Cellular H2 Research Articles

Maize catalases are recruited by a virus to modulate viral multiplication and infection.

Given the detrimental effects of excessive reactive oxygen species (ROS) accumulation in plant cells, various antioxidant mechanisms have evolved to maintain cellular redox homeostasis, encompassing both enzymatic components (e.g., catalase, superoxide dismutase) and non-enzymatic ones. Despite extensive research on the role of antioxidant systems in plant physiology and responses to abiotic stresses, the potential exploitation of antioxidant enzymes by plant viruses to facilitate viral infection remains insufficiently addressed. Herein, we demonstrate that maize catalases (ZmCATs) exhibited up-regulated enzymatic activities upon sugarcane mosaic virus (SCMV) infection. ZmCATs played crucial roles in SCMV multiplication and infection by catalysing the decomposition of excess cellular H2 O2 and promoting the accumulation of viral replication-related cylindrical inclusion (CI) protein through interaction. Peroxisome-localized ZmCATs were found to be distributed around SCMV replication vesicles in Nicotiana benthamiana leaves. Additionally, the helper component-protease (HC-Pro) of SCMV interacted with ZmCATs and enhanced catalase activities to promote viral accumulation. This study unveils a significant involvement of maize catalases in modulating SCMV multiplication and infection through interaction with two viral factors, thereby enhancing our understanding regarding viral strategies for manipulating host antioxidant mechanisms towards robust viral accumulation.

A Single-Atom Manganese Nanozyme Mn-N/C Promotes Anti-Tumor Immune Response via Eliciting Type I Interferon Signaling.

Tumor microenvironment (TME)-induced nanocatalytic therapy is a promising strategy for cancer treatment, but the low catalytic efficiency limits its therapeutic efficacy. Single-atom catalysts (SACs) are a new type of nanozyme with incredible catalytic efficiency. Here, a single-atom manganese (Mn)-N/C nanozyme is constructed. Mn-N/C catalyzes the conversion of cellular H2O2 to ∙OH through a Fenton-like reaction and enables the sufficient generation of reactive oxygen species (ROS), which induces immunogenic cell death (ICD) of tumor cells and significantly promotes CD8+T anti-tumor immunity. Moreover, RNA sequencing analysis reveals that Mn-N/C treatment activates type I interferon (IFN) signaling, which is critical for Mn-N/C-mediated anti-tumor immune response. Mechanistically, the release of cytosolic DNA and Mn2+ triggered by Mn-N/C collectively activates the cGAS-STING pathway, subsequently stimulating type I IFN induction. A highly efficient single-atom nanozyme, Mn-N/C, which enhances anti-tumor immune response and exhibits synergistic therapeutic effects when combined with the anti-PD-L1 blockade, is proposed.

Hydrogen sulfide signalling in neurodegenerative diseases.

The gaseous neurotransmitter hydrogen sulfide (H2 S) exerts neuroprotective efficacy in the brain via post-translational modification of cysteine residues by sulfhydration, also known as persulfidation. This process is comparable in biological impact to phosphorylation and mediates a variety of signalling events. Unlike conventional neurotransmitters, H2 S cannot be stored in vesicles due to its gaseous nature. Instead, it is either locally synthesized or released from endogenous stores. Sulfhydration affords both specific and general neuroprotective effects and is critically diminished in several neurodegenerative disorders. Conversely, some forms of neurodegenerative disease are linked to excessive cellular H2 S. Here, we review the signalling roles of H2 S across the spectrum of neurodegenerative diseases, including Huntington's disease, Parkinson's disease, Alzheimer's disease, Down syndrome, traumatic brain injury, the ataxias, and amyotrophic lateral sclerosis, as well as neurodegeneration generally associated with ageing.

