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Biomedical subjects

J Miller

Publications and source records attributed to J Miller.

At least 19 recordsLinked to original sources
PubMed1992

The importance of arginine 102 for the substrate specificity of Escherichia coli malate dehydrogenase.

The malate dehydrogenase from Escherichia coli has been specifically altered at a single amino acid residue by using site-directed mutagenesis. The conserved Arg residue at amino acid position 102 in the putative substrate binding site was replaced with a Gln residue. The result was the loss of the high degree of specificity for oxaloacetate. The difference in relative binding energy for oxaloacetate amounted to about 7 kcal/mol and a difference in specificity between oxaloacetate and pyruvate of 8 orders of magnitude between the wild-type and mutant enzymes. These differences may be explained by the large hydration potential of Arg and the formation of a salt bridge with a carboxylate group of oxaloacetate.

Amino Acid Sequence↗
PubMed1992

Brain-derived neurotrophic factor augments rotational behavior and nigrostriatal dopamine turnover in vivo.

Brain-derived neurotrophic factor (BDNF), a member of the nerve growth factor (NGF)-related family of neutrophins, promotes the survival and differentiation of cultured nigral dopamine neurons. Two-week infusions of BDNF were made above the right pars compacta of the substantia nigra in adult rats. Systemic injection of these animals with (+)-amphetamine, a dopamine-releasing drug, induced 3 or 4 body rotations per minute directed away from the nigral infusion site. Neither supranigral NGF nor neocortical BDNF infusions induced rotational behavior. Systemic injections of the postsynaptic dopamine receptor agonist apomorphine did not induce rotations in these animals, demonstrating a presynaptic dopamine neuron locus for BDNF action. In support of this, neostriatal levels of the dopamine metabolite homovanillic acid (HVA) were elevated by 28%, and the HVA/dopamine and dihydroxyphenylacetic acid (DOPAC)/dopamine ratios were elevated by 56% and 34%, respectively, in the BDNF-infused brain hemisphere. BDNF augmented striatal concentrations of HVA and DOPAC and the metabolite/dopamine ratios to even greater extents after (+)-amphetamine injection, when peak rotational effects occurred. Intrastriatal infusions of BDNF produced fewer rotations per minute (1-2.5) after (+)-amphetamine and smaller elevations in HVA and the HVA/dopamine ratio (15% and 30%, respectively) than after supranigral delivery. Neither striatal dopamine, gamma-aminobutyric acid, nor acetylcholine high-affinity uptake or the synthetic enzymes for these neurotransmitters was altered by BDNF. These behavioral and neurochemical effects demonstrate an action of BDNF on dopamine neurons in vivo and are consistent with a potential role for BDNF in the treatment of Parkinson disease.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1992

Helical model of nucleation and propagation to account for the growth of type I collagen fibrils from symmetrical pointed tips: a special example of self-assembly of rod-like monomers.

A model was developed to account for the recent observations indicating that type I collagen fibrils assembled in vivo grow from symmetrical pointed tips. The essential features of the model are (i) a distinctive structural nucleus forms at each end of a growing fibril and growth of the fibril then proceeds by propagation of the two structural nuclei, (ii) the two structural nuclei have similar spiral or helical conformations, and (iii) assembly of each structural nucleus requires two kinds of specific binding steps defined as 3.4 D-period and 0.4 D-period overlaps, but propagation of the nucleus requires only the 3.4 D-period binding step.

Collagen↗
PubMed1992

Attachment of a single fluorescent label to peptides for determination by capillary zone electrophoresis.

Complicated electropherograms are produced in the separation of fluorescently labeled peptides. Incomplete labeling of epsilon-amino groups on lysine residues results in the production of 2n-1 reaction products, where n is the number of alpha and epsilon amino groups in the peptide. A single label is attached to the peptide by first taking the peptide through one cycle of the Edman degradation reaction. All epsilon-amino groups are converted to the phenyl thiocarbamyl and the cleavage step exposes one alpha-amino group at the N-terminus of the peptide; the fluorescent label is attached to the N-terminus.

Amino Acid Sequence↗
PubMed1992

The CD28 ligand, B7, enhances IL-2 production by providing a costimulatory signal to T cells.

Previous studies demonstrated that a human pre-B acute lymphoblastic leukemia cell line, NALM-6, failed to stimulate a primary MLR, despite expression of class II MHC and adhesion molecules. Here we demonstrate that this is the result of the fact that NALM-6 cells do not express the ligand for CD28, namely B7. NALM-6 transfectants that expressed high levels of B7 gained the capacity to stimulate IL-2 production by class II MHC molecule-specific alloreactive T cells and to costimulate a polyclonal population of purified T cells cultured with immobilized anti-CD3 mAb. In the presence of PMA, NALM-6 cells transfected with B7 polyclonally stimulated T cells in a cyclosporine A-resistant fashion, a property previously attributed only to agonistic anti-CD28 mAb. The gain of these functions could not be explained solely by an increased capacity of the transfectants to form conjugates with T cells, suggesting that the CD28/B7 interaction transduces a costimulatory signal in T cells.

