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M D Hoffman

Publications and source records attributed to M D Hoffman.

69 records · Page 4Linked to original sources
PubMed1987

Comparison of the roles of calmodulin and protein kinase C in activation of the human neutrophil respiratory burst.

The roles of calmodulin and protein kinase C in the activation of the human neutrophil respiratory burst were characterized pharmacologically. The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) did not inhibit superoxide anion generation by neutrophils stimulated for 30 minutes with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) or 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA). However, H-7 did depress superoxide production during the first 5 minutes following stimulation. In contrast, the specific calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and the dual calmodulin antagonist/protein kinase C inhibitor trifluoperazine (TFP) were potent inhibitors of the response throughout the 30 minute incubation. Stimulation of neutrophils with submaximal doses of FMLP or PMA failed to promote inhibition of the respiratory burst by H-7 or H-9, but did stimulate a respiratory burst response which was not inhibited by TFP or W-7. These results suggest that while protein kinase C may play a role in the initiation of the respiratory burst response, propagation of the response is dependent on calmodulin-dependent processes. The inability of TFP and W-7 to inhibit superoxide anion generation in response to submaximal stimulatory doses of FMLP or PMA suggests that calmodulin-independent processes may also be involved in activation of the respiratory burst.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1987

Inhibition of human neutrophil activation by the allergic mediator release inhibitor, CI-922: differential inhibition of responses to a variety of stimuli.

The allergic mediator release inhibitor 3,7-dimethoxy-4-phenyl-N-1H-tetrazol-5-yl-4H-furo[3,2-b]indole-2- carboxamide, L-arginate (CI-922) is a potent inhibitor of human neutrophil functions in vitro. Over a concentration range from 1 to 100 mumol CI-922 inhibits the chemotactic response of neutrophils to the synthetic chemotaxin N-formyl-methionyl-leucyl-phenylalanine (FMLP). CI-922 also inhibits respiratory and secretory responses of neutrophils in response to agents that stimulate phospholipase C-dependent phosphoinositide hydrolysis to generate the second messengers inositol 1,4,5, trisphosphate and 1,2 diacylglycerol, including: the plasma membrane receptor-specific ligands FMLP and C5a; serum-opsonized zymosan; concanavalin A; and the guanine nucleotide regulatory protein-specific stimulus guanosine-5'-0-(3-thiotriphosphate) (GTP gamma S). CI-922 also inhibits neutrophil functions stimulated by the calcium ionophore A23187. In contrast, CI-922 does not inhibit neutrophil responses to protein kinase C-specific stimuli such as phorbol 12-myristate 13-acetate (PMA) or L-alpha-1,2 dioctanoylglycerol (DiC8). CI-922 also fails to inhibit the synergistic activation of the respiratory burst by suboptimal concentrations of PMA and calcium ionophore A23187. The observation that CI-922 inhibits neutrophil responses to a variety of soluble and particulate stimuli, excluding protein kinase C-specific stimuli, allows us to postulate the site of action of the compound. We propose that CI-922 inhibits neutrophil activation at a site distal to signal transduction through the guanine nucleotide regulatory protein required for second messenger generation but proximal to phosphorylation reactions mediated by protein kinase C and calmodulin-dependent protein kinases.

Azoles↗
PubMed1986

The protein kinase C inhibitors H-7 and H-9 fail to inhibit human neutrophil activation.

The protein kinase C inhibitors 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) and N-(2-aminoethyl)-5-isoquinolinesulfonamide (H-9) were examined for their ability to inhibit human neutrophil activation. At concentrations up to 100 micromolar, these compounds failed to inhibit either respiratory burst or the secretory response of neutrophils stimulated with particulate (serum-opsonized zymosan) or soluble (A23187, FMLP, PMA) stimuli. In contrast, the calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) inhibited both oxygen radical generation and lysosomal enzyme release in response to the same stimuli. These results suggest that calmodulin-dependent enzymes, rather than protein kinase C, may be essential for neutrophil activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1985

Changes in isometric function following rhythmic exercise.

Seven male subjects exercised for 1, 3, 10 and 20 min on a cycle ergometer at 20, 60 and 80% VO2max, and then held to fatigue a sustained contraction of the quadriceps at 40% maximal voluntary contraction in order to determine what influence various levels of dynamic exercise would have on isometric function of the same group of muscles. Muscle temperature was measured before and within 15 s of the completion of the cycling to determine whether changes in muscle temperature might influence the subsequent isometric performance. Isometric endurance was shorter as the severity of the cycling increased beyond 20% VO2max, and as the duration of cycling increased up to 10 min. There were discrete linear relationships between muscle temperature and isometric endurance associated with cycling at 60% and 80% VO2max. There was a direct inverse relationship between quadriceps strength after cycling and muscle temperature, yet a significant reduction in strength occurred only after cycling at 80% VO2max. These results suggest that the encroachment on endurance and strength are controlled by different mechanisms. The heart rates during the isometric contractions were dependent on the preceding rhythmic exercise and decreased after exercise at 60 or 80% VO2max. In contrast, the blood pressure always increased during the isometric contractions, reaching similar values at the point of fatigue, regardless of the severity of the previous rhythmic exercise. These data provide additional evidence that separate mechanisms control changes in heart rate and blood pressure.