RHY‐1 Promotes Hydrogen Sulfide Tolerance

Once known best for its toxic effects, recent work has shown that hydrogen sulfide (H2 S) has many roles as a cellular messenger. For example, both endogenously produced and exogenously supplied H2 S protect against cellular damage and death associated with ischemia/reperfusion injury in mammals. The mechanistic relationship between beneficial and toxic effects of H2 S is not understood. We have developed C. elegans as a model to understand the mechanistic basis of H2 S effects in animals. In addition to facile genetic and genomic tools, using C. elegans provides the ability to precisely control both genotype and cellular H2 S exposure. C. elegans grown in 50 ppm H2 S are long-lived and resistant to hypoxia-induced disruption of protein homeostasis. The early transcriptional response to H2 S requires the C. elegans orthologue of the hypoxia-inducible transcription factor, hif-1. HIF-1 promotes survival in H2 S, at least in part, by upregulating expression of sqrd-1, which encodes the sulfide-quinone oxidoreductase. SQRD-1 catalyzes the first step in the mitochondrial oxidation of H2 S. In an unbiased forward genetic screen, we found that expression of rhy-1 suppresses lethality of hif-1 mutant animals in 50 ppm H2 S. RHY-1 is an integral-membrane ER protein with predicted acyltransferase (ACYL3) activity. The ACYL3 family is large and conserved across species from bacteria to primates, but the function of these enzymes is largely unstudied. RHY-1 was first characterized as a negative regulator of HIF-1, and our data indicate that it also promotes survival in H2 S independently of HIF-1. To understand RHY-1 function we have used biotinylation by antibody recognition (BAR) to identify proteins that interact with RHY-1. We show that RHY-1 directly associates with CYSL proteins, which are orthologues of cystathionine β-synthase. Mutations in cysl-1 abrogate RHY-1 function to regulate HIF-1 and to promote survival independently of HIF-1. We have also identified a novel methyltransferase, RIPS-1, that is required for RHY-1 to promote survival in H2 S but which is dispensable for the proper regulation of HIF-1. Together, our data show that RHY-1 promotes survival in H2 S through a novel mechanism which can bypass the only known catabolic pathway for H2 S in vivo. Understanding the mechanism of RHY-1 activity in H2 S will begin to elucidate the relationship between H2 S signaling and toxicity, and could lead to new therapeutic strategies to modulate endogenous H2 S levels.

Stimuli-Responsive Manganese Single-Atom Nanozyme for Tumor Therapy via Integrated Cascade Reactions.

The single-atom enzyme (SAE) is a novel type of nanozyme that exhibits extraordinary catalytic activity. Here, we constructed a PEGylated manganese-based SAE (Mn/PSAE) by coordination of single-atom manganese to nitrogen atoms in hollow zeolitic imidazolate frameworks. Mn/PSAE catalyzes the conversion of cellular H2 O2 to . OH through a Fenton-like reaction; it also promotes the decomposition of H2 O2 to O2 and continuously catalyzes the conversion of O2 to cytotoxic . O2- via oxidase-like activity. The catalytic activity of Mn/PSAE is more pronounced in the weak acidic tumor environment; therefore, these cascade reactions enable the sufficient generation of reactive oxygen species (ROS) and effectively kill tumor cells. The prominent photothermal conversion property of the amorphous carbon can be utilized for photothermal therapy. Hence, Mn/PSAE exhibits significant therapeutic efficacy through tumor microenvironment stimulated generation of multiple ROS and photothermal activity.

The role of oxidative stress in the growth of the indoor mold Cladosporium cladosporioides under water dynamics.

Moisture is one of the critical abiotic factors that can affect mold growth. Indoor humidity is typically fluctuating, which renders a transient water supply for mold growth. Understanding mold growth under water dynamics and its underlying mechanisms can help in the development of novel and sustainable mold prevention strategies. In this study, pre-germination and germinated spores of Cladosporium cladosporioides were exposed to daily wet-dry cycles with different combinations of wetting and drying duration. Afterward, growth delay, cellular H2 O2 concentration, and catalase (CAT) activity were measured and compared. We found that underdaily wet-dry cycles, the longer the growth delay was observed, the higher the cellular H2 O2 concentration was detected, with the 12-12 wet-dry cycle (12-hour wet and 12-hour dry) showing the longest growth delay and highest cellular H2 O2 production. A positive correlation between cellular H2 O2 concentration and growth delay was suggested by Pearson correlation coefficient and linear regression analysis (P<.0001, R2 =0.85). Furthermore, under dailywet-dry cycles, molds derived from pre-germination spores generally exhibited shorter growth delay, lower cellular H2 O2 concentration, and higher CAT activity than molds developed from germinated spores. These results together suggest that the growth delay of C.cladosporioides under water dynamics is associated with oxidative stress.

