Vital involvement of a natural killer cell activation receptor in resistance to viral infection.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Effects of peroxynitrite on pulmonary edema and the oxidative state.

Excess nitric oxide (NO) generation in the presence of superoxide anion (O2-) leads to the formation of peroxynitrite which may result in lung injury. Oxidant-mediated lung injury has a critical role in pulmonary diseases. We therefore determined whether peroxynitrite causes lung fluid accumulation, lipid peroxidation, and formation of nitrotyrosine using an isolated perfused rat lung model. The lung weight gain during bolus peroxynitrite infusion increased in a dose-dependent manner over a range of 3 to 30 mumole. Concomitantly, bronchoalveolar lavage Ficoll also increased, indicative of increased endothelial permeability. Peroxynitrite increased the production of thiobarbituric acid reactive substances, an index of lipid peroxidation. Furthermore, nitrotyrosine levels in lung tissue rose with increased concentration of peroxynitrite, as determined by Western blot using antinitrotyrosine antibodies. These results suggest that peroxynitrite, formed from NO and O2-, leads to increased pulmonary fluid accumulation, possibly through lipid peroxidation and/or nitration of cell membrane proteins.

Activation of the erythropoietin receptor is not required for internalization of bound erythropoietin.

Erythropoietin (EPO) is required for the survival and expansion of red blood cell progenitor cells and supports continued differentiation of these committed progenitors to mature red blood cells. After binding to its cognate receptor, EPO promotes receptor homodimerization, activation of receptor-associated JAK2, subsequent receptor tyrosine phosphorylation, and transduction of signal. EPO is also internalized and degraded in lysosomes. The contribution of EPO-induced receptor internalization to modulation of EPO signals has not been determined. To examine this question, we generated a panel of hematopoietic cell lines containing progressively truncated isoforms of the erythropoietin receptor (EPO-R) and determined the rate and extent of EPO internalization and receptor downregulation. We demonstrated that a membrane-proximal domain of the cytoplasmic tail of the EPO-R was the minimal region required for EPO-induced receptor internalization. This cytoplasmic domain is also the minimal domain required for activation of JAK2, a cytosolic tyrosine kinase essential for the function of the EPO-R. However, neither EPO activation of cytosolic JAK2 tyrosine kinase activity nor tyrosine phosphorylation of the EPO-R cytoplasmic tail was required for EPO-induced receptor downregulation. Both functional and nonfunctional cell surface receptor isoforms were internalized equally. These results suggest that, for downregulation of cell surface ligand occupied EPO-R and possibly for signaling receptors of the cytokine receptor superfamily in general, internalization of cell surface ligand occupied receptors may follow a pathway distinct from signaling receptors of the receptor tyrosine kinase (RTK) family.

A thioreduction pathway tethered to the membrane for periplasmic cytochromes c biogenesis; in vitro and in vivo studies.

The c-type cytochromes are distinguished from other heme proteins by the covalent ligation of two heme vinyl groups to two cysteine residues on the apoprotein (at a CXXCH domain). The present study was undertaken to elucidate the roles and topological locations of two of the proteins necessary for cytochrome c biogenesis, the HelX and Ccl2 proteins in the Gram-negative bacteria Rhodobacter capsulatus. From their primary sequence, each of these proteins has a CXXC motif that could be involved in the reduction of the cysteine residues of the apocytochromes c, a prerequisite for covalent ligation to the heme. Results of site-directed mutagenesis of HelX and Ccl2 demonstrate that each cysteine residue is required for the in vivo function of the protein. We demonstrate that the native HelX in R. capsulatus is tethered to the cytoplasmic membrane via its uncleaved signal sequence. Ccl2 is tethered by a single transmembrane domain present in the C terminus with the N-terminal two-thirds of the protein in the periplasm. Thus, both CXXC motifs are exposed to the periplasm. The complete HelX protein and the soluble N-terminal portion of Ccl2 (called Ccl2*) were overproduced and purified from periplasmic fractions. The Ccl2* signal sequence is efficiently processed. In vitro studies with these purified proteins indicate that although neither can reduce insulin, HelX can reduce the Ccl2 cysteine residues and the Ccl2 cysteine residues are oxidized by an apocytochrome c peptide containing the CXXCH domain. Revertants of an helX deletion mutant were isolated that regain the ability to make c-type cytochromes (and thus grow photosynthetically); some of these suppressor strains are enhanced for photosynthetic growth by the addition of thio-reducing agents. In contrast, revertants of a ccl2 deletion strain could not be isolated under any condition. These results suggest that the HelX and Ccl2 proteins form a thioreduction pathway (HelX-->Ccl2-->apocytochrome c) whereby Ccl2 function may be highly specific for apocytochromes c while HelX may act as a more general reductant of proteins with vicinal cysteines.

