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Biomedical subjects

M Dworkin

Publications and source records attributed to M Dworkin.

At least 19 recordsLinked to original sources
PubMed1999

Fibrils as extracellular appendages of bacteria: their role in contact-mediated cell-cell interactions in Myxococcus xanthus.

Social behavior in the myxobacterium Myxococcus xanthus involves epicellular, peritrichous appendages called fibrils. These are polysaccharide organelles containing a set of tightly adhering proteins. It is proposed that cell-cell contact is perceived by the fibrils and is mediated by the action of a fibrillar ADP-ribosyl transferase. Fibrils or fibril-like organelles have also been found on a variety of other gram-negative bacteria and at least one archaeon, and may mediate cell-cell contact between the bacteria themselves or between the bacteria and their eukaryotic host cells.

Animals↗
PubMed1998

Factors leading to delay in diagnosis of invasive cervical cancer.

Objective: Screening for cervical cancer has been one of the major successes in American health care. The reasons for screening failure have not been well described. We have encountered 24 patients in the past year on our gynecologic oncology service who have presented with grossly visible stage IB or higher cervical cancers. A questionnaire was developed to determine why these patients presented with invasive cervical cancer, a theoretically preventable disease.Methods: All patients seen at our hospital center with grossly visible cervical cancer were offered entry into this prospective study, approved by the IRB. After informed consent was obtained, a questionnaire was administered to the patients by a physician. The questions involved sexual and gynecologic history, symptom review, social history, and personal concerns involving gynecologic exams. Twenty-four patients were approached for the study and 20 were enrolled. Four patients were unable to participate secondary to medical or social conditions that did not allow them to complete the questionnaire.Results: Of the 20 patients enrolled in this study, 14 of 20 (70%) presented with bleeding irregularities. Upon further questioning, 19 of 20 (95%) reported a history of abnormal bleeding. Only 3 of 20 (15%) patients reported a history of an abnormal Papanicolaou smear, and 7 of 20 (35%) reported not having had a Papanicolaou in the past 5 years. Two patients had never had a Papanicolaou smear. Seventeen of 20 (85%) of all patients in the study had medical insurance at the time of diagnosis, and only 2 patients reported lack of insurance as the reason they had not been seen by a gynecologist in the past 2 years. The majority of the study patients, 17 of 20 (85%) had contact with medical personnel in the 2 years prior to diagnosis. Sixteen of the 20 patients had been seen by a physician, 5 of 20 had surgery or other invasive procedures, and 6 of 20 had been seen in an emergency department. Only 5 of 20 patients knew that a Papanicolaou smear is a screening test for cervical cancer. Five of the 20 thought it to be a test for infections, and 10 of 20 did not know the purpose of a Papanicolaou smear. The main reason women gave for not having had a Papanicolaou smear in the past 2 years was that they felt healthy and had no symptoms (12/20).Conclusion: The majority of patients in our study (17/20) reported contact with a physician within 2 years of diagnosis of their invasive cervical cancer. It appears that failure to screen and lack of understanding regarding Papanicolaou smear screening, rather than lack of access to medical care, is a major cause for delay in diagnosis of cervical cancer.

Journal Article↗
PubMed1997

ADP-ribosylation by the extracellular fibrils of Myxococcus xanthus.

The isolated, extracellular fibrils of the myxobacterium, Myxococcus xanthus, are capable of carrying out ADP-ribosylation. The substrate for the ADP-ribosylation is reactive with monoclonal antibody 2105, which has been shown to be directed specifically against the integral fibril proteins. The extracellular fibrils thus contain both the ADP-ribosyl transferase and the substrate for the ribosylation. This process may play a role in the contact-mediated cell-cell interactions that are an important part of the social behaviour of M. xanthus.

Adenosine Diphosphate↗
PubMed1997

Identification and characterization of Myxococcus xanthus mutants deficient in calcofluor white binding.