Functional roles of norCB in Pseudomonas aeruginosa ATCC 9027 under aerobic conditions.

Nitric oxide (NO) reductase (NorCB) of Pseudomonas aeruginosa is an essential enzyme that metabolizes NO and alleviates anaerobic NO toxicity during denitrification processes under anaerobic conditions. However, the molecular functions of norCB in the presence of oxygen are poorly understood. This study utilized norCB knockout from P. aeruginosa ATCC 9027 to analyze the resulting phenotypic changes of ΔnorCB in comparison to the wild-type parental strain (WT) and the complementary strain (ΔnorCB-com). The results demonstrated an increase in planktonic growth and biofilm formation by ΔnorCB compared to WT and ΔnorCB-com in the presence of isothiazolones under aerobic conditions. Deletion of norCB led to increased swimming ability and decreased pyocyanin production. Inactivation of norCB also led to an increase of cellular H2 O2 concentration due to decreased activity of its catalases. In addition, the deletion of norCB also influenced the relative expressions of several other genes, including norD, nirS, hmgA, and hpd. These findings provide preliminary evidence that norCB in P. aeruginosa plays an essential role in bacterial life process under aerobic conditions and improves the application of denitrification in the next step.

Extended hypoxia-mediated H2 S production provides for long-term oxygen sensing.

Numerous studies have shown that H2 S serves as an acute oxygen sensor in a variety of cells. We hypothesize that H2 S also serves in extended oxygen sensing. Here, we compare the effects of extended exposure (24-48hours) to varying O2 tensions on H2 S and polysulphide metabolism in human embryonic kidney (HEK 293), human adenocarcinomic alveolar basal epithelial (A549), human colon cancer (HTC116), bovine pulmonary artery smooth muscle, human umbilical-derived mesenchymal stromal (stem) cells and porcine tracheal epithelium (PTE) using sulphur-specific fluorophores and fluorometry or confocal microscopy. All cells continuously produced H2 S in 21% O2 and H2 S production was increased at lower O2 tensions. Decreasing O2 from 21% to 10%, 5% and 1% O2 progressively increased H2 S production in HEK293 cells and this was partially inhibited by a combination of inhibitors of H2 S biosynthesis, aminooxyacetate, propargyl glycine and compound 3. Mitochondria appeared to be the source of much of this increase in HEK 293 cells. H2 S production in all other cells and PTE increased when O2 was lowered from 21% to 5% except for HTC116 cells where 1% O2 was necessary to increase H2 S, presumably reflecting the hypoxic environment in vivo. Polysulphides (H2 Sn , where n=2-7), the key signalling metabolite of H2 S also appeared to increase in many cells although this was often masked by high endogenous polysulphide concentrations. These results show that cellular H2 S is increased during extended hypoxia and they suggest this is a continuously active O2 -sensing mechanism in a variety of cells.

The Molecular Proceedings of Biological Hydrogen Turnover.

