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Biomedical subjects

J E Manning

Publications and source records attributed to J E Manning.

At least 37 records · Page 2Linked to original sources
PubMed1995

Pure paraflagellar rod protein protects mice against Trypanosoma cruzi infection.

The paraflagellar rod proteins (PAR) purified from Trypanosoma cruzi epimastigotes were shown to protect mice against an otherwise lethal challenge inoculum of 10(3) bloodstream-form trypomastigotes. The injection route used for immunization was shown to have a marked impact on the development of protective immunity. Mice receiving subcutaneous (s.c.) injections of PAR proteins had reduced bloodstream parasitemias and showed 100% survival following challenge. In contrast, mice immunized via the intraperitoneal (i.p.) route developed parasitemia levels equivalent to those of unimmunized controls and did not survive infection. Western blotting (immunoblotting) demonstrated that sera from both i.p. and s.c. immunized mice reacted specifically with PAR proteins; however, the antibody titer of the i.p. immunized mice was approximately 64-fold greater than that of the s.c. immunized mice, suggesting that the protective response in the s.c. immunized mice is cell mediated rather than humoral.

Animals↗
PubMed1995

Cardiopulmonary resuscitation.

Research into the physiologic changes that occur during cardiac arrest and resuscitation have led to important changes in our approach to resuscitation of the cardiac arrest victim. Methods that improve coronary perfusion pressure, coupled with direct or indirect measures of coronary perfusion, are actively being sought to improve resuscitation rates and outcomes. These studies have broadened the therapeutic options available to clinicians treating the cardiac arrest victim. Although significant improvements in hospital discharge rates and neurologic outcome have not been realized as yet, a firm basis for future studies has been established. Overall, the most significant intervention that the clinician can presently perform is early and prompt defibrillation of the patient in ventricular fibrillation.

Animals↗
PubMed1994

Identification of immunodominant epitopes in Trypanosoma cruzi trypomastigote surface antigen-1 protein that mask protective epitopes.

The gene that encodes trypomastigote surface Ag-1 (TSA-1), a major surface Ag of the bloodstream trypomastigote stage of Trypanosoma cruzi, was expressed in a baculovirus expression system. To determine the epitope(s) in TSA-1 that was recognized during T. cruzi infection and after immunization with TSA-1, subregions of the TSA-1 gene were expressed in a bacterial expression system. As seen by Western blotting, both mice and rabbits immunized with recombinant TSA-1 protein, as well as T. cruzi-infected mice, developed strong immune responses to the carboxyl-proximal region of TSA-1, but show no reaction to the amino-proximal portion of TSA-1. When mice were immunized with either recombinant TSA-1 protein or the carboxyl-proximal region of TSA-1, they did not survive challenge with 10(3) bloodstream trypomastigotes. However, 70% of the mice immunized with the amino-proximal portion of TSA-1 survived challenge with 10(3) bloodstream trypomastigotes. Thus, the immune responses elicited by recombinant TSA-1 or the carboxyl-proximal portion of TSA-1 are nonprotective during T. cruzi infection. In contrast, vaccination with the amino proximal region of TSA-1 elicits a protective immune response. These results suggest that responses to immunodominant epitope(s) within the carboxyl-proximal portion of TSA-1 mask epitopes within the amino-proximal portion that are capable of stimulating host-protective immune responses. It is suggested that immunodominant regions in surface molecules such as TSA-1 may provide a mechanism for the parasite to evade the host immune response by directing the response away from epitopes that have the potential to elicit a reaction that is damaging to the parasite.

Animals↗
PubMed1994

Dosage compensation in Drosophila: the X-chromosomal binding of MSL-1 and MLE is dependent on Sxl activity.

In Drosophila, dosage compensation, i.e. the equalization of levels of X-linked gene products in the two sexes, is achieved by the hypertranscription of most X-linked genes in males relative to females. The products of at least four genes, collectively termed male-specific lethal (msl) genes, are required for this process and, at least in the case of three of them, mediate this function through an association with the X chromosome in males. We have studied some of the parameters that affect the association of the msl-1 gene product and found that its presence is dependent on the wild-type function of the other three genes, leading to the conclusion that these gene products contribute to the formation of a multi-subunit complex. Furthermore, the X-chromosomal association of the msl-1 and mle gene products is negatively correlated with the level of function of the master regulatory gene Sxl and can assume either a mosaic or a uniform distribution in the tissues of mutant XX individuals. Surprisingly, we also found that the association of these two msl gene products with the two X chromosomes in females of certain mutant genotypes does not result in the hypertranscription of X-linked genes or in any apparent reduction in viability.

