Intensive insulin therapy not only normalizes the elevated blood glucose levels that develop in nondiabetics and diabetics who are battling critical illnesses but also, and remarkably, increases the survival of these individuals (N Engl J Med 2001;345:1359-67 and J Clin Invest 2004;114:1187-95). To begin to determine the presently unknown mechanism for this lifesaving effect, we hypothesized that insulin therapy decreases lung inflammation and acute lung injury seriously ill patients. We chose to initially test this premise using a standard rat model in which lung inflammation and acute lung injury develop following insufflation of cytokines (interleukin-1 and/or interferon-γ) that are increased in lungs of patients with acute lung injury. We found that insulin therapy decreases lung neutrophil recruitment (lung lavage neutrophil counts) and acute lung injury (lung lavage LDH concentrations) in lungs of cytokine insufflated rats. In addition, insulin therapy also decreased xanthine oxidoreductase (XOR) activity in mononuclear phagocytes recovered by lavage from lungs of cytokine insufflated rats. The latter observation is relevant because our prior studies suggest that mononuclear phagocyte XOR activity may contribute to neutrophil increases in lungs of rats insufflated with cytokines (Am J Respir Cell Mol Biol 2004;30:479-90). In parallel, we found that inducing hyperglycemia (without administering insulin) increased the number of neutrophils recoverable from lungs of rats insufflated with cytokines. These preliminary findings suggest that one possible effect of intensive insulin therapy is to reduce lung inflammation and acute lung injury. This mechanism might contribute to the reduced mortality of critically ill patients who receive intensive insulin therapy.

For unknown reasons, serum ferritin levels increase in patients at risk for and with acute lung injury (ALI). To improve understanding of the relationship between serum ferritin alterations and the development of ALI, we investigated the effect of iron deficiency on the serum ferritin response of rats subjected to hemorrhage. We found that rats fed an iron-deficient diet for 6 weeks had decreased hemoglobin, hematocrit, liver total iron, liver total iron-binding capacity, and liver ferritin concentrations but the same serum ferritin concentrations as rats fed a control diet. Following hemorrhage, serum ferritin concentrations increased rapidly and progressively in rats fed a control diet. Along with increases in serum ferritin concentrations, control diet rats subjected to hemorrhage also had increased lung lavage leukocyte numbers, lung myeloperoxidase activities (lung inflammation), and lung lavage protein concentrations (lung leak) compared to control diet fed rats subjected to sham treatment. By comparison, the serum ferritin concentrations, lung inflammation, and lung leak of hemorrhaged rats fed an iron-deficient diet were decreased compared to hemorrhaged rats fed a control diet. These findings indicate that serum ferritin concentrations increase and acute lung injury develops following hemorrhage in rats fed a control, but not an iron-deficient, diet. A relatively brief exposure to an iron-deficient diet reduces hemorrhage-induced ALI.

Aldehyde oxidase (AOX) is a member of the xanthine oxidase (XO) family of molybdenum hydroxylase, iron-sulfur flavoproteins and is involved in the metabolism of a wide range of native and xenobiotic compounds. The potentially toxic reduced oxygen intermediates (ROI), hydrogen peroxide (H2O2) and superoxide anion (O-2(.-)), are generated when reduced AOX becomes oxidized by molecular oxygen, raising the possibility for involvement of AOX in pathophysiology. Indeed, ROI generation by AOX has been directly implicated in hepatic ethanol toxicity. A cDNA encoding human AOX has been cloned, sequenced, and identified as AOX1. AOX1 was proposed as a candidate for an autosomal recessive form of amyotrophic lateral sclerosis (ALS2) because a YAC carrying AOX1 was mapped to the ALS2 locus and was expressed in microglial cells of the spinal cord. As a source of H2O2, AOX could mediate motor neuron degeneration. To provide a basis for further analysis of AOX1 in pathophysiology, and to examine the relationship of the human AOX1 gene to the gene for human xanthine dehydrogenase (XDH), we have studied the chromosomal locus encoding AOX1 in humans. In the present communication, we have analyzed P1 artificial chromosomes containing AOX1. Our refined chromosomal mapping by FISH locates AOX1 very centromere proximal in the 2q33 region at 2q32.3-2q33.1. We present the first complete structural map of an AOX gene and provide direct evidence that human XDH and AOX1 are related by a gene duplication event. In addition, 1500 bp of upstream DNA containing the putative AOX1 promoter were sequenced and expressed. In contrast to the amino acid coding regions, AOX1 and XDH promoter sequences exhibit marked divergence that reflects the differential activation of these closely related genes. Evidence is presented that AOX may be polygenic in humans as it is in plants, Dipterans, and mice.