Over the past two decades, the bioinorganic chemistry of hydrogenases has attracted much interest from basic and applied research. Hydrogenases are highly efficient metalloenzymes that catalyze the reversible reduction of protons to molecular hydrogen (H2) in all domains of life. Their iron- and nickel-based cofactors represent promising blueprints for the design of biomimetic, synthetic catalysts. In this Account, we address the molecular proceedings of hydrogen turnover with [FeFe]-hydrogenases. The active site cofactor of [FeFe]-hydrogenases ("H-cluster") comprises a unique diiron complex linked to a [4Fe-4S] cluster via a single cysteine. Since it was discovered that a synthetic analogue of the diiron site can be incorporated into apoprotein in vitro to yield active enzyme, significant progress has been made toward a comprehensive understanding of hydrogenase catalysis. The diiron site carries three to four carbon monoxide (CO) and two cyanide (CN-) ligands that give rise to intense infrared (IR) absorption bands. These bands are sensitive reporters of the electron density across the H-cluster, which can be addressed by infrared spectroscopy to follow redox and protonation changes at the cofactor. Synthetic variation of the metal-bridging dithiolate ligand at the diiron site, as well as site-directed mutagenesis of amino acids, provides access to the proton pathways toward the cofactor. Quantum chemical calculations are employed to specifically assign IR bands to vibrational modes of the diatomic ligands and yield refined H-cluster geometries. Here, we provide an overview of recent research on [FeFe]-hydrogenases with emphasis on experimental and computational IR studies. We describe advances in attenuated total reflection Fourier transform infrared spectroscopy (ATR FTIR) and protein film electrochemistry, as well as density functional theory (DFT) calculations. Key cofactor species are discussed in terms of molecular geometry, redox state, and protonation. Isotope editing is introduced as a tool to evaluate the cofactor geometry beyond the limits of protein crystallography. In particular, the role of proton-coupled electron transfer (PCET) in the generation of catalytically relevant redox species is addressed. We propose that site-selective protonation of the H-cluster biases surplus electrons either to the [4Fe-4S] cluster or to the diiron site. Protonation of the [4Fe-4S] cluster prevents premature reduction at the diiron site and stabilizes a reactive, terminal hydride. The observed H-cluster species are assigned to rapid H2 conversion or to reactions possibly involved in activity regulation and cellular H2 sensing. In the catalytic cycle of [FeFe]-hydrogenases, an H-cluster geometry is preserved that features a bridging CO ligand. PCET levels the redox potential for two steps of sequential cofactor reduction. The concept of consecutive PCET at a geometrically inert cofactor with tight control of electron and proton localization may inspire the design of a novel generation of biomimetic catalysts for the production of H2 as a fuel.

Potential role for Streptococcus gordonii-derived hydrogen peroxide in heme acquisition by Porphyromonas gingivalis.

Streptococcus gordonii, an accessory pathogen and early colonizer of plaque, co-aggregates with many oral species including Porphyromonas gingivalis. It causes α-hemolysis on blood agar, a process mediated by H2 O2 and thought to involve concomitant oxidation of hemoglobin (Hb). Porphyromonas gingivalis has a growth requirement for heme, which is acquired mainly from Hb. The paradigm for Hb heme acquisition involves the initial oxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb), followed by heme release and extraction through the actions of K-gingipain protease and/or the HmuY hemophore-like protein. The ability of S.gordonii to mediate Hb oxidation may potentially aid heme capture during co-aggregation with P.gingivalis. Hemoglobin derived from zones of S.gordonii α-hemolysis was found to be metHb. Generation of metHb from oxyHb by S.gordonii cells was inhibited by catalase, and correlated with levels of cellular H2 O2 production. Generation of metHb by S.gordonii occurred through the higher Hb oxidation state of ferrylhemoglobin. Heme complexation by the P.gingivalis HmuY was employed as a measure of the ease of heme capture from metHb. HmuY was able to extract iron(III)protoporphyrin IX from metHb derived from zones of S.gordonii α-hemolysis and from metHb generated by the action of S.gordonii cells on isolated oxyHb. The rate of HmuY-Fe(III)heme complex formation from S.gordonii-mediated metHb was greater than from an equivalent concentration of auto-oxidized metHb. It is concluded that S.gordonii may potentially aid heme acquisition by P.gingivalis by facilitating metHb formation in the presence of oxyHb.

The significance of glutathione and ascorbate in modulating the retrograde high light response in Arabidopsis thaliana leaves.

