Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

1. Rat pancreatic islets were isolated and then maintained in culture for 2-4 days before being incubated in groups of 100 in the presence of different glucose (0-20 mM) or CaCl2 (1.2-4.2 mM) concentrations, or with uncoupler. 2. Increases in extracellular glucose concentration resulted in increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase in the islets, with half-maximal effects around 5-6 mM-glucose. Increasing extracellular glucose from 3 to 20 mM resulted in a 4-6-fold activation of pyruvate dehydrogenase within 2 min. 3. The total enzyme activity was unchanged, and averaged 0.4 m-unit/100 islets at 37 degrees C. 4. These changes in active pyruvate dehydrogenase were broadly similar to changes in insulin secretion by the islets. 5. Increasing extracellular Ca2+ or adding uncoupler also activated pyruvate dehydrogenase to a similar degree, but only the former was associated with increased insulin secretion.     

Basic residues at the C-gate of DNA gyrase are involved in DNA supercoiling

DNA gyrase is a type II topoisomerase that is responsible for maintaining the topological state of bacterial and some archaeal genomes. It uses an ATP-dependent two-gate strand-passage mechanism that is shared among all type II topoisomerases. During this process, DNA gyrase creates a transient break in the DNA, the G-segment, to form a cleavage complex. This allows a second DNA duplex, known as the T-segment, to pass through the broken G-segment. After the broken strand is religated, the T-segment is able to exit out of the enzyme through a gate called the C-gate. Although many steps of the type II topoisomerase mechanism have been studied extensively, many questions remain about how the T-segment ultimately exits out of the C-gate. A recent cryo-EM structure of Streptococcus pneumoniae GyrA shows a putative T-segment in close proximity to the C-gate, suggesting that residues in this region may be important for coordinating DNA exit from the enzyme. Here, we show through site-directed mutagenesis and biochemical characterization that three conserved basic residues in the C-gate of DNA gyrase are important for DNA supercoiling activity, but not for ATPase or cleavage activity. Together with the structural information previously published, our data suggest a model in which these residues cluster to form a positively charged region that facilitates T-segment passage into the cavity formed between the DNA gate and C-gate.     

Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

Each of the two highly conserved tryptophan residues in hTNF (positions 28 and 114) was converted into phenylalanine by site-directed mutagenesis and the mutant proteins were partially purified. A cytotoxicity assay on mouse L929 cells showed only a slight reduction in biological activity, strongly suggesting that neither of the two amino acids is involved in the active site.     

Arginine residues are involved in the transport function of bilitranslocase

Specific guanido group reagents inhibit bilitranslocase transport activity in rat liver plasma membrane vesicles. Their reaction is shown to be affected by sulfobromophthalein, Thymol blue and bilirubin, which are translocated by bilitranslocase across the plasma membrane. It is concluded that the transport function of bilitranslocase depends on arginine residues, which are involved in the interaction with the molecules to be translocated.     

Injury toNitella internodal cells alters microtubule organization but microtubules are not involved in the wound response

Summary The arrangement and relative stability of cortical microtubules during and after wound induction in internodal cells ofNitella flexilis andNitella pseudoflabellata were examined by immunofluorescence and by microinjection of fluorescently tagged tubulin. The formation of cellulosic wall appositions (wound walls), induced by treatment with 5×10^–2MCaCl₂, was identicalin young, growing cells and older non-growing internodes, suggesting that the initial microtubule pattern, which differs in growing and non-growing cells, does not influence wound wall formation. Depolymerization of microtubules with oryzalin did not alter wound wall morphology and microtubules were not detected during wound wall formation. After cessation of wound wall growth, microtubules were once again found in the wound site but these were always randomly oriented, even in young cells where the surrounding microtubules were organized into transverse arrays. Microtubules were similarly randomized in chloroplast-free windows induced by laser irradiation. Analysis of microtubule organization in living cells revealed that the microtubules in wound sites are less stable than the microtubules of adjacent transversely oriented arrays. The results indicate that although wounding can alter the relative stability and spatial organization of cortical microtubules, microtubules are neither involved in vesicle transport nor the construction of cellulosic wound walls.Abbreviations AFW artificial fresh water - BSA bovine serum albumin - DMSO dimethyl sulfoxide - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline     

Tyrosine residues are essential for the activity of the human placental taurine transporter

