Study of the Individual Cytochrome b5 and Cytochrome b5 Reductase Domains of Ncb5or Reveals a Unique Heme Pocket and a Possible Role of the CS Domain

NADH cytochrome b₅ oxidoreductase (Ncb5or) is found in animals and contains three domains similar to cytochrome b₅ (b₅), CHORD-SGT1 (CS), and cytochrome b₅ reductase (b₅R). Ncb5or has an important function, as suggested by the diabetes and lipoatrophy phenotypes in Ncb5or null mice. To elucidate the structural and functional properties of human Ncb5or, we generated its individual b₅ and b₅R domains (Ncb5or-b₅ and Ncb5or-b₅R, respectively) and compared them with human microsomal b₅ (Cyb5A) and b₅R (Cyb5R3). A 1.25 Å x-ray crystal structure of Ncb5or-b₅ reveals nearly orthogonal planes of the imidazolyl rings of heme-ligating residues His⁸⁹ and His¹¹², consistent with a highly anisotropic low spin EPR spectrum. Ncb5or is the first member of the cytochrome b₅ family shown to have such a heme environment. Like other b₅ family members, Ncb5or-b₅ has two helix-loop-helix motifs surrounding heme. However, Ncb5or-b₅ differs from Cyb5A with respect to location of the second heme ligand (His¹¹²) and of polypeptide conformation in its vicinity. Electron transfer from Ncb5or-b₅R to Ncb5or-b₅ is much less efficient than from Cyb5R3 to Cyb5A, possibly as a consequence of weaker electrostatic interactions. The CS linkage probably obviates the need for strong interactions between b₅ and b₅R domains in Ncb5or. Studies with a construct combining the Ncb5or CS and b₅R domains suggest that the CS domain facilitates docking of the b₅ and b₅R domains. Trp¹¹⁴ is an invariant surface residue in all known Ncb5or orthologs but appears not to contribute to electron transfer from the b₅R domain to the b₅ domain.     

Temporal and tissue specific gene expression patterns of the zebrafish kinesin-1 heavy chain family,kif5s,during development

Homo- and heterodimers of Kif5 proteins form the motor domain of Kinesin-1, a major plus-end directed microtubule motor. Kif5s have been implicated in the intracellular transport of organelles, vesicles, proteins, and RNAs in many cell types. There are three mammalian KIF5s. KIF5A and KIF5C proteins are strictly neural in mouse whereas, KIF5B is ubiquitously expressed. Mouse knockouts indicate crucial roles for KIF5 in development and human mutations in KIF5A lead to the neurodegenerative disease Hereditary Spastic Paraplegia. However, the developmental functions and the extent to which individual kif5 functions overlap have not been elucidated. Zebrafish possess five kif5 genes: kif5Aa, kif5Ab, kif5Ba, kif5Bb, and kif5C. Here we report their tissue specific expression patterns in embryonic and larval stages. Specifically, we find that kif5As are strictly zygotic and exhibit neural-specific expression. In contrast, kif5Bs exhibit strong maternal contribution and are ubiquitously expressed. Lastly, kif5C exhibits weak maternal expression followed by enrichment in neural populations. In addition, kif5s show distinct expression domains in the larval retina.     

Identification and characterization of a novel chitinase-like gene cluster (AgCht5) possibly derived from tandem duplications in the African malaria mosquito, Anopheles gambiae