Calcofluor white is a fluorescent dye that binds to glycans and can be used to detect extracellular polysaccharide in Myxococcus xanthus and many other bacteria. We observed that an esg mutant showed less binding to calcofluor white than wild-type cells. Unlike S-motility mutants that share this phenotypic characteristic, the esg mutant exhibited S motility. This led us to identify a collection of nine new transposon insertion mutants, designated Cds (for calcofluor white binding deficient and S motile), which exhibited a phenotype similar to that of the esg strain. The Cds phenotype was found in 0.6% of the random insertion mutants that were screened. The Cds mutants were also found to be defective in cell-cell agglutination and developmental aggregation. Extracellular matrix fibrils composed of roughly equal amounts of polysaccharide and protein have been shown to be involved in agglutination, and electron microscopic examination showed that esg and the other Cds mutants lack the wild-type level of fibrils. Analysis of total M. xanthus carbohydrate demonstrated that polysaccharide content increased by about 50% when wild-type cells entered stationary phase. This induction was reduced or eliminated in all of the Cds mutants. The degree of polysaccharide deficiency in the Cds mutants correlated with the degree of loss of agglutination and dye binding as well as with the severity of the developmental aggregation defect. Preliminary genetic characterization demonstrated that the transposon insertion mutations in three of the Cds mutants (SR53, SR171, and SR200) were loosely linked. The results of this study suggest that many genes are involved in the production of calcofluor white binding polysaccharide material found in the extracellular matrix and that the polysaccharide is fibrillar. These results are also consistent with the findings of earlier studies which indicated that fibrils function to join agglutinating cells and to form multicellular fruiting aggregates.

Agglutination↗
PubMed1996

Tyrosine phosphorylation in Myxococcus xanthus, a multicellular prokaryote.

Tyrosine phosphorylation is an extremely rare event in prokaryotes, occurring almost exclusively in multicellular eukaryotes. We have identified, for the first time, by the use of antiphosphotyrosine monoclonal antibody and Western blot (immunoblot) analysis, two tyrosine-phosphorylated membrane proteins in the multicellular prokaryote Myxococcus xanthus. The pattern of tyrosine phosphorylation was shown to change during development, indicating a possible role for this regulatory modification during two stages of development, i.e., aggregation and sporulation. Furthermore, the altered pattern of tyrosine phosphorylation observed in a variety of signaling mutants was shown to differ from that observed in the wild type, suggesting further the possible involvement of tyrosine phosphorylation during the development program.

Antibodies, Monoclonal↗
PubMed1996

Mutants of Myxococcus xanthus dsp defective in fibril binding.

The dsp mutant of Myxococcus xanthus lacks extracellular fibrils and as a result is unable to undergo cohesion, group motility, or development (J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5765-5770, 1983; J. W. Arnold and L. J. Shimkets, J. Bacteriol. 170:5771-5777, 1983; R. M. Behmlander and M. Dworkin, J. Bacteriol. 173:7810-7821, 1991; L. J. Shimkets, J. Bacteriol. 166:837-841, 1986; L. J. Shimkets, J. Bacteriol. 166:842-848, 1986). However, cohesion and development can be phenotypically restored by the addition of isolated fibrils (R. M. Behmlander, Ph.D. thesis, University of Minnesota, Minneapolis, 1994; B.-Y. Chang and M. Dworkin, J. Bacteriol. 176:7190-7196, 1994). As part of our attempts to examine the interaction of fibrils and cells of M. xanthus, we have isolated a series of secondary mutants of M. xanthus dsp in which cohesion, unlike that of the parent strain, could not be rescued by the addition of isolated fibrils. Cells of M. xanthus dsp were mutagenized either by ethyl methanesulfonate or by Tn5 insertions. Mutagenized cultures were enriched by selection of those cells that could not be rescued, i.e., that failed to cohere in the presence of isolated fibrils. Seven mutants of M. xanthus dsp, designated fbd mutants, were isolated from 6,983 colonies; these represent putative fibril receptor-minus mutants. The fbd mutants, like the parent dsp mutant, still lacked fibrils, but displayed a number of unexpected properties. They regained group motility and the ability to aggregate but not the ability to form mature fruiting bodies. In addition, they partially regained the ability to form myxospores. The fbd mutant was backcrossed into the dsp mutant by Mx4 transduction. Three independently isolated transconjugants showed essentially the same properties as the fbd mutants--loss of fibril rescue of cohesion, partial restoration of myxospore morphogenesis, and restoration of group motility. These results suggest that the physical presence of fibrils is not necessary for group motility, myxospore formation, or the early aggregative stage of development. We propose, however, that the perception of fibril binding is required for normal social behavior and development. The dsp fbd mutants (from here on referred to as fbd mutants) open the possibility of isolating and characterizing a putative fibril receptor gene.