Treatment of human placental brush-border membrane vesicles with four tyrosine group-specific reagents, N-acetylimidazole, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), tetranitromethane and p-nitrobenzesulfonyl fluoride, inhibited NaCl gradient-driven taurine uptake in these vesicles without affecting the vesicle integrity. The relative potency of these reagents to inhibit the transporter was in the following order: tetranitromethane greater than NBD-Cl greater than p-nitrobenzenesulfonyl fluoride greater than N-acetylimidazole. The inhibition by N-acetylimidazole was reversible with hydroxylamine and the inhibition by NBD-Cl was reversible with 2-mercaptoethanol. Kinetic analysis of taurine uptake in control and in N-acetylimidazole-treated membrane vesicles revealed that the inhibition was primarily due to a reduction in the maximal velocity. There was no change in the affinity of the transporter for taurine in control and treated vesicles. The transporter could be protected from the N-acetylimidazole-induced inhibition by Na+. The dependence of taurine uptake rate on extravesicular Na+ concentration was sigmoidal and analysis of the data revealed that two Na+ ions were involved per transport of one taurine molecule. It is concluded that tyrosine residues are essential for optimal transport function of the human placental taurine transporter and that these critical tyrosine residues are located at or near the Na+-binding site of the transporter.     

Tyrosine hydroxylase and insulin-like growth factor II but not insulin are adjacent in the teleost species barramundi, Lates calcarifer

In humans, the genes encoding tyrosine hydroxylase (TH), insulin and insulin-like growth factor II (IGF-II) form an extremely tight linkage group on chromosome 11p15. Characterisation of the homologous genomic region of a teleost, the barramundi Lates calcarifer , revealed tight linkage of the TH and IGF-II genes, and the absence of the gene encoding insulin.     

Prostaglandin cyclooxygenase products but not thromboxane A(2) are involved in the pathogenesis of ewthromelalgia in thrombocythaemia

Fluid of artificial blisters from erythromelalgic skin areas in primary thrombocythaemia contained a high amount of prostaglandin-E-like activity. Dazoxiben did not alleviate the erythromelalgia in patients with primary thrombocythaemia despite complete inhibition of platelet malondialdehyde and thromboxane B(2) synthesis and no inhibition of prostaglandin-E-like material. During a 10-day dazoxiben treatment period, persistent erythromelalgia was associated with a significant shortened mean platelet life span of 3.2 days. During subsequent treatment with low dose acetylsalicylic acid daily complete relief of erythromelalgia was associated with inhibition of platelet prostaglandin endoperoxide production and correction of platelet mean life span to normal, 7.9 days. These observations indicate that prostaglandin E(2), or another prostaglandin endoperoxide metabolite, is involved in the pathogenesisof erythromelalgia. The presented study does not give one single clue as to the origin (platelet, vessel wall or other) of the prostanoid, but very likely originates from platelets because a very low dose of acetylsalicylic acid (250 to 500 mg every other day), which irreversibly inhibits platelet cyclooxygenase, is highly effective in the prevention of erythromelalgia in thrombocythaemia.     

Multiple bromodomain genes are involved in restricting the spread of heterochromatic silencing at the Saccharomyces cerevisiae HMR-tRNA boundary

The transfer RNA gene downstream from the HMR locus in S. cerevisiae functions as part of a boundary (barrier) element that restricts the spread of heterochromatic gene silencing into the downstream region of chromosome III. A genetic screen for identifying additional genes that, when mutated, allow inappropriate spreading of silencing from HMR through the tRNA gene was performed. YTA7, a gene containing bromodomain and ATPase homologies, was identified multiple times. Previously, others had shown that the bromodomain protein Bdf1p functions to restrict silencing at yeast euchromatin-heterochromatin boundaries; therefore we deleted nonessential bromodomain-containing genes to test their effects on heterochromatin spreading. Deletion of RSC2, coding for a component of the RSC chromatin-remodeling complex, resulted in a significant spread of silencing at HMR. Since the bromodomain of YTA7 lacks a key tyrosine residue shown to be important for acetyllysine binding in other bromodomains, we confirmed that a GST-Yta7p bromodomain fusion was capable of binding to histones in vitro. Epistasis analysis suggests that YTA7 and the HMR-tRNA function independently to restrict the spread of silencing, while RSC2 may function through the tRNA element. Our results suggest that multiple bromodomain proteins are involved in restricting the propagation of heterochromatin at HMR.     

