Application of electroporation for transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Bacillus, Staphylococcus, Enterococcus and Propionibacterium

Plasmid DNA was introduced by electroporation into Bacillus, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, Propionibacterium and Staphylococcus as an alternative to competent-cell or protoplast transformation. Plasmid-containing transformants were recovered in these recipients at frequencies ranging from 10(1) to 10(5) transformants micrograms-1 of pGK12. Several parameters of the protocol, including DNA concentration, voltage, plating regimen and electroporation buffers were evaluated to determine conditions that improved transformation frequencies for Lactobacillus acidophilus. Using optimized conditions, the following plasmids were introduced into L. acidophilus: pAMB1, pC194, pGB354, pGKV1, pSA3, pTRK13, pTV1 and pVA797. The ability to transfer plasmid DNA via eletroporation will greatly facilitate the application of recombinant DNA methodology and transposon technology to Gram-positive bacteria for cloning and analysis of significant genes.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

Genetic structure of the Mongols as derived from the ABO, MN, Rh, EsD, GLO1, PGM1, AcP, 6-PGD, Hp, Gc, Tf, C'3 and ChE2 loci

According to integral characterization of gene frequencies of the investigated loci AB0, MN, Rh, GLO1, PGM1, EsD, AcP, 6-PGD, Hp, Tf, Gc, C'3 and ChE2, Mongolian population has high level of polymorphism, with the exception of haplotypes R" (cdE) and Ry(CdE) at the Rh locus and TfB0-1 at the Tf locus. The data on biochemical and immunological polymorphic gene markers analysed in the population of Mongolia show that the Mongolians have some distinctive features, in comparison with the mean-in-the-world characteristics: high frequencies of the B genes at the AB0 locus; D, E, R1 and R2 at the Rh locus; GLO11, PGDc, TfDChi, E2(C5+), PGM1(1+); low frequencies of the genes A(AB0), R0(Rh), AcPc, Hp1, Gc2, C'3F, PGM 1(2-); the rest of the genes at the above-mentioned loci and the genes of the locus MN have the mean-in-the-world frequencies.     

Survey of distribution of substance P, vasoactive intestinal polypeptide, cholecystokinin, neurotensin, Met-enkephalin, bombesin and PHI in the spinal cord of cat, dog, sloth and monkey

Levels of substance P (sP), peptide-histidine-isoleucine (PHI), vasoactive intestinal polypeptide (VIP), cholecystokinin (CCK), neurotensin (NT), bombesin (BOM) and methionine-enkephalin (Met-Enk) like immunoreactivity were measured in cat, dog, primate and sloth cervical, thoracic, lumbar and sacral dorsal and ventral horns and dorsal root ganglia. The levels of peptides in the cat sacral cord and the principal peaks of immunoreactivity on a 10-60% acetonitrile gradient on a C18 reverse phase high performance liquid chromatography (HPLC) were sP (sP1-11: 369 ng/g), PHI (PHI: 271 ng/g), VIP (VIP1-28: 210 ng/g), Met-Enk (Met1-5 and extended forms: 257 ng/g), BOM (BOM1-10 and GRP1-27: 20 ng/g), CCK (CCK-8: 15 ng/g) and NT (NT1-13: 10 ng/g). Consideration of the rostrocaudal levels revealed an approximately even distribution with the exception of VIP and PHI which showed sacral/cervical ratios of 79 and 63. For sP, Met-Enk and BOM dorsal/ventral ratios were greater than 1 at all spinal levels. For VIP, PHI and CCK these ratios were greater than 1 only in the sacral cord. Dorsal root ganglion (DRG) levels of sP, VIP, PHI were readily measurable in single ganglia and covaried with the respective levels in the dorsal cord. Pooled samples of spinal ganglia and the trigeminal ganglia revealed that the relative levels of peptide immunoreactivity were: sP (25 ng/g); VIP (26 ng/g); PHI (28 ng/g); Met-Enk (6 ng/g); CCK (2 ng/g); NT (1 ng/g); and BOM (1 ng/g).     

