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Biomedical subjects

D Kaiser

Publications and source records attributed to D Kaiser.

At least 55 records · Page 3Linked to original sources
PubMed1999

Apoptosis induced by lack of hemodynamic forces is a general endothelial feature even occuring in immortalized cell lines.

Confluent monolayers of primary endothelial cells display a high viability and an apparently constant cell density. However upon prolonged cultivation the monolayer degenerates with increasing numbers of senescent cells finally representing the whole culture. Recently we showed that lack of hemodynamic forces induces apoptosis in organ cultures as well as in confluent monolayers of human umbilical cord vein endothelial cells (HUVEC). The apoptosis started at a low level and was counteracted by a continuous proliferation of the remaining cells. Here we show that the induction of apoptosis by lack of hemodynamic forces is a general characteristic of vascular endothelial cells, valid for endothelial cells from various organs and species: human umbilical cord vein endothelial cells (HUVEC), human microvascular placental endothelial cells (HPEC) and bovine aorta endothelial cells (BAEC). Furthermore apoptosis due to the lack of hemodynamic forces can also be induced in various endothelial cell lines: EA.hy 926 derived from HUVEC and PBMEC-A1 derived from PBMEC. However degeneration of confluent monolayers does not occur with these cell lines even in monolayers kept for several weeks. This indicates that the degeneration of normal endothelial cell monolayers is caused by depletion of the proliferation potential of the endothelial cells.

Animals↗
PubMed1998

The guanosine nucleotide (p)ppGpp initiates development and A-factor production in myxococcus xanthus.

Guanosine 3'-di-5'-(tri)di-phosphate nucleotides [(p)ppGpp], synthesized in response to amino acid limitation, induce early gene expression leading to multicellular fruiting body formation in Myxococcus xanthus. A mutant (DK527) that fails to accumulate (p)ppGpp in response to starvation was found to be blocked in development prior to aggregation. By use of a series of developmentally regulated Tn5lac transcriptional fusion reporters, the time of developmental arrest in DK527 was narrowed to within the few hours of development, the period of starvation recognition. The mutant is also defective in the production of A-factor, an early extracellular cell-density signal. The relA gene from Escherichia coli, which encodes a ribosome-dependent (p)ppGpp synthetase, rescues this mutant. We also demonstrate that inactivation of the M. xanthus relA homolog blocks development and the accumulation of (p)ppGpp. Moreover, the wild-type allele of Myxococcus relA rescues DK527. These observations support a model in which accumulation of (p)ppGpp, in response to starvation, initiates the program of fruiting body development, including the production of A-factor.

4-Butyrolactone↗
PubMed1998

Alignment enhances the cell-to-cell transfer of pilus phenotype.

Social gliding motility of Myxococcus xanthus requires polar type IV pili. Tgl mutants lack pili and lack social motility. However, both defects can be rescued phenotypically, but not genotypically, when tgl+ donor and tgl- recipient cells make physical contact with each other. What is the cellular and molecular basis of this transfer of phenotype, which is called stimulation? Stimulation does not occur in liquid nor in soft (0.5%) agar; however, on a more firm surface (1.0% agar) cells stimulate each other efficiently. Microscopy revealed that cells placed on 1.0% agar readily became aligned whereas they remained poorly aligned on 0.5% agar. It was observed, moreover, that stimulation is greatly reduced when donor and recipient cells lack the ability to move because of mutations in any of several different motility genes. Microscopy showed that motile cells became highly aligned, and can even align nonmotile cells. Using a stimulation assay based on the assembly of pili, a strong correlation was found between conditions that promote cell alignment and the speed or extent of stimulation. Because pili are assembled only at the end(s) of a cell, and a parallel alignment of cells in contact brings them end-to-end, we would suggest that end-to-end contacts are important for stimulation of pilus assembly.

Bacterial Proteins↗
PubMed1998

Correlation of femoral and lumbar DXA and calcaneal ultrasound, measured in situ with intact soft tissues, with the in vitro failure loads of the proximal femur.

The objective of this study was to determine experimentally the sex-specific correlation of femoral and lumbar DXA and calcaneal ultrasound, measured in situ, with the in vitro failure loads of the proximal femur. Fifty-eight cadavers with intact skin and soft tissues (34 male, aged 81.2 +/- 8.7 years; 24 female, aged 83.7 +/- 10.6 years) were examined. The bone mass of the proximal femur and the lumbar spine were determined using dual-energy X-ray absorptiometry and the ultrasonic properties of the calcaneus with quantitative ultrasound. Afterwards, the right femora were excised 18 cm distal to the minor trochanter, and their load to failure determined with a material testing machine. Femoral fracture loads were significantly higher in males than in females, both before and after correcting for body height and weight. Femoral neck bone mineral density (BMD) was significantly correlated with femoral failure loads (r = 0.65 all specimens, 0.57 males (0.64 after excluding trochanteric fractures) and 0.77 females; p < 0.001). The correlations with the ultrasonic Stiffness Index of the calcaneus were in a similar range (r = 0.67 all specimens, 0.48 males (0.64 after excluding trochanteric fractures) and 0.65 females; p < 0.001). The correlations between femoral failure loads and the spinal BMD were lower (r = 0.40, p < 0.01), particularly in males (r = 0.30, not significant). In contrast to previous experimental investigations on excised bones, our results are consistent with clinical studies that have reported that ultrasound and femoral DXA have a similar ability to predict the risk of hip fracture.