Motifs of two small residues can assist but are not sufficient to mediate transmembrane helix interactions

Both experimental and statistical searches for specific motifs that mediate transmembrane helix-helix interactions showed that two glycine residues separated by three intervening residues (GxxxG) provide a framework for specific interactions. Further work suggested that other motifs of small residues can mediate the interaction of transmembrane domains, so that the AxxxA-motif could also drive strong interactions of alpha-helices in soluble proteins. Thus, all these data indicate that a motif of two small residues in a distance of four might be enough to provide a framework for transmembrane helix-helix interaction. To test whether GxxxG is equivalent to (small)xxx(small), we investigated the effect of a substitution of either of the two Gly residues in the glycophorin A GxxxG-motif by Ala or Ser using the recently developed GALLEX system. The results of this mutational study demonstrate that, while a replacement of either of the two Gly by Ala strongly disrupts GpA homo-dimerization, the mutation to Ser partly stabilizes a dimeric structure. We suggest that the Ser residue can form a hydrogen bond with a backbone carbonyl group of the adjacent helix stabilizing a preformed homo-dimer. While (small)xxx(small) serves as a useful clue, the context of adjacent side-chains is essential for stable helix interaction, so each case must be tested.     

Tyrosine residues 654 and 670 in beta-catenin are crucial in regulation of Met-beta-catenin interactions

beta-Catenin, a key component of the canonical Wnt pathway, is also regulated by tyrosine phosphorylation that regulates its association to E-cadherin. Previously, we reported its association with the hepatocyte growth factor (HGF) receptor Met at the membrane. HGF induced Met-beta-catenin dissociation and nuclear translocation of beta-catenin, which was tyrosine-phosphorylation-dependent. Here, we further investigate the Met-beta-catenin interaction by selectively mutating several tyrosine residues, alone or in combination, in beta-catenin. The mutants were subcloned into FLAG-CMV vector and stably transfected into rat hepatoma cells, which were treated with HGF. All single or double-mutant-transfected cells continued to show HGF-induced nuclear translocation of FLAG-beta-catenin except the mutations affecting 654 and 670 simultaneously (Y654/670F), which coincided with the lack of formation of beta-catenin-TCF complex and DNA synthesis, in response to the HGF treatment. In addition, the Y654/670F-transfected cells also showed no phosphorylation of beta-catenin or dissociation from Met in response to HGF. Thus, intact 654 and 670 tyrosine residues in beta-catenin are crucial in HGF-mediated beta-catenin translocation, activation and mitogenesis.     

Mycoplasma genitalium mg200 and mg386 genes are involved in gliding motility but not in cytadherence

Isolation and characterization of transposon-generated Mycoplasma genitalium gliding-deficient mutants has implicated mg200 and mg386 genes in gliding motility. The proposed role of these genes was confirmed by restoration of the gliding phenotype in deficient mutants through gene complementation with their respective mg386 or mg200 wild-type copies. mg200 and mg386 are the first reported gliding-associated mycoplasma genes not directly involved in cytadherence. Orthologues of MG200 and MG386 proteins are also found in the slow gliding mycoplasmas, Mycoplasma pneumoniae and Mycoplasma gallisepticum, suggesting the existence of a unique set of proteins involved in slow gliding motility. MG200 and MG386 proteins share common features, such as the presence of enriched in aromatic and glycine residues boxes and an acidic and proline-rich domain, suggesting that these motifs could play a significant role in gliding motility.     

Organ boundary NAC‐domain transcription factors are implicated in the evolution of petal fusion

• Research rationale: Evolution of fused petals (sympetaly) is considered to be an important innovation that has repeatedly led to increased pollination efficiency, resulting in accelerated rates of plant diversification. Although little is known about the underlying regulation of sympetaly, genetic pathways ancestrally involved in organ boundary establishment (e.g. CUP SHAPED COTYLEDON [CUC] 1–3 genes) are strong candidates. In sympetalous petunia, mutations in the CUC1/2‐like orthologue NO APICAL MERISTEM (NAM) inhibit shoot apical meristem formation. Despite this, occasional ‘escape shoots’ develop flowers with extra petals and fused inter‐floral whorl organs.
• Central methods: To To determine if petunia CUC‐like genes regulate additional floral patterning, we used virus‐induced silencing (VIGS) following establishment of healthy shoot apices to re‐examine the role of NAM in petunia petal development, and uniquely characterise the CUC3 orthologue NH16.
• Key results: Confirming previous results, we found that reduced floral NAM/NH16 expression caused increased petal–stamen and stamen–carpel fusion, and often produced extra petals. However, further to previous results, all VIGS plants infected with NAM or NH16 constructs exhibited reduced fusion in the petal whorl compared to control plants.
• Main conclusions: Together with previous data, our results demonstrate conservation of petunia CUC‐like genes in establishing inter‐floral whorl organ boundaries, as well as functional evolution to affect the fusion of petunia petals.

