Spore germination and germinant receptor genes in wild strains of Bacillus subtilis.

AIMS: To compare the germination of laboratory and wild strains of Bacillus subtilis. METHODS AND RESULTS: The spore germination of B. subtilis 168 (subsp. subtilis) was compared with that of the laboratory strain W23 (subsp. spizizenii) and desert-sourced isolates, including one member of subsp. subtilis (RO-NN-1), strains TU-B-10, RO-E-2, N10 and DV1-B-1, (all subsp. spizizenii), the B. mojavensis strain RO-H-1 and a B. subtilis natto strain. All germinated in L-alanine, although some were slower, and some 10-fold less sensitive to germinant. All germinated in calcium dipicolinate (CaDPA). Germination in asparagine, glucose, fructose + KCl was slow and incomplete in many of the strains, and decoating RO-NN-1 and W23 spores did not restore germination rates. Comparing the sequences of B. subtilis strains 168, RO-NN-1, W23, TU-B-10 and DV1-B-1, the operons encoding GerA, B and K germinant receptors were intact, although the two additional operons yndDEF and yfkQRST had suffered deletions or were absent in several spizizenii strains. CONCLUSIONS: Wild strains possess an efficient germination machinery for L-alanine germination. AGFK germination is often less efficient, the gerB genes more diverged, and the two germinant receptor operons of unknown function have been lost from the genome in many subsp. spizizenii strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The two major subspecies of B. subtilis have conserved GerA receptor function, confirming its importance, at least in the natural environments of these strains.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein.

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD) cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Novel method of intralesional cidofovir injection into laryngotracheal papillomata.

Laryngeal papillomatosis is characterised by multiple papillomata affecting the upper respiratory tract. This condition is difficult to treat due to its recurrent nature. Treatment often involves surgical debulking. A number of non-surgical treatments have been reported. Intralesional cidofovir, a cytosine nucleoside analogue with antiviral activity, has been used in an attempt to manage the condition. We present a novel technique of administering cidofovir in a case of recurrent laryngotracheal papillomata.

ICChI, a glycosylated chitinase from the latex of Ipomoea carnea.

A multi-functional enzyme ICChI with chitinase/lysozyme/exochitinase activity from the latex of Ipomoea carnea subsp. fistulosa was purified to homogeneity using ammonium sulphate precipitation, hydrophobic interaction and size exclusion chromatography. The enzyme is glycosylated (14-15%), has a molecular mass of 34.94 kDa (MALDI-TOF) and an isoelectric point of pH 5.3. The enzyme is stable in pH range 5.0-9.0, 80 degrees C and the optimal activity is observed at pH 6.0 and 60 degrees C. Using p-nitrophenyl-N-acetyl-beta-D-glucosaminide, the kinetic parameters K(m), V(max), K(cat) and specificity constant of the enzyme were calculated as 0.5mM, 2.5 x 10(-8)mol min(-1)microg enzyme(-1), 29.0 s(-1) and 58.0mM(-1)s(-1) respectively. The extinction coefficient was estimated as 20.56 M(-1)cm(-1). The protein contains eight tryptophan, 20 tyrosine and six cysteine residues forming three disulfide bridges. The polyclonal antibodies raised and immunodiffusion suggests that the antigenic determinants of ICChI are unique. The first fifteen N-terminal residues G-E-I-A-I-Y-W-G-Q-N-G-G-E-G-S exhibited considerable similarity to other known chitinases. Owing to these unique properties the reported enzyme would find applications in agricultural, pharmaceutical, biomedical and biotechnological fields.

Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea.

A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.

Indicain, a dimeric serine protease from Morus indica cv. K2.

A high molecular mass serine protease has been purified to homogeneity from the latex of Morus indica cv. K2 by the combination of techniques of ammonium sulfate precipitation, hydrophobic interaction chromatography, and size-exclusion chromatography. The protein is a dimer with a molecular mass of 134.5 kDa and with two monomeric subunits of 67.2 kDa and 67.3 (MALDI-TOF), held by weak bonds susceptible to disruption on exposure to heat and very low pH. Isoelectric point of the enzyme is pH 4.8. The pH and temperature optima for caseinolytic activity were 8.5 and 80 degrees C, respectively. The extinction coefficient (epsilon280(1%)) of the enzyme was estimated as 41.24 and the molecular structure consists of 52 tryptophan, 198 tyrosine and 42 cysteine residues. The enzyme activity was inhibited by phenylmethylsulfonylflouride, chymostatin and mercuric chloride indicating the enzyme to be a serine protease. The enzyme is fairly stable and similar to subtilases in its stability toward pH, strong denaturants, temperature, and organic solvents. Polyclonal antibodies specific to enzyme and immunodiffusion studies reveal that the enzyme has unique antigenic determinants. The enzyme has activity towards broad range of substrates comparable to those of subtilisin like proteases. The N-terminal residues of indicain (T-T-N-S-W-D-F-I-G-F-P) exhibited considerable similarity to those of other known plant subtilases, especially with cucumisin, a well-characterized plant subtilase. This is the first report of purification and characterization of a subtilisin like dimeric serine protease from the latex of M. indica cv. K2. Owing to these unique properties the reported enzyme would find applications in food and pharma industry.

