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J Smit

Publications and source records attributed to J Smit.

At least 73 records · Page 4Linked to original sources
PubMed1992

Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein.

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98,132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest.

Amino Acid Sequence↗
PubMed1991

Expression in Caulobacter crescentus of the phosphate-starvation-inducible porin OprP of Pseudomonas aeruginosa.

The gene for the phosphate-starvation-inducible outer membrane protein OprP, of Pseudomonas aeruginosa was introduced into Caulobacter crescentus CB2A on a plasmid vector. As is the case in P. aeruginosa and Escherichia coli the oprP gene was inducible under conditions of limiting phosphate in C. crescentus. However, the maximal medium concentration of phosphate which still permitted induction of OprP was lower in C. crescentus (50 microM) than in P. aeruginosa (200 microM). Induction of OprP was coincident with the process of stalk elongation, known to occur in C. crescentus under phosphate starvation conditions. When induced, OprP was localized to the cell envelope and became a major membrane protein, indicating that the Pseudomonas promoter was efficiently recognized in C. crescentus and that the gene product was targeted to the appropriate region of the cell. Our data provide support for the hypothesis that the mechanism for regulation of phosphate-starvation-inducible genes is highly conserved amongst the eubacteria.

Bacteria↗
PubMed1991

Characterization of caulobacters isolated from wastewater treatment systems.

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.

Bacteria↗
PubMed1991

A transducing bacteriophage for Caulobacter crescentus uses the paracrystalline surface layer protein as a receptor.

The bacteriophage phi Cr30, a transducing phage for Caulobacter crescentus strains, required the paracrystalline surface (S) layer for infectivity. Wild-type strains were phage resistant when rsaA, the gene for the 130K S-layer protein, was interrupted with an antibiotic resistance cassette. Strains that had lost the S layer by mutation were phage resistant, as were mutants that produce an S layer but which do not attach the structure to the cell surface. Phage sensitivity was restored to 130K-protein-deficient strains by introducing rsaA on a plasmid. Spontaneous phage-resistant strains produced expected phenotypes as follows (in order of decreasing frequency): S-layer cell attachment defects, no S layer, or an S layer that was wild type in appearance.

Bacteria, Aerobic↗
PubMed1991

Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus.

Caulobacters are adherent prosthecate bacteria that are members of bacterial biofouling communities in many environments. Investigation of the cell surface carbohydrates produced by two strains of the freshwater Caulobacter crescentus, CB2A and CB15A, revealed a hitherto undetected extracellular polysaccharide (EPS) or capsule. Isolation and characterization of the EPS fractions showed that each strain produced a unique neutral EPS which could not be readily removed from the cell surface by washing. Monosaccharide analysis showed that the main CB2A EPS contained D-glucose, D-gulose, and D-fucose in a ratio of 3:1:1, whereas the CB15A EPS fraction contained D-galactose, D-glucose, D-mannose, and D-fucose in approximately equal amounts. Methylation analysis of the main CB2A EPS showed the presence of terminal glucose and gulose groups, 3-linked fucosyl, and two 3,4-linked glucosyl units, thus confirming the pentasaccharide repeating unit indicated by 1H nuclear magnetic resonance analysis. Similar studies of the CB15A EPS revealed a tetrasaccharide repeating unit consisting of terminal galactose, 4-linked fucosyl, 3-linked glucosyl, and 3,4-linked mannosyl residues. EPS was not detectable by thin-section electron microscopy techniques, including some methods designed to preserve or enhance capsules, nor was the EPS readily detected on the cell surface by scanning electron microscopy when conventional fixation techniques were used; however, a structure consistent with EPS was revealed when samples were prepared by cryofixation and freeze-substitution methods.

Bacterial Adhesion↗
PubMed1991

Transformation of freshwater and marine caulobacters by electroporation.

We performed plasmid electrotransformation of Caulobacter crescentus strains and obtained up to 3 x 10(8) transformants per micrograms of pKT230. The presence and integrity of the paracrystalline protein surface (S) layer influenced electroporation; caulobacters lacking the S layer were electrotransformed 10 times more efficiently than caulobacters possessing the S layers. A procedure yielding 1,500 transformants per micrograms of pKT230 was developed for a marine caulobacter. Electroporation was used in combination with several genetic techniques, including introduction of ligation mixtures, suicide transposon mutagenesis, gene replacement, and plasmid electrotransfer from Escherichia coli to caulobacters.

