infiltration in nude mouse explants. CONCLUSION: The hypothesized biological properties of COX-2 inhibition did not influence lesion number or size in the nude mouse model of endometriosis.

Properties of BK(Ca) channels formed by bicistronic expression of hSloalpha and beta1-4 subunits in HEK293 cells.

Large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels are sensitive to both voltage and internal [Ca(2+)] and are found in many tissues. Their physiological roles range from causing relaxation of smooth muscle to regulating the frequency of action potential firing. There is considerable variation between different tissues in their Ca(2+)- and voltage-dependence. Much of this variation results from the association of the pore-forming alpha subunit (hSloalpha) with different beta subunits leading to altered channel properties. Since hSloalpha alone produces functional BK(Ca) channels, we have used a bicistronic expression method to ensure that both alpha and beta subunits are expressed, with the beta subunit being in excess. Using this method we have investigated the effect of four beta subunits (beta1 to beta4) on cloned BK(Ca) channels. The four beta subunits were individually cloned into a vector that had hSloalpha cDNA inserted downstream of an internal ribosome entry site. The constructs were transiently transfected into HEK293 cells together with a construct that expresses green fluorescent protein, as a marker for transfection. Fluorescent cells expressed BK(Ca) channels whose currents were recorded from inside-out or outside-out patches. The currents we measured using this expression system were similar to those expressed in Xenopus oocytes by Brenner et al. (Brenner, R., Jegla, T.J., Wickenden, A., Liu, Y., Aldrich, R.W. 2000. Cloning and functional expression of novel large-conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem.275:6453-6461.)

Cloning and characterization of two splice variants of human phosphodiesterase 11A.

Phosphodiesterase 11A (PDE11A) is a recently identified family of cAMP and cGMP hydrolyzing enzymes. Thus far, a single splice variant designated as PDE11A1 has been reported. In this study, we identify and characterize two additional splice variants of PDE11A, PDE11A2 and PDE11A3. The full-length cDNAs are 2,141 bp for PDE11A2 and 2205 bp for PDE11A3. The ORF of PDE11A2 predicts a protein of 576 aa with a molecular mass of 65.8 kDa. The ORF of PDE11A3 predicts a protein of 684 aa with a molecular mass of 78.1 kDa. Comparison of the PDE11A2 sequence with that of PDE11A1 indicates an additional 86 aa at the N terminus of PDE11A2. Part of this sequence extends the potential cGMP binding region (GAF domain) present in PDE11A1. Compared with PDE11A2, PDE11A3 has an additional 108 N-terminal amino acids. Sequence analysis of PDE11A3 indicates the presence of another GAF domain in this region. This diversification of regulatory sequences in the N-terminal region of PDE11A splice variants suggests the interesting possibility of differential regulation of these enzymes. Recombinant PDE11A2 and -A3 proteins expressed in the Baculovirus expression system have the ability to hydrolyze both cAMP and cGMP. The K(m) values for cAMP hydrolysis are 3.3 microM and 5.7 microM for PDE11A2 and PDE11A3, respectively. The K(m) values for cGMP hydrolysis are 3.7 microM and 4.2 microM for PDE11A2 and PDE11A3, respectively. Both PDEs showed a V(max) ratio for cAMP/cGMP of approximately 1.0. PDE11A2 is sensitive to dipyridamole, with an IC(50) of 1.8 microM, and to zaprinast, with an IC(50) of 28 microM. PDE11A3 demonstrated similar pattern of inhibitor sensitivity with IC(50) values of 0.82 and 5 microM for dipyridamole and zaprinast, respectively.

Interleukin 6 induction in the canine myocardium after cardiopulmonary bypass.

OBJECTIVE: Interleukin 6 is a proinflammatory cytokine with a plasma concentration that has been noted to increase in response to cardiopulmonary bypass. The source of interleukin 6 after cardiopulmonary bypass is unknown. This study examined the myocardium as a potential source of interleukin 6 in this context. METHODS: Dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After rewarming, they were reperfused with the chest open for either 3 (n = 4) or 6 (n = 4) hours, at the end of which myocardial samples were obtained. Four additional animals undergoing open thoracotomy without bypass served as time-matched controls. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization were used to examine the myocardium for the induction of interleukin 6 and intercellular adhesion molecule-1. RESULTS: Northern blot analysis and reverse transcriptase-polymerase chain reaction demonstrated a marked increase in myocardial interleukin 6 messenger RNA in 3 of 4 dogs at 3 hours after bypass and 3 of 4 dogs at 6 hours after bypass, which was not present in sham-bypass control animals. Northern blots at 3 hours after cardiopulmonary bypass also demonstrated myocardial intercellular adhesion molecule-1 induction. In situ hybridization studies confirmed that cardiac myocytes were a principal source of interleukin 6 messenger RNA early after cardiopulmonary bypass. Northern blots of messenger RNA extracted from isolated neutrophils and mononuclear leukocytes obtained from blood samples before bypass, at the end of bypass, and 3 hours after bypass failed to demonstrate interleukin 6 induction. CONCLUSION: Despite protection with cold cardioplegic arrest, the myocardium was a significant source of interleukin 6 synthesis after cardiopulmonary bypass. Local production of interleukin 6 may play a pivotal role in postoperative myocardial function.