Is there an association between blood group O and epistaxis?

INTRODUCTION: Blood group O is associated with lower expression of von Willebrand factor suggesting a relative bleeding tendency. A lower admission rate for epistaxis among Asians compared with Caucasians has also been noted, with one explanation being higher prevalence of blood group O among Caucasians. This study investigates whether blood group O is over-represented in patients admitted with epistaxis. METHODS: A retrospective study was conducted, using computerised hospital in-patient and blood bank databases to identify Caucasians admitted with epistaxis between January 2000 and December 2005 inclusive. The control group consisted of 500 consecutive patients who had a primary total hip arthroplasty and 500 consecutive patients who gave birth within the delivery suite. RESULTS: 1261 Caucasians admitted with epistaxis were identified. Among epistaxis patients, 50.44 per cent were blood group O but among the control group this was 45.10 per cent (chi-square test p = 0.008). CONCLUSION: Blood group O appears over-represented in Caucasian patients admitted with epistaxis, compared with the control population, raising the possibility that blood group O is a risk factor for epistaxis.

Membrane reinsertion of a myristoyl-peptidyl anchored extracellular domain growth hormone receptor.

The actions of GH are mediated through a cell surface cytokine receptor. We previously demonstrated that naturally occurring truncated membrane bound GH receptors (GHRs) can block GH receptor signaling. We have now investigated whether recombinant extracellular GHR can be conjugated to a myristoylated-peptide (mp) tail and inserted into cell membranes to modulate GHR signaling. Recombinant human extracellular domain (1-241) GHR was expressed in Escherichia coli, purified, and refolded from cell lysate. The free C-terminal cysteine was then reduced and conjugated to an activated preformed mp tail. The properties of the purified tailed GHR (GHR-mp) were then compared with those of the untailed purified GHR 1-241. Fluorescence-activated cell sorter analysis and cell surface binding assays demonstrated that GHR-mp inserted into the cell surface membranes of CHO cells, whereas untailed GHR 1-241 showed no insertion. In a cell-based bioassay GHR-mp partially inhibited wild-type GHR signaling, whereas GHR 1-241 had no effect. Truncated extracellular domain GHR can, when specifically modified with a membrane-localizing mp unit, insert into cell surface membranes and modulate GHR signaling.

Is admission for epistaxis more common in Caucasian than in Asian people? A preliminary study.

OBJECTIVES: Epistaxis is a common ENT complaint. Although casual observation suggested that it is more common in Caucasian, compared with Asian people, a literature search failed to find any studies investigating ethnicity and epistaxis. The aim of this study was to identify any differences in emergency admission rates for epistaxis between Asian and Caucasian people. DESIGN: Retrospective observational study using hospital computerised data (HISS). SETTING: Large University Hospital accepting ENT emergencies. PARTICIPANTS: All Asian and Caucasian patients admitted under ENT care as an emergency (1 January 2000 to 30 November 2005), split into two groups: one composed of epistaxis patients, the other of all other ENT emergency admissions. MAIN OUTCOMES MEASURES: The proportions of Asian and Caucasian patients among the two patient groups, either epistaxis admissions or other ENT emergency admissions. RESULTS: The proportions of Asian and Caucasian patients in the group admitted with emergency epistaxis were 7.1% (100/1410) and 92.9% (1310/1410) respectively. However, the proportions of Asian and Caucasian patients in the group composed of any other ENT emergency were 13.2% (729/5515) and 86.8% (4786/5515), respectively (chi-squared P < 0.01). CONCLUSIONS: Caucasian people form an unexpectedly large, and Asians a smaller proportion of emergency epistaxis admissions. The possibility of an ethnic risk factor for epistaxis warrants further investigation.

How do spores germinate?

Spore germination, as defined as those events that result in the loss of the spore-specific properties, is an essentially biophysical process. It occurs without any need for new macromolecular synthesis, so the apparatus required is already present in the mature dormant spore. Germination in response to specific chemical nutrients requires specific receptor proteins, located at the inner membrane of the spore. After penetrating the outer layers of spore coat and cortex, germinant interacts with its receptor: one early consequence of this binding is the movement of monovalent cations from the spore core, followed by Ca2(+) and dipicolinic acid (DPA). In some species, an ion transport protein is also required for these early stages. Early events - including loss of heat resistance, ion movements and partial rehydration of the spore core - can occur without cortex hydrolysis, although the latter is required for complete core rehydration and colony formation from a spore. In Bacillus subtilis two crucial cortex lytic enzymes have been identified: one is CwlJ, which is DPA-responsive and is located at the cortex-coat junction. The second, SleB, is present both in outer layers and at the inner spore membrane, and is more resistant to wet heat than is CwlJ. Cortex hydrolysis leads to the complete rehydration of the spore core, and then enzyme activity within the spore protoplast resumes. We do not yet know what activates SleB activity in the spore, and neither do we have any information at all on how the spore coat is degraded.