Bacteria↗
PubMed1991

Perceptions of caring: do patients and nurses agree?

A recent study of home care nurses and their patients showed that perceptions of nurse caring behaviors often vary among the two groups. Understanding these differences can help agencies allocate resources and nurses offer more appropriate care.

Behavior↗
PubMed1991

A method of tagging specific-purpose linkers with an antibiotic-resistance gene for linker mutagenesis using a selectable marker.

A method is described for tagging double-stranded linkers with an antibiotic-resistance gene permitting the direct selection of specific linker insertions during random linker-insertion mutagenesis; after selection, the antibiotic-resistance marker is removed leaving the linker-encoded restriction site in the target gene. The method is simple, relying on commercially available linkers and DNA-modifying enzymes, but retains considerable target site flexibility. The tagged linkers can be inserted at blunt-ended sites created in the target gene with DNase I or at sites created by restriction enzyme digestion leaving blunt ends, 5'-CG or 5'-GATC extensions. The advantages of such an approach over the use of standard antibiotic-resistance gene cartridges is discussed.

Base Sequence↗
PubMed1990

Behavioral and neuroendocrine response to psychosocial stress in male rats: the effects of the 5-HT 1A agonist ipsapirone.

The effect of the 5-HT 1A agonist ipsapirone on the behavior, plasma catecholamine, and corticosterone levels was studied in male Wistar rats during the psychosocial stress of confrontation with a confined dominant opponent 24 hr after defeat. The effect of the drug was also studied during a predefeat confrontation with the confined (would-be dominant) rat. Blood samples were withdrawn via a permanent heart catheter. The drug (5 mg/kg, ip) or vehicle was given 30 min before transportation to the experimental room. Ipsapirone had no major effects on the plasma hormone concentrations and had no influence upon the behavioral response to the confined rat. At the postdefeat test ipsapirone led to a significant increase of immobility, whereas both rearing and time spent sniffing the cage were diminished. Postdefeat psychosocial stress resulted in an increase of the hormone, particularly catecholamine levels. These responses were further elevated by the drug. The presence of high corticosterone levels in the home cage after postdefeat ipsapirone treatment leads to the hypothesis that postsynaptic 5-HT 1A receptor hypersensitivity develops after the social stress of defeat.

Agonistic Behavior↗
PubMed1990

Influence of apo E polymorphism on the response to simvastatin treatment in patients with heterozygous familial hypercholesterolemia.

In a group of 120 patients with heterozygous familial hypercholesterolemia (FH) the influence of the apolipoprotein E (apoE) polymorphism on pre-treatment plasma lipid levels and on the response to treatment with simvastatin was studied. The apoE phenotype distribution did not differ significantly between the FH group and a sample group of the Dutch population. Differences in pre-treatment lipid levels were not related to the apoE polymorphism in this FH population. After 12 weeks use of a daily dose of 40 mg simvastatin, the plasma total cholesterol, low density lipoprotein (LDL)-cholesterol and plasma triglyceride levels were reduced on average by 33%, 38% and 19%, respectively. At the same time high density lipoprotein (HDL)-cholesterol concentration increased on average by 7%. In the combined FH patient group (males and females) a considerable interindividual variation in response to simvastatin was observed, but was not related to the apoE polymorphism. However, considering males and females separately, we found that female FH patients with the apoE3E3 phenotype responded better on simvastatin treatment with respect to LDL-cholesterol than male FH patients with the apoE3E3 phenotype.

Adult↗
PubMed1990

High-level expression vectors for Caulobacter crescentus incorporating the transcription/translation initiation regions of the paracrystalline surface-layer-protein gene.

