Genetic identification of two distinct DNA polymerases, DnaE and PolC, that are essential for chromosomal DNA replication in Staphylococcus aureus

We isolated and characterized temperature-sensitive mutants for two genes, dnaE and polC, that are essential for DNA replication in Staphylococcus aureus. DNA replication in these mutants had a slow-stop phenotype when the temperature was shifted to a non-permissive level. The dnaE gene encodes a homolog of the alpha-subunit of the DNA polymerase III holoenzyme, the replicase essential for chromosomal DNA replication in Escherichia coli. The polC gene encodes PolC, another catalytic subunit of DNA polymerase, which is specifically found in gram-positive bacteria. The wild-type dnaE or polC gene complemented the temperature-sensitive phenotypes of cell growth and DNA replication in the corresponding mutant. Single mutations resulting in amino-acid exchanges were identified in the dnaE and polC genes of the temperature-sensitive mutants. The results indicate that these genes encode two distinct DNA polymerases which are both essential for chromosomal DNA replication in S. aureus. The number of viable mutant cells decreased at non-permissive temperature, suggesting that inactivation of DnaE and PolC has a bactericidal effect and that these enzymes are potential targets of antibiotics.     

Albutensin A, an ileum-contracting peptide derived from serum albumin, acts through both receptors for complements C3a and C5a

Albutensin A is an ileum-contracting peptide derived from serum albumin. The sequences of bovine, human and porcine albutensin A are ALKAWSVAR, AFKAWAVAR, and AFKAWSLAR, respectively. These albutensin A homologs all exhibited biphasic ileal contractions in the longitudinal strips of guinea pig ileum. The order of potency in the contraction was porcine > bovine > human homologs. The ileal contraction profiles were similar to those of oryzatensin and casoxin C, agonist peptides for complement C3a receptors derived from rice albumin and bovine -casein, respectively. All three homologs of albutensin A have homology with the COOH-terminal sequences of complements C3a and C5a, which are essential for their activities; porcine albutensin A showed the highest homology. Indeed, porcine albutensin A was confirmed to act through both C3a and C5a receptors by a radioreceptor assay and cross-desensitization in the ileal contraction. In addition, bovine and human homologs also showed affinity for both receptors. This study suggests that a bioactive peptide acting through both C3a and C5a receptors is released by the proteolytic cleavage of serum proteins other than complement components.     

Preparation of (2S,4R)‐2,4‐thiazolidinedicarboxylic acid by separation of diastereoisomeric salts with organic amines

L ‐Cysteine was condensed with glyoxylic acid monohydrate in acetic acid at 30°C to give (4R)‐2,4‐thiazolidinedicarboxylic acid [(4R)‐TDA] as a mixture of two diastereoisomers, (2R,4R)‐ and (2S,4R)‐TDA. An attempt was made to separate (2S,4R)‐TDA from the diastereoisomeric salts of (4R)‐TDA with 1‐propylamine, 2‐methyl‐2‐propylamine, benzylamine, and (R)‐ and (S)‐1‐phenylethylamines [(R)‐ and (S)‐PEA]. The salts of (2S,4R)‐TDA were preferentially crystallized as less soluble diastereoisomeric salts. When the salt with (R)‐PEA was employed, the separation was successfully achieved to afford optically pure (2S,4R)‐TDA in a yield of 41%, based on the starting amount of the diastereoisomeric mixture of (4R)‐TDA. Chirality 11:326–329, 1999. © 1999 Wiley‐Liss, Inc.     

Application of rapid freezing followed by freeze-substitution acrolein fixation for cytochemical studies of the rat stomach

Summary A method involving rapid freezing followed by substitution fixation was developed, using acrolein as a fixative. This was then applied to several cytochemical stainings, and showed well preserved and clear cell structures. Membranes were apparently negatively stained and the ultrastructure of mitochondria, rough endoplasmic reticulum and Golgi apparatus was clearly discernible. The mitochondrial and cytoplasmic matrices were stained rather densely compared with routine chemically fixed preparations, implying a good preservation of matrix substrances. Periodic acid-thiocarbohydrazide-silver proteinate staining was applied to the present method. The mucous granules of surface covering epithelial cells indicated fine staining of bipartite structure and the Golgi apparatus of mucous cells showed clear staining differences based on polarity. Postembedding lectin-ferritin and immunocytochemical stainings were applicable to the present preparations and stable stainings of secretory granules were obtained. A low temperature embedding material, Lowicryl K4M, was also examined. The cell preservation of these samples was not as good as those embedded in Epon, but the rough endoplasmic reticulum and Golgi apparatus of chief cells were stained with anti-pepsinogen antibody as were the secretory granules. The present method was also applicable to light microscopy.     