Animals↗
PubMed1994

Autocrine growth stimulation of human renal Wilms' tumour G401 cells by a gastrin-like peptide.

The role of gastrin in the control of growth of renal G401 cells isolated from a human nephroblastoma (Wilms' tumour) was investigated. G401 cell growth was enhanced in the presence of exogenous gastrin. Addition of anti-gastrin antibodies to serum-free medium significantly inhibited the growth of G401 cells. G401 cells contained the equivalent of 4.3 pg/10(6) cells of gastrin, and serum-free medium collected over 48 hr from G401 cells contained the equivalent of 38 ng/10(6) cells of gastrin, as determined by radioimmunoassay. Growth of G401 cells was inhibited in a concentration-related way by a variety of gastrin/CCK receptor antagonists. Devazepide and proglumide were, respectively, the most and the least potent inhibitors of G401 cell growth (potency order devazepide > L-365,260 = lorglumide > loxiglumide > benzotript > proglumide). These gastrin/CCK receptor antagonists had similar growth-inhibitory activities in human colonic adenocarcinoma HCT-116 cells. Growth of HCT-116 cells was stimulated to a lesser extent, as compared with G401 cells, by exogenous gastrin, and endogenous gastrin was not detectable in HCT-116 cells. The results are consistent with a role for a gastrin-like peptide in the control of growth of a renal cell line. The data suggest that gastrin/CCK receptor antagonists warrant further investigation as therapeutic agents for the control of gastrin-responsive tumours derived from outside, as well as inside, the gastrointestinal tract, including tumours derived from the kidney.

Benzamides↗
PubMed1994

Expression and evolution of members of the Trypanosoma cruzi trypomastigote surface antigen multigene family.

The trypomastigote specific surface antigens of Trypanosoma cruzi are encoded by a supergene family which includes the TSA family. The TSA family is characterized by the presence of a 27-bp tandem repeat array in the coding region. Here, we report the characterization and analysis of the three TSA family members in the Esmeraldo strain of the parasite. In this strain 2 distinct telomeric members are expressed abundantly as 3.7-kb mRNAs, while the remaining member is located at an internal chromosomal site and is expressed at less than 2% of the level seen for the telomeric members. Based on hybridization to DNA separated by PFGE, 3 chromosomes of sizes 1.8 Mb, 0.98 Mb, and 0.90 Mb each contain one of the telomeric members. In addition, the two smaller chromosomes also contain the single internal member. Since both chromosomes contain similar TSA family members, and vary only slightly in size, we suggest that they are homologues. Comparisons of the nucleotide sequences of the different members of the family show that the internal gene differs from the telomeric genes primarily in sequences found 3' of the repeat array. These comparisons also reveal that the three genes are analogous, supporting the hypothesis that short segments between the family members are exchanged by gene conversion events. We propose that similar conversion events between members of different gene families may generate some of the diversity found within the supergene family.

Amino Acid Sequence↗
PubMed1994

An appraisal of the virulence factors associated with streptococcal endocarditis.

Platelet aggregation is believed to be a virulence factor in infective endocarditis. Other factors may be adhesion to components of thrombotic vegetations, particularly platelets, fibronectin and fibrinogen. Two strains from the Streptococcus sanguis group (SSG) were chosen for comparative study on the basis that one aggregated both human and rat platelets and the other lacked this capacity. Both strains caused endocarditis in the rat model but the aggregating strain was found in higher numbers in the excised vegetations. The nonaggregating strain was unable to bind to human or rat platelets but could bind insoluble fibronectin, insoluble fibrinogen and platelet-fibrin clots from both sources, albeit to a lesser extent than the aggregating strain. These results suggest that whereas adhesion to, and aggregation of, platelets are not essential events in the initiation of the pathogenesis of experimental endocarditis, they may be factors contributing to virulence.

Animals↗
PubMed1994

A comparative study of the aggregation of human, rat and rabbit platelets by members of the Streptococcus sanguis group.

