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Results for “Generational stabilization”

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PubMed1988

Sensory interaction with central 'generators' during respiration in the dogfish.

The activity in sensory and motor nerves of the gills was recorded from selected branches of the vagus nerve in decerebrate dogfish, Scyliorhinus canicula. Vagal motoneuronal activity was observed at the start of the rapid pharyngeal contraction and was followed by sensory nerve activity which preceded the slow expansion phase. Rhythmical vagal motoneuronal activity was still present after all movements had been prevented by curare paralysis although the frequency of the rhythm was higher than in the ventilating fish. Electrical stimulation of vagal sensory fibres had 3 effects on the ventilatory movements. (1) It evoked a reflex contraction of several gill muscles after a latency of about 11 ms. (2) It could reset the respiratory cycle because a stimulus given during expansion delayed the onset of the subsequent contraction. (3) The stimulus could entrain the rhythm if it was given continuously at a frequency close to that of ventilation. The vagal motor rhythm was disrupted by trigeminal nerve stimulation in the paralyzed fish but not if the motor rhythm was being entrained by vagal nerve stimulation. Vagal sensory activity may be important, therefore, in maintaining the stability of the generating circuits.

Animals↗

PubMed1976

Rhythmic swimming activity in neurones of the isolated nerve cord of the leech.

1. Repeating bursts of motor neurone impulses have been recorded from the nerves of completely isolated nerve cords of the medicinal leech. The salient features of this burst rhythm are similar to those obtained in the semi-intact preparation during swimming. Hence the basic swimming rhythm is generated by a central oscillator. 2. Quantitative comparisons between the impulse patterns obtained from the isolated nerve cord and those obtained from a semi-intact preparation show that the variation in both dorsal to ventral motor neurone phasing and burst duration with swim cycle period differ in these two preparations. 3. The increase of intersegmental delay with period, which is a prominent feature of swimming behaviour of the intact animal, is not seen in either the semi-intact or isolated cord preparations. 4. In the semi-intact preparation, stretching the body wall or depolarizing an inhibitory motor neurone changes the burst duration of excitatory motor neurones in the same segment. In the isolated nerve cord, these manipulations also change the period of the swim cycle in the entire cord. 5. These comparisons suggest that sensory input stabilizes the centrally generated swimming rhythm, determines the phasing of the bursts of impulses from dorsal and ventral motor neurones, and matches the intersegmental delay to the cycle period so as to maintain a constant body shape at all rates of swimming.

Action Potentials↗

PubMed1990

Functional interaction among catalytic residues in subtilisin BPN'.

Variants of the serine protease, subtilisin BPN', in which the catalytic triad residues (Ser-221, His-64, and Asp-32) are replaced singly or in combination by alanine retain activities with the substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPF-pna) that are at least 10(3) to 10(4) above the non-enzymatic rate [Carter, P., Wells, J.A. Nature (London) 322:564-568, 1988]. A possible source of the residual activity was the hydrogen bond with the N delta 2 of Asn-155 that helps to stabilize the oxyanion generated in the tetrahedral transition state during amide bond hydrolysis by the wild-type enzyme. Replacing Asn-155 by Gly (N155G) lowers the turnover number (kcat) for sAAPF-pna by 150-fold with virtually no change in the Michaelis constant (KM). However, upon combining the N155G and S221A mutations to give N155G:S221A, kcat is actually 5-fold greater than for the S221A enzyme. Thus, the catalytic role of Asn-155 is dependent upon the presence of Ser-221. The residual activity of the N155G:S221A enzyme (approximately 10(4)-fold above the uncatalyzed rate) is not an artifact because it can be completely inhibited by the third domain of the turkey ovomucoid inhibitor (OMTKY3), which forms a strong 1:1 complex with the active site. The mutations N155G and S221A individually weaken the interaction between subtilisin and OMTKY3 by 1.8 and 2.0 kcal/mol, respectively, and in combination by 2.1 kcal/mol. This is consistent with disruption of stabilizing interactions around the reactive site carbonyl of the OMTKY3 inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

PubMed1994

Generation of contractile force by cultured Dupuytren's disease and normal palmar fibroblasts.