Assessing actual contribution of IF1, inhibitor of mitochondrial FoF1, to ATP homeostasis, cell growth, mitochondrial morphology, and cell viability

F(o)F(1)-ATP synthase (F(o)F(1)) synthesizes ATP in mitochondria coupled with proton flow driven by the proton motive force (pmf) across membranes. It has been known that isolated IF1, an evolutionarily well conserved mitochondrial protein, can inhibit the ATP hydrolysis activity of F(o)F(1). Here, we generated HeLa cells with permanent IF1 knockdown (IF1-KD cells) and compared their energy metabolism with control cells. Under optimum growth conditions, IF1-KD cells have lower cellular ATP levels and generate a higher pmf and more reactive oxygen species. Nonetheless, IF1-KD cells and control cells show the same rates of cell growth, glucose consumption, and mitochondrial ATP synthesis. Furthermore, contrary to previous reports, the morphology of mitochondria in IF1-KD cells appears to be normal. When cells encounter sudden dissipation of pmf, the cytoplasmic ATP level in IF1-KD cells drops immediately (~1 min), whereas it remains unchanged in the control cells, indicating occurrence of futile ATP hydrolysis by F(o)F(1) in the absence of IF1. The lowered ATP level in IF1-KD cells then recovers gradually (~10 min) to the original level by consuming more glucose than control cells. The viability of IF1-KD cells and control cells is the same in the absence of pmf. Thus, IF1 contributes to ATP homeostasis, but its deficiency does not affect the growth and survival of HeLa cells. Only when cells are exposed to chemical ischemia (no glycolysis and no respiration) or high concentrations of reactive oxygen species does IF1 exhibit its ability to alleviate cell injury.     

Aerobic Biotransformation of N-Nitrosodimethylamine and N-Nitrodimethylamine by Benzene-, Butane-, Methane-, Propane-, and Toluene-Fed Cultures

N-Nitrosodimethylamine (NDMA) is an emerging contaminant of concern. N-nitrodimethylamine (DMNA) is a structural analog to NDMA. NDMA and DMNA have been found in drinking water, groundwater, and other media and are of concern due their toxicity. The authors evaluated biotransformation of NDMA and DMNA by cultures enriched from contaminated groundwater growing on benzene, butane, methane, propane, or toluene. Maximum specific growth rates of enriched cultures on butane (μ_max = 1.1 h^?1) and propane (μ_max = 0.65 h^?1) were 1 to 2 orders of magnitude higher than those presented in the literature. Growth rates of mixed cultures grown on benzene (μ_max = 1.3 h^?1), methane (μ_max = 0.09 h^?1), and toluene (μ_max = 0.99 h^?1) in these studies were similar to those presented in the literature. NDMA biotransformation rates for methane oxidizers (υ_max = 1.4 ng min^?1 mg^?1) and toluene oxidizers (υ_max = 2.3 ng min^?1 mg^?1) were comparable to those presented in the literature, whereas the biotransformation rate for propane oxidizers (υ_max = 0.37 ng min^?1 mg^?1) was lower. NDMA biotransformation rates for benzene oxidizers (υ_max = 1.02 ng min^?1 mg^?1) and butane oxidizers (υ_max = 1.2 ng min^?1 mg^?1) were comparable to those reported for other primary substrates. These studies showed that DMNA biotransformation rates for benzene (υ_max = 0.79 ng min^?1 mg^?1), butane (υ_max = 1.0 ng min^?1 mg^?1), methane (υ_max = 2.1 ng min^?1 mg^?1), propane (υ_max = 1.46 ng min^?1 mg^?1), and toluene (υ_max = 0.52 ng min^?1 mg^?1) oxidizers were all comparable. These studies highlight potential bioremediation methods for NDMA and DMNA in contaminated groundwater.     