Insect chitinase 5 (Cht5), a well-characterized enzyme found in the molting fluid and/or integument, is classified as a group I chitinase and is usually encoded by a single gene. In this study, a Cht5 gene cluster consisting of five different chitinase-like genes (AgCht5-1, AgCht5-2, AgCht5-3, AgCht5-4 and AgCht5-5) was identified by a bioinformatics search of the genome of Anopheles gambiae. The gene models were confirmed by cloning and sequencing of the corresponding cDNAs and gene expression profiles during insect development were determined. All of these genes are found in a single cluster on chromosome 2R. Their open reading frames (ORF) range from 1227 to 1713 bp capable of encoding putative proteins ranging in size from 409 to 571 amino acids. The identities of their cDNA sequences range from 52 to 66%, and the identities of their deduced amino acid sequences range from 38 to 53%. There are four introns for AgCht5-1, two for AgCht5-2 and AgCht5-3, only one for AgCht5-4, but none for AgCht5-5 in the genome. All five chitinase-like proteins possess a catalytic domain with all of the conserved sequence motifs, but only AgCht5-1 has a chitin-binding domain. Phylogenetic analysis of these deduced proteins along with those from other insect species suggests that AgCht5-1 is orthologous to the Cht5 proteins identified in other insect species. The differences in expression patterns of these genes at different developmental stages further support that these genes may have distinct functions. Additional searching of the genomes of two other mosquito species led to the discovery of four Cht5-like genes in Aedes aegypti and three in Culex quinquefasciatus. Thus, the presence of a Cht5 gene cluster appears to be unique to mosquito species and these genes may have resulted from gene tandem duplications.     

Catabolism and disposition of exogenous 5-hydroxytryptamine in cockroach,Periplaneta americana,tissues

The concentrations of tryptophan, 5-hydroxytryptophan, 5-hydroxytryptamine, N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were determined in the cerebral ganglia, haemolymph and Malpighian tubules of the cockroach Periplaneta americana, using high performance liquid chromatography with electrochemical detection. Injected 5-hydroxytryptamine was rapidly removed from the haemolymph with a concomitant elevation of circulating N-acetyl 5-hydroxytryptamine and little accumulation of 5-hydroxytryptamine in the cerebral ganglia. N-acetyl 5-hydroxytryptamine and N-acetyl dopamine were also rapidly removed from the haemolymph. Incubation of haemolymph from 5-hydroxytryptamine-injected insects and glucosidase or phosphatase, indicated that most of the injected 5-hydroxytryptamine had been converted to a sugar conjugate of N-acetyl 5-hydroxytryptamine. Whole haemolymph did not catabolize 5-hydroxytryptamine or N-acetyl 5-hydroxytryptamine whereas Malpighian tubules N-acetylated both 5-hydroxytryptamine and dopamine and metabolized N-acetyl 5-hydroxytryptamine. Injection of p-chlorophenylalanine (200 and 500 μg/g) had no effect on 5-hydroxytryptamine concentrations in the cockroach cerebral ganglia.     

Involvement of Arabidopsis CPR5 in thermotolerance

The constitutive expressor of pathogenesis-related genes 5 (CPR5) plays a role in pathogen defence responses, programmed cell death, cell wall biogenesis, seed generation and senescence regulation in plants. Here, we investigated the functional characteristics of CPR5 to long-term heat stress in Arabidopsis with different genotypes: wild type (WT), cpr5 mutant and cpr5/CPR5 complementary transgenic plant. The cpr5 mutant showed increased susceptibility to long-term heat stress, displaying significant decreases in hypocotyl elongation, seedling and inflorescence survival, membrane integrity and photosystem II activity (Fv/Fm) during heat stress. However, the thermotolerance was recovered when cpr5 mutant was transformed with a CPR5 gene. H₂O₂ accumulation and lipid peroxidation were lower in cpr5/CPR5 plants and WT than in cpr5 mutants after exposure to 36?°C for 5?days. The alleviated oxidative damage was associated with increased activities of superoxide dismutase, catalase, and ascorbate peroxidase. Furthermore, the induced expression of HSP17.6A-CI, HSP101 and HSP70B under long-term heat stress was more substantial in cpr5/CPR5 plants and WT than in cpr5 mutants. These findings suggest that CPR5 plays an important role in thermotolerance of Arabidopsis by regulating the activities of antioxidant enzymes and the expressions of heat shock protein genes.     