Movement↗
PubMed1994

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus.

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis, supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Chromatography, Gas↗
PubMed1994

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch.

The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.

Chromatography, Gas↗
PubMed1994

Endogenous ADP-ribosylation during development of the prokaryote Myxococcus xanthus.

We examined endogenous ADP-ribosylation of proteins during the development of the prokaryote Myxococcus xanthus. In vivo and in vitro endogenous ADP-ribosylation of M. xanthus proteins was detected and the profile of modified proteins changed during development. Adenosine and nicotinamide inhibited ADP-ribosylation. Nicotinamide stimulated cells at low density to develop, in a manner similar to that previously observed with adenosine. Higher concentrations of nicotinamide inhibited aggregation. The in vivo effects of nicotinamide on developing M. xanthus cells correlate with its in vitro effects on ADP-ribosylation and the developmental profile of putative ADP-ribosylation substrates. These results suggest that ADP-ribosylation may regulate developmental proteins in M. xanthus.

Adenosine↗
PubMed1994

Biochemical and structural analyses of the extracellular matrix fibrils of Myxococcus xanthus.

It is characteristic of myxobacteria to produce large amounts of extracellular material. This report demonstrates that this material in Myxococcus xanthus is fibrillar and describes the structure and chemical composition of the fibrils. The extracellular matrix fibrils are the mediators of cell-cell cohesion in M. xanthus. As such, the fibrils play an important role in the cell-cell interactions that form the basis for the social and developmental lifestyle of this organism. The fibrils are composed of protein and carbohydrate in a 1.0:1.2 ratio. Combined, the two fractions accounted for greater than 85% of the mass of isolated fibrils, and the fibrils were found to compose up to 10% of the dry weight of cells grown at high density on a solid surface. The polysaccharide portion of the fibrils was shown to be composed of five different monosaccharides: galactose, glucosamine, glucose, rhamnose, and xylose. Glucosamine, one of the component monosaccharides of the fibrils and a known morphogen for M. xanthus, inhibited cohesion to a level near that of Congo red (the positive control for cohesion inhibition). Glucose and xylose also inhibited cohesion but less than did glucosamine. Analysis of the morphology of the fibrils, the periodicities within the distribution of fibril diameters observed by field emission scanning electron microscopy, and the observation of fibrils on hydrated cells strongly suggested that the extracellular matrix of M. xanthus was indeed arranged as fibrils. Furthermore, results suggested that the fibrils were constructed as carbohydrate structures with associated proteins.

Bacterial Adhesion↗
PubMed1994

Integral proteins of the extracellular matrix fibrils of Myxococcus xanthus.

The extracellular matrix fibrils of Myxococcus xanthus are mediators of cell-cell cohesion and as such are required for the maintenance of the social lifestyle characteristic of these prokaryotes. The fibrils have also been implicated as factors involved in contact-mediated cell interactions and in signal exchange. The fibrils are extracellular carbohydrate structures with associated proteins. All of the major proteins associated with the fibrils react with monoclonal antibody 2105 and can be removed from the fibrils only by boiling with sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. For consistency with their integral association with the fibrils, we have designated this class of proteins as integral fibrillar proteins class 1 (IFP-1). IFP-1 comprises five major proteins whose molecular sizes range from 66 to 14 kDa. All of the proteins in IFP-1 have been purified from isolated fibrils by electroelution after size separation on SDS-PAGE gels. Analysis of the purified proteins suggested that the forms with different molecular sizes result from the aggregation of a single small-molecular-size subunit. Fingerprint analysis and amino acid composition profiles confirmed the identity among the different members of IFP-1. The sequence of the 31 amino-terminal amino acids of the 31-kDa form of IFP-1 (IFP-1:31) was determined. There was no significant homology to other known protein sequences. During development there is a dramatic shift in the banding pattern of IFP-1 proteins without any apparent overall loss of total protein.