Molecular and immunological analysis of an ABC transporter complex required for cytochrome c biogenesis.

The helABC genes are predicted to encode an ATP-binding cassette (ABC) transporter necessary for heme export for ligation in bacterial cytochrome c biogenesis. The recent discoveries of homologs of the helB and helC genes in plant mitochondrial genomes suggest this is a highly conserved transporter in prokaryotes and some eukaryotes with the HelB and HelC proteins comprising the transmembrane components. Molecular genetic analysis in the Gram-negative bacterium Rhodobacter capsulatus was used to show that the helABC and helDX genes are part of an operon linked to the secDF genes. To facilitate analysis of this transporter, strains with non-polar deletions in each gene, epitope and reporter-tagged HelABCD proteins, and antisera specific to the HelA and HelX proteins were generated. We directly demonstrate that this transporter is present in the cytoplasmic membrane as an HelABCD complex. The HelB and HelC but not HelD proteins are necessary for the binding and stability of the HelA protein, the cytoplasmic subunit containing the ATP-binding region. In addition we show that the HelA protein co-immunoprecipitates with either the HelC or HelD proteins. Thus, the HelABCD heme export complex is distinguished by the presence of four membrane-associated subunits and represents a unique subfamily of ABC transporters.

Sympathetic nerve influence on alveolar type II cell ultrastructure.

Pulmonary surfactant release from lamellar bodies in alveolar type II cells in response to exogenous and circulating catecholamines is well documented but sympathetic influences from direct nerve stimulation are not as extensively studied. In order to study sympathetic nerve influence on release, ultrastructure of type II cells was examined following one min stimulation of the stellate ganglion (SGS) which sends sympathetic fibers to lung parenchyma. The type II cell secretion was assessed by measuring volume density of lamellar bodies, interpreting decreased volume density to indicate increased secretion according to accepted stereologic principles. The present study showed lamellar body volume density was reduced 20.8% in type II cells of SGS animals. Multivesicular body volume density was increased 150% by SGS, but Golgi bodies were unchanged. These data are consistent with type II cell origin of at least some of the increased saturated phospholipid recovered from airspaces in previous SGS studies, and support the view that sympathetic nerve activity may contribute to surfactant homeostasis under some conditions.

Cytochromes c biogenesis in a photosynthetic bacterium requires a periplasmic thioredoxin-like protein.

Rhodobacter capsulatus is a Gram-negative photosynthetic bacterium that requires c-type cytochromes for photosynthetic electron transport. Our studies demonstrate that the gene helX is required for the biogenesis of c-type cytochromes in R. capsulatus. A helX chromosomal deletion mutant cannot grow photosynthetically, due to a deficiency of all c-type cytochromes. The predicted amino acid sequence of the helX gene product (176 residues) is related to that of thioredoxin and shares active-site homology with protein disulfide isomerase. Cytochrome c2-alkaline phosphatase gene fusions are used to show that HelX is not required for the transcription, translation, or secretion of apocytochrome c2. HelX-alkaline phosphatase and HelX-beta-galactosidase gene fusions are used to demonstrate that HelX is a periplasmic protein, which is consistent with the presence of a typical signal sequence in HelX. Based on these results, we propose HelX functions as a periplasmic disulfide oxidoreductase that is essential for cytochromes c biogenesis. This role is in accordance with the observation that both heme and the cysteines of apocytochromes c (Cys-Xaa-Yaa-Cys-His) must be in the reduced state for covalent linkage between the two moieties to occur.

DNA gyrase activities from Rhodobacter capsulatus: analysis of target(s) of coumarins and cloning of the gyrB locus.

