Protein structure refinement: Streptomyces griseus serine protease A at 1.8 A resolution.

The crystal structure of the bacterial serine protease from Streptomyces griseus (SGPA) has been refined at 1.8 Å resolution by a restrained parameter least-squares procedure (Konnert, 1976) to a conventional R factor of 0.139 for 12662 statistically significant reflections [I > 3σ(I)]. The number of variable parameters in the final model was 5912 which included positional and individual thermal parameters of the enzyme, and positions, B factors and occupancies of 175 solvent molecules. The algorithm used for this refinement allows for the simultaneous restraint on bond distances and distances related to interbond angles, the coplanarity of atoms in planar groups, the conservation of chirality of asymmetric centres, non-bonded contact distances, conformational torsional angles and individual isotropic temperature factors.The refined structure of SGPA differs from ideal bond lengths by an overall root-mean-square deviation of 0.02 Å; the corresponding value for angle distances is 0.038 Å. Comparison of the phase angles for the shell of data, 8.0 to 2.8 Å, between the multiple isomorphous replacement phases (Brayer et al., 1978a) and the refined phases, indicates an overall difference (r.m.s.) of 56.6 °. The average conformational angle of the peptide bond (ω) is 179.7 ° (root-mean-square deviation ± 2.5 °) for the 180 peptide bonds of SGPA. Of the 175 solvent molecules included during the course of the refinement, 22 with occupancies ranging from 1.00 to 0.38 are located in the active site and the substrate binding region. It was not until these water molecules were included in the refinement process that the active Ser195 adopted its final conformation (χ¹ = ?77 °). The resulting distance from O^γ of Ser195 to N^ε2 of His57 is 3.1 Å, which, when taken with the observed distortion from linearity (50 °), indicates a rather weak interaction.     

ACTION OF THE NEUROTOXIN KAINIC ACID ON HIGH AFFINITY UPTAKE OF l-GLUTAMIC ACID IN RAT BRAIN SLICES

Kainic acid is a linear competitive inhibitor (K_is 250 μm ) of the ‘high affinity’ uptake of l -glutamic acid into rat brain slices. Kainic acid inhibits the ‘high affinity’ uptake of l -glutamic, d -aspartic and l -aspartic acids to a similar extent. Kainic acid is not actively taken up into rat brain slices and is thus not a substrate for the ‘high affinity’ acidic amino acid transport system or any other transport system in rat brain slices. Kainic acid (300 μm ) does not influence the steady-state release or potassium-stimulated release of preloaded d -aspartic acid from rat brain slices. Kainic acid binds to rat brain membranes in the absence of sodium ions in a manner indicating binding to a population of receptor sites for l -glutamic acid. Only quisqualic and l -glutamic acid inhibit kainic acid binding in a potent manner. The affinity of kainic acid for these receptor sites appears to be some 4 orders of magnitude higher than for the ‘high affinity’l -glutamic acid transport carrier. Dihydrokainic acid is approximately twice as potent as kainic acid as an inhibitor of ‘high affinity’l -glutamic acid uptake but is some 500 times less potent as an inhibitor of kainic acid binding and at least 1000 times less potent as a convulsant of immature rats on intraperitoneal injection. Dihydrokainic acid might be useful as a ‘control uptake inhibitor’ for the effects of kainic acid on ‘high affinity’l -glutamic acid uptake since it appears to have little action on excitatory receptors. N-Methyl-d -aspartic acid is a potent convulsant of immature rats, but does not inhibit kainic acid binding or ‘high affinity’l -glutamic acid uptake. N-Methyl-d -aspartic acid might be useful as a ‘control excitant’ that activates different excitatory receptors to kainic acid and does not influence ‘high affinity’l -glutamic acid uptake.     

Mapping of chloroplast genes involved in chloroplast ribosome biogenesis in Chlamydomonas reinhardtii

Summary Chloroplast gene mutations which confer antibiotic resistance on chloroplast ribosomes of the green alga Chlamydomonas reinhardtii have been tested for allelism and mapped by recombination analysis of progeny from biparental zygote clones. Thirty-one independently isolated streptomycin resistant mutants have chloroplast ribosomes which are resistant to this drug in an assay based on misreading of isoleucine in response to a poly U template, and comprise one nuclear and four chloroplast gene loci. Four mutants resistant to spectinomycin, and three mutants resistant to neamine and kanamycin, which have chloroplast ribosomes resistant to their respective antibiotics in poly U directed phenylalanine incorporation, appear to map in a single chloroplast gene locus. Representative alleles of this nr/spr locus, the four streptomycin resistance loci, and two chloroplast gene loci for erythromycin resistance, have been analyzed in a series of parallel crosses to establish the following map order for these seven genes in the chloroplast genome: er-u-la-er-u-37-nr-u-2-1/spr-u-1-H-4-sr-u-2-23-sr-u-2-60-sr-u-sm3-sr-u-sm2. These seven genes may constitute a ribosomal region within the chloroplast genome of Chlamydomonas comparable to the ribosomal gene clusters in bacteria.     