Benzofuranoid neolignans from Aniba simulans

Forteen neolignans, isolated from the benzene extract of Aniba simulans (Lauraceae) trunk wood, included the hitherto undescribed (2S, 3S, 5R)-5-allyl-5,7-dimethoxy-2-(3′,4′,5′-trimethoxyphenyl)-3-methyl-2,3,5,6-tetra-hydro-6-oxobenzofuran, (2R,3S,5R) -5-allyl-5-methoxy-2-(3′-methoxy-4′,5′-methylenedioxyphenyl)-3-methy1-2,3,5, 6-tetrahydro-6-oxobenzofuran, (2S,3S)-6-O-allyl -5-methoxy-2-(3′-methoxy-4′-5′-methylenedioxyphenyl)-3-methyl-2,3-dihydrobenzofuran, (2R,3S)-6-O-allyl-5-methoxy-2- (3′-methoxy-4′,5′-methylenedioxyphenyl)-3-methyl-2,3-dihydrobenzofuran and 7-allyl-6-hydroxy-5-methoxy-2-(3′-methoxy-4,5′ -methylenedioxyphenyl)-3-methylbenzofuran.     

Inhibitors of sterol biosynthesis. Syntheses of 14α-alkyl substituted 15-oxygenated sterols

The chemical syntheses of a number of 14α-alkyl substituted 15-oxygenated sterols have been pursued to permit evaluation of their activity in the inhibition of the biosynthesis of cholesterol and other biological effects. Described herein are the first chemical syntheses of 14α-ethyl-5α-cholest-7-en-3β-ol-15-one, bis-3β,15α-acetoxy-14α-ethyl-5α-cholest-7-ene, 3β-acetoxy-14α-ethyl-5α-cholest-7-en-15β-ol, 14α-ethyl-5α-cholest-7-en-3β,15β-diol, 14α-ethyl-5α-cholest-7-en-3β,15α-diol, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15α-ol, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15β-ol, bis-3β,15α-hexadecanoyloxy-14α-ethyl-5α-cholest-7-ene, 3β-hexadecanoyloxy-14α-ethyl-5α-cholest-7-en-15-one, 3α-benzoyloxy-14α-ethyl-5α-cholest-7-en-15-one, 14α-ethyl-5α-cholest-7-en-3α-ol-15-one, 14α-ethyl-5α-cholest-7-en-15-on-3β-yl pyridinium sulfate, 14α-ethyl-5α-cholest-7-en-15-on-3β-yl potassium sulfate (monohydrate), 14α-ethyl-5α-cholest-7-en-15-on-3α-yl pyridinium sulfate, 14α-ethyl-5α-cholest-7-en-15-on-3α-yl potassium sulfate (monohydrate), 3β-ethoxy-14α-ethyl-5α-cholest-7-en-15-one, 3β-acetoxy-14α-n-propyl-5α-cholest-7-en-15-one, 14α-n-propyl-5α-cholest-7-en-3β-ol-15-one, bis-3β, 15α-acetoxy-14α-n-propyl-5α-cholest-7-ene, 3β-acetoxy-14α-n-propyl-5α-cholest-7-en-15β-ol, 14α-n-propyl-5α-cholest-7-en-3β, 15α-diol, 14α-n-propyl-5α-cholest-7-en-3β, 15β-diol, 14α-n-butyl-5α-cholest-7-en-3β-ol-15-one, 3β-acetoxy-14-α-n-butyl-5α-cholest-7-en-15-one, bis-3β,15α-acetoxy-14α-n-butyl-5α-cholest-7-ene, 3β-acetoxy-14α-n-butyl-5α-cholest-7-en-15β-ol, 14α-n-butyl-5β-cholest-7-en-3β, 15β-diol, and 14α-n-butyl-5α-cholest-7-en-3β, 15α-diol.     