We purified aldehyde oxidase (AO) from rabbit livers and found that AO produced deoxyribonucleic acid (DNA) single strand nicks in vitro. Acetaldehyde, benzaldehyde, and certain purine bases were effective substrates for AO catalyzed DNA strand nicking. DNA strand nicking did not occur with the reducing substrates nicotinamide-adenine dinucleotide or dithionite that produce superoxide anion (O2'(-)). Inclusion of electron transport inhibitors, potassium cyanide, ferricyanide or menadione, prevented AO catalyzed nicking. AO induced DNA strand nicking was dependent upon hydrogen peroxide (H2O2) formation and most likely generation of hydroxyl radical (HO'). The present observations may be pertinent to the recently proposed involvement of AO in inherited juvenile familial amyotrophic lateral sclerosis (JFALS) and other oxygen radical mediated diseases.

Denver, Tokyo, and Salt Lake City investigators recently published different complimentary deoxyribonucleic acid (cDNA) sequences for human liver xanthine dehydrogenase/xanthine oxidase (XD/XO). The gene encoding the Denver cDNA was subsequently linked to juvenile familial amyotrophic lateral sclerosis (JFALS) at chromosome 2q33 and has been proposed as the ALS2 locus, The present investigation was undertaken to elucidate the differences between the three cDNA sequences, and we provide evidence that the Denver cDNA encodes aldehyde oxidase (AO): first, the Denver cDNA sequence diverged significantly from the Tokyo and Salt Lake City cDNA sequences which were very similar; second, the deduced protein sequence from the Denver cDNA was very similar to the amino acid sequence of purified rabbit liver AO protein; third, the deduced Denver protein sequence was 76% identical to the encoded 101 amino acid long peptides from partial cDNAs for rabbit and rat AO and 81.7% identical to 300 amino acids from an incomplete cDNA encoding bovine AO; fourth, the Denver gene was expressed in liver, kidney, lung, pancreas, prostate, testes, and ovary while the Tokyo XD gene was expressed predominantly in liver and small intestine; fifth, the Denver gene was previously mapped to chromosome 2q33 which is syntenic to the mouse AO locus on chromosome 1, Our results have revealed dramatic similarities in protein and DNA sequence in the human molybdenum hydroxylases, have uncovered unanticipated complexity in the human molybdenum hydroxylase genes, and advance the potential for AO derived oxygen radicals in JFALS and other human diseases.

We determined that mitochondrial respiration reduced cytosolic oxidant stress in vivo and scavenged extramitochondrial superoxide anion (O2-.) in vitro. First, Saccharomyces cerevisiae deficient in both the cytosolic antioxidant cupro-zinc superoxide dismutase (Cu,Zn-SOD) and electron transport (Rho0 state) grew poorly (P < 0.05) in 21% O2 compared with parent yeast and yeast deficient only in electron transport or Cu,Zn-SOD, whereas anaerobic growth was the same (P > 0.05) in all yeast. Second, isolated yeast and mammalian mitochondria scavenged extramitochondrial O2-. generated by xanthine/xanthine oxidase. Yeast mitochondria scavenged 42% more (P < 0.05) extramitochondrial O2-. during pyruvate/malate-induced respiration than in the resting state. Addition of either antimycin (respiratory chain inhibitor) or FCCP (respiratory chain uncoupler) prevented increased O2-. scavenging. Mitochondria isolated from yeast deficient in the mitochondrial manganous superoxide dismutase (Mn-SOD) increased (P < 0.05) O2-. scavenging 56% during respiration. This apparent SOD activity, expressed in units of SOD activity per milligram of mitochondrial protein, was the same (9 +/- 0.6 vs. 10 +/- 1.0; P = 0.43) as the O2-. scavenging of mitochondria with Mn-SOD, suggesting that respiration-dependent mitochondrial O2-. scavenging was nonenzymatic. Finally, isolated rat liver and lung mitochondria also increased (P < 0.05) O2-. scavenging during respiration. We speculate that respiring mitochondria, via the protonmotive pump, present a polarized, proton-rich surface that enhances nonenzymatic dismutation of extramitochondrial O2-. and that this is a previously unrecognized function of mitochondrial respiration with potential physiological ramifications.