Amino Acid Sequence↗
PubMed1994

Isolated fibrils rescue cohesion and development in the Dsp mutant of Myxococcus xanthus.

Extracellular fibrils are involved in cell cohesion and cell development in Myxococcus xanthus. One group of social motility mutants, Dsp, is unable to produce extracellular fibrils; these mutants also lose the abilities to cohere and to develop. Extracellular fibrils isolated from vegetative wild-type cells and added to Dsp cells fully restored the abilities of these cells to cohere and to undergo normal morphological development. The fibrils thus mimic the ability of intact, wild-type cells to carry out the same rescue. Optimal cohesion rescue by fibrils required calcium and magnesium ions, did not require protein synthesis, but was energy dependent, i.e., sodium azide and sodium cyanide blocked rescue. Cohesion rescue was also blocked by the diazo dye Congo red. Cohesion rescue is genus specific, i.e., isolated fibrils did not cause the cohesion of Pseudomonas aeruginosa, Bacillus subtilis, Proteus mirabilis, Escherichia coli, or the related myxobacterium Stigmatella aurantiaca. Developmental rescue of Dsp by isolated fibrils included aggregation, fruiting body formation, and myxospore morphogenesis. Developmental gene expression in the Dsp mutant was only partially rescued by the isolated fibrils.

Bacterial Adhesion↗
PubMed1994

Territorial interactions between two Myxococcus Species.

It is unusual to find fruiting bodies of different myxobacteria occupying the same territory on natural samples. We were thus interested in determining whether myxobacteria establish territorial dominance and, if so, what the mechanism of that interaction is. We had previously observed that vegetative swarms of Myxococcus xanthus and Stigmatella aurantiaca placed close to each other on an agar surface initially merged but eventually separated. Further studies indicated that these two species also formed separate fruiting bodies when mixed together on developmental agar (unpublished observation). We examined the interactions between two more closely related myxobacteria, M. xanthus and M. virescens, in greater detail. When mixtures of a kanamycin-resistant strain of M. xanthus and a kanamycin-sensitive strain of M. virescens were placed together under developmental conditions, the cells sorted themselves out and established separate fruiting body territories. In addition, differential viable counts of a mixture of the two species during development indicated that each strain was producing an extracellular component that inhibited the growth and development of the other. Nevertheless, finally, M. virescens invariably outcompeted M. xanthus at all input ratios of M. xanthus/M. virescens tested. This is consistent with the observation that M. virescens is by far the more commonly encountered of the two species. The properties of the inhibitory substance from M. virescens are consistent with the possibility that it is a bacteriocin. Our working hypothesis is that the bacteriocin plays a role in the establishment of myxobacterial territoriality. If so, this is an example of an ecological function of bacteriocins.

Bacteriocins↗
PubMed1993

Effect of blood perfusion on the ablation of liver parenchyma with high-intensity focused ultrasound.

This paper discusses the effect of blood perfusion on the ablation of rat liver tissue with high-intensity focused ultrasound (HIFU). For this study a practical method has been developed, in which the liver blood flow can be reduced by ligation of the hepatic artery and portal vein. During the treatment the rat liver was mobilized out of the abdomen and the blood flow was measured using both the radioactive microsphere method and a laser Doppler blood-flow monitor. The results show that the hepatic blood flow was about 23 ml/100 g min-1 via the hepatic artery and about 227 ml/100 g min-1 via the portal vein. The total liver blood flow was reduced by 98% when both the hepatic artery and portal vein were ligated. Comparative lesions were made on the same liver lobes of rats with both normal and reduced blood flow using a focused ultrasound beam of 1.7 MHz, 67-425 W cm-2 spatially averaged focal intensity ISAL and 2-20 s exposure duration. A marked difference has been found between the lesion dimensions obtained with normal blood flow and that with reduced blood flow. For exposures at 169 W cm-2 the lesion diameter with normal blood flow was reduced by 14% for 3 s exposure duration compared to that obtained with both hepatic artery and portal vein ligated, while the reduction was more than 20% for longer durations.