Bacterial DNA gyrase is composed of two subunits, gyrase A and B, and is responsible for negatively supercoiling DNA in an ATP-dependent manner. The coumarin antibiotics novobiocin and coumermycin are known inhibitors of bacterial DNA gyrase in vivo and in vitro. We have cloned, mapped, and partially sequenced Rhodobacter capsulatus gyrB which encodes the gyrase B subunit that is presumably involved in binding to coumarins. DNA gyrase activities from crude extracts of R. capsulatus were detected and it was shown that the R. capsulatus activity is (1) inhibited by novobiocin and coumermycin, (2) ATP-dependent and, (3) present in highly aerated and anaerobically grown cells. We previously observed that when R. capsulatus coumermycin-resistant strains are continuously recultured on media containing coumermycin they sometimes acquired mutations in hel genes (i.e., cytochromes c biogenesis mutations). We discuss the possibility that coumarins may inhibit cytochromes c biogenesis as a second target in R. capsulatus via hel (i.e., a putative ATP-dependent heme exporter).

Bacterial cytochromes c biogenesis.

We report the primary sequence analyses of two loci, hel and ccl, whose gene products are required specifically for the biogenesis of c-type cytochromes in the Gram-negative photosynthetic bacterium Rhodobacter capsulatus. Genetic and molecular analyses show that the hel locus contains at least four genes, helA, helB, helC, and orf52, and the ccl locus contains two genes, ccl1 and ccl2, that are essential for cytochromes c biogenesis. HelA is homologous to a class of proteins called ABC transporters and helA, helB, and helC are proposed to encode an export complex. Cytochrome c2-alkaline phosphatase gene fusions were used to show that apocytochrome c2 synthesis and secretion are not affected by the hel and ccl defects. Ccl1 and Ccl2 possess typical signal sequences to direct them to the periplasm. The periplasmic orientation of Ccl1 was confirmed using a Ccl1-alkaline phosphatase gene fusion. The Ccl1-alkaline phosphatase gene fusion analysis also demonstrated that Ccl1 does not require hel genes for its synthesis and secretion. Ccl1 is homologous to proteins encoded by chloroplast and mitochondrial genes, suggesting analogous functions in these organelles. Taken together, these results support the hypothesis that the hel-encoded proteins are required for the export of heme to the periplasm where it is subsequently ligated to the c-type apocytochromes.

A bacterial homolog to HPRT.

The deduced 182 amino acid sequence of an open reading frame in the photosynthetic bacterium Rhodobacter capsulatus shows significant similarity to the hypoxanthine-guanine phosphoribosyltransferases of other organisms. This similarity includes conserved amino acid residues involved in Lesch-Nyhan syndrome.

A bacterial homolog to the mitochondrial enoyl-CoA hydratase.

A 257-amino acid (aa) open reading frame in the photosynthetic bacterium, Rhodobacter capsulatus, shows significant homology to the mitochondrial enoyl-CoA hydratase (290 aa). This similarity in size and sequence suggests that R. capsulatus oxidizes fatty acids using specific components, more like the mitochondrial system than the multifunctional component system of Escherichia coli.

Neurogenic pulmonary edema in a pulmonary normotensive model.

In the present study our aim was to determine whether or not neurogenic pulmonary edema would develop from a brief pulse of intracranial pressure (ICP) in the absence of any obvious pulmonary hypertension. There were three groups of cats: sham-operated controls, ICP only, and ICP plus variable occlusion of the pulmonary artery. Partial occlusion of the pulmonary artery was carried out by placing a ligature around the pulmonary trunk and mechanically constricting the artery to maintain pulmonary arterial pressure (PAP) and left atrial pressure (LAP) at pre-ICP levels. In sham-operated animals the extravascular lung water/blood free dry weight ratio (EVLW/BFDW) was 3.26 +/- 0.07 and broncho-alveolar lavage (BAL) protein, 6.49 +/- 0.62 mg/g lung. ICP-only caused a rise in PAP, left atrial pressure, and EVLW/BFDW to 3.67 +/- 0.08 (P less than 0.05). ICP with partial occlusion of the pulmonary artery prevented any rise in PAP or LAP while EVLW/BFDW rose to 3.67 +/- 0.10 (P less than 0.05) and BAL protein was 8.37 +/- 1.27 mg/g lung. Our results show that EVLW/BFDW can increase with neurogenic pulmonary edema in cats in the absence of an obvious increase in pulmonary arterial or left atrial pressure.