A number of plasmid vectors were constructed for high-level gene expression in the dimorphic gram-negative bacterium Caulobacter crescentus. These vectors incorporate the transcription and translation initiation regions of the C. crescentus CB15A rsaA gene, which codes for the abundantly synthesized protein comprising the bacterium's paracrystalline surface layer. The expression vectors are based on the broad-host-range IncQ plasmid RSF1010 (R300B) and incorporate the rsaA promoter and transcription start site. Some vectors also contain translation initiation information; these can result in the addition of as little as a single glycine residue to the protein encoded by the cloned segment. The vectors can be introduced into C. crescentus by electroporation at high frequency (ranging up to 10(6)-10(7) electroporants/micrograms DNA with surface-layer-deficient mutant C. crescentus CB2A) or conjugal transfer. They range in size from 10 to 12 kb, specify either chloramphenicol or kanamycin resistance, and possess the restriction sites EcoRI, BamHI, KpnI, and SstI for cloning genes downstream of the rsaA gene sequences. For a number of the vectors, the complete nucleotide sequence is known. A comparison was made between the expression of an endoglucanase gene from these plasmids in C. crescentus CB2A and CB15A and similar constructions under the control of lacZ alpha transcription and translation initiation signals carried on a pUC9 vector in an Escherichia coli host. The two expression systems compared favorably; cell lysates prepared from C. crescentus CB2A exhibited 40% of the endoglucanase activity of similarly prepared lysates from E. coli JM101. Lysates prepared from C. crescentus CB15A exhibited only 8% of the endoglucanase activity of E. coli lysates.

Amino Acid Sequence↗
PubMed1990

Behavioural physiology of serotonergic and steroid-like anxiolytics as antistress drugs.

Pharmacological studies are useful tools to understand the neurobiological basis of behavioural and physiological stress mechanisms. Ipsapirone, a 5-HT1A autoreceptor agonist is a representative of novel anxiolytics without the disadvantages of benzodiazepam-like drugs. Behavioural, physiological and neuroendocrine studies in the rat are reviewed which were aimed to investigate the antistress properties of ipsapirone during reexposure to various conditioned emotional stress situations. It is demonstrated that in certain situations, probably due to a stress-induced sensitisation of postsynaptic 5-HT1A receptors, anxiolytic doses of the drug may show prostress (anxiogenic) behavioural and neuroendocrine effects. Furthermore, brain corticosteroid receptors, probably interacting with the serotonergic transmission, are involved in anxiogenic/prostress processes. In this respect antagonists of the brain mineralocorticoid-like (type I) receptors may be important antistress drugs of the future.

Animals↗
PubMed1990

Attachment of the adhesive holdfast organelle to the cellular stalk of Caulobacter crescentus.

Caulobacters attach to surfaces in the environment via their holdfasts, attachment organelles located at the base of the flagellum in swarmer cells and later at the end of the cellular stalk in the stalked cells which develop from the swarmer cells. There seems to be little specificity with respect to the types of surfaces to which holdfasts adhere. A notable exception is that the holdfast of one cell does not adhere to the cell surface of another caulobacter, except by joining holdfasts, typically forming "rosettes" of stalked cells. Thus, the localized adhesion of the holdfasts to the cells is in some way a specialized attachment. We investigated this holdfast-cell attachment by developing an adhesion screening assay and analyzing several mutants of Caulobacter crescentus CB2A selected to be defective in adhesion. One class of mutants made a normal holdfast by all available criteria, yet the attachment to the cell was very weak, such that the holdfast was readily shed. Another class of mutants made no holdfast at all, but when mixed with a wild-type strain, a mutant of this class participated in rosette formation. The mutant could also attach to the discarded holdfast produced by a shedding mutant. In addition, when rosettes composed of holdfast-defective and wild-type cells were examined, an increase in the number of holdfast-defective cells was correlated with a decrease in the ability of the holdfast material at the center of the rosette to bind colloidal gold particles. Gold particles are one type of surface to which holdfasts adhere well, suggesting that the stalk end and the colloidal gold particles occupy the same sites on the holdfast substance. Taken together, the data support the interpretation that there is a specialized attachment site for the holdfast at the base of the flagellum which later becomes the end of the stalk, but not a specialized region of the holdfast for attachment to this site. Also, attachment to the cell is accomplished by bond formations that occur not only at the time of holdfast production. Thus, we propose that the attachment of the holdfast to the cell is a true adhesion process and that the stalk tip and base of the flagellum must have compositions distinctly different from that of the remainder of the caulobacter cell surface.

Bacteria↗
PubMed1990

Identification of genes affecting production of the adhesion organelle of Caulobacter crescentus CB2.