Isolation and characterization of a Δ1-pyrroline-5-carboxylate synthetase (NtP5CS) from Nitraria tangutorum Bobr. and functional comparison with its Arabidopsis homologue

Several functional and regulatory proteins play important roles in controlling plant stress tolerance. Proline (Pro) is one of the most accumulated osmolytes correlated with tolerance to stresses. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS) is a rate-limiting enzyme in Pro biosynthesis. In the present study, we isolated the cDNA for a P5CS gene (NtP5CS) from the halophyte Nitraria tangutorum. Phylogenetic analysis and subcellular localization analysis of NtP5CS-GFP protein in onion cells showed that NtP5CS was a new P5CS gene and was involved in Pro synthesis in N. tangutorum. Expression of the NtP5CS gene was induced by salt stress, dehydration, and high and low temperatures. Escherichia coli overexpressing AtP5CS or NtP5CS exhibited better growth in all treatments, including high salinity, high alkalinity, dehydration, osmotic, heat and cold stresses. Additionally, NtP5CS recombinant E. coli cells grew better than did AtP5CS recombinant cells in response to abiotic stresses. Our data demonstrate that the P5CS from a halophytic species functions more efficiently than its homologue from a glycophytic species in improving the stress tolerance of E. coli.     

Protein-protein interactions in the systems of cytochromes P450 3A4 and 3A5

Molecular interactions between protein partners of the monooxygenase system involved in drug biotransformation (cytochromes P450 3A4, 3A5 and cytochrome b ₅) have been investigated. Human cytochromes P450 3A4 and 3A5 (CYP3A4 and CYP3A5) form complexes with various forms of cytochrome b ₅, including microsomal (b ₅ mc) and mitochondrial (b ₅ om) forms of this protein, as well as two chimeric constructs (b ₅(om-mc),(b ₅(mc-om). Interestingly, significant differences were observed only during interactions with b ₅ om. Electroanalytical characteristics of electrodes with immobilized hemoproteins have been determined for CYP3A4, CYP3A5, b ₅ mc, b ₅ om, b ₅ mc(om-mc), and b ₅ mc(mc-om). The electrochemical analysis revealed that these proteins are characterized by close reduction potentials ranged from ?0.435 V to ?0.350 V (vs. Ag/AgCl). Cytochrome b ₅ mc stimulated the electrocatalytic activity of CYP3A4.     

A novel Δ-pyrroline-5-carboxylate synthetase gene of Medicago truncatula plays a predominant role in stress-induced proline accumulation during symbiotic nitrogen fixation

Proline accumulates in environmentally stressed plant cells including those of legume roots and nodules, but how its level is regulated is poorly understood. Δ¹-Pyrroline-5-carboxylate synthetase (P5CS), the committed-step enzyme of proline biosynthesis, is encoded by two duplicated genes in many plants. Here, we isolated MtP5CS3, a third gene, from Medicago truncatula, whose predicted polypeptide sequence is highly similar to those of previously isolated MtP5CS1 and MtP5CS2 except an extra amino-terminal segment. MtP5CS3 was strongly expressed under salinity and drought in shoots and nodulating roots, while MtP5CS1 was constitutive and MtP5CS2 induced by abscisic acid. Under salinity, MtP5CS3 promoter was more active than those of MtP5CS1 and MtP5CS2, as shown by GUS fusions. Translationally fused MtP5CS1-GFP was localized in the cytoplasm, whereas significant proportions of MtP5CS2-GFP and MtP5CS3-GFP were co-localized with rubisco small subunit protein-fused RFP in transformed hairy root cells. Under salinity, RNA silencing of MtP5CS1 or MtP5CS2 strongly induced MtP5CS3 expression, while that of MtP5CS3 decreased free proline content and nodule number. Consistently, Mtp5cs3, a loss-of-function mutant, accumulated much less proline, formed fewer nodules, and fixed nitrogen significantly less efficiently than the wild type under salinity. Thus, MtP5CS3 plays a critical role in regulating stress-induced proline accumulation during symbiotic nitrogen fixation.     