Retrograde signals from the chloroplast control expression of nuclear genes. A large fraction of these genes is affected rapidly upon light intensity shifts. This study was designed to address the interdependence of signaling pathways involved in the rapid high light response and redox and reactive oxygen species signaling by exploiting the glutathione and ascorbate deficient mutants pad2 and vtc1. In the first set of experiments the transcriptional response of the two transcription factors ERF6 and ERF105 that had previously been shown to rapidly respond to light was shown to be deregulated in the pad2 mutant but not in the vtc1 background. The transcriptional response after combining the low-to-high light transfer with methylviologen pretreatment further demonstrated the significance of glutathione in strongly modulating the retrograde response. Transcripts encoding small heat shock proteins (HSP17.4, HSP176a, HSP20-like1 and HSP20-like2) and the lipid transfer protein LTP3 were taken as markers responding to the combinatorial treatment in wild type, and most strongly in pad2 in high light or upon methylviologen treatment. A correlation with H2 O2 accumulation was not observed. It is concluded that glutathione-dependent processes participate in light-triggered rapid gene regulation independent on cellular H2 O2 .

Superoxide serves as a putative signal molecule for plant cell division: overexpression of CaRLK1 promotes the plant cell cycle via accumulation of O2- and decrease in H2 O2.

Reactive oxygen species (ROS) exert both positive and negative effects on plant growth and development and therefore receive a great deal of attention in current research. A hot pepper, Capsicum annuum receptor-like kinase 1 (CaRLK1) was ectopically expressed in Nicotiana tabacum BY-2 cell and Nicotiana benthamiana plants. This ectopic expression of CaRLK1 enhanced cell division and proliferation in both heterologous systems. Apparently, CaRLK1 is involved in controlling the cell cycle, possibly by inducing expressions of cyclin B1, cyclin D3, cyclin-dependent protein kinase 3, condensin complex subunit 2 and anaphase-promoting complex subunit 11 genes. CaRLK1 overexpression also increased transcript accumulation of NADPH oxidase genes, generation of O2- and catalase (CAT) activity/protein levels. In parallel, it decreased cellular H2 O2 levels and cell size. Treatment with Tiron or diphenyleneiodonium (DPI) both decreased the cell division rate and O2- concentrations, but increased cellular H2 O2 levels. Tobacco BY-2 cells overexpressing CaRLK1 were more sensitive to amino-1,2,4-triazole (3-AT), a CAT inhibitor, than control cells, suggesting that the increased H2 O2 levels may not function as a signal for cell division and proliferation. Overexpression of CaRLK1 stimulated progression of the cell cycle from G0 /G1 phase into the S phase. It is concluded that the CaRLK1 protein plays a pivotal role in controlling the level of O2- as signaling molecule which promotes cell division, concomitant with a reduction in H2 O2 by the induction of CAT activity/protein.

Human Umbilical Cord Wharton's Jelly Stem Cell Conditioned Medium Induces Tumoricidal Effects on Lymphoma Cells Through Hydrogen Peroxide Mediation.

Several groups have reported that human umbilical cord Wharton's jelly stem cells (hWJSCs) possess unique tumoricidal properties against many cancers. However, the exact mechanisms as to how hWJSCs inhibit tumor growth are not known. Recent evidence suggests that exposure of cancer cells to high hydrogen peroxide (H2 O2 ) levels from H2 O2 -releasing drugs causes their death. We therefore explored whether the tumoricidal effect of hWJSCs on lymphoma cells was mediated via H2 O2 . We first exposed lymphoma cells to six different molecular weight cut-off (MWCO) concentrates of hWJSC-conditioned medium (hWJSC-CM) (3, 5, 10, 30, 50, 100 kDa) for 48 h. Since, the 3 kDa-MWCO concentrate showed the greatest cell inhibition we then investigated whether the tumoricidal effect of the specific 3 kDa-MWCO concentrate on two different lymphoma cell lines (Ramos and Toledo) was mediated via accumulation of H2 O2 . We used a battery of assays (MTT, propidium iodide, mitochondria membrane potential, apoptosis, cell cycle, oxidative stress enzymes, hydrogen peroxide, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation) to test this mechanism. The hWJSC-CM-3 kDa MWCO concentrate significantly decreased cell viability and mitochondrial membrane potential and increased cell death and apoptosis in both lymphoma cell lines. There were significant increases in superoxide dismutase with concomitant decreases in glutathione peroxidase, catalase, and thioredoxin peroxidase activities. H2 O2 levels, mitochondrial superoxide, hydroxyl radical, peroxynitrile anion, and lipid peroxidation were also significantly increased in both lymphoma cell lines. The results suggested that the hWJSC-CM-3 kDa MWCO concentrate regulates cellular H2 O2 leading to a tumoricidal effect and may thus be a promising anti-lymphoma agent. J. Cell. Biochem. 117: 2045-2055, 2016. © 2016 Wiley Periodicals, Inc.