Thermodynamics of hydrolysis of disaccharides. Cellobiose, gentiobiose, isomaltose, and maltose

The thermodynamics of the enzymatic hydrolysis of cellobiose, gentiobiose, isomaltose, and maltose have been studied using both high pressure liquid chromatography and microcalorimetry. The hydrolysis reactions were carried out in aqueous sodium acetate buffer at a pH of 5.65 and over the temperature range of 286 to 316 K using the enzymes beta-glucosidase, isomaltase, and maltase. The thermodynamic parameters obtained for the hydrolysis reactions, disaccharide(aq) + H2O(liq) = 2 glucose(aq), at 298.15 K are: K greater than or equal to 155, delta G0 less than or equal to -12.5 kJ mol-1, and delta H0 = -2.43 +/- 0.31 kJ mol-1 for cellobiose; K = 17.9 +/- 0.7, delta G0 = -7.15 +/- 0.10 kJ mol-1 and delta H0 = 2.26 +/- 0.48 kJ mol-1 for gentiobiose; K = 17.25 +/- 0.7, delta G0 = -7.06 +/- 0.10 kJ mol-1, and delta H0 = 5.86 +/- 0.54 kJ mol-1 for isomaltose; and K greater than or equal to 513, delta G0 less than or equal to -15.5 kJ mol-1, and delta H0 = -4.02 +/- 0.15 kJ mol-1 for maltose. The standard state is the hypothetical ideal solution of unit molality. Due to enzymatic inhibition by glucose, it was not possible to obtain reliable values for the equilibrium constants for the hydrolysis of either cellobiose or maltose. The entropy changes for the hydrolysis reactions are in the range 32 to 43 J mol-1 K-1; the heat capacity changes are approximately equal to zero J mol-1 K-1. Additional pathways for calculating thermodynamic parameters for these hydrolysis reactions are discussed.     

Loss of SMEK, a novel, conserved protein, suppresses MEK1 null cell polarity, chemotaxis, and gene expression defects

MEK/extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase signaling is imperative for proper chemotaxis. Dictyostelium mek1(-) (MEK1 null) and erk1(-) cells exhibit severe defects in cell polarization and directional movement, but the molecules responsible for the mek1(-) and erk1(-) chemotaxis defects are unknown. Here, we describe a novel, evolutionarily conserved gene and protein (smkA and SMEK, respectively), whose loss partially suppresses the mek1(-) chemotaxis phenotypes. SMEK also has MEK1-independent functions: SMEK, but not MEK1, is required for proper cytokinesis during vegetative growth, timely exit from the mound stage during development, and myosin II assembly. SMEK localizes to the cell cortex through an EVH1 domain at its N terminus during vegetative growth. At the onset of development, SMEK translocates to the nucleus via a nuclear localization signal (NLS) at its C terminus. The importance of SMEK's nuclear localization is demonstrated by our findings that a mutant lacking the EVH1 domain complements SMEK deficiency, whereas a mutant lacking the NLS does not. Microarray analysis reveals that some genes are precociously expressed in mek1(-) and erk1(-) cells. The misexpression of some of these genes is suppressed in the smkA deletion. These data suggest that loss of MEK1/ERK1 signaling compromises gene expression and chemotaxis in a SMEK-dependent manner.     

Synthetic inhibitors of trypsin, plasmin, kallikrein, thrombin, C1r-, and C1 esterase.

p-Carbethoxyphenyl episol-guanidinocaproate and p-(p'-guanidinobenzoyloxy)-phenyl derivatives were prepared, and their inhibitory effects on trypsin, plasmin, plasma kallikrein, thrombin, C1r- and C1 esterase were examined. Among the various inhibitors tested, p-nitrophenyl p'-guanidinobenzoate, N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzoyl glycolate and N,N-dimethylamino p-(p'-guanidinobenzoyloxy)-benzilcarbonyloxy glycolate were the most effective inhibitors of trypsin, plasmin, plasma kallikrien and thrombin, and they strongly inhibited the esterolytic activities of C1r- and C1 esterase.     

Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

Serologic prevalence of Toxoplasma gondii in field mice, Microtus fortis, from Yuanjiang, Hunan Province, People's Republic of China

Antibodies to Toxoplasma gondii were investigated in serum samples of field mice, Microtus fortis, from Yuanjiang, Hunan Province, People's Republic of China. The modified agglutination test (MAT) incorporating formalin-fixed whole tachyzoites and mercaptoethanol was used to determine antibodies. Antibodies to T. gondii (MAT > or = 1:20) were found in 36 (29%) of 124 trapped mice. The antibody titers of positive sera (percentage in parentheses) were 1:20 (8.9), 1:40 (3.2), 1:80 (3.2), 1:160 (1.6), 1:320 (1.6), 1:640 (1.6), 1:1,280 (1.6), 1: 2,560 (0.8), and > 1:2,560 (6.5). No antibody to T. gondii was found in 104 sera of laboratory-bred M. fortis infected with Schistosoma japonicum between 1 and 45 days after infection.     