     

Amino acid substitutions in GyrA of Burkholderia glumae are implicated in not only oxolinic acid resistance but also fitness on rice plants

Oxolinic acid (OA) resistance in field isolates of Burkholderia glumae, a causal agent of bacterial grain rot, is dependent on an amino acid substitution at position 83 in GyrA (GyrA83). In the present study, among spontaneous in vitro mutants from the OA-sensitive B. glumae strain Pg-10, we selected OA-resistant mutants that emerged at a rate of 5.7 x 10(-10). Nucleotide sequence analysis of the quinolone resistance-determining region in GyrA showed that Gly81Cys, Gly81Asp, Asp82Gly, Ser83Arg, Asp87Gly, and Asp87Asn are observed in these OA-resistant mutants. The introduction of each amino acid substitution into Pg-10 resulted in OA resistance, similar to what was observed for mutants with the responsible amino acid substitution. In vitro growth of recombinants with Asp82Gly was delayed significantly compared to that of Pg-10; however, that of the other recombinants did not differ significantly. The inoculation of each recombinant into rice spikelets did not result in disease. In inoculated rice spikelets, recombinants with Ser83Arg grew less than Pg-10 during flowering, and growth of the other recombinants was reduced significantly. On the other hand, the reduced growth of recombinants with Ser83Arg in spikelets was compensated for under OA treatment, resulting in disease. These results suggest that amino acid substitutions in GyrA of B. glumae are implicated in not only OA resistance but also fitness on rice plants. Therefore, GyrA83 substitution is thought to be responsible for OA resistance in B. glumae field isolates.     

Identification of the amino acid residues involved in the hemolytic activity of the Cucumaria echinata lectin CEL-III

Background

CEL-III is a hemolytic lectin isolated from the sea cucumber Cucumaria echinata that shows Ca^2 +-dependent Gal/GalNAc-binding specificity. This lectin is composed of two carbohydrate-recognition domains (domains 1 and 2) and an oligomerization domain (domain 3) that facilitates CEL-III assembly in the target cell membrane to form ion-permeable pores.

Methods

Several amino acid residues in domain 3 were replaced by alanine, and hemolytic activity of the mutants was examined.

Results

K344A, K351A, K405A, K420A and K425A showed marked increases in activity. In particular, K405A had activity that was 360-fold higher than the wild-type recombinant CEL-III and 3.6-fold higher than the native protein purified from sea cucumber. Since these residues appear to play roles in the stabilization of domain 3 through ionic and hydrogen bonding interactions with other residues, the mutations of these residues presumably lead to destabilization of domain 3, which consequently induces the oligomerization of the protein through association of domain 3 in the membrane. In contrast, K338A, R378A and R408A mutants exhibited a marked decrease in hemolytic activity. Since these residues are located on the surface of domain 3 without significant interactions with other residue, they may be involved in the interaction with components of the target cell membrane.

Conclusions

Several amino acid residues, especially basic residues, are found to be involved in the hemolytic activity as well as the oligomerization ability of CEL-III.

General significance

The results provide important clues to the membrane pore-forming mechanism of CEL-III, which is also related to that of bacterial pore-forming toxins.     

NK cells, but not NKT cells, are involved in Pseudomonas aeruginosa exotoxin A-induced hepatotoxicity in mice

Pseudomonas aeruginosa exotoxin A (PEA) causes T cell- and Kupffer cell (KC)-dependent liver injury in mice. TNF-alpha as well as IL-18 and perforin are important mediators of liver damage following PEA injection. In this study, we focus on the role of NK and NKT cells in PEA-induced liver toxicity. Depletion of both NK and NKT cells by injection of anti-NK1.1 Ab as well as depletion of NK cells alone by anti-asialo GM1 Ab protected mice from PEA-induced hepatotoxicity, whereas mice lacking only NKT cells were susceptible. Additionally, we observed infiltration of NK cells, T cells, and neutrophils into liver parenchyma after injection of PEA. The number of NKT cells, however, remained unchanged. The increase in intrahepatic NK cells depended on KCs and the TNF-alpha-dependent up-regulation of the adhesion molecule VCAM-1 in the liver, but not on NKT cells. PEA also augmented the cytotoxicity of hepatic NK cells against typical NK target cells (YAC-1 cells). This effect depended on KCs, but not on TNF-alpha or NKT cells. Furthermore, only weak expression of MHC class I was detected on hepatocytes, which was further down-regulated in PEA-treated mice. This could explain the susceptibility of hepatocytes to NK cell cytolytic activity in this model. Our results demonstrate that NK cells, activated and recruited independently of NKT cells, contribute to PEA-induced T cell-dependent liver injury in mice.     

Disulfide-linked integrase oligomers involving C280 residues are formed in vitro and in vivo but are not essential for human immunodeficiency virus replication

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.