Blood Pressure↗
PubMed1985

Nonrandom catabolism of proteins in the formation of antigenic peptide fragments.

To examine the role of protein catabolism in the formation of antigenic peptide fragments, human fibrinopeptide-immune guinea pig T cells were stimulated with the large native molecule, human fibrinogen. Two different systems were tested. In the first, we determined responses by human fibrinopeptide B (hFPB)-immune T cells, to which strain (St.) 2 guinea pigs are responders and St. 13 are nonresponders, and by human fibrinopeptide A (hFPA)-immune T cells to which St. 13 are responders and St. 2 are nonresponders. Of interest in this comparison is that both hFPA and hFPB are amino terminal peptides on the A and B chain of fibrinogen, respectively, and are readily cleaved by thrombin during fibrin formation and by other trypsin-like enzymes, leaving a carboxyl terminal Arg. Thus, if fibrinogen catabolism occurred, both antigenic peptides should be equally represented for availability in T cell responses. It was found that hFPB-immune St. 2 T cells responded to fibrinogen, but no response was observed with hPFA-immune St. 13 T cells cultured with fibrinogen. To rule out that there was a general catabolic defect in St. 13 antigen-presenting cells, fibrinogen was presented by (2 X 13)F1 macrophages to fibrinopeptide-immune parental T cells. Again it was found that F1 macrophages could present fibrinogen to hFPB-immune T cells but failed to present hFPA. In another comparison, responses with fibrinogen were also determined with des-ARg-hFPB, which lacks the carboxyl terminal Arg of hFPB, to which St. 13 are responders and St. 2 are nonresponders. The advantage of this comparison is that both antigenic determinants are contained within the same small peptide. St. 13 des-Arg-hFPB-immune T cells failed to respond in vitro by culture with human fibrinogen, suggesting that these antigenic determinants are not produced from larger peptides or proteins containing those determinants. To rule out the possibility that this was only an in vitro phenomenon, guinea pigs were immunized with the larger protein, the B chain of fibrinogen, and the immune T cells were examined for responses to fibrinopeptides derived from the B chain. Immune St. 2 T cells responded to hFPB but not to des-Arg-hFPB, whereas St. 13 T cells remained unresponsive with both peptides. These results indicate that proteolysis of larger proteins to form small antigenic peptides is not a random event and that not all potential antigenic determinants contained in a protein are produced during antigen processing.

Animals↗
PubMed1984

Identification of macrophage receptors for angiotensin: a potential role in antigen uptake for T lymphocyte responses?

To determine if macrophages express receptors for peptide antigens, guinea pig peritoneal exudate cells (PEC) were examined for their uptake of the octapeptide angiotensin II (AII). PEC were incubated with [3H]AII, with or without nonradioactive AII as a cold inhibitor, for varying lengths of time before harvesting to determine the cell-associated [3H]AII counts per minute. The PEC-associated [3H]AII increased from 90 to 120 min of incubation, then plateaued on additional incubation to 3.5 hr. Inclusion of nonradiolabeled AII into the culture decreased the cell-associated [3H]AII by 80 to 90% at all time points. The uptake of [3H]AII was temperature-sensitive, with maximum cell-associated [3H]-AII occurring at 37 degrees C and reduced uptake occurring at 4 degrees C. The association of [3H]AII with PEC was specific and saturable, and the inhibitory dose for reducing the cell-associated [3H]AII by 50% with nonradiolabeled AII was around 6 X 10(-6) M. Various AII analogs were also employed as inhibitors to determine the fine specificity of [3H]AII binding, and only those analogs with nonaromatic amino acid substitutions for the carboxyl terminal Phe8 showed reduced inhibitory activity, indicating that Phe8 is important for binding. Scatchard analysis of binding indicated that two classes of receptors interacted with AII: a low number of receptors with Ka approximately equal to 3.5 X 10(8) M-1, and a large number of relatively low affinity of binding showing a Ka approximately equal to 5 X 10(5) M-1. The cellular binding activity was associated with isolated PEC plasma membranes, and after density gradient fractionation of solubilized membranes, AII binding activity was primarily associated with molecules of m.w. of around 50,000. PEC were separated into macrophages and lymphocytes by adherence, and all of the [3H]AII binding activity was associated with the macrophage-enriched cells. These results show that macrophages express specific receptors for AII and related peptides that are responsible for most of the uptake of AII by macrophages. We discuss the relevance of this receptor for the immunologically important uptake of AII by macrophages, and a potential physiologic role in angiotensin-converting enzyme production.

