scholarly journals Characterization of the yrbA Gene ofBacillus subtilis, Involved in Resistance and Germination of Spores

1999 ◽  
Vol 181 (16) ◽  
pp. 4986-4994 ◽  
Author(s):  
Hiromu Takamatsu ◽  
Takeko Kodama ◽  
Tatsuo Nakayama ◽  
Kazuhito Watabe
Keyword(s):  
Optical Density ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Consensus Sequences ◽  
Insertional Inactivation ◽  
Coat Layer

ABSTRACT Insertional inactivation of the yrbA gene ofBacillus subtilis reduced the resistance of the mutant spores to lysozyme. The yrbA mutant spores lost their optical density at the same rate as the wild-type spores upon incubation with l-alanine but became only phase gray and did not swell. The response of the mutant spores to a combination of asparagine, glucose, fructose, and KCl was also extremely poor; in this medium yrbA spores exhibited only a small loss in optical density and gave a mixture of phase-bright, -gray, and -dark spores. Northern blot analysis of yrbA transcripts in varioussig mutants indicated that yrbA was transcribed by RNA polymerase with ςE beginning at 2 h after the start of sporulation. The yrbA promoter was localized by primer extension analysis, and the sequences of the −35 (TCATAAC) and −10 (CATATGT) regions were similar to the consensus sequences of genes recognized by ςE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins solubilized from intact yrbA mutant spores showed an alteration in the protein profile, as 31- and 36-kDa proteins, identified as YrbA and CotG, respectively, were absent, along with some other minor changes. Electron microscopic examination ofyrbA spores revealed changes in the spore coat, including a reduction in the density and thickness of the outer layer and the appearance of an inner coat layer-like structure around the outside of the coat. This abnormal coat structure was also observed on the outside of the developing forespores of the yrbA mutant. These results suggest that YrbA is involved in assembly of some coat proteins which have roles in both spore lysozyme resistance and germination.

Bacillus subtilis spoVIF (yjcC) gene, involved in coat assembly and spore resistance

Microbiology ◽  
2003 ◽  
Vol 149 (10) ◽  
pp. 3011-3021 ◽  
Author(s):  
Ritsuko Kuwana ◽  
Satoko Yamamura ◽  
Hiromi Ikejiri ◽  
Kazuo Kobayashi ◽  
Naotake Ogasawara ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Electron Microscopic Examination ◽  
Coat Proteins ◽  
Electron Microscopic ◽  
Systematic Screening ◽  
Consensus Sequences ◽  
Transmission Electron ◽  
Insertional Inactivation ◽  
Spore Coat Proteins

In systematic screening four sporulation-specific genes, yjcA, yjcB, yjcZ and yjcC, of unknown function were found in Bacillus subtilis. These genes are located just upstream of the cotVWXYZ gene cluster oriented in the opposite direction. Northern blot analysis showed that yjcA was transcribed by the SigE RNA polymerase beginning 2 h (t 2) after the onset of sporulation, and yjcB, yjcZ and yjcC were transcribed by the SigK RNA polymerase beginning at t 4 of sporulation. The transcription of yjcZ was dependent on SigK and GerE. The consensus sequences of the appropriate sigma factors were found upstream of each gene. There were putative GerE-binding sites upstream of yjcZ. Insertional inactivation of the yjcC gene resulted in a reduction in resistance of the mutant spores to lysozyme and heat. Transmission electron microscopic examination of yjcC spores revealed a defect of sporulation at stage VI, resulting in loss of spore coats. These results suggest that YjcC is involved in assembly of spore coat proteins that have roles in lysozyme resistance. It is proposed that yjcC should be renamed as spoVIF.

scholarly journals Expression of Aleutian Mink Disease Parvovirus Capsid Proteins in a Baculovirus Expression System for Potential Diagnostic Use

1994 ◽  
Vol 6 (1) ◽  
pp. 23-29 ◽  
Author(s):  
Wai-Hong Wu ◽  
Marshall E. Bloom ◽  
Bradley D. Berry ◽  
Michael J. McGinley ◽  
Kenneth B. Platt
Keyword(s):  
Microscopic Examination ◽  
Expression System ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Baculovirus Expression System ◽  
Immunoblot Analysis ◽  
Capsid Proteins ◽  
Electron Microscopic ◽  
Nuclear Polyhedrosis