Aggregation of platelets by bacteria is a potential factor in the pathogenesis of infective endocarditis. Twenty-five strains from the Streptococcus sanguis group, including 15 recent isolates from cases of endocarditis, were compared for their ability to aggregate human and rat platelets over periods of 15 and 25 min, respectively. In each case, 76% of strains caused aggregation; the median time to onset of aggregation was longer for human platelets (12 min) than for rat platelets (1 min). Strains unable to aggregate human platelets included three from cases of endocarditis. There was no correlation between the ability to aggregate human and rat platelets, although the majority of strains (60%) aggregated both. Tests on representative strains for their ability to aggregate rabbit platelets gave results similar to those for rat platelets, including a median time of 1 min to onset of aggregation. The differences in the ability of individual bacterial strains to aggregate human and animal platelets indicate that caution is needed in extrapolating in-vitro observations to the in-vivo situation.

Animals↗
PubMed1993

Aortic arch versus central venous epinephrine during CPR.

STUDY OBJECTIVE: To determine if delivery of epinephrine to the peripheral arterial system by an aortic arch catheter is more effective than central venous epinephrine administration during cardiac resuscitation. DESIGN: Randomized, nonblinded, controlled trial. TYPE OF PARTICIPANTS: Sixteen mongrel canines (25 to 31 kg). INTERVENTIONS: Animals had aortic arch pressure, and right atrial pressure, superior vena cava infusion, and descending aortic arch infusion catheters placed using fluoroscopy. After ten minutes of ventricular fibrillation, three DC countershocks were delivered over one minute. If unsuccessful, CPR at 120 compressions per minute was begun, and at 60 seconds of CPR, epinephrine (1 mg/50 mL normal saline) was administered either through the superior vena cava or the aortic arch catheter followed by one more minute of CPR. Defibrillation then was attempted and, if unsuccessful, further resuscitative efforts followed advanced cardiac life support guidelines, except route and dose of epinephrine remained the same. MEASUREMENTS AND MAIN RESULTS: Aortic arch pressure, right atrial pressure, and coronary perfusion pressure (diastolic aortic arch pressure minus diastolic right atrial pressure) were recorded continuously. Aortic arch pressure and coronary perfusion pressure increased more rapidly and to a greater magnitude with aortic arch-epinephrine than superior vena cava-epinephrine. Coronary perfusion pressure doubled by ten seconds in seven of eight in the aortic arch-epinephrine group versus none in the superior vena cava-epinephrine group. Aortic arch pressure and coronary perfusion pressure increases consistently plateaued within 60 seconds after aortic arch-epinephrine but not after superior vena cava-epinephrine. Return of spontaneous circulation was faster (P < .05) in the aortic arch-epinephrine group. Maximal coronary perfusion pressure after epinephrine correlated with the coronary perfusion pressure immediately before epinephrine administration in both groups, but more strongly in the aortic arch-epinephrine group (P = .0001). CONCLUSION: For an equivalent dose of epinephrine, aortic arch administration produces a more rapid response and more rapid peak effect than central venous administration. The combination of aortic arch-epinephrine administration and aortic pressure monitoring may be useful when initial standard resuscitative measures have not been successful.

Animals↗
PubMed1993

Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells.

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes.

Amnion↗
PubMed1993

The male-specific lethal-one (msl-1) gene of Drosophila melanogaster encodes a novel protein that associates with the X chromosome in males.

Male-specific lethal-one (msl-1) is one of four genes that are required for dosage compensation in Drosophila males. To determine the molecular basis of msl-1 regulation of dosage compensation, we have cloned the gene and characterized its products. The predicted msl-1 protein (MSL-1) has no significant similarity to proteins in the current data bases but contains an acidic N terminus characteristic of proteins involved in transcription and chromatin modeling. We present evidence that the msl-1 protein is associated with hundreds of sites along the length of the X chromosome in male, but not in female, nuclei. Our findings support the hypothesis that msl-1 plays a direct role in increasing the level of X-linked gene transcription in male nuclei.

Amino Acid Sequence↗
PubMed1992

Evidence for two distinct major protein components, PAR 1 and PAR 2, in the paraflagellar rod of Trypanosoma cruzi. Complete nucleotide sequence of PAR.