Immobilized alpha-melanocyte stimulating hormone 10-13 (GKPV) inhibits tumor necrosis factor-alpha stimulated NF-kappaB activity.

alpha-MSH is an anti-inflammatory peptide which signals by binding to the melanocortin-1 receptor (MC1R) and elevating cyclic AMP in several different cells and tissues. The carboxyl terminal peptides of alpha-MSH (KPV/GKPV) are the smallest minimal sequences that prevent inflammation, but it is not known if they operate via MC1R or cyclic AMP. The aim of this study was to examine the intracellular signaling potential of the GKPV peptide sequence when immobilized to polystyrene beads via a polyethylene glycol moiety. Beads containing an immobilized GKPV peptide were investigated for their ability to inhibit proinflammatory tumor necrosis factor-alpha (TNF-alpha) stimulated activation of NF-kappaB in HBL cells stably transfected with an NF-kappaB-luciferase reporter construct. Peptide functionalized beads were compared with the ability of soluble peptide alone (alpha-MSH or GKPV) or non-functionalized beads to inhibit TNF-alpha stimulated activation of NF-kappaB. GKPV peptide functionalized beads significantly inhibited NF-kappaB-luciferase activity in comparison to beads containing no peptide moiety in one of two growths conditions investigated. Soluble alpha-MSH and GKPV peptides were also confirmed to inhibit NF-kappaB-luciferase. The present study suggests that the carboxyl terminal MSH peptide acts via a cell receptor-based mechanism and furthermore may support the potential use of such immobilized ligands for anti-inflammatory therapeutic use.

Failure of moxidectin to control benzimidazole-, levamisole- and ivermectin-resistant Teladorsagia circumcinda in a sheep flock.

Control of a benzimidazole-, levamisole- and ivermectin-resistant population of Teladorsagia circumcincta was attempted using moxidectin administered orally at the manufacturer's recommended dose rate of 200 microg/kg bodyweight. Ewes were dosed after lambing with the aim of controlling their periparturient rise in faecal egg output and lambs were dosed at six-week intervals throughout the summer. This regimen failed to suppress the establishment of significant numbers of infective helminth larvae on the pasture, resulting in unsatisfactory lamb production. Oral dosing with moxidectin was effective in removing adult female burdens of ivermectin-resistant T. circumcincta, but the effect of the drug did not persist against the resistant helminth population.

A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism.

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains. In addition, the cleavage of a specific fluorogenic peptide substrate by rabbit m-calpain is inhibited by a synthetic peptide that corresponds to residues 10-17 of rabbit skeletal muscle AMP deaminase; this peptide contains a sequence (K-E-L-D-D-A) that is present in the fourth subdomain A of rabbit calpastatin, suggesting that the N-terminus of AMP deaminase shares with calpastatin a regulatory sequence that might exert a protective role against the fragmentation-induced activation of AMP deaminase. These observations suggest that a calpain-like proteinase present in muscle removes from AMP deaminase a domain that holds the enzyme in an inactive conformation and which also contains a regulatory region that protects against unregulated proteolysis. We conclude that proteolysis of AMP deaminase is the basis of the large ammonia accumulation that occurs in skeletal muscle subjected to strong tetanic contraction or passing into rigor mortis.

Spore germination.

Despite being relatively insensitive to environmental insult, the spore is responsive to low concentrations of chemical germinants, which induce germination. The process of bacterial spore germination involves membrane permeability changes, ion fluxes and the activation of enzymes that degrade the outer layers of the spore. A number of components in the spore that are required for the germination response have been identified, including a spore-specific family of receptor proteins (the GerA family), an ion transporter and cortex lytic enzymes. The germinant traverses the outer layers of the spore and interacts with its receptor in the inner membrane to initiate the cascade of germination events, but the molecular details of this signal transduction process remain to be identified.

New World leishmaniasis from Spain.

A 69 year old man living in Spain contracted mucocutaneous leishmaniasis involving the nose. The infecting organism was Leishmania infantum, which only rarely causes the New World form of the disease. The source of infection was probably a neighbour's dog. The patient began treatment with liposomal amphotericin B but died of pneumonia two months later.

Transcriptional responses during outgrowth of Bacillus subtilis endospores.