Contractile fibroblasts are believed to be responsible for palmar fascia contracture in Dupuytren's Disease. An in vitro collagen lattice model was used to examine the contractile properties of Dupuytren's fibroblasts from 10 patients undergoing partial fasciectomy, and palmar fascia fibroblasts from 6 patients undergoing carpel tunnel release. Dupuytren's and palmar fascia fibroblasts cultured within a stabilized collagen lattice acquired morphological characteristics similar to those of 'myofibroblasts' in Dupuytren's diseased fascia. Both types of fibroblasts generated contractile forces that resulted in rapid collagen lattice contraction after release of the lattice from points of stabilization. Generation of contractile force by the fibroblasts was inhibited by disruption of the actin cytoskeleton, lack of cells, or serum removal. Afferent neuropeptides (substance P, galanin and neurokinin A) did not promote lattice contraction. These results demonstrate that normal palmar fascia fibroblasts can modulate into Dupuytren's-like fibroblasts and that cultured fibroblasts, from either Dupuytren's diseased or normal palmar fascia, can generate contractile forces that are transmitted to extracellular matrix. In addition, fibroblast contraction is an actin based process which requires specific factor(s) present in serum. It is suggested that in Dupuytren's disease extracellular cues trigger the modulation of fibroblasts to Dupuytren's fibroblasts and the promotion of contractile forces responsible for palmar fascia contrature.

Actins↗

PubMed1987

Cytoplasmic bar-like structures of alveolar type II cells: an ultrastructural study in freshly isolated cells from rat lungs.

Bar-like structures are tubular cytoplasmic inclusions found in situ in pulmonary epithelial type II cells of several animal species. The physiological significance and mode of formation of these inclusions are not fully established. In this paper, we describe bar-like structures as found in freshly isolated type II cells from rat lungs. Pulmonary cells were dissociated from the tissue with elastase and separated on a discontinuous density gradient of Percoll. The complete isolation procedure yielded 17 X 10(6) type II cells per animal (purity = 80%). Either from the crude cell suspensions or the purified preparations, only a small fraction of the type II cell population harbored the inclusions (less than 5%). It is shown that the bounding membranes of the bar-like structures can derive from the endoplasmic reticulum, the nuclear membrane, or the Golgi apparatus. Occasional connections with lamellar bodies were observed, and different levels of complexity in the bar-like structures were also found. The apparent rigid conformation and the orientation of the bar-like structures were taken as evidence for a role of the cytoskeleton in their formation. Because the inclusions do not appear to be new organelles or cellular structures performing a specific function, we propose that their formation may be a transient and limited cellular event in normal cells. However, the stabilization and the generation of the osmiophilic structures, as well as their overproduction, may reflect alterations of the normal physiology of the type II cells.

Animals↗

PubMed1995

Mechanical properties of the extracellular matrix influence fibronectin fibril assembly in vitro.

Mechanical properties of the extracellular matrix (ECM) are proposed to influence cell behavior and biological activity. The influence of the mechanical environment on fibronectin fibril assembly was evaluated. Fibroblasts were cultured in hydrated collagen gels with two distinctly different mechanical properties. Cells cultured within a stabilized collagen gel generate stress that is transmitted throughout the matrix (stressed gel). In contrast, cells that are cultured within a collagen gel that is floating freely in media do not generate stress (relaxed gel). Fibroblasts in the stressed collagen gel develop large bundles of actin microfilaments and associated fibronectin fibrils, while fibroblasts within relaxed gels do not form stress fibers or assemble fibronectin into fibrils. In addition, we have evaluated the mechanism of fibronectin fibril assembly employed by fibroblasts cultured within a stressed three-dimensional collagen matrix and the role of fibronectin fibrils in transmission of cell-generated forces to the surrounding matrix. Fibronectin fragments (70-kDa amino terminal fragment, 110-kDa cell-adhesive fragment, and GRGDS peptide) and a monoclonal antibody body blocked fibronectin fibril assembly in stressed three-dimensional collagen gels. These results suggest that the features of fibronectin required for fibronectin fibril assembly by cells in collagen gels is similar to those required by cells cultured on a planar substratum. Although fibronectin fibril assembly was blocked by these inhibiting fragments and antibody, the cells displayed prominent actin bundles and developed isometric tension, indicating that stress fiber formation and contractile force transmission is not dependent on the presence of fibronectin fibrils.

Actins↗

PubMed1990

Cellular interactions in metastasis.