High light-induced hydrogen peroxide production in Chlamydomonas reinhardtii is increased by high CO2 availability.

The production of reactive oxygen species (ROS) is an unavoidable part of photosynthesis. Stress that accompanies high light levels and low CO2 availability putatively includes enhanced ROS production in the so-called Mehler reaction. Such conditions are thought to encourage O2 to become an electron acceptor at photosystemI, producing the ROS superoxide anion radical (O2·-) and hydrogen peroxide (H2 O2 ). In contrast, here it is shown in Chlamydomonas reinhardtii that CO2 depletion under high light levels lowered cellular H2 O2 production, and that elevated CO2 levels increased H2 O2 production. Using various photosynthetic and mitochondrial mutants of C.reinhardtii, the chloroplast was identified as the main source of elevated H2 O2 production under high CO2 availability. High light levels under low CO2 availability induced photoprotective mechanisms called non-photochemical quenching, or NPQ, including state transitions (qT) and high energy state quenching (qE). The qE-deficient mutant npq4 produced more H2 O2 than wild-type cells under high light levels, although less so under high CO2 availability, whereas it demonstrated equal or greater enzymatic H2 O2 -degrading capacity. The qT-deficient mutant stt7-9 produced the same H2 O2 as wild-type cells under high CO2 availability. Physiological levels of H2 O2 were able to hinder qT and the induction of state2, providing an explanation for why under high light levels and high CO2 availability wild-type cells behaved like stt7-9 cells stuck in state1.

Hydrogen isotope fractionation during H2/CO2 acetogenesis: hydrogen utilization efficiency and the origin of lipid‐bound hydrogen

ABSTRACTHydrogen metabolism was studied in the anaerobic bacterium, Sporomusa sp. strain DMG 58, by measuring natural abundance levels of deuterium in H2, H2O, and individual fatty acids during acetogenic growth on H2/CO2. Four cultures were grown, each in medium with a distinct hydrogen‐isotopic composition (δD‐H2O). The δD value of H2 was quantified in the residual gas exiting the growth chambers and found to decrease concurrently with net H2 consumption, indicating rapid isotope exchange between H2 and H2O. An isotopic mass balance was used to constrain the efficiency with which H2 was activated by the cell and the reducing equivalents catabolized, which we term the H2 utilization efficiency. Results indicate that H2 utilization efficiency in these cultures is less than 20% during the growth phase, and less than 2% after the growth phase. The gross rate of cellular H2 activation was similar in the growth phase and afterward. Biomass harvested at the end of each experiment was used to analyse the D/H of individual membrane lipids. Values of δD were highly correlated between lipids and water (δD‐lipids = 0.59 × δD‐water – 381‰; R2 = 0.995), indicating the source of lipid hydrogen is in isotopic equilibrium with water. Results are consistent with two possibilities: (i) water is the sole source of hydrogen to lipids, and the fractionation during biosynthesis is significantly larger than previously observed (α = 0.59), or (ii) hydrogen from H2 is incorporated into lipids, but only after reaching isotopic equilibrium with H2O. Fatty acids were strongly depleted in deuterium relative to all other organisms studied thus far, and such large depletions may prove useful as biomarkers for studying H2 cycling in anoxic environments as well as in the geological record.

Sustained photobiological hydrogen gas production upon reversible inactivation of oxygen evolution in the green alga Chlamydomonas reinhardtii.