In vitro propagation of taro,with spermine,arginine, and ornithine

In vitro growth and multiplication of taro [Colocasia esculenta var. antiquorum cv. Keladi Birah] was improved considerably, when primary shoot apices were cultured on two modifications of Linsmaier and Skoog [1965] medium, containing 5.5 mg 1^–1 naphthaleneacetic acid and 0.2 mg 1^–1 kinetin or 1.85 mg 1^–1 naphthaleneacetic acid and 2 mg 1^–1 kinetin and supplemented with 10^–4 or 10^–3 mol·1^–1 of polyamine spermine or either of the precursors of polyamine putrescine—arginine and ornithine. Plantlets were regenerated directly from primary shoot apices, axillary buds and protocorm-like bodies [PLB]. Frequency of plantlet regeneration, rate of development and growth in height of main plantlets were enhanced by the addition of arginine and ornithine to the media. Secondary plantlet formation from axillary buds and PLB were promoted by spermine and arginine respectively.     

The behavior of isolated hearts of the grasshopper,Chortophaga viridifasciata,and the moth,Samia walkeri,in solutions with different concentrations of sodium,potassium, calcium,and magnesium

1. The blood of Chortophaga viridifasciata was analyzed. The average concentrations of inorganic cations expressed as milligrams per cent are: sodium, 250.66; potassium, 13.52; calcium, 11.40; and magnesium, 51.15. The osmotic pressure of the blood at 0°C. is 10.7 atmospheres. Protein and non-protein nitrogen, expressed as milligrams per cent, are 253.4 and 140.0, respectively. 2. The blood of Samia walkeri has an osmotic pressure of 13.36 atmospheres at 0°C. Its protein nitrogen is 628.58, and its non-protein nitrogen, 441.20 milligrams per cent. 3. The effects of isotonic chloride solutions of sodium, potassium, calcium, and magnesium and of distilled water on the heart beat of these two species were determined. The heart of the grasshopper failed to beat in isotonic solutions of KCl, MgCl₂, or in distilled water. For both insects, sodium was found to be the least toxic ion. In the case of the grasshopper, calcium ranks next in order. In the case of the moth, potassium ranks next after sodium and is followed by calcium and magnesium. 4. The ratio of sodium to potassium in milligrams per cent, necessary for maintaining the normal heart beat of Chortophaga viridifasciata is 3 to 1, but it may be increased to at least 34 to 1 without any appreciable effects. The ratio of potassium to calcium necessary for maintaining the normal heart beat of this insect is 1 to 1, and may be increased to as much as 3 to 1. 5. The ratio of sodium to potassium, in milligrams per cent, necessary for maintaining the normal heart beat of Samia walkeri was found to be equal to or to exceed 1 to 13.8. The sodium content may be increased so that the ratio of sodium to potassium is 34 to 1 without any toxic effects. The ratios of potassium to calcium required for normal heart beat in this insect may be 1 to 1, 2 to 1, or 3 to 1. 6. The hearts of the grasshoppers beat normally in isotonic solutions having an osmotic pressure of 10.7 atmospheres. They beat equally well in solutions having an osmotic pressure of 13.4 atmospheres. The hearts of the cynthia pupae beat normally in isotonic solutions having an osmotic pressure of 13.36 atmospheres. However, they also beat normally in solutions having an osmotic pressure of 10.02 atmospheres. Therefore, although the blood of the cynthia moth and of the grasshopper have different osmotic pressures, their hearts are tolerant to solutions having the same tonicity. Because of this, and since the ratios of potassium to calcium necessary for maintaining normal heart beats of both insects are the same, solutions favorable to the grasshopper may also be favorable to the cynthia moth.     