Recently, point mutations in superoxide dismutase 1 (SOD1) have been shown to lead to a subset of autosomal dominantly inherited familial amyotrophic lateral sclerosis (ALS). These findings have led to the hypothesis that defects in oxygen radical metabolism may be involved in the pathogenesis of ALS. Therefore, we decided to analyze other enzymes involved in oxygen radical metabolism for possible involvement in other forms of ALS. We report here analysis of two genes encoding the molybdenum hydroxylases aldehyde oxidase (AO) and xanthine dehydrogenase/ oxidase (XDH) for involvement in ALS. Of particular interest, one gene identified as encoding aldehyde oxidase is shown to map to 2q33, a region recently shown to contain a gene responsible for a familial form of ALS with autosomal recessive inheritance (FALS-AR). The AO gene appears to be located within 280,000 bp of simple sequence repeat marker D2S116, which shows no recombination with the FALS-AR locus. The AO gene is highly expressed in glial cells of human spinal cord. In addition, we mapped a gene for XDH to 2p22, a region previously shown to contain a highly homologous but different form of XDH. Neither of these XDH genes appears to be highly expressed in human spinal cord. This evidence suggests that AO may be a candidate gene for FALS-AR.

The tissue specificity of the intra-acrosomal protein SP-10 was examined by Northern blot and polymerase chain reaction (PCR) analysis. Messenger RNA from 36 tissues in the female baboon (Papio papio) was isolated, separated on agarose gels, transferred to nylon, and probed with either SP-10, beta-actin, or cyclophilin cDNA. Northern blots, which were processed at both low and high stringency, showed SP-10 to be expressed exclusively in the testis. The mRNA from each tissue was also reverse transcribed, and both SP-10 and beta-actin were amplified by PCR from the resulting cDNA. Ethidium bromide-stained agarose gels of the SP-10 PCR products showed three clear bands from the testis but no co-migrating bands from the other tissues. Southern blots of the PCR products showed that only the three bands in the testis were related to SP-10. The data demonstrate that the SP-10 gene products are testis specific, a characteristic essential for a contraceptive vaccine candidate molecule.

To address the possibility that electron transport is a biologically significant source of superoxide anion (O2 radical anion) during exposure to hyperoxia in vivo, we constricted Saccharomyces cerevisiae strains with selective disruptions in the gene encoding the mitochondrial manganese-containing superoxide dismutase (Mn-SOD) and/or genes encoding proteins critical for complexes in electron transport. We hypothesized that complete absence of electron transport would restore growth in hyperoxia to a Mn-SOD-deficient yeast. We found that yeast deficient in Mn-SOD activity failed to grow normally in hyperoxia (95% O2, 5% CO2). In contrast, Mn-SOD-deficient yeast with complete absence of electron transport (the Rho0 state) grew normally in hyperoxia. By comparison, Mn-SOD-deficient yeast which were deficient only in cytochrome-c-oxidase, the terminal step in electron transport, had only partially restored growth in hyperoxia. Our results indicate that electron transport is a major source of O2 radical anion in vivo, and that the principal site of this O2 radical anion production is proximal to the cytochrome-c-oxidase complex.

In this study, cDNAs encoding the intraacrosomal protein SP‐10 were cloned and sequenced from baboon (Papio papio) and macaque (Macaca fasicularis) testis libraries and the sequence compared to that of human SP‐10. Two alternatively spliced SP‐10 cDNAs were obtained from both baboon and macaque testis libraries. The two cDNAs in each species contained open reading frames encoding proteins of exactly 285 and 251 amino acids. A 98% homology between baboon and macaque SP‐10 was found at the protein and DNA levels. An 85% and 89% homology between baboon and macaque SP‐10 and human SP‐10 was present at the protein and DNA level, respectively. A mouse intraacrosomal protein, MSA‐63, considered to be an SP‐10 homologue, exhibited an overall 53% homology to nonhuman primate SP‐10 and a 60% homology to human SP‐10 at the protein level. Polymerase chain reaction analysis of testis mRNA confirmed the existence of two alternately spliced SP‐10 mRNAs in both nonhuman primates. Primer extension analysis indicated a common major transcriptional start site in baboon, macaque, and human SP‐10 67 nucleotides 5′ to the ATG codon. The amino acid sequence data for nonhuman primate SP‐10s suggest that antibodies generated by vaccinating baboons and macaques with human SP‐10 will likely recognize nonhuman primate SP‐10, supporting the testing of an SP‐10 contraceptive vaccine based on human SP‐10 in these nonhuman primate models. © 1993 Wiley‐Liss, Inc.