The work describes a novel approach for sustained photobiological production of H(2) gas via the reversible hydrogenase pathway in the green alga Chlamydomonas reinhardtii. This single-organism, two-stage H(2) production method circumvents the severe O(2) sensitivity of the reversible hydrogenase by temporally separating photosynthetic O(2) evolution and carbon accumulation (stage 1) from the consumption of cellular metabolites and concomitant H(2) production (stage 2). A transition from stage 1 to stage 2 was effected upon S deprivation of the culture, which reversibly inactivated photosystem II (PSII) and O(2) evolution. Under these conditions, oxidative respiration by the cells in the light depleted O(2) and caused anaerobiosis in the culture, which was necessary and sufficient for the induction of the reversible hydrogenase. Subsequently, sustained cellular H(2) gas production was observed in the light but not in the dark. The mechanism of H(2) production entailed protein consumption and electron transport from endogenous substrate to the cytochrome b(6)-f and PSI complexes in the chloroplast thylakoids. Light absorption by PSI was required for H(2) evolution, suggesting that photoreduction of ferredoxin is followed by electron donation to the reversible hydrogenase. The latter catalyzes the reduction of protons to molecular H(2) in the chloroplast stroma.

Nonlysosomal, pre-Golgi degradation of unassembled asialoglycoprotein receptor subunits: a TLCK- and TPCK-sensitive cleavage within the ER.

The human asialoglycoprotein receptor subunit H2a is cotranslationally inserted into the ER membrane. When expressed together with subunit H1 in mouse fibroblasts part forms a hetero-oligomer that is transported to the cell surface, but when expressed alone it is all rapidly degraded. Degradation is insensitive to lysosomotropic agents and the undegraded precursor is last detected in the ER region of the cell. Small amounts of an intermediate 35-kD degradation product can be detected (Amara, J. F., G. Lederkremer, and H. F. Lodish. 1989. J. Cell Biol. 109:3315). We show here that the oligosaccharides on both precursor H2a and the 35-kD fragment are Man6-9GlcNAc2, structures typically found in pre-Golgi compartments. Subcellular fractionation shows that the intermediate degradation product does not cofractionate with the lysosomal enzyme beta-galactosidase, but is found in a part of the ER that contains ribosomes. Thus the intermediate degradation product is localized in the ER, indicating that the initial degradation event does take place in the ER. All degradation of H2a, including the initial endoproteolytic cleavage generating the 35-kD intermediate, is blocked by the protease inhibitors N-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone. These drugs do not inhibit ER-to-Golgi transport of H1. Depleting the cells of ATP or inhibiting protein synthesis allows the initial endoproteolytic cleavage to occur, but blocks further degradation of the 35-kD intermediate; thus we can convert all cellular H2 into the 35-kD intermediate. Approximately 50% of H2b, a splicing variant differing from H2a by a five amino acid deletion, can be transported to the cell surface, and the rest appears to be degraded by the same pathway as H2a, both when expressed alone in fibroblasts and together with H1 in HepG2 cells. Addition of N-tosyl-L-lysine chloromethyl ketone or N-tosyl-L-phenylalanine chloromethyl ketone blocks degradation of the approximately 50% that is not transported, but does not affect the fraction of H2b that moves to the Golgi region. Thus, a protein destined for degradation will not be transported to the Golgi region if degradation is inhibited.

In situ H2 production and utilization by natural populations of N2-fixing blue-green algae

Contemporaneous in situ acetylene-reduction, 15N2-fixation, and 3H2-exchange assays reveal parallel patterns of N2 fixation and H2 utilization in natural populations of the blue-green algae Anabaena and Aphanizomenon. As spring and summer blooms progress, increasing ratios of acetylene reduction versus 15N2 fixation closely follow elevated rates of cellular H2 utilization. Both acetylene-reduction and H2-utilization rates were largely attributable to blue-green algae as opposed to associated bacteria and other phytoplankton. It is concluded that elevated H2 utilization reflects increased H2 production via nitrogenase. This can be substantiated by monitoring rising acetylene-reduction versus 15N2-fixation ratios during bloom development. Simultaneous deployment of the above techniques provides evidence for (a) in situ H2 production and (b) seasonal trends in rates of H2 production among natural blue-green algal populations.