A 2.3-kb cDNA clone encoding Aleutian mink disease parvovirus (ADV) structural proteins VP1 and VP2 was inserted into the polyhedron gene of Autographa calijbmica nuclear polyhedrosis virus (AcNPV) and expressed by the recombinant virus, AcADV-1, in Spodoptera frugiperda-9 cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western immunoblot analysis (WIA) indicated that synthesis of both VP1 and VP2 was being directed by AcADV-1. Fluorescence microscopic examination of AcADV-1 -infected S. frugiperda-9 cells indicated that the recombinant protein was present within the nucleus of the cells, and electron microscopic examination of these cells revealed the presence of small particles 23–25 nm in diameter. Structures resembling empty ADV capsids could be purified on CsCl density gradients, thus indicating that the ADV proteins were self-assembling. The antigenicity of recombinant VP1 and VP2 was evaluated by WIA. Sera collected from 16 mink prior to infection with ADV did not react with VP1 and VP2. Ten sera collected from mink with counter current immunoelectrophoresis (CIE) titers greater than 4 (log2) reacted with VP1 and VP2 in WIA. Two of 6 sera with CIE titers of 4 and 1 of 14 sera with CIE titers <4 reacted with the recombinant proteins. These results suggest that baculovirus recombinant ADV capsid proteins may be useful as diagnostic antigens.

Electrophoretic and electron microscopic examination of the adductor and diductor muscles of an articulate brachiopod, Terebratalia transversa

1982 ◽  
Vol 60 (4) ◽  
pp. 550-559 ◽  
Author(s):  
William P. Eshleman ◽  
Jerrel L. Wilkens ◽  
Michael J. Cavey
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Light Chains ◽  
Electron Microscopic ◽  
Electrophoretic Patterns ◽  
Transmission Electron ◽  
Adductor Muscles ◽  
Regulation Of Contraction

The proteins of the striated adductor muscles, smooth adductor muscles, and diductor muscles of the articulate brachiopod Terebratalia transversa have been examined by sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Electrophoretic patterns indicate the presence of paramyosin in all of these valve muscles. Tentative identification has also been made of the proteins responsible for actin and for myosin regulation of contraction (troponin–tropomyosin and myosin light chains, respectively). The myofilaments of the striated adductor cells, smooth adductor cells, and diductor cells have been characterized by transmission electron microscopy. The smooth adductor cells and the diductor cells exhibit very thick myofilaments which are fusiform in shape, exceptionally long, and axially banded. Morphological features of these thick myofilaments are consistent with those of paramyosin filaments found in other muscles and myoepithelia. Although the striated adductor cells contain paramyosin, it is not manifest in the thick myofilaments.

scholarly journals Some studies on the composition and surface properties of oil bodies from the seed cotyledons of safflower (Carthamus tinctorius) and linseed (Linum ustatissimum)

1980 ◽  
Vol 190 (3) ◽  
pp. 551-561 ◽  
Author(s):  
C R Slack ◽  
W S Bertaud ◽  
B D Shaw ◽  
R Holland ◽  
J Browse ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Electron Microscopic Examination ◽  
Carthamus Tinctorius ◽  
Oil Body ◽  
Oil Bodies ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Glycerolipid Composition ◽  
Induced Aggregation