The metastatic cascade is a sequence of events that must be completed for metastases to be established. The realization that tumors are heterogeneous, consisting of many different subpopulations differing in many characteristics, and the belief that there are selective events in the metastatic process have led several laboratories to isolate and characterize variants with both high and low metastatic potential. Typically, the highly metastatic variants have been able to form distant metastases when implanted into the subcutis. Such lines have been popular for studies of metastatic mechanisms and anti-metastatic therapy, but they may be atypical examples, and thus not the best experimental models. Recent studies indicate that normal tissue influences metastasis such that many tumors metastasize only if placed in the orthotopic site. Furthermore, some cells that do not metastasize individually are able to do so in conjunction with other variant subpopulations. Thus, mixtures of tumor cells in the tissue of origin can express a more malignant character. We review possible mechanisms for such influential interactions, as well as the role of cellular interactions in generating heterogeneity and stabilizing tumor characteristics.

Animals↗

PubMed1988

Isolation and characterization of Saccharomyces cerevisiae mutants resistant to T-2 toxin.

T-2 toxin, a trichothecene mycotoxin, inhibits the growth of Saccharomyces cerevisiae. We have isolated nine spontaneous S. cerevisiae mutants resistant to this toxin. The mutants were distinguished from the wild type according to their degree of resistance to T-2 toxin on media with dextrose or glycerol as the carbon source. Generation time, mutation stability and level of cross-resistance to roridin A, another trichothecene, were determined for each mutant. The T-2 toxin resistant mutants were further characterized by subsequent tests involving cross-resistance and collateral sensitivity to chlorampenicol, neomycin, paromomycin, ethidium bromide and thiolutin. Mutants have been placed into three subgroups and the mechanism of T-2 toxin resistance in each group has been postulated. Mutant HK1 is the first S. cerevisiae isolate resistant to roridin A. One particular isolate, mutant HK11, carries a single recessive nuclear mutation. This mutation was termed ttt (for T-2 toxin resistant).

Antifungal Agents↗

PubMed1978

Transfer of genetic information via isolated amphibian metaphase chromosomes.

The metaphase chromosome transfer system of McBride and Ozer (1973) has been adapted to a haploid, euploid, frog cell line. Genes coding for a deoxypyrimidine kinase and an enzyme responsible for a thymidine-specific saturable transport system have each been transferred at frequencies between 10(-6) and 10(-5) transferents per cell treated. Revertants for each of these two genes were observed at frequencies between 10(-8) and 10(-7) revertants per cell tested. Selfing controls showed no transferents. Two colonies were obtained in which cotransfer of both genes may have occurred. Activities of the transferred genes were assayed by incorporation of [3H]thymidine into alkali-stable, acid-precipitable material. Growth properties of 13 transferents in various media were also determined and presence of the appropriate enzymes inferred. These transferents were tested for stability early (25 generations) after transfer and were found to be stable. All 13 transferents possess the normal haploid number of chromosomes (n = 13) with no cytologically detectable chromosomal fragments.

Animals↗

PubMed1994

Dynamic role of microfilaments in intestinal chloride secretion.

The importance of microfilaments in the regulation of chloride (Cl-) secretion by the human intestinal cell line T84 was investigated using the cytoskeletal probe phalloidin to bind and stabilize F-actin. Phalloidin was found to inhibit secretion mediated by cyclic adenosine monophosphate (cAMP) and the sustained secretory response to the calcium (Ca+2) ionophore ionomycin but not to affect the transient Ca+2-mediated response to carbachol and histamine. Fluorescent microscopic examination of F-actin revealed regionally restricted microfilament remodeling in cAMP- and ionomycin-treated cells. Normal regulation of apical Cl- and basolateral potassium (K+) channel functions was evident in phalloidin-loaded cells. It is concluded that prevention of cytoskeletal remodeling by actin stabilization inhibits the generation of a sustained Cl- secretory response by a mechanism that does not involve Cl- or K+ channels. Depolymerization of F-actin plays an integral role in the regulation of intestinal Cl- secretion.

Actin Cytoskeleton↗

PubMed1992

Round table on epidemiology and control of fox rabies.

The current epizootic of rabies in Europe has as its main host the fox. Oral vaccination of the fox population has proven to be particularly effective. It is clear that the major components for a successful vaccination programme are a potent and stable vaccine, and an effective baiting system; the latter should attract the target animal but no non-target species. Recently, vaccines of increased stability have been generated; amongst these is a vaccinia recombinant virus which expresses rabies virus glycoprotein. Consequently, both attenuated live virus vaccines and a recombinant vaccine are available for routine field vaccination of the fox population.