Absorptiometry, Photon↗
PubMed1998

How and why myxobacteria talk to each other.

In response to starvation, myxobacteria build multicellular fruiting bodies that contain many thousands of cells and that have particular species-specific shapes. To coordinate fruiting body development, the myxobacterial cells signal to each other. The timing of and cellular responses to these signals help to give form to the fruiting body. Following identification of several signal molecules, important transcriptional regulators and other signals have recently been identified. Steps on signal transduction pathways have also been defined.

Bacterial Proteins↗
PubMed1998

Prediction of vertebral failure loads from spinal and femoral dual-energy X-ray absorptiometry, and calcaneal ultrasound: an in situ analysis with intact soft tissues.

The objective of the current study was to determine the correlation of spinal and femoral dual-energy X-ray absorptiometry (DXA) and calcaneal ultrasound, measured in situ with intact soft tissues, with the in vitro failure loads of lumbar vertebral bodies. Forty-nine cadavers with intact skin and soft tissues (32 men aged 82.1 +/- 9.0 years, 17 women aged 83.1 +/- 10.1 years) were examined. The bone mineral content (BMC), the projectional area, and the bone mineral density (BMD) of the lumbar spine and proximal femur were determined with DXA, and the ultrasonic properties of the calcaneus with quantitative calcaneal ultrasound. The fourth lumbar vertebra was then excised with adjacent intervertebral disks and its mechanical failure load determined, using a materials testing machine. Absolute fracture loads were significantly higher in men than in women, but they were similar after adjusting for body weight and height. Spinal DXA was significantly associated with vertebral failure load (r = 0.62 combined; r = 0.54 men; r = 0.58 women). Femoral DXA (r = 0.46) and calcaneal ultrasound (r = 0.48) showed somewhat lower correlation coefficients, with the speed of sound (SOS) being able to add predictive information in a stepwise regression model. Normalizing the vertebral failure loads to body weight and height reduced the correlations, with only spinal DXA yielding a significant relationship. Our data suggest that previous in vitro studies may have overestimated the association between spinal DXA and vertebral failure loads, presumably because measurements were performed on excised bones, but not in situ in the presence of soft tissue inhomogeneity. The results indicate that, even in a population of old age and under in situ conditions, spinal DXA may still be somewhat better than femoral DXA and calcaneal ultrasound in predicting vertebral failure loads.

Absorptiometry, Photon↗
PubMed1998

The pilH gene encodes an ABC transporter homologue required for type IV pilus biogenesis and social gliding motility in Myxococcus xanthus.

Type IV pilus genes have been shown to be required for social gliding motility in Myxococcus xanthus. We report the discovery of four additional pil genes: pilD, a homologue of type IV prepilin leader peptidases; and pilG, pilH and pilI, which have no known homologues in other type IV pilus systems. pilH encodes an ATP-binding cassette (ABC) transporter homologue, the first such homologue to be required for the biogenesis of any bacterial pilus type. pilG and pilI are co-transcribed with pilH and appear to be functionally related to pilH. Null mutants of pilG, pilH and pilI all lack social motility, are deficient in pilus production, have elevated sporulation efficiencies and display similar developmental abnormalities. In addition, all three mutations reduced the amount of PilA found in the supernatant after cells were sedimented from liquid culture. We suggest that the products of these three genes form a single ABC exporter complex, in which pilI is an integral membrane protein with membrane-spanning domains, and pilG is an accessory factor. The complex may participate in pilus assembly and/or the export of PilA pilin.

ATP-Binding Cassette Transporters↗
PubMed1998

Targeted mutagenesis of sigma54 activator proteins in Myxococcus xanthus.