De novo kinetochore assembly requires the centromeric histone H3 variant

Kinetochores mediate chromosome attachment to the mitotic spindle to ensure accurate chromosome segregation. Budding yeast is an excellent organism for kinetochore assembly studies because it has a simple defined centromere sequence responsible for the localization of >65 proteins. In addition, yeast is the only organism where a conditional centromere is available to allow studies of de novo kinetochore assembly. Using a conditional centromere, we found that yeast kinetochore assembly is not temporally restricted and can occur in both G1 phase and prometaphase. We performed the first investigation of kinetochore assembly in the absence of the centromeric histone H3 variant Cse4 and found that all proteins tested depend on Cse4 to localize. Consistent with this observation, Cse4-depleted cells had severe chromosome segregation defects. We therefore propose that yeast kinetochore assembly requires both centromeric DNA specificity and centromeric chromatin.     

The acute hypoxic ventilatory response: testing the adaptive significance in human populations

The acute Hypoxic Ventilatory Response (HVR) is an important component of human hypoxia tolerance, hence presumably physiological adaptation to high altitude. We measured the isocapnic HVR (L min(-1) %(-1)) in two genetically divergent low altitude southern African populations. The HVR does not differ between African Xhosas (X) and Caucasians (C) (X:-0.34+/-0.36; C:-0.42+/-0.33; P > 0.34), but breathing patterns do. Among all Xhosa subjects, size-independent tidal volume was smaller (X: 0.75+/-0.20; C: 1.11+/-0.32 L; P < 0.01), breathing frequency higher (X: 22.2+/-5.7; C: 14.3+/-4.2 breaths min(-1); P < 0.01) and hypoxic oxygen saturation lower than among Caucasians (X: 78.4+/-4.7%; C: 81.7+/-4.7%; P < 0.05). The results remained significant if subjects from Xhosa and Caucasian groups were matched for gender, body mass index and menstrual cycle phase in the case of females. The latter also employed distinct breathing patterns between populations in normoxia. High repeatability (intra-class correlation coefficient) of the HVR in both populations (0.77-0.87) demonstrates that one of the prerequisites for natural selection, consistent between-individual variation, is met. Finally, we explore possible relationships between inter-population genetic distances and HVR differences among Xhosa, European, Aymara Amerindians, Tibetan and Chinese populations. Inter-population differences in the HVR are not attributable to genetic distance (Mantel Z-test, P = 0.59). The results of this study add novel support for the hypothesis that differences in the HVR, should they be found between other human populations, may reflect adaptation to hypoxia rather than genetic divergence through time.     

Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging concepts in cell signaling

The observation that purified yeast glutamine synthetase is rapidly inactivated in a thiol-containing buffer yet retains activity in crude extracts containing the same thiol led to our discovery of an enzyme that protects against oxidation in a thiol-containing system. This novel antioxidant enzyme was shown to reduce hydroperoxides and, more recently, peroxynitrite with the use of electrons provided by a physiological thiol like thioredoxin. It defined a family of proteins, present in organisms from all kingdoms, that was named peroxiredoxin (Prx). All Prx enzymes contain a conserved Cys residue that undergoes a cycle of peroxide-dependent oxidation and thiol-dependent reduction during catalysis. Mammalian cells express six isoforms of Prx (Prx I to VI), which are classified into three subgroups (2-Cys, atypical 2-Cys, and 1-Cys) based on the number and position of Cys residues that participate in catalysis. The relative abundance of Prx enzymes in mammalian cells appears to protect cellular components by removing the low levels of peroxides produced as a result of normal cellular metabolism. During catalysis, the active site cysteine is occasionally overoxidized to cysteine sulfinic acid. Contrary to the general belief that oxidation to the sulfinic state is an irreversible process in cells, studies on the fate of the overoxidized Prx species revealed a mechanism by which the catalytically active thiol form is recovered. This sulfinic reduction is a slow, ATP-dependent process that is specific to 2-Cys Prx isoforms. This reversible overoxidation may represent an adaptation unique to eukaryotic cells that accommodates the intracellular messenger function of H₂O₂, but experimental validation of such speculation is yet to come.     

Phospholipase A2 in astrocytes

Astrocytes comprise the major cell type in the central nervous system (CNS) and they are essential for support of neuronal functions by providing nutrients and regulating cell-to-cell communication. Astrocytes also are immune-like cells that become reactive in response to neuronal injury. Phospholipases A₂ (PLA ₂) are a family of ubiquitous enzymes that degrade membrane phospholipids and produce lipid mediators for regulating cellular functions. Three major classes of PLA ₂ are expressed in astrocytes: group IV calcium-dependent cytosolic PLA ₂ (cPLA₂), group VI calcium-independent PLA ₂ (iPLA₂), and group II secretory PLA ₂ (sPLA₂). Upregulation of PLA ₂ in reactive astrocytes has been shown to occur in a number of neurodegenerative diseases, including stroke and Alzheimer’s disease. This review focuses on describing the effects of oxidative stress, inflammation, and activation of G protein-coupled receptors on PLA ₂ activation, arachidonic acid (AA) release, and production of prostanoids in astrocytes.     