Animals↗

PubMed1998

Recordings of postsubiculum head direction cells following lesions of the laterodorsal thalamic nucleus.

Areas of the rodent limbic system are important for solving spatial tasks and accurate navigation. Previous studies have identified cells in the postsubiculum (PoS) and the lateral dorsal thalamus (LDN) which discharge as a function of the animal's head direction in the horizontal plane. These two brain areas are reciprocally connected with one another. To determine the contribution of the LDN to the functioning of PoS head direction cells, we lesioned the LDN and recorded single units in the PoS. We report here that lesions of the LDN had little effect upon the firing properties of PoS HD cells. In addition, HD cells from lesioned animals showed normal responses to two environmental manipulations: (1) when the salient visual cue was rotated the preferred firing directions of PoS HD cells shifted a similar amount and (2) cells frequently ceased firing, or had reductions in their peak firing rate, when the animal was restrained and passively rotated through the preferred firing direction. These results indicate that the LDN does not play a substantive role in either the generation or the stability of the HD cell signal in the PoS.

Action Potentials↗

PubMed1985

Genetic transformation of the phytopathogenic bacteria, Erwinia chrysanthemi.

Erwinia chrysanthemi is an enterobacterium whose phytopathogenicity is due to its pectinolytic and cellulolytic activities. The CaCl2 mediated transformation procedure was successfully applied to two E. chrysanthemi wild type strains. The highest efficiency of transformation of E. chrysanthemi with pBR322 was found using 0.1 M CaCl2, 0.1 M MgCl2 treated cells and a heat pulse at 30 degrees C for 6 min. This yielded about 600 transformants per microgram of pBR322 DNA and 2.3 X 10(-6) per viable cell. Plasmid stability after twenty generations was lower than in E. coli: only 40-60% of the cells retained the plasmid in the absence of selective pressure. Based on this result, cloning in E. chrysanthemi with pBR322 vectors should therefore be possible, making it a potential host for cloning any gene for biomedical or industrial purposes.

Calcium Chloride↗

PubMed1998

Important and emerging beta-lactamase-mediated resistances in hospital-based pathogens: the Amp C enzymes.

Resistance to third-generation cephalosporins mediated by beta-lactamases is an increasing problem for clinical therapeutics. A wide range of Enterobacteriaceae produce these AmpC enzymes (Bush-Jacoby-Medeiros group 1), including Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia spp., and Serratia marcescens. Resistance via this mechanism has been shown to be statistically correlated with the use of some third-generation cephalosporins, and the infections caused by these stably derepressed enzyme-producing species seem to occur most frequently in the seriously ill. More recently the genes encoding this enzyme have been documented on plasmids capable of transfer into other species such as Klebsiella pneumoniae. Fourth-generation cephalosporins, with stability and low affinity for the Amp C beta-lactamases and the ability to penetrate rapidly into the periplasmic space of Gram-negative organisms, offer a viable alternative in the treatment of these infections or as empiric regimens. Furthermore, these compounds (example: cefpirome) possess greater potency against the frequently occurring Gram-positive cocci such as oxacillin-susceptible staphylococci and the streptococci (including some penicillin-resistant strains) as compared to previously used anti-pseudomonal cephalosporias, ceftazidime.

Bacterial Proteins↗

PubMed1996

A model-independent, nonlinear extrapolation procedure for the characterization of protein folding energetics from solvent-denaturation data.

We have characterized the guanidine-induced denaturation of hen egg white lysozyme within the 30-75 degrees C temperature range on the basis of equilibrium fluorescence measurements, unfolding assays, kinetic fluorescence measurements, and differential scanning calorimetry. Analysis of the guanidine denaturation profiles according to the linear extrapolation method yields values for the denaturation Gibbs energy which are about 15 kJ/mol lower than those derived from differential scanning calorimetry. Our results strongly suggest that this discrepancy is not due to deviations from the two-state denaturation mechanism. We propose a new method for the determination of denaturation Gibbs energies from solvent-denaturation data (the constant-delta G extrapolation procedure). It employs several solvent-denaturation profiles (obtained at different temperatures) to generate the protein stability curve at zero denaturant concentration within the -8 to 8 kJ/mol delta G range. The method is model-independent and provides a practical, nonlinear alternative to the commonly employed linear extrapolation procedure. The application of the constant-delta G method to our data suggests that the guanidine-concentration dependence of the denaturation Gibbs energy is approximately linear over an extended concentration range but, also, that strong deviations from linearity may occur at low guanidine concentrations. We tentatively attribute these deviations to the abrupt change of the contribution to protein stability that arises from pairwise charge-charge electrostatic interactions. This contribution may be positive, negative, or close to zero, depending on the pH value and the charge distribution on the native protein surface [Yang, A.-S., & Honig, B. (1993) J. Mol. Biol. 231, 459-474], which may help to explain why disparate effects have been found when studying protein denaturation at low guanidine concentrations. Kinetic m values for lysozyme denaturation depend on temperature, in a manner which appears consistent with Hammond behavior.