1. The average oil-body diameter in intact cells of developing linseed (Linum usitatissimum) and safflower (Carthamus tinctorius) cotyledons was similar (about 1.4 micrometer), and there was little change in size after oil bodies were isolated and repeatedly washed. 2. The glycerolipid composition of washed oil bodies from both developing and mature cotyledons of the two species was similar; oil bodies from ten different batches of cotyledons contained 4.3 +/- 0.16 mumol of 3-sn-phosphatidylcholine and 25.2 +/- 1.7 mumol of diacylglycerol per 1000 mumol of triacylglycerol. During four successive washings of a once-washed oil-body preparation, the proportion of diacylglycerol to triacylglycerol remained constant and that of 3-sn-phosphatidylcholine to triacylglycerol decreased by only 20%. 3. The protein content of thrice-washed oil bodies from the two species was similar, about 2.4% of the weight of glycerolipids, and appeared to be independent of the stage of cotyledon maturity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that the protein of purified oil bodies from the two species consisted mainly of only four polypeptides and that two of the polypeptides from each species had apparent mol.wts. of 17500 and 15500. Similar patterns of polypeptides were obtained after the hydrolysis of the 15500-mol.wt. polypeptides from linseed and safflower oil bodies by Staphylococcus aureus V8 proteinase, whereas the proteolysis of the 17500-mol.wt. polypeptides from the two species produced different patterns of polypeptides. 4. The 3-sn-phosphatidylcholine in oil-body preparations was hydrolysed about 85% by bee-venom phospholipase A2 without any apparent coalescence of the oil bodies. Incubation with lipase from Rhizopus arrhizus caused rapid coalescence of the oil bodies, and this lipase appeared to initially hydrolyse diacylglycerols in preference to triacylglycerol. 5. Oil bodies from both species were almost completely dispersed in suspensions of pH between 7.1 and 8.3, but formed large aggregates at pH values between 6.7 and 3.9; pH-induced aggregation caused no coalescence. Aggregates formed under acidic conditions were dispersed by re-adjusting the pH of suspensions to 8.3. 6. A freeze-etch electron-microscopic examination of isolated oil bodies indicated that these organelles were bounded by some form of membrane with a particle-free outer surface.

scholarly journals EFFECTS OF HUMAN PLATELET LYSATES WITHOUT ADDITIONAL GROWTH FACTORS ON THE PROTEIN PROFILES OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELL CULTURE MEDIA

2017 ◽  
Vol 10 (17) ◽  
pp. 54
Author(s):  
Hazriani Ra ◽  
Lisa Amir ◽  
Ratna Farida
Keyword(s):  
Culture Media ◽  
Umbilical Vein ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Test Results ◽  
Control Groups ◽  
Cell Culture Media ◽  
Endothelial Cell Culture ◽  
Fetal Bovine

Objectives: The objective of this study is to evaluate the effect of HPLs with no additional GFs on the HUVEC protein profile. Methods: HUVEC cultures were examined in groups as follows: Fetal bovine serum (FBS), 2%-HPL with a GF, and 2%- and 5%-HPL without a GF which were analyzed with a sodium dodecyl sulfate-polyacrylamide gel electrophoresis test. Results: The intensity, thickness, and molecular weight of HUVEC band proteins cultured without a GF were not significantly different compared to the control groups (FBS or HPL with a GF). Conclusions: No difference was found in the HUVEC protein profile after they were cultured with FBS and HPLs, with or without GFs.

scholarly journals Characterization of Proteins in the Outer Membrane Preparation of a Murine Pathogen, Helicobacter bilis

2001 ◽  
Vol 69 (5) ◽  
pp. 3502-3506 ◽  
Author(s):  
Zhongming Ge ◽  
Peter Doig ◽  
James G. Fox
Keyword(s):  
Outer Membrane ◽  
Wide Spectrum ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Membrane Preparation ◽  
Laboratory Animals ◽  
Helicobacter Bilis ◽  
H Pylori

ABSTRACT Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins fromH. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.

Plant NAD-Dependent Glutamate Dehydrogenase. Purification, Molecular Properties and Metal Ion Activation of the Enzymes from Lemna minor and Pisum sativum

1980 ◽  
Vol 35 (3-4) ◽  
pp. 213-221 ◽  
Author(s):  
Heinz-Walter Scheid ◽  
Adelheid Ehmke ◽  
Thomas Hartmann
Keyword(s):  
Amino Acid ◽  
Pisum Sativum ◽  
Glutamate Dehydrogenase ◽  
Gel Electrophoresis ◽  
Metal Ion ◽  
Lemna Minor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Equilibrium ◽  
Electron Microscopic