Cardiopulmonary response to an induced pulse in intracranial pressure.

Increased intracranial pressure may result in the Cushing response. We applied a short pulse of pressure to the cranial cavity of anesthetized cats which were intubated, curarized, ventilated, and the cranium exposed to an 80- to 100-msec pulse of pressure at 5.3 atm. The following significant increases developed: Intracranial pressure rose from 7.4 +/- 1.5 to 150.6 +/- 19.4 mm Hg, systolic arterial peak pressure from 130.7 +/- 8.1 to 299.0 +/- 11.4, pulmonary peak pressure from 18.9 +/- 1.9 to 42.9 +/- 4.9. Alveolar lavage protein in controls was 6.7 +/- 0.4 mg/g lung compared to 11.9 +/- 2.0 in the experimental group. Extravascular lung water/dry weight ratios increased from 3.36 +/- 0.04 in controls to 3.51 +/- 0.09 but varied inversely with pulmonary systolic peak pressure (r = 0.59). These results showed that a pulse of pressure applied to the cranium of cats produced lung edema which was inversely related to pulmonary artery pressures.

Stellate ganglion stimulation with alpha and beta adrenergic blockade: effect on lung lipids and compliance in cats.

Stellate ganglion stimulation (SGS) can alter lung lipids and reduce static lung compliance, although the mechanisms remain unclear. Phentolamine and propranolol were administered to anesthetized cats prior to stimulation in order to investigate SGS effects on lung lipids and compliance mediated via alpha and beta adrenergic pathways. Analysis of lung lavage revealed that SGS alone decreased cholesterol and the cholesterol/DSPC ratio which might be expected to decrease lung compliance. Alpha and beta blockade alone resulted in no changes from control in cholesterol or DSPC. Alpha blockade plus SGS yielded increased rather than decreased cholesterol and DSPC, while beta blockade prevented any change. A reduction in both static and dynamic lung compliance caused by SGS also was blocked by both alpha and beta blockade. Thus both the alpha and beta blockade prevented the SGS-induced decreases in cholesterol, cholesterol/DSPC ratio, and lung compliance. Furthermore, alpha blockade plus SGS resulted in increased TPL as well as cholesterol and DSPC. The data are consistent with the view that DSPC and cholesterol are released into the subphase by beta adrenergic mechanisms, and that their relative amounts may influence surface properties.

Beta adrenergic receptors and glucagon in seizures from exposure to oxygen at high pressure (OHP).

Previous studies have shown that sympathetic factors and blood glucose are of importance in the development of seizures and lung damage from OHP. In the present study we examined the influence of beta sympathetic agonists and blocking agents and glucagon on OHP toxicity. Rats were exposed to 6 ATA OHP and examined for time-to-seizure and lung damage. Pretreatment with propranolol increased the time-to-seizure by 70% and practolol by 50% without altering gross lung appearance or lung wet wt/dry wt. Propranolol and practolol also prevented brain glycogen depletion prior to seizure which otherwise occurred in subconvulsive exposure to OHP. Isoproterenol and glucagon pretreatment had no effect on time-to-seizure but isoproterenol did increase lung injury. Both practolol and propranolol block the beta-receptor influence on adenyl cyclase-stimulated second messenger production, while both isoproterenol and glucagon activate adenyl cyclase to produce second messenger. Our results may suggest a possible role for second messenger in mediating some of the acute toxic effects of OHP on the CNS.

Traumatic head injury and pulmonary damage.

Head-injury victims may develop pulmonary complications such as hemorrhagic edema, congestion, and hyperemia. In the present experiments we examined changes in lung surface tension, cholesterol, and phospholipids of alveolar lavage recovered from cats experimentally exposed to a sudden lethal blow to the occiput by captive bolt. Minimum surface tension and cholesterol from traumatized cats which developed convulsive seizures immediately after the blow were more than doubled while no change was seen in traumatized cats without seizures. Total phospholipids and disaturated phosphatidyl choline in either group were not increased over control levels. However, the ratio of cholesterol to total phospholipids increased significantly in traumatized cats with seizures, but did not increase in the cats without seizures. Lung wash protein was higher than in controls in both groups of traumatized cats. This work suggests that at least part of the pulmonary changes following this mechanical head injury may be mediated through a change in the alveolar surface lining.