Transposon (Tn5) mutagenesis was used to identify regions in the genome involved with production, regulation, or attachment to the cell surface of the adhesive holdfast of the freshwater bacterium Caulobacter crescentus CB2. A total of 12,000 independently selected transposon insertion mutants were screened for defects in adhesion to cellulose acetate; 77 mutants were detected and examined by Southern blot hybridization mapping methods and pulsed-field gel electrophoresis. Ten unique sites of Tn5 insertion affecting holdfast function were identified that were clustered in four regions of the genome. Representative mutants of the 10 Tn5 insertion sites were examined by a variety of methods for differences in their phenotype leading to the loss of adhesiveness. Four phenotypes were identified: no holdfast production, production of a smaller or an altered holdfast, production of a holdfast that was unable to remain attached to the cell, and a fourth category in which a possible alteration of the stalk was related to impaired adhesion of the cell. With the possible exception of the last class, no pleiotropic mutants (those with multiple defects in the polar region of the cell) were detected among the adhesion-defective mutants. This was unexpected, since holdfast deficiency is often a characteristic of pleiotropic mutants obtained when selecting for loss of other polar structures. Overall, the evidence suggests that we have identified regions containing structural genes for the holdfast, genes involved with proper attachment or positioning on the caulobacter surface, and possibly regions that regulate the levels of holdfast production.

Bacterial Adhesion↗
PubMed1990

Electromyogram power spectra frequencies associated with motor unit recruitment strategies.

The isolated contributions of motor unit recruitment and firing rate variations to the median frequency of the electromyogram's power density spectrum were determined. Orderly stimulation of the cat gastrocnemius motor units via nerve electrodes gave rise to linearly increasing median frequency regardless of the action potential firing rate of the active motor units. Increase in the discharge rate of all the motor units resulted in nearly constant median frequency. It was concluded that the increasing average conduction velocity during motor unit recruitment is the major contributor to variations in the electromyogram median frequency. The possibility of using the median frequency as the index to identify the recruitment control strategies employed by various muscles during increasing force contraction is suggested.

Action Potentials↗
PubMed1989

Metabolic activity of microcarrier attached liver cells after intraperitoneal transplantation during severe liver insufficiency in the rat.

Short- and long-term effects of intraperitoneally transplanted microcarrier attached liver cells (MAL) have been studied in two experimental models of severe liver insufficiency in the rat: subtotal hepatectomy (HX) and acute liver ischemia. Intraperitoneal transplantation of MAL immediately after subtotal hepatectomy resulted in a significantly lower plasma ammonia level, a higher caffeine clearance, a higher urea production and a significantly smaller loss in body weight in comparison to sham transplanted control rats. Since thymidine kinase activity in the regenerating host liver was only significantly stimulated at t = 48 h it is concluded that the observed metabolic effects are mainly due to the metabolic activity of the transplanted MAL, although a small stimulative effect of MAL-TX on host liver regeneration cannot be excluded. In the course of acute liver ischemia, MAL transplantation results in delayed development of acute hepatic encephalopathy (HE), judged by clinical grading, EEG spectral analysis and Visual Evoked Response (VER) parameters. Furthermore, MAL transplantation is associated with less increased levels of plasma ammonia during acute liver ischemia.

Alanine Transaminase↗
PubMed1988

Discrimination between RNA circles, interlocked RNA circles and lariats using two-dimensional polyacrylamide gel electrophoresis.

Two-dimensional polyacrylamide gel electrophoresis can be used to identify structural forms of RNA such as linear RNA, circular RNA, interlocked circles and lariats. The procedure is based upon the characteristic migration behaviour of the degradation products derived from the intact structures present already before the start of the experiment or formed during or after electrophoresis in the first dimension. After autoradiography to detect the positions of the radiolabeled RNA molecules, circles broken during electrophoresis of the first dimension give rise to horizontal lines touching the diagonal formed by linear RNAs at a point corresponding to the length of the RNA circle from which it was derived. Products derived from interlocked RNA circles by breakage after completion of the first dimension appear on a vertical line underneath the intact complex and consist of free RNA circles and their linear derivatives. Broken lariats give rise to two lines depending on the location of the break. Lariats with broken tails are present on a line to a position that corresponds to the length of their tail and that runs parallel to the diagonal formed by linear products. Lariats with a broken eye form a line running from the position of the intact product to the diagonal formed by the linear RNAs.

Electrophoresis, Polyacrylamide Gel↗