Animals↗
PubMed1991

A development-specific protein in Myxococcus xanthus is associated with the extracellular fibrils.

We have been using monoclonal antibodies (MAbs) as probes to study developmentally relevant cell surface antigens (CSA) that may be required for cellular interactions in Myxococcus xanthus. Three independently isolated MAbs, G69, G357, and G645, isolated by Gill and Dworkin recognize a CSA detectable only on developing cells (J. S. Gill and M. Dworkin, J. Bacteriol. 168:505-511, 1986). The CSA is made within the first 30 min of submerged development and increases until myxosporulation. The CSA is also produced at low levels after 24 h in shaken-starved cultures and during glycerol sporulation. No antigen can be detected in lysed, vegetative cells, and expression of the antigen is blocked in the presence of rifampin or chloramphenicol. The antigen is expressed in submerged, developmental cultures of asg, bsg, csg, dsg, and mgl mutants and is not expressed in a dsp mutant. All of the three MAbs immunoprecipitate the same protein of approximately 97,000 Da from lysed developmental cells. Competitive immunoprecipitations suggest that they recognize at least two different epitopes on the CSA. The epitopes recognized by MAbs G69, G357, and G645 are sensitive to protease digestion, whereas the epitopes recognized by MAbs G357 and G645 are resistant to periodate oxidation. The epitope recognized by MAb G69 is sensitive to periodate oxidation. Fractionation of lysed developing cells shows that most of the antigen is localized in the pellet after centrifugation at 100,000 x g. To determine whether the antigen is expressed on the cell surface, we labeled developing whole cells with either MAb G69, G357, or G645 and gold-labeled anti-mouse immunoglobulin G. Low-voltage scanning electron microscopy of labeled cells shows that the antigen is associated with the fibrillar matrix that surrounds the cells and that the antigen is retained on isolated, developmental fibrils from M. xanthus. The CSA has been designated dFA-1, for developmental fibrillar antigen 1.

Antibodies, Monoclonal↗
PubMed1991

Effects of glucosamine on lysis, glycerol formation, and sporulation in Myxococcus xanthus.

Glucosamine (GlcN), which has previously been shown to rescue fruiting body formation, lysis, and sporulation in a developmental mutant (G. Janssen and M. Dworkin, Dev. Biol. 112:194-202, 1985), induced lysis in vegetative and developing wild-type cells and inhibited fruiting body formation. It also resulted in a transient, intracellular increase in the concentration of glycerol, a known sporulation inducer, and sporulation of the surviving cells. Phospholipase activity, which was shown to be normally developmentally regulated, increased 7.6-fold after treatment of vegetative cells with 50 mM GlcN. Likewise, autocidal activity, which normally increased 18 to 24 h after the initiation of development, increased 20% when vegetative or developing cells were exposed to GlcN. Two mutants resistant to GlcN-induced lysis (MD1021 and MD1022) were isolated and showed neither an increase in autocide production nor an increase in phospholipase activity in response to added GlcN. MD1021 was developmentally deficient, and GlcN rescued fruiting body formation as well as phospholipase activity and autocide production. We propose that GlcN exerts its lytic effect by regulating the activity of phospholipase enzymes that release autocides, compounds that are believed to be responsible for developmental autolysis. GlcN-induced sporulation was found to depend on several factors: the initial cell density, the amount of lysis induced by GlcN, and the presence of tan-phase variants. An initial cell density of greater than 2 x 10(5) cells per ml was required to support GlcN-induced sporulation, and sporulation did not occur unless 50 to 75% of these cells had lysed. Mutants that were resistant to GlcN-induced lysis also did not sporulate in the presence of GlcN. The effects of GlcN on developing cells depended on the concentration of GlcN added; the addition of low concentrations of GlcN resulted in enhancement of sporulation, while higher concentrations resulted in the inhibition of sporulation. The ultrastructure of GlcN-induced spores resembled that of spores induced by the exogenous addition of glycerol, in contrast to spores isolated from mature fruiting bodies. A model by which GlcN may regulate both lysis and sporulation is presented.

Drug Resistance, Microbial↗