Calorimetry↗

PubMed2025

Chemoselective Tagging of Protein Methacrylation.

Protein lysine methacrylation (Kmea) is a recently identified post-translational modification whose biofunction remains poorly understood. Until now, there has been no chemical labeling method for Kmea modification, which has severely hindered the discovery and functional studies of methacrylated proteins. Here, we developed a photocatalytic thia-Michael reaction system for the chemoselective labeling of protein methacrylation. By exploiting the dual effect of steric hindrance and the stability of the generated C-center radical, the reaction interference of the structural isomer crotonylation can be efficiently avoided. Based on this reaction, a multifunctional water-soluble benzenethiol-azide probe azDSH was designed and synthesized, and a workflow for the specific labeling, enrichment, and identification of Kmea proteins was developed. Proteomic identification of histone and nuclear protein extracts and whole-cell lysate revealed a number of novel Kmea proteins and modification sites besides histones, such as HMGB1, TdIF2, UHRF1, HNRPD, BRWD1, TAF1, TACC1, and SETD3, providing new targets for the study of epigenetic regulation. This study provides an effective method for the analysis of protein methacrylation modifications in biological systems.

Humans↗

PubMed1983

Crystallographic studies on apocarboxypeptidase A and the complex with glycyl-L-tyrosine.

The crystal structures of zinc-free carboxypeptidase A (apocarboxypeptidase A) and the complex of glycyl-L-tyrosine with apocarboxypeptidase A are described and compared to the corresponding structures of the zinc-containing enzyme. Only small conformational changes in the zinc ligands accompany removal of the metal. Interactions between the tyrosine residue of glycyl-L-tyrosine and apocarboxypeptidase A are similar to those observed in the complex with the holoenzyme. However, in the absence of zinc, the carbonyl oxygen of the glycyl moiety now receives a hydrogen bond from the side chain of arginine-127. Although not as yet observed, a similar shift of the carbonyl oxygen of a susceptible bond from the zinc to arginine-127 could stabilize tetrahedral intermediates generated during the hydrolysis of substrates by carboxypeptidase.

Apoenzymes↗

PubMed1996

Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination.

DNA molecules undergoing transformation into yeast are highly recombinogenic, even when diverged. We reasoned that transformation-associated recombination (TAR) could be employed to clone large DNAs containing repeat sequences, thereby eliminating the need for in vitro enzymatic reactions such as restriction and ligation and reducing the amount of DNA handling. Gently isolated human DNA was transformed directly into yeast spheroplasts along with two genetically marked (M1 and M2) linearized vectors that contained a human Alu sequence at one end and a telomere sequence at the other end (Alu-CEN-M1-TEL and Alu-M2-TEL). Nearly all the M1-selected transformants had yeast artificial chromosomes (YACs) containing human DNA inserts that varied in size from 70 kb to > 600 kb. Approximately half of these had also acquired the unselected M2 marker. The mitotic segregational stability of YACs generated from one (M1) or two (M1 and M2) vector(s) was comparable, suggesting de novo generation of telomeric ends. Since no YACs were isolated when rodent DNAs or a vector lacking an Alu sequence was used, the YACs were most likely the consequence of TAR between the repeat elements on the vector(s) and the human DNA. Using the BLUR13 Alu-containing vector, we demonstrated that human DNA could be efficiently cloned from mouse cells that contained a single human chromosome 16. The distribution of cloned DNAs on chromosome 16 was determined by fluorescence in situ hybridization. We propose that TAR cloning can provide an efficient means for generating YACs from specific chromosomes and subchromosome fragments and that TAR cloning may be useful for isolating families of genes and specific genes from total genome DNA.

Animals↗