The Bacillus subtilis 168 genome contains an array of alternative sigma factors, many of which play important roles in reprogramming expression during stress and sporulation. The role of the different sigma factors during outgrowth, when the germinated endospore is converted back to a vegetative cell, is less well characterized. The activity of the alternative sigma factors sigmaB, sigmaD and sigmaH during endospore outgrowth was analysed by Northern blotting and lacZ reporter assays. While sigmaD and sigmaH were transcriptionally active during outgrowth, sigmaB-dependent transcription was not observed until after the first cell division, when growth slowed. Using an IPTG-controllable copy of sigA, an optimal level of expression was required to maintain growth rate at the end of outgrowth. The genes encoding the putative extracytoplasmic function (ECF) sigma factors sigmaI, sigmaV, sigmaW, sigmaZ and YlaC were insertionally inactivated using pMUTIN4. These strains, together with sigM and sigX mutants, were tested to determine their role and measure their expression during endospore outgrowth. Transcripts or beta-galactosidase activity were observed for each of the ECF sigma factors early after germination. With the exception of MJH003 (sigM), which showed an exacerbated salt stress defect, inactivation of the ECF sigma factor genes did not affect outgrowth in the conditions tested.

Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19.

Plantaricin C19, an anti-Listeria bacteriocin, was successfully purified by adsorption to and release from producing cells at low pH combined with reverse phase high-performance liquid chromatography (HPLC). The purification resulted in a 900-fold increase in specific activity with a yield of 15% of the original activity. Mass spectrometry analysis gave a molecular weight of 3845.3. Protein microsequencing identified 36 amino acids. Plantaricin C19 is rich in both hydrophobic and basic amino acids in good accordance with its basic and hydrophobic character. Comparison of the amino acid sequence of plantaricin C19, with the sequence of some other anti-Listeria bacteriocins produced with lactic acid bacteria, revealed that plantaricin C19 has in its N-terminal region the consensus sequence--YYGNGL--(uniquely with Valine instead of Leucine as found in all other bacteriocins), identifying plantaricin C19 as a pediocin-like bacteriocin. Plantaricin C19 exerted a bacteriostatic action on sensitive cells of Listeria grayi IP 6818 in BHI broth. No loss of intracellular K+, Mg2+ or UV-absorbing materials was observed. Adsorption of plantaricin C19 on L. grayi CIP 6818 decreased in the presence of salts.

A new class of glutaminase from Aspergillus oryzae.

The koji mold Aspergillus oryzae is able to produce glutaminase which converts glutamine to glutamic acid, one of the most important flavor components in soy sauce. We present here the isolation and the complete nucleotide sequence of the glutaminase- encoding gene from A. oryzae U212, an industrial strain used in Thailand. N-terminal and internal amino acid sequences were determined from purified glutaminase. A 700-bp fragment was amplified by PCR using oligonucleotide primers designed from partial amino acid sequences. This PCR fragment was used as a homologous probe for screening an A. oryzae genomic DNA library. RT-PCR showed that the gene contained seven short introns. Sequence analysis revealed an open reading frame that encodes a protein of 690 amino-acid residues with a predicted molecular mass of 76 kDa. The N-terminal and internal amino acid sequences of the deduced protein exactly matched the ones determined from the purified protein. Comparison of the amino acid sequence with glutaminase sequences from other origins showed that A. oryzae glutaminase shares little homology with those of bacteria, eukaryote and mammals. The A. oryzae glutaminase gene was expressed in A. nidulans to confirm the presence of a functional glutaminase gene in the cloned DNA. To our knowledge, this is the first reported glutaminase gene cloned from filamentous fungi.

GerN, an endospore germination protein of Bacillus cereus, is an Na(+)/H(+)-K(+) antiporter.

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na(+)-sensitive phenotype of an Escherichia coli mutant that is deficient in Na(+)/H(+) antiport activity (strain KNabc). GerN also reduced the concentration of K(+) required to support growth of an E. coli mutant deficient in K(+) uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na(+)/H(+) antiport activity, with a K(m) for Na(+) estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li(+), but not K(+), served as a substrate. GerN-mediated Na(+)/H(+) antiport was further demonstrated in everted vesicles as energy-dependent accumulation of (22)Na(+). GerN also used K(+) as a coupling ion without completely replacing H(+), as indicated by partial inhibition by K(+) of H(+) uptake into right-side-out vesicles loaded with Na(+). K(+) translocation as part of the antiport was supported by the stimulatory effect of intravesicular K(+) on (22)Na(+) uptake by everted vesicles and the dependence of GerN-mediated (86)Rb(+) efflux on the presence of Na(+) in trans. The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na(+)/H(+)-K(+) antiport. GerN-mediated Na(+)/H(+)-K(+) antiport was much more rapid than GerN-mediated Na(+)/H(+) antiport.