Molecular cloning and characterization of a distinct human phosphodiesterase gene family: PDE11A.

We report here the cloning, expression, and characterization of human PDE11A1, a member of a distinct cyclic nucleotide phosphodiesterase (PDE) family. PDE11A exhibits

Isolation and characterization of a novel, human neuronal nitric oxide synthase cDNA.

A splice variant of nNOS has recently been identified in both rat and mouse which contains an in-frame insertion of 34 conserved amino acids between the N-terminal oxygenase and the C-terminal reductase domains. In the present study we report the isolation and characterization of a similar, but not identical (76% amino acid identity), human variant (nNOSmu) which arises from the splicing in of an additional exon between exons 16 and 17 of the human nNOS gene. Furthermore, we describe two additional splice variants which, if translated, would give rise to truncated forms of nNOS lacking the C-terminal reductase domain. These additional variants and nNOSmu; have a distinct and more restricted expression pattern compared to nNOS. Further studies are required to elucidate the possible physiological roles of these novel human nNOS splice variants in NO signaling.

Molecular cloning and expression of human cGMP-binding cGMP-specific phosphodiesterase (PDE5).

A human PDE5 cDNA has been isolated which contains an open reading frame encoding an 875 amino acid, 100,012 Da polypeptide, the expression of which yields a protein of the predicted size and is capable of hydrolyzing cGMP. The deduced amino acid sequence is very similar (95%) to that of bovine PDE5, and comprises a conserved cGMP-binding domain and catalytic domain. Northern analysis reveals a major and minor transcript of approximately 9 kb and approximately 8 kb respectively, thus indicating the existence of at least two splice variants, the major form being readily detected in bladder, colon, lung, pancreas, placenta, prostate, small intestine, and stomach.

Intercellular adhesion molecule-1 regulation in the canine lung after cardiopulmonary bypass.

OBJECTIVE(S): Neutrophil sequestration in the lung after cardiopulmonary bypass has been shown to be dependent on the adhesion molecule CD18. Thus we sought to determine whether endothelial expression of intercellular adhesion molecule-1 (a ligand for CD18) in pulmonary capillaries mediates neutrophil adhesion in this setting. METHODS: Seven adult mongrel dogs underwent 90 minutes of hypothermic cardiopulmonary bypass with 60 minutes of cardioplegic arrest. After warming, dogs were reperfused for up to 9 hours and lung biopsy specimens were obtained. Lung tissue was examined by Northern and Western blot analysis and by immunohistologic methods. Three sham-operated dogs served as time-matched controls. RESULTS: Northern blots demonstrated increased expression of intercellular adhesion molecule-1 messenger ribonucleic acid within 5 minutes of cessation of bypass (or approximately 30 minutes after aortic crossclamp release), which persisted at 9 hours of recovery and was not present in controls. Western blots showed intercellular adhesion molecule-1 protein expression before bypass but a measurable increase in intercellular adhesion molecule-1 protein in four of seven dogs in the bypass group by the ninth hour of recovery. Pulmonary neutrophil accumulation 9 hours after cardiopulmonary bypass was greater in those dogs with an increased intercellular adhesion molecule-1 protein expression. Immunoelectron microscopy demonstrated the pulmonary capillary endothelium capable of increased intercellular adhesion molecule-1 protein expression at the 9-hour time point. CONCLUSIONS: Cardiopulmonary bypass resulted in intercellular adhesion molecule-1 induction in the canine lung during recovery. An increased expression of intercellular adhesion molecule-1 protein in the lung was associated with an increased accumulation of neutrophils in affected animals. Thus intercellular adhesion molecule-1 expression may serve as a mechanism that predisposes the lungs to inflammatory cell-mediated injury postoperatively.