We isolated cDNAs encoding xanthine dehydrogenase (XD; xanthine:NAD+ oxidoreductase, EC 1.1.1.204) from a human liver cDNA library. The complete nucleotide sequence of human XD was determined; the deduced amino acid sequence encoded a protein of 1336 amino acid residues of M(r) 147,782. Human XD possessed many of the signature sequences typical of XDs from flies and rodents, including an unusual cysteine distribution, a potential 2Fe/2S binding site, and a putative molybdopterin cofactor binding domain. Analysis of potential NAD binding sites suggested a simple hypothesis for the conversion of human XD into the oxygen metabolite forming xanthine oxidase (XO; xanthine:oxygen oxidoreductase, EC 1.1.3.22). Using a human XD complementary RNA hybridization probe, we found a 5100-base RNA in human liver by RNA blot-hybridization analysis. This RNA exhibited tissue-specific distribution that may be pertinent to XD- and XO-mediated oxygen radical injury in ischemia/reperfusion and inflammation. A second 4500-base RNA was detected in some tissues and may arise through differential transcription termination.

PROBLEM:To develop a method to measure a recombinant sperm protein, SP-10, during scale up and purification for a contraceptive vaccine formulation.METHOD:A quantitative assay method for the human intraacrosomal protein SP-10 was developed utilizing the format of indirect capture enzyme-linked immunosorbent assay (ELISA). A SP-10 specific monoclonal antibody mAb, MHS-10, was used as the capture antibody. Two recognition reagents, a rabbit polyclonal anti-SP-10 antisera (pAb) and a biotin-labeled mAb, MHS-10, were used as the recognition antibodies, respectively. A SP-10 recombinant fusion protein consisting of 125 SP-10 amino acids linked to glutathione transferase was used as a working SP-10 standard. The coefficient of variance for the assay system using the rabbit pAb was in the range of 0.099 to 0.157, and for the assay system using the biotinylated mAb MHS-10 was in the range of 0.081 to 0.084.RESULTS:Employing biotinylated MHS-10 as the recognition antibody, it was found that the native SP-10 molecule had more than one MHS-10 epitope. The concentration of SP-10 in a pool of human sperm extracts was found to be approximately 1% of the total proteins, assayed by both of the recognition antibody systems.CONCLUSIONS:The assay system described is useful to monitor the yield of recombinant SP-10 during scale-up production of the SP-10 vaccine.

We show here that SNF1 and SSN6 are required for derepression of the glucose-repressible yeast genes COX6 and CYC1, which encode the mitochondrial proteins cytochrome c oxidase subunit VI and iso-1-cytochrome c, respectively. In an snf1 mutant genetic background, the transcription of both COX6 and CYC1 continued to be repressed after cells were shifted into derepressing media. In an ssn6 mutant genetic background, both COX6 and CYC1 were expressed constitutively at high levels in repressing media. SSN6 acted epistatically to SNF1 in the regulation of both cytochrome genes. These findings are similar to previous findings on the effects of SNF1 and SSN6 on SUC2 expression in Saccharomyces cerevisiae and are consistent with a model proposing that SNF1 exerts its effect through SSN6 on COX6 and CYC1.

cDNAs coding for the intra-acrosomal protein SP-10 were cloned and characterized as a first step in understanding the expression of this antigen during spermatogenesis. Three overlapping SP-10-specific cDNAs were isolated from a human testes cDNA expression library. These cDNAs hybridized to a 1.35-kb mRNA that was present in human testes but was not found in liver or placenta. Complete sequencing of these cDNAs, designated SP-10-5, SP-10-8, and SP-10-10, produced an 1117-bp sequence containing a 265-amino acid-coding region for the SP-10 protein. Hydrophobicity plots generated from the deduced amino acid sequence showed a very hydrophobic amino terminus characteristic of a signal peptide. Sequence data showed that three different amino acid repeats occurred a total of 16 times in the central third of the SP-10 protein. Interestingly, cDNA SP-10-10 has an internal 57-base pair (19 amino acids) in-frame deletion that is not present in SP-10-5, suggesting that alternative splicing generates more than one SP-10 mRNA. The SP-10 protein appears to be a unique acrosomal protein, based on previous immunohistological data and the observation that SP-10 cDNA sequences did not show any significant homology to other sequences found in the Genbank, National Biomedical Research Foundation, or Swiss sequence banks. A recombinant SP-10 fusion protein was produced in an Escherichia coli expression vector and used to generate a polyclonal antiserum. This antiserum stained the acrosomal cap in situ and reacted with a similar set of peptides on Western blots as did a monoclonal antibody to SP-10.