Evidence that the caterpillar salivary enzyme glucose oxidase provides herbivore offense in solanaceous plants

The insect salivary enzyme glucose oxidase (GOX) can inhibit wound-inducible nicotine production in tobacco, Nicotiana tabacum. We examined whether salivary gland extracts of Helicoverpa zea lacking active GOX could still suppress nicotine in tobacco, Nicotiana tabacum, and whether GOX could suppress wound-inducible defenses of another Solanaceous plant, tomato Lycopersicon esculentum. Tobacco leaves were wounded with a cork borer and treated with water, salivary gland extracts with active GOX (SxG), or salivary gland extracts with inactive GOX (SxI). After three days, leaves treated with SxG had significantly less nicotine than all other wounded treatments. Neonates that fed on the terminal leaves of tobacco plants treated with SxG had significantly higher survival than neonates that fed on leaves treated with either SxI or water. This evidence supports the assertion that GOX is the salivary factor responsible for the suppression of tobacco plant nicotine production by H. zea saliva. Results for the NahG tobacco plants, which lack salicylic acid (SA) due to a transgene for bacterial SA hydroxylase, indicate that suppression of nicotine by GOX does not require SA. However, tobacco leaves that were wounded and treated with SxG had significantly higher levels of the SA-mediated PR-1a protein than leaves treated with SxI or water. Leaves of tomato plants wounded with scissors and then treated with SxG had trypsin inhibitor levels that were moderately lower than plants wounded and treated with purified GOX, water, or SxI. However, all the wounded tomato leaves irrespective of treatment resulted in lower caterpillar growth rates than the non-wounded tomato leaves. Glucose oxidase is the first insect salivary enzyme shown to suppress wound-inducible herbivore defenses of plants.     

Nitric oxide control of large veins in the toad <Emphasis Type="Italic">Bufo marinus</Emphasis>

This study examined the nitric oxide (NO) control of the vascular smooth muscle of the ventral abdominal vein and vena cava of the toad, Bufo marinus, by using anatomical and physiological approaches. Nicotinamide adenine di-nucleotide phosphate-diaphorase histochemistry and immunohistochemistry using endothelial nitric oxide synthase (NOS) and neural NOS antibodies produced no evidence for endothelial NOS in the veins, but, neural NOS-immunoreactive perivascular nerves were present. Acetylcholine (10^–5 M) caused a vasodilation in both veins that was endothelium-independent, and which was blocked by the soluble guanylyl cyclase inhibitor, ODQ (10^–5 M). The NOS inhibitors, L-NNA (10^–4 M) and L-NAME (10^–4 M), did not significantly reduce the vasodilatory effect of acetylcholine in the veins; this suggested that the vasodilation was not due to NO. However, in the presence of phenoxybenzamine (10^–7–10^–8 M), L-NNA significantly reduced the vasodilatory effect of acetylcholine in the veins. This unusual response is due to phenoxybenzamine partially inactivating the muscarinic receptor pool in the veins. In addition, the neural NOS inhibitor, vinyl-L-NIO (10^–5 M), significantly reduced the acetylcholine-mediated vasodilation in the presence of phenoxybenzamine. The results show that in toad veins, nitrergic nerves rather than an endothelial NO system are involved in NO-mediated vasodilation.     

Distinct signaling pathways for induction of type II NOS by IFNgamma and LPS in BV-2 microglial cells

Nitric oxide (NO) release upon microglial cell activation has been implicated in the tissue injury and cell death in many neurodegenerative diseases. Recent studies have indicated the ability of interferon-gamma (IFNgamma) and lipopolysaccharides (LPS) to independently induce type II nitric oxide synthase (iNOS) expression and NO production in BV-2 microglial cells. However, a detailed comparison between the signaling pathways activating iNOS by these two agents has not been accomplished. Analysis of PKC isoforms revealed mainly the presence of PKCdelta, iota and lambda in BV-2 cells. Although both IFNgamma and LPS could specifically enhance the tyrosine phosphorylation of PKCdelta, treatment with IFNgamma induced a steady increase of phospho-PKCdelta for up to 1h, whereas treatment with LPS elevated phospho-PKCdelta levels only transiently, with peak activity at 5 min. Rottlerin, a specific inhibitor for PKCdelta, dose-dependently inhibited IFNgamma- and LPS-induced NO production. Despite the common involvement of PKCdelta, IFNgamma- but not LPS-induced NO production involved extracellular signal-regulated kinases (ERK1/2) cascade and IFNgamma-induced phosphorylation of ERK1/2 was mediated through PKC. On the other hand, LPS- but not IFNgamma-induced NO production was through stimulation of NF-kappaB activation and nuclear translocation to interact with DNA. These results demonstrated distinct signaling pathways for induction of iNOS by IFNgamma and LPS in BV-2 microglial cells.