Spectrophotometric determination of lipases, lysophospholipases, and phospholipases

Spectrophotometric techniques for determining the activities of lipases, lysophospholipases, and phospholipases are reviewed. These methods involve the use of thioester substrate analogs as well as omega-nitrophenyl derivatives of the corresponding lipids. The most promising results are obtained with the thioester substrate analogs. Mono- and diacylglycerol lipases are assayed by using rac-1-S-decanoyl-1-mercapto-2,3-propanediol and rac-1,2-S,O-didecanoyl-1-mercapto-2,3-propanediol, respectively. Phospholipases A1 and A2 are determined by using rac-1,2-S,O-didecanoyl-3-phosphocholine-1-mercapto-2,3-propanediol and 2-hexadecanoylthio-1-ethyl-phosphocholine, respectively. Lysophospholipases are measured by using 2-hexadecanoylthio-1-ethyl-phosphocholine. Phospholipase C is assayed with rac-1-S-phosphocholine-2,3-O-didecanoyl-1-mercapto-2,3-propanediol. Thioester substrate analog assay procedures are more rapid, sensitive, convenient, continuous, and less expensive than the classical radiochemical techniques.     

The electron affinities of deprotonated adenine, guanine, cytosine, uracil, and thymine

Electron attachment rates and gas phase acidities for the canonical tautomers of the nucleobases and electron affinities for thymine, deprotonated thymine, and cytosine are reported The latter are from a new analysis of published photoelectron spectra. The values for deprotonated thymine are (all in eV) keto-N1-H, 3.327(5); enol-N3-H, 3.250(5), enol-C2OH, 3.120(5) enol-N1-H, 3.013(5), and enol-C4OH,3.123(5). The values for deprotonated cytosine, keto-N1-H, 3.184(5); trans-NH-H, 3.008(5); cis-NH-H, 3.039(5); and enol-N1-H, 2.750(5) and enol-O-H, 2.950(5). The gas phase acidities from these values are obtained from these values using experimental or theoretical calculations of bond dissociation energies. Kinetic and thermodynamic properties for thermal electron attachment to thymine are obtained from mass spectrometric data. We report an activation energy of 0.60 eV and electron affinity of thymine, 1.0(1) eV.     

Molecular interactions of a soluble gibberellin receptor, GID1, with a rice DELLA protein, SLR1, and gibberellin

GIBBERELLIN INSENSITIVE DWARF1 (GID1) encodes a soluble gibberellin (GA) receptor that shares sequence similarity with a hormone-sensitive lipase (HSL). Previously, a yeast two-hybrid (Y2H) assay revealed that the GID1-GA complex directly interacts with SLENDER RICE1 (SLR1), a DELLA repressor protein in GA signaling. Here, we demonstrated, by pull-down and bimolecular fluorescence complementation (BiFC) experiments, that the GA-dependent GID1-SLR1 interaction also occurs in planta. GA(4) was found to have the highest affinity to GID1 in Y2H assays and is the most effective form of GA in planta. Domain analyses of SLR1 using Y2H, gel filtration, and BiFC methods revealed that the DELLA and TVHYNP domains of SLR1 are required for the GID1-SLR1 interaction. To identify the important regions of GID1 for GA and SLR1 interactions, we used many different mutant versions of GID1, such as the spontaneous mutant GID1s, N- and C-terminal truncated GID1s, and mutagenized GID1 proteins with conserved amino acids replaced with Ala. The amino acid residues important for SLR1 interaction completely overlapped the residues required for GA binding that were scattered throughout the GID1 molecule. When we plotted these residues on the GID1 structure predicted by analogy with HSL tertiary structure, many residues were located at regions corresponding to the substrate binding pocket and lid. Furthermore, the GA-GID1 interaction was stabilized by SLR1. Based on these observations, we proposed a molecular model for interaction between GA, GID1, and SLR1.     

Overproduction, purification, and characterization of EBNA1, the origin binding protein of Epstein-Barr virus.