Influence of blood glucose on convulsive seizures from hyperbaric oxygen.

Previous evidence suggests a causal relationship between blood glucose levels and the development of generalized epileptiform seizures. In the present study rats were pretreated with glucose, alloxan, or insulin prior to exposure to 6 atmospheres absolute (ATA) oxygen in a hyperbaric chamber. The results showed that the administration of glucose prior to oxygen exposure increased the time-to-seizure by 90% and alloxan by 110%, whereas in contrast insulin decreased the time-to-seizure by 55%. Blood glucose levels were consistently elevated in rats following oxygen exposure. A trend towards reduced lung damage by glucose and alloxan pretreatment was suggested by the data, although no changes were significant. Our results showed that prior administration of glucose or alloxan offered partial protection from oxygen toxicity in rats, whereas insulin generally augmented the reaction.

Protection from oxygen-induced seizures by clonazepam and propylene glycol.

General anesthetics, ganglionic blocking agents, anticonvulsants, and antioxidants have been shown to afford protection from seizures caused by exposure to hyperbaric oxygen. In the present study cats were exposed to 5 ATA oxygen in pairs in a hyperbaric chamber until both the control and pretreated cat convulsed or for a maximum 120 min exposure. Small amounts of four common antiepileptic agents and propylene glycol in amounts far less than previously reported (0.1 to 0.2 ml/kg) were initially tested for potential anticonvulsant activity. Two agents, clonazepam and propylene glycol, offered significant protection in delaying the onset of seizures whereas carbamazepine, valproic acid, and trimethadione appeared to hasten the onset of seizure activity. The time to seizures was increased nearly five times by clonazepam and over three times by very small amounts of propylene glycol.

Stellate ganglion influence on static compliance and surface layer lipids in cat lungs.

In order to determine the effects of stellate ganglion stimulation (SGS) on static lung compliance, pressure-volume relationships were measured on lungs in situ in cats anesthetized with ketamine. The lungs were inflated with 100% oxygen to 100, 93.75, 87.5, 81.25, 75, 50, and 25% TLC as determined at 20 cm H2O transpulmonary pressure. After a maximal filling, lungs were inflated twice to each volume, alternating unstimulated and SGS conditions. Static lung compliance was decreased by SGS at 93.75 (5.7 +/- 0.4 (SE) vs. 5.4 +/- 0.4 ml/cm H2O; P less than 0.05) and 87.5% TLC (6)6 +/- 0.6 vs 6.0 +/- 0.5 ml/cm H2O; P less than 0.05). In order to assess possible effects of SGS on surfactant that might be related to the loss of compliance, lung lipids and proteins obtained during SGS were quantitated. Total phospholipid (TPL) increased from 3.22 +/- 0.33 to 4.16 +/- 0.23 mg/g lung after 1 min 7V SGS (P less than 0.05) and to 4.58 +/- 0.61 mg/g lung after 4 min 20V (P less than 0.05). The ratio of cholesterol (chol) to disaturated phosphatidylcholine (DSPC) decreased after 4 and 6 min, and may have resulted in increased surface tension forces at high lung volumes. The data from this study lend support to previous findings which showed that the compliance decrease during SGS may be related to increased surface tension forces.

Absence of stellate ganglion influence on alveolar-duct constriction.

Previous work has shown decreased lung compliance with high wash fluid surface tensions, but normal ratios of lung weight to body weight during stellate ganglion stimulation (SGS) in cats and monkeys. In the present study, effects of SGS on diameters of alveolar ducts were determined in spontaneously breathing cats under ketamine anesthesia. Compliance and pulmonary resistance were also measured. A fluorocarbon cooled to - 100 degrees C was poured onto an exposed lung lobe after rapidly opening the chest after sham operation and during SGS. Examination of the outer 2 mm of the lungs by light microscopy revealed no alveolar-duct constriction by SGS, although compliance decreased by 15%. Pulmonary resistance was unchanged. Alveolar-duct diameters were 157 +/- 4.3 micron (mean +/- SEM) in control animals and 163 +/- 4.6 micron in SGS cats. Absence of alveolar-duct constriction suggests that peripheral airway constriction did not contribute to the decrease in compliance during SGS.