Receptor-mediated DNA delivery approaches to human gene therapy.

The current non-viral approaches to gene therapy are presented with a particular emphasis on receptor-mediated DNA delivery and the recent advances made that make this approach an attractive alternative to viral-based vector systems. The general strategy for receptor-mediated DNA delivery involves condensing DNA via electrostatic interaction with a polycation, e.g. polylysine, chemically linked to a cell binding ligand, e.g. transferrin. The potency of this delivery system has recently been improved by incorporating an endosome disrupting (endosomolytic) agent into the DNA-complex, such as adenovirus particles, which enhances release into the cytoplasm. Therefore, the approach now is to mimic viral entry mechanisms, e.g. by incorporating a synthetic peptide with endosomolytic activity, and, in effect, manufacture a synthetic virus.

The Xenopus U7 snRNA-encoding gene has an unusually compact structure.

A subclone containing a single Xenopus borealis U7 snRNA-encoding gene has been microinjected into X. laevis oocyte nuclei to examine its expression using [32P]GTP as an in vivo label. Only two U7 snRNA bands were detected after incubation, and subsequent fractionation of the oocyte showed that only the larger transcript is present in the nucleus. The sequence of this functional U7 gene shows that, in addition to the coding region, it contains, in the appropriate locations, the 3'-box and proximal sequence element (PSE) which are typical of Pol II-transcribed snRNA genes. Surprisingly, the Xenopus U7 gene contains two adjacent octamer-binding motifs located only 12 and 24 bp upstream from the PSE, instead of the usual location around 150-200 bp upstream. No other cis-acting elements appear to be present. A 5' deletion analysis shows that the transcription level of this U7 gene remains constant if sequences upstream of the two octamer motifs are removed, yet is undetectable when an additional 34 bp containing both octamers and the PSE are removed. This confirms that the Xenopus U7 gene is the most compact snRNA-encoding gene isolated to date. A comparison of U7 sequences shows there is a much greater conservation in the 5' half of the molecule, which contains sequences that base-pair with target pre-mRNA, than in the 3' half which can form a single stem-loop structure that varies in size.

A transcriptional analysis of the gene encoding mouse U7 small nuclear RNA.

Expression of the U7 gene, encoding mouse U7 snRNA, following microinjection into Xenopus oocytes is both accurate and efficient, giving rise to mature U7 snRNA and a precursor with an 8-nucleotide (nt) 3' extension. The mouse U7 gene promoter, which is similar to that of the vertebrate major U genes comprising a DSE, a PSE and a 3' box, with the same spatial arrangement, is as efficient as the Xenopus U2 gene promoter in this assay. A deletion analysis of the mouse U7 gene identified sequences downstream from the 3' box, within the region (nt +74 to +196), which seem to have a negative regulatory effect upon the frequency of transcription initiation and are also required for accurate 3' end formation. Sequences in the nt -1699 to -431 region also seemed to have a negative effect on the level of transcription. In addition, sequences upstream from the PSE, within the nt -65 to -421 region, are necessary for accurate and efficient synthesis of mature U7 snRNA. Finally, the mouse U7 snRNA may not form a functional snRNP in Xenopus oocytes due to defective snRNP assembly and/or nuclear import.

Analysis of a gene cluster coding for the Xenopus laevis U7 snRNA.

A cluster of Xenopus laevis U7 snRNA genes has been isolated and sequenced. The gene structure is more compact than, but otherwise comparable to, the major U snRNA genes since the distal sequence element (DSE) is located only 4 nt upstream of the PSE. The corresponding RNA is present in the oocyte and accumulates early in oogenesis.

Sequence and expression of a mouse U7 snRNA type II pseudogene.

The U7 snRNP functions in the 3' processing of histone pre-mRNAs of the replication variant class. The U7 snRNA is apparently the only RNA component of this minor snRNP particle. So far, the only U7 snRNA genes that have been isolated are a cluster of 5 from sea urchin. The function of the sea urchin U7 snRNP has been examined by microinjection experiments using Xenopus oocytes and the expression of these sea urchin U7 snRNA genes has been analysed by microinjection into sea urchin eggs. In our studies of U7 snRNA genes in mouse, we have isolated one of only 3 detectable U7 snRNA pseudogenes in the mouse genome and have determined its nucleotide sequence. Sequence comparisons and transcription analysis following microinjection of the gene into Xenopus oocytes supports our assignment of this isolate as a U7 snRNA type II pseudogene.