The baculovirus expression system was used to overproduce the Epstein-Barr virus nuclear antigen, EBNA1, in insect cells. EBNA1 overproduced via baculovirus expression (baculoEBNA1) was followed during purification to homogeneity using its ability to specifically retain the family of repeats of the latent origin of replication, oriP, onto nitrocellulose filters. A two-column procedure was developed which yields more than 1 mg of homogeneous baculoEBNA1 from 9 x 10(8) insect cells (1.5 liters). Pure baculoEBNA1 had no detectable ATPase or helicase activity. BaculoEBNA1 was labeled with [32P]orthophosphate in vivo, and analysis showed detectable levels of phosphoserine; no phosphothreonine or phosphotyrosine could be detected. The baculoEBNA1 appeared dimeric in solution, and a stoichiometry of 56 baculoEBNA1 monomers per 24 EBNA1 binding sites in oriP suggests baculoEBNA1 binds its consensus site as a dimer. The binding of baculoEBNA1 to the dyad symmetry element of oriP (Kd approximately 2 nM) required more baculoEBNA1 and appeared less stable than the binding of baculoEBNA1 to the family of repeats in oriP (Kd approximately 0.2 nM).     

The Effect of Mustard Gas on Salivary Trace Metals (Zn,Mn, Cu,Mg, Mo,Sr, Cd,Ca, Pb,Rb)

We have determined and compared trace metals concentration in saliva taken from chemical warfare injures who were under the exposure of mustard gas and healthy subjects by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) for the first time. The influence of preliminary operations on the accuracy of ICP-OES analysis, blood contamination, the number of restored teeth in the mouth, salivary flow rate, and daily variations in trace metals concentration in saliva were also considered. Unstimulated saliva was collected at 10:00–11:00 a.m. from 45 subjects in three equal groups. The first group was composed of 15 healthy subjects (group 1); the second group consisted of 15 subjects who, upon chemical warfare injuries, did not use Salbutamol spray, which they would have normally used on a regular basis (group 2); and the third group contained the same number of patients as the second group, but they had taken their regular medicine (Salbutamol spray; group 3). Our results showed that the concentration of Cu in saliva was significantly increased in the chemical warfare injures compared to healthy subjects, as follows: healthy subjects 15.3± 5.45(p.p.b.), patients (group 2) 45.77±13.65, and patients (Salbutamol spray; group 3) 29 ±8.51 (P <0.02). In contrast, zinc was significantly decreased in the patients, as follows: healthy subjects 37 ± 9.03(p.p.b.), patients (group 2) 12.2 ± 3.56, and patients (Salbutamol spray; group 3) 20.6 ±10.01 (P < 0.01). It is important to note that direct dilution of saliva samples with ultrapure nitric acid showed the optimum ICP-OES outputs.     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

SLLP1, a unique,intra-acrosomal,non-bacteriolytic,c lysozyme-like protein of human spermatozoa

We report the presence of a unique, non-bacteriolytic, c (chicken or conventional type) lysozyme-like protein, SLLP1, in the acrosome of human sperm. C lysozymes are bacteriolytic and can also bind to N-acetylglucosamines linked by beta-1,4 glycosidic bonds. Most of the invariant residues (17 out of 20), including all the cysteines, were conserved in SLLP1, but the two catalytic residues E35 and D52 of c lysozymes were replaced with T and N, respectively. The full-length cDNA encodes a protein of 215 aa with a predicted protease cleavage site between A87 and K88. The processed form of SLLP1, which showed an exon-intron organization similar to human c lysozyme, was the major isoform in the acrosome of ejaculated sperm. As expected, based on its sequence, the mature protein secreted from yeast showed no bacteriolytic activity. A significant decrease (54%, P < or = 0.001) in the number of sperm bound to zona-free hamster eggs was observed in the presence of antisera to recombinant SLLP1. SLLP1 mRNA (size, approximately 1 kb) appeared to be expressed only in the testis and in the Burkitt lymphoma Raji cell line. The gene SPACA3 encodes SLLP1 and contains five exons at locus 17q11.2. Because of its typical c lysozyme-like sequence, genomic organization, conservation of putative substrate-binding sites even in the absence of catalytic residues, and localization in the acrosomal matrix, we hypothesize that, after acrosome reaction, SLLP1 could be a potential receptor for the egg oligosaccharide residue N-acetylglucosamine, which is present in the extracellular matrix over the egg plasma membrane, within the perivitelline space, pores of zona pellucida, and cumulus layers.