The previously identified major protein components of the paraflagellar rod in Trypanosoma cruzi, PAR 1 and PAR 2, were analyzed to determine if they are distinct proteins or different conformations of a single polypeptide as has been suggested for other trypanosomatids. Amino acid sequence analysis showed PAR 1 and PAR 2 to be two distinct polypeptides. Antibodies specific against either PAR 1 or PAR 2 were shown to each react with a distinct band in Western blots of paraflagellar isolates of T. cruzi and other trypanosomatids if rigorous protease inhibition was used. The PAR 2 message was isolated and characterized by Northern blot and nucleic acid sequence analysis. Preliminary analysis of the PAR 2 gene indicates that PAR 2 is a member of a multigene family with all members residing on a single chromosome.

Amino Acid Sequence↗
PubMed1992

The non-dosage compensated LSP1-alpha gene of Drosophila melanogaster lies immediately downstream of the dosage compensated L12 gene.

The X-linked gene LSP1-alpha of Drosophila melanogaster, expressed in the third larval instar, does not exhibit dosage compensation at its normal locus but does compensate when it is relocated to ectopic sites on the X chromosome. A transcription unit designated L12, which is active in the second larval instar and capable of encoding a putative protein of 28.5 kDa, lies immediately downstream from LSP1-alpha. We have determined that L12 is dosage compensated by measuring the steady-state level of its transcript in male and female larvae. The difference in response of these two adjacent genes should be taken into consideration when models of the mechanism of dosage compensation are formulated.

Animals↗
PubMed1992

Selective aortic arch perfusion during cardiac arrest: a new resuscitation technique.

STUDY OBJECTIVES: To demonstrate the technique of selective aortic arch perfusion during cardiac arrest and to observe the hemodynamic effects of volume infusion and aortic epinephrine administration. DESIGN: Sequential series, nonrandomized, noncontrolled. TYPE OF PARTICIPANTS: Fourteen mongrel dogs weighing 21 to 36 kg. INTERVENTIONS: Animals had midaortic arch pressure, right atrial pressure, and descending aortic arch balloon occlusion catheters placed. After ten minutes of ventricular fibrillation, balloon inflation and aortic arch infusions were initiated as follows: group 1 (six), 30 mL/kg/min of 0.9% NaCl for two minutes; group 2 (four), 30 mL/kg/min of oxygenated lactated Ringer's with 2 mg/L epinephrine for two minutes, followed by CPR; and group 3 (four), 20 mL/kg/min of oxygenated perfluorochemicals with 4 mg/L epinephrine for one minute, then CPR. MEASUREMENTS AND MAIN RESULTS: Midaortic arch pressure, right atrial pressure, and coronary perfusion pressure each rose significantly in all groups. Midaortic arch pressure and coronary perfusion pressure increases were greater in groups 2 and 3 than in group 1. In groups 1 and 2, right atrial pressure increases at end-selective aortic arch perfusion were excessive as midaortic arch pressure and right atrial pressure increased linearly and similarly after 20 to 30 seconds. In groups 2 and 3, CPR-diastolic midaortic arch pressure and coronary perfusion pressure after selective aortic arch perfusion were good and similar to midaortic arch pressure and coronary perfusion pressure at end-selective aortic arch perfusion. CONCLUSION: Selective aortic arch perfusion is technically feasible, but excessive right atrial pressure increases limit maximal infusion rates and volumes. Selective aortic arch perfusion infusates with epinephrine produce greater midaortic arch pressure and coronary perfusion pressure during infusion than infusate without epinephrine. Controlled studies are needed to determine if selective aortic arch perfusion improves resuscitation outcome.

Animals↗
PubMed1991

A method of identifying and isolating a unique member of a multigene family: application to a trypanosome surface antigen gene.

A chimeric oligonucleotide was constructed using DNA sequences from two distal regions of a cDNA which encodes a major surface antigen (TSA-1) of Trypanosoma cruzi. Conditions were found that allowed the chimeric oligonucleotide to hybridize only to a 5.4 kb EcoRI fragment in a Southern blot of total genomic DNA. The 5.4 kb EcoRI genomic DNA fragment has previously been shown to be located at a telomeric site, thus the studies described here directly demonstrate that the TSA-1 gene is telomeric in location. It is also shown that the chimeric oligonucleotide can be used to selectively identify recombinant lambda phage which harbor the TSA-1 gene using standard library screening procedures. Since these studies demonstrate that a chimeric oligonucleotide can be used to identify in both Southern blots and library screens a single member among the more than sixty members of the TSA-1 gene family, it seems likely that chimeric oligonucleotides may be of general use in studies involving repetitive DNA sequence families.

Animals↗