Myxococcus xanthus DNA segments related to the highly conserved central sequence of sigma54 activator proteins have been investigated. A genetic technique designed to inactivate a gene that encodes such an activator by inserting a plasmid-borne internal fragment of the putative gene has been tested. When the internal fragment inserted by homologous recombination into the corresponding chromosomal locus, the expected duplication of the gene was observed by Southern hybridization. The single restriction fragment characteristic of each segment was replaced in the insertion strains by two hybridizing fragments, and one of these fragments hybridized with the kanamycin resistance gene of the plasmid vector. The combined molecular weights of the two fragments from the insertion strains were equal to the molecular weight of the original fragment plus the expected molecular weight contributed by the vector. In the duplication, one copy is expected to have an N-terminal deletion and the other copy is expected to have a C-terminal deletion. In most cases, the net result should be loss of activator function. If an activator is essential for vegetative growth, then it should not be possible to obtain the insertion strain by plasmid integration. Indeed, integrants for three of the segments were not obtained in repeated trials; however, a plausible explanation for these results other than lethality can be offered. Of the seven insertions validated by Southern hybridization, four strains exhibited defects in the development of fruiting bodies. One of these failed to develop in submerged culture, though it developed normally on agar. The other three showed arrested development of fruiting bodies, each at a morphologically different stage of aggregation. One of the mutants may be defective in the reception pathway of A-signal.

Bacterial Proteins↗
PubMed1998

Contact stimulation of Tgl and type IV pili in Myxococcus xanthus.

Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.

Bacterial Proteins↗
PubMed1997

sigma54, a vital protein for Myxococcus xanthus.

The rpoN gene encoding the transcription factor sigma54 in Myxococcus xanthus has been cloned using a heterologous rpoN probe. The sequence of the cross-hybridizing DNA confirmed the existence of an ORF 1518 bp long that encodes a well conserved member of the sigma54 family of sigma factors. Low- as well as high-stringency hybridizations detected only a single rpoN gene in the M. xanthus chromosome. In other bacteria, sigma54 is an alternative sigma, and null mutants are viable. However, all attempts to construct a strain containing a null mutation in the M. xanthus rpoN have been unsuccessful. Partial diploids of rpoN+/rpoN null are viable. Recombination experiments with such partial diploids showed the impossibility of constructing, either by segregation or by transduction, a viable null haploid under any of a wide range of growth conditions. The product of the rpoN gene, sigma54, therefore appears to be essential for growth in M. xanthus.

Amino Acid Sequence↗
PubMed1997

Lack of hemodynamic forces triggers apoptosis in vascular endothelial cells.

The endothelial cells (EC) of the vascular system are regularly exposed to a range of hemodynamic forces with great impact on cellular structures and functions. A shear-stress-dependent modulation has been reported for various endothelial functions, and first attempts were made to characterize the nature of the shear-stress-sensitive receptor. Apoptosis has been induced in cultures of vascular EC upon application of various stimuli characteristic of traumatic or pathogenic events. However, no link between apoptosis and mechanical stimulation has been reported so far-neither for vascular EC nor for other types of cells. Here we show that the lack of hemodynamic forces triggers apoptosis in endothelial cells, hinting at mechanical forces as essential stimuli for the maintenance of blood vessels.

Apoptosis↗
PubMed1997

The Myxococcus xanthus pilT locus is required for social gliding motility although pili are still produced.

Social gliding motility in Myxococcus xanthus depends on the presence of Type IV pili. To begin to examine the role of pili in social motility, 17 mutants were identified which had lost social motility, but still expressed pili. Four of these mutants carry point mutations which mapped to a locus upstream of the recently identified pilS, pilR, and pilA genes. Sequencing of this locus revealed a gene with homology to pilT from Pseudomonas Aeruginosa. Sequencing of the four point mutations revealed that they occurred within the M. xanthus pilT locus. A markerless deletion within M. xanthus pilT, similar to the four point mutations, disrupted social gliding behaviour but did not interfere with pilus formation or pilus-dependent cell-cell agglutination. Using time-lapse videomicroscopy, residual social motility was observed in dsp- strains (known to be deficient in fibril but not pilus production); this was not observed in a delta pilT dep- double mutant. Two genes flanking pilT were also sequenced, and found to have homology to pilB and pilC from P. aeruginosa. Markerless deletions within these genes caused both pilus and social-motility defects. These results indicate that M. xanthus pilB and pilC are required for pilus biogenesis, while pilT is required for assembled pili to play their role in social motility. Thus, pilB, pilT, pilC, pilS, pilR and pilA form a contiguous cluster of pil genes required for social motility.

Adenosine Triphosphatases↗
PubMed1997

Heart rate change as a function of age, tidal volume and body position when breathing using voluntary cardiorespiratory synchronization.