Abstract Glutamate dehydrogenase (ʟ-glutamate: NAD+ oxidoreductase (deaminating) EC 1.4.1.2) has been purified to homogeneity from Lemna minor and seeds of Pisum sativum. As established by polyacrylamide gel electrophoresis the Pisum-enzyme constitutes a multiple pattern of seven char­ge isoenzymes whereas the Lemna enzyme shows one single protein band. Molecular weights of 230 000 were calculated for both enzymes by sedimentation equilibrium measurements (Pisum-enzyme) and comparative gel filtration (Lemna-enzyme). Sodium dodecyl sulfate gel electrophoresis and electron microscopic observations revealed that both enzymes are composed of four identical subunits (molecular weight 58 500) arranged in a tetraedric structure. The amino acid compositions of both enzymes are similar to those of various hexameric glutamate dehydrogenases. The N-terminal amino acid of the Pisum-enzyme is alanine. Both enzymes require Ca2+ for maximal catalytic activity. For the Lemna-enzyme the K0.5 values for Ca2+ are 22 µᴍ (NADH-dependent reaction) and 4 µᴍ (NAD+ -dependent reaction), respectively. Ca2+ which to some extent can be replaced by Zn2+ does not affect the enzyme aggregation but seems to govern a reversible equilibrium between catalytically active and inactive enzyme forms.

scholarly journals The population, protein profile and ultrastructure of Ascaridia galli in chicken treated using Areca catechu crude aqueous extract

2019 ◽  
Vol 44 (4) ◽  
pp. 392
Author(s):  
W. W. Mubarokah ◽  
W. Nurcahyo ◽  
J. Prastowo ◽  
K. Kurniasih
Keyword(s):  
Aqueous Extract ◽  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Anthelmintic Activity ◽  
Positive Control ◽  
Negative Control ◽  
Ascaridia Galli ◽  
Areca Catechu ◽  
Crude Aqueous Extract

The study aimed at investigating the population, the protein profile and the ultrastructure of adult worms in the intestine of domestic chicken treated using Areca catechu crude aqueous extract. Fifty domestic female chickens of 6 weeks of age were assigned to 5 groups. Group A (negative control) was not given any treatment and any drug. Groups B, C and D were given the treatment at the doses of 26 mg/mL, 53 mg/mL and 79 mg/mL, respectively. Group E (positive control) was given Pyrantel®. Necropsy was conducted to all of the chickens 14 days after the treatment. Adult worms were collected and counted. The worms used in Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and Scanning Electron Microscopy (SEM) were those collected from the jejunum of the chickens in the groups A, B and C. The biggest number of the worms was found in the jejunum. The results of electrophoresis showed that the dose 53 mg/mL gave fewer protein bands than the negative control (21:12 ratio), while the results of the SEM showed that there was cuticle damage and anterior labia abrasion at the dose of 53 mg/mL. The Areca catechu crude aqueous extract showed anthelmintic activity potential by reducing the number of the adult worms, lowering their protein profile and damaging the A. galli worms in the intestine.

Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

1992 ◽  
Vol 4 (2) ◽  
pp. 249 ◽  
Author(s):  
A Paliwal ◽  
B Malaviya ◽  
VP Kamboj
Keyword(s):  
Menstrual Cycle ◽  
Protein Profile ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Blue Staining ◽  
Protein Patterns ◽  
Coomassie Blue Staining ◽  
Coomassie Blue ◽  
Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

scholarly journals Isolation of the intercellular glycoproteins of desmosomes.

1981 ◽  
Vol 90 (1) ◽  
pp. 243-248 ◽  
Author(s):  
G Gorbsky ◽  
M S Steinberg
Keyword(s):  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insoluble Residue ◽  
Differential Centrifugation ◽  
Electron Microscopic ◽  
Nonionic Detergent ◽  
Cell Layers ◽  
Membrane Attachment

To characterize the desmosome components that mediate intercellular adhesion and cytoskeletal-plasma membrane attachment, we prepared whole desmosomes and isolated desmosomal intercellular regions (desmosomal "cores") from the living cell layers of bovine muzzle epidermis. The tissue was disrupted in a nonionic detergent at low pH, sonicated, and the insoluble residue fractionated by differential centrifugation and metrizamide gradient centrifugation. Transmission electron microscopic analyses reveal that a fraction obtained after differential centrifugation is greatly enriched in whole desmosomes that possess intracellular plaques. Metrizamide gradient centrifugation removes most of the plaque material, leaving the intercellular components and the adjoining plasma membranes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis coupled with methods that reveal carbohydrate-containing moieties on gels demonstrate that certain proteins present in whole desmosomes are glycosylated. These glycoproteins are specifically and greatly enriched in the desmosome cores of which they are the principal protein constituents, and thus may function as the intercellular adhesive of the desmosome.

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