Correlation between the optimal growth pressures of four Shewanella species and the stabilities of their cytochromes c 5

Shewanella species live widely in deep-sea and shallow-water areas, and thus grow piezophilically and piezosensitively. Piezophilic and psychrophilic Shewanella benthica cytochrome c ₅ (SB cytc ₅) was the most stable against guanidine hydrochloride (GdnHCl) and thermal denaturation, followed by less piezophilic but still psychrophilic Shewanella violacea cytochrome c ₅ (SV cytc ₅). These two were followed, as to stability level, by piezosensitive and mesophilic Shewanella amazonensis cytochrome c ₅ (SA cytc ₅), and piezosensitive and psychrophilic Shewanella livingstonensis cytochrome c ₅ (SL cytc ₅). The midpoint GdnHCl concentrations of SB cytc ₅, SV cytc ₅, SL cytc ₅, and SA cytc ₅ correlated with the optimal growth pressures of the species, the correlation coefficient value being 0.93. A similar trend was observed for thermal denaturation. Therefore, the stability of each cytochrome c ₅ is related directly to its host’s optimal growth pressure. Phylogenetic analysis indicated that Lys-37, Ala-41, and Leu-50 conserved in piezosensitive SL cytc ₅ and SA cytc ₅ are ancestors of the corresponding residues in piezophilic SB cytc ₅ and SV cytc ₅, Gln, Thr, and Lys, respectively, which might have been introduced during evolution on adaption to environmental pressure. The monomeric Shewanella cytochromes c ₅ are suitable tools for examining protein stability with regard to the optimal growth pressures of the source species.     

Cathelicidin represents a new target for manipulation of skin inflammation in Netherton syndrome

Netherton syndrome (NS) is a severe ichthyosis caused by inactivating mutations in the SPINK5 gene encoding the serine protease inhibitor LEKTI. Spink5^−/− mice recapitulate NS and die perinatally from extensive dehydration as a result of a severe defect of the epidermal barrier. We showed that deletion of Klk5 in Spink5^−/− rescues neonatal lethality (Furio et al., 2015). However, Spink5^−/−Klk5^−/− mice developed skin shedding and inflammation during the first week from birth and the majority (70%) succumbed on P7. The remaining mice lived short (i.e. mean survival was 5 months) indicating alternative inflammatory pathways. Since cathelicidin is increased in Spink5^−/− epidermis, we investigated whether it could be implicated in NS pathology. Ablation of Camp in Spink5^−/− suppressed epidermal inflammation and restored abnormal epidermal differentiation, nevertheless, it failed to inhibit overdesquamation and Spink5^−/−Camp^−/− succumbed perinatally due to skin barrier defect, similarly to Spink5^−/−. Joint invalidation of Klk5 and Camp significantly extended survival of Spink5^−/−Klk5^−/−Camp^−/− mice. We provide evidence that cathelicidin is implicated in NS-associated skin inflammation in vivo. Therefore, marketed products that are known to reduce cathelicidin expression could be repurposed for the management of NS.     

Cloning and expression of the Apa LI, Nsp I, Nsp HI, Sac I, Sca I, and Sap I restriction-modification systems in Escherichia coli

The genes encoding the ApaLI (5′-G^TGCAC-3′), NspI (5′-RCATG^Y-3′), NspHI (5′-RCATG^Y-3′), SacI (5′-GAGCT^C-3′), SapI (5′-GCTCTTCN1^-3′, 5′-^N4GAAGAGC-3′) and ScaI (5′-AGT^ACT-3′) restriction-modification systems have been cloned in E.?coli. Amino acid sequence comparison of M.ApaLI, M.NspI, M.NspHI, and M.SacI with known methylases indicated that they contain the ten conserved motifs characteristic of C5 cytosine methylases. NspI and NspHI restriction-modification systems are highly homologous in amino acid sequence. The C-termini of the NspI and NlaIII (5′-CATG-3′) restriction endonucleases share significant similarity. 5mC modification of the internal C in a SacI site renders it resistant to SacI digestion. External 5mC modification of a SacI site has no effect on SacI digestion. N4mC modification of the second base in the sequence 5′-GCTCTTC-3′ blocks SapI digestion. N4mC modification of the other cytosines in the SapI site does not affect SapI digestion. N4mC modification of ScaI site blocks ScaI digetion. A DNA invertase homolog was found adjacent to the ApaLI restriction-modification system. A DNA transposase subunit homolog was found upstream of the SapI restriction endonuclease gene.