Voluntary cardiorespiratory synchronization (VCRS), where inspiration and expiration follows a fixed number of heart beats, was used to investigate heart rate change (HRC) in 16 subjects (grouped as 20-30 and 46-57 years of age) as a function of age, body position, and respiratory tidal volumes of 500 and 1000 ml. In phase I, the subjects, following a tone, inspired for two heart beats and expired for three beats. In phase II, the older group's breathing pattern was 2/2, 2/3, 3/3, 4/4, 5/5, and 6/6 inspiratory and expiratory heart beats, respectively, per respiratory cycle. Comparing the younger and older groups, we found a statistically significant (p < 0.05) decrease in HRC for beat 1, 2, and 4 of the respiratory cycle in the supine position, and also for beat 2 in the sitting position, but no significant change with tidal volume. The ratio of the younger to older group's beat 2 HRC was 2.54 sitting and 3.54 supine. Increasing the number of beats per respiratory cycle from four to 12 resulted in the HRC growing from 1.8 to 7.5 beats over the respiratory cycle, showing the importance of respiratory rate in measuring HRC. VCRS can easily observe the influence of each phase of the respiratory cycle on the HRC and show the unique phase dependent changes that occur.

Adult↗
PubMed1997

The tgl gene: social motility and stimulation in Myxococcus xanthus.

Mutations in the tgl locus inactivate social gliding motility in Myxococcus xanthus and block production of pili. The tgl locus is distinctive among the genes for social motility because social gliding and pili can be restored transiently to tgl mutant cells by mixing them with tgl+ cells, a process known as stimulation. The tgl locus was cloned with a linked insertion of transposon Tn5 by using the kanamycin resistance encoded by that transposon. A 16-kb segment of chromosomal DNA complemented the social motility defect when introduced into tgl mutant cells to form a tandem duplication tgl+/tgl heterozygote. To delimit the autonomous tgl transcription unit, subfragments of this 16-kb piece were integrated at the ectopic Mx8 prophage attachment site. A 1.7-kb DNA fragment was identified which, when integrated at the Mx8 site, simultaneously rescued social motility and pilus production. The ability to stimulate tgl mutants was also rescued by the 1.7-kb fragment. Because rescue of stimulation from an mgl-deficient donor strain which cannot swarm was observed, this demonstrates that a stimulation donor requires a tgl+ allele but does not require the capacity to swarm actively. The nucleotide sequence of the 1.7-kb fragment revealed two protein coding regions, open reading frame A and open reading frame B (ORFB). ORFB is the tgl gene, because a 613-bp DNA fragment which includes 75% of ORFB rescues tgl-1, -2, and -3 mutants and because disruption of ORFB by deletion or insertion of transposon Tn5lac constitutes a tgl mutation.

Amino Acid Sequence↗
PubMed1997

Identification and localization of the Tgl protein, which is required for Myxococcus xanthus social motility.

Tgl protein is required for the production of the type IV pili found at a pole of the Myxococcus xanthus cell. These pili are essential for social motility. Evidence is presented that Tgl is a membrane protein, based on experiments with polyclonal antibody specific for Tgl that was raised against the fusion proteins beta-galactosidase-Tgl and TrpE-Tgl. Immunoaffiity-purified antibody reacted with a protein in M. xanthus having an apparent molecular mass of 27.5 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the sequence of the tgl gene translates into a polypeptide of 27 kDa. Although these numbers are close, it is likely that the primary tgl translation product is processed and modified in M. xanthus. The N terminus has a signal peptidase II recognition sequence, cleavage of which is expected to remove 19 amino acid residues. When the tgl gene is expressed in Escherichia coli, the protein product consistently migrates faster in the gel than mature Tgl expressed in M. xanthus, suggesting a second modification by addition which slows migration of the protein from M. xanthus. Tgl, as detected by its specific antibody, sediments with the membrane fraction of cells. It can be extracted with detergents but not with salt or by the addition of chelators for divalent cations. In an equilibrium gradient, Tgl bands at the buoyant density of membranes and with the NADH-oxidase activity. Intact cells failed to bind anti-Tgl antibody, and less than 2% of the total Tgl is released in soluble form from the periplasm. Yet, cells that had been osmotically shocked and treated with paraformaldehyde were able to react with the specific antibody--a reaction absent from cells with a deletion of the tgl transcription unit. Assuming that osmotic shock disrupts the outer membrane, the fractionation and localization data imply that Tgl is attached to the inner or outer membranes, from which it is exposed to the intermembranous space. Tgl is necessary for synthesis of pili in M. xanthus and is the only pilus protein that can be donated by other cells (stimulation). Tgl contains six tandem copies of the tetratrico peptide repeat structural motif. Its membrane localization, capacity for stimulation, and content of tetratrico structural repeats together suggest that Tgl may be necessary for the assembly of pilin subunits into filaments.

Amino Acid Sequence↗
PubMed1997

Regulation of expression of the pilA gene in Myxococcus xanthus.

Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) during vegetative growth and fruiting-body development was examined. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and was used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated; however, the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.

Bacterial Proteins↗