Angiotensin II↗
PubMed1984

Macrophage processing of peptide antigens: identification of an antigenic complex.

In this report, we present data from our examination of the fate of the octapeptide antigen angiotensin II (AII) after processing by guinea pig macrophages. Macrophages were cultured with [125I]-AII or [3H]-AII for various times, and the AII products in the culture supernatant fluid and solubilized cell lysate (macrophage-associated AII) were analyzed by chromatography on Sephadex G-25. With [3H]-AII, both the cell-associated and cellfree AII was catabolized to three distinct peptide fragments: Peak B, containing seven to eight amino acids; Peak B1, with five to six amino acids; and Peak C, containing four to five amino acids. Surprisingly, in the cells another major form of AII was obtained, Peak A, which showed an m.w. of 50,000 to 70,000. Formation of Peak A was blocked by inclusion of non-radiolabeled AII into the incubation mixture, and could be formed by repulsing fresh macrophages with isolated Peaks B and B1, and to only a minor extent with Peak C. Peak A was formed within an hour of culture of macrophages with [3H]-AII, and remained stably cell-associated through a day of culture. In a pulse-chase type of experiment, it was shown that macrophages rapidly exocytose cell-associated Peaks B and C, and Peak B1 less rapidly, whereas Peak A is retained and is the predominant cell-associated form of AII after a day in culture. Incubation of [3H]-AII with isolated macrophage plasma membranes also resulted in Peak A formation, showing that the complex can be formed by a membrane event not requiring cellular uptake of peptide. The complex of AII in Peak A was acid-stable, and approximately 20% of Peak A was recovered after boiling in SDS. Because Peak A is the primary form of AII retained by macrophages, it was important to determine if this complex was antigenic for T cells. AII-immune T cells cultured with Peak A showed significant proliferation that was specific for AII. Thus, Peak A appears to represent a stable complex of AII with macrophage membrane structures that provide an immunologically relevant form of the antigen. These results are discussed with respect to a pathway for macrophage processing of peptide antigens to form a stable membrane-bound complex that is important for T cell responses.

Amino Acid Sequence↗
PubMed1982

T lymphocyte recognition of peptide antigens: evidence favoring the formation of neoantigenic determinants.

To more precisely define the nature of exogenous antigenic determinants recognized by T cells, the response to fibrinopeptide fragment B beta 7-14 and a peptide of the inverted amino acid sequence of B beta 7-14 was examined. Strain 2 guinea pig T cells immunized with B beta 7-14 showed in vitro proliferative responses with B beta 7-14, but failed to respond to the inverted B beta 7-14 peptide. Moreover, the inverted B beta 7-14 peptide was nonimmunogenic and failed to prime strain 2 T cells for responses to native or inverted B beta 7-14. These results suggest that T cell recognition of peptide antigens involves more than simple interactions with amino acid side chains and that the ordering of the amino acids within the peptide is critical. One interpretation of these results is that T cells exhibit polarity in reading of antigenic determinants and peptides become associated with self in some fashion to form a neoantigenic determinant. To test this possibility, a Gly residue was added to the carboxyl end of B beta 7-14 (B beta 7-15), which is the likely site of attachment to self. It was found that strain 13 guinea pigs, which are totally unresponsive to B beta 7-14, produced T cell responses to B beta 7-15. This observation is consistent with the interpretation that Gly spaces the B beta 7-14 away from self to form an antigenic determinant complementary to strain 13 T cell antigen recognition structures. These results are discussed with respect to several mechanisms for immune response gene control of T cell responses.

Amino Acid Sequence↗
PubMed1982

Characterization of a 75,000 mol. wt glycoprotein synthesized by guinea-pig T-lymphocytes: a possible homologue of Lyt-1 antigen.

A xenoantiserum raised in rabbits by immunization with strain 13 guinea-pig antigen-activated T-lymphocytes was previously found to recognize a non-immunoglobulin, 75,000 mol. wt glycoprotein synthesized by guinea-pig T-cells. This protein, p75, has been further characterized to determine its biochemical properties and its expression by various cell types. p75 was found to be a single-chain protein which could be bound by the lectin Lens culinaris hemagglutinin. It has an apparent mol. wt slightly greater than mu-chain as assessed by SDS-polyacrylamide gel electrophoresis and could not be precipitated by anti-guinea-pig immunoglobulin reagents. It exhibited considerable charge heterogeneity during isoelectric focusing and was not affected by neuraminidase treatment, p75 was synthesized by thymus, spleen and lymph node cells, by antigen-stimulated T-cells from strain 13 and strain 2 guinea-pigs, and by guinea-pig B-cell L2C leukemia lines, but not by normal B-lymphocytes or macrophages. No differences between the isoelectric focusing patterns of p75 molecules isolated from different cell types could be demonstrated. The chemical properties of p75 and its expression by the cell types so far examined indicate that p75 is a possible candidate for the guinea-pig homologue of the murine Lyt-1 antigen.