Glycoconjugate cytochemistry of the rat fundic gland using lectin/colloidal-gold conjugates and Lowicryl K4M

Summary The fundic gland of the rat stomach was studied using the low-temperature embedding resin Lowicryl K4M and postembedding staining with lectin/colloidal-gold (CG) conjugates. Intense labeling with Ricinus communis agglutinin I was observed not only in mucous-producing cells but also in parietal cells. In contrast, Helix pomatia agglutinin (HPA) only labeled mucous neck cells and intermediate cells between mucous neck cells and chief cells. The other epithelial cells present in the rat fundic gland showed virtually no reaction with this lectin. Our results indicate that HPA might be a marker lectin of mucous neck cells and their derivatives. The combination of embedding in the hydrophilic resin Lowicryl K4M and postembedding staining with lectin-CG conjugates provided satisfactory staining results, and made it possible to visualize the precise distribution of terminal glycoconjugates in intracellular components as well as on the plasma membrane.     

Glycoconjugate cytochemistry of the rat fundic gland using lectin/colloidal-gold conjugates and Lowicryl K4M. Helix pomatia lectin is a specific marker for mucous neck cells in fundic glands of the rat gastric mucosa

The fundic gland of the rat stomach was studied using the low-temperature embedding resin Lowicryl K4M and postembedding staining with lectin/colloidal-gold (CG) conjugates. Intense labeling with Ricinus communis agglutinin I was observed not only in mucous-producing cells but also in parietal cells. In contrast, Helix pomatia agglutinin (HPA) only labeled mucous neck cells and intermediate cells between mucous neck cells and chief cells. The other epithelial cells present in the rat fundic gland showed virtually no reaction with this lectin. Our results indicate that HPA might be a marker lectin of mucous neck cells and their derivatives. The combination of embedding in the hydrophilic resin Lowicryl K4M and postembedding staining with lectin-CG conjugates provided satisfactory staining results, and made it possible to visualize the precise distribution of terminal glycoconjugates in intracellular components as well as on the plasma membrane.     

Crystallization and preliminary x-ray investigation of soybean beta-amylase.

Beta-Amylase [1, 4-alpha-D-glucan maltohydrolase, EC 3.2.1.2] has been purified from defatted soybean meal by fractional precipation with ammonium sulfate, ion-exchange chromatography on CM- and DEAE-Sephadex and gel filtration chromatography on Sephadex G-100. Two different components of beta-amylase were crystallized from ammonium sulfate solutions, and the homogeneity of each preparation was confirmed by sedimentation and disc electrophoretic analyses. Both components of soybean beta-amylase formed large single crystals (trigonal crystal system) from 40--50 per cent saturated ammonium sulfate solution buffered at pH 5.4 on dialyzing concentrated protein solution in the apparatus of Zeppezauer et al. Preliminary X-ray diffraction data gave a hexagonal lattice with unit cell dimensions a=86.1 A and c=144.4 A. The space group corresponds to P3121 or P3221, and one asymmetric unit contains one molecule of beta-amylase, assuming a crystal density of 1.25 g/ml and a molecular weight of the enzyme of 60,000 daltons. In this case, the crystal has a volume of 2.53 A-3 per atomic mass unit, and the percentage of protein in the crystal is about 52.     

A monoclonal antibody, PGM34, against 6-sulfated blood-group H type 2 antigen, on the carbohydrate moiety of mucin

Mucin, a major component of mucus, is a highly O-glycosylated, high-molecular-mass glycoprotein extensively involved in the physiology of gastrointestinal mucosa. To detect and characterize mucins derived from site-specific mucous cells, we developed a monoclonal antibody, designated PGM34, by immunizing a mouse with purified pig gastric mucin. The reactivity of PGM34 with mucin was inhibited by periodate treatment of the mucin, but not by trypsin digestion. This suggests that PGM34 recognizes the carbohydrate portion of mucin. To determine the epitope, oligosaccharide-alditols obtained from pig gastric mucin were fractionated by successive gel-filtration, ion-exchange, and normal-phase HPLC, and tested for reactivity with PGM34. Two purified oligosaccharide-alditols that reacted with PGM34 were obtained. Their structures were determined by NMR spectroscopy as Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Fucalpha1-2Galbeta1-3)GalNAc-ol and Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta1-6(Galbeta1-3)GalNAc-ol. None of the defucosylated or desulfated forms of these oligosaccharides reacted with PGM34. Thus, the epitope of PGM34 was determined as the Fucalpha1-2Galbeta1-4GlcNAc(6SO(3)H)beta- sequence. Immunohistochemical examination of rat gastrointestinal tract showed that PGM34 stained surface mucous cells close to the generative cell zone in the gastric fundus and goblet cells in the small intestine, but only slightly stained antral mucous cells in the stomach. These data, taken together, show that PGM34 is a very useful tool for elucidating the role of mucins with characteristic sulfated oligosaccharides.