Animals↗
PubMed1982

Evidence for covert cellular interaction sites expressed by activated T lymphocytes.

In this report we examined the effects of trypsin treatment of immune guinea pig T lymphocytes on their binding to syngeneic macrophages. Immune T cells positively selected by culture for 1 wk with antigen and treated with trypsin showed dramatic binding to macrophages in the absence of additional antigen. This binding was rapid and greatest at 1 hr after the addition of trypsin-treated T cells to macrophages. Thereafter, the bound T cells dissociated from the macrophages and did not rebind. Dissociation of macrophage-bound T cells appeared to be an active process that could be inhibited by blocking T cell protein synthesis. Macrophage binding by trypsin-treated T cells was dependent on prior in vitro stimulation; similar treatment of immune T cells taken directly from the animal did not augment binding, and treatment of T cells cultured without antigen resulted in only modest binding. In addition, the trypsin-induced binding phenomenon showed elements of genetic restrictions. Trypsin treatment of immune T cells selected by culture for 1 wk with antigen resulted in preferential binding to syngeneic macrophages. In addition, immune F1 T cells selected by culture with antigen-pulsed parental macrophages preferentially bound to macrophages of the haplotype used for selection. Evidence is presented suggesting that the trypsin-induced binding was not simply due to antigen carry-over from the trypsinized lymphocytes and favoring the possibility that this binding may be antigen-independent. These results suggest that antigen-activated T cells express covert interaction sites for macrophages and that these sites are actively covered-up by other membrane components.

Animals↗
PubMedDate not supplied

Cardiorespiratory fitness and training in quadriplegics and paraplegics.

With the growing interest in exercise and sport and the significance of cardiovascular disease in the spinal cord injured population, the role of endurance training in improving cardiovascular health is of particular interest. Ordinary daily activities of those with spinal cord injury are usually not adequate to maintain cardiovascular fitness, and lack of participation in a regular activity programme may result in a debilitative cycle. As this occurs, there is a reduction in functional work capacity which may limit independence, and the reduction in cardiovascular fitness may increase the risk for cardiovascular disease. Work capacity in those with spinal cord injury is limited by loss of functional muscle mass and sympathetic control. Sympathetic nervous system impairment limits control of regional blood flow and cardiac output, and maximum heart rate following cervical lesions may be reduced to 110 to 130 beats/min. However, endurance training in quadriplegics and paraplegics can elicit improvements in exercise performance similar to those observed in able-bodied individuals. Review of 13 cardiorespiratory training studies involving spinal cord injured subjects revealed average improvements of 20% in VO2max and 40% in physical work capacity after 4 to 20 weeks of training. Based upon the positive results of these studies, the general endurance training guidelines for the normal population appear to also be appropriate for the spinal cord injured population. These guidelines can be followed during participation in a number of different activities and sports including wheelchair pushing, arm crank ergometry, aerobic swimming, ambulation training, canoeing and wheelchair basketball. There is no evidence that intense training and competition is harmful, but special areas of risk as a result of impairments in sensation, cardiovascular function, autonomic function and temperature regulation must be considered. The long term benefits of endurance training in those with spinal cord injury has not been adequately studied, but there is suggestion that similar physiological and psychological changes may occur as in able-bodied individuals.

Cardiovascular System↗
PubMedDate not supplied

BRCA1 germline mutations in Cypriot breast cancer patients from 26 families with family history.

Germline mutations in the BRCA1 gene are causative for a variable number of hereditary breast/ovarian cancers. The data presented in this study are based on genetic analysis of the BRCA1 gene in 49 DNA samples from breast cancer patients with a positive family history. A combination of manual direct DNA sequencing and SSCP analysis was used to screen the entire coding region of BRCA1. Overall 13 variants were detected which included 5 missense mutations, 3 polymorphisms and 5 intronic changes. Further genetic analysis of the 13 variants was carried out using 50 control DNA samples. Our results showed that 12 out of the 13 variants detected in the DNA of the patients group, were also present in the control group. It appears that the Greek Cypriot families studied so far have an unexpectebly low frequency of deleterious mutations in the BRCA1 gene. This is the first report on BRCA1 mutation analysis in Cyprus.

Adult↗