Antigen-Presenting Cells↗
PubMed1992

Antigen presentation enhanced by the alternatively spliced invariant chain gene product p41.

During biosynthesis, class II molecules of the major histocompatibility complex are associated with a nonpolymorphic protein called invariant chain, Ii, which facilitates folding of class II molecules and their exit from the endoplasmic reticulum, interferes with their association with peptide and directs their post-Golgi transport (refs 7-9). If Ii blocks class II loading with endogenous antigens in the endoplasmic reticulum and/or directs class II molecules to the exogenous antigen-loading compartment, then the co-expression of Ii should enhance the ability of class II molecules to present exogenous antigens to T cells. But data supporting a role for Ii in class II-restricted antigen presentation are controversial. Here we show that Ii can facilitate exogenous antigen presentation for a subset of antigens. Although all known functions of Ii have been ascribed to the principal form of Ii, p31, we find that in most cases antigen presentation is facilitated only by the alternatively spliced, minor form of Ii, p41.

Animals↗
PubMed1992

Differential induction of transcription factors that regulate the interleukin 2 gene during anergy induction and restimulation.

T cell activation requires two distinct signals. The first is delivered through the antigen-specific T cell receptor (TCR), and the second is provided by costimulatory molecule(s) present on the surface of the antigen-presenting cell (APC). Stimulation of T helper type 1 T cell clones through the TCR in the absence of the costimulatory activity results in a lack of interleukin 2 (IL-2) secretion and proliferation, and the induction of a long-lived state of nonresponsiveness, termed anergy. In this study, we have examined the transcription factors involved in IL-2 gene expression that are expressed after stimulation of normal T cell clones through the TCR with and without engagement of the necessary costimulatory molecule(s). Antigen-specific activation of the clones results in the induction of a similar pattern of transcription factors that have been previously shown to regulate IL-2 expression. In contrast, antigen presentation by chemically fixed APC, a condition that results in T cell anergy, induces neither NF-AT nor one of the two NF-kappa B binding factors. Thus, the failure to express IL-2 during the induction of T cell anergy may be attributed to the absence of these two transcription factors. When anergized T cells are restimulated with antigen and conventional APC, they induce the transcription factors associated with IL-2 expression, but they fail to synthesize measurable IL-2. Taken together, these data indicate that the control of IL-2 gene expression during anergy induction and during normal stimulation of anergized cells are distinct, and suggest the presence of additional regulatory elements in the IL-2 gene.

Animals↗
PubMed1992

Invariant chain can function as a chaperone protein for class II major histocompatibility complex molecules.

During biosynthesis, class II major histocompatibility complex molecules are intimately associated with invariant chain (Ii). The Ii-class II association has been shown to block peptide-class II binding and to affect the ultimate conformation of class II expressed on the cell surface. To assess the biochemical basis for the effects of Ii on class II, we have analyzed the biosynthesis of class II in EL4 cells transfected with I-Ad with and without Ii. In these studies, we found that Ii had a profound effect on the biosynthesis of I-Ad. In the absence of Ii, class II could form dimers efficiently, but these dimers appeared to be misfolded and this altered conformation resulted in the loss of some monoclonal antibody epitopes and inefficient transport from the endoplasmic reticulum to the Golgi. In addition, class II that was transported through the Golgi accumulated an abnormally increased molecular mass associated with N-linked glycosylation. Subsequent transfection of Ii into these cells resulted in recovery of normal class II conformation, causing a restoration of monoclonal antibody epitopes, efficient intracellular transport, and normal glycosylation. Together, these data indicate that Ii can have a profound effect on the folding, transport, and modification of class II molecules and suggest that one function of Ii may be to act as a class II-specific chaperone.

Animals↗
PubMed1992

Heart, heart-lung, and lung transplantation in the first year of life.

Seventeen infants less than 1 year of age have undergone heart (12), heart-lung (3), and lung (2) transplantation for end-stage cardiopulmonary disease. The infants undergoing heart transplantation had a mean age of 4.5 months (range, 19 days to 12 months) with the diagnosis of cardiomyopathy in 4 and congenital heart disease in 8. Four of the 8 patients (50%) had hypoplastic left heart syndrome. Actuarial survival at 1 and 2 years was 74% and compared favorably with the survival of older children at 1 and 2 years of 82% and 69%. The linearized rejection rate was less in infants as compared with children more than 1 year of age (0.61 versus 1.48 episodes per 100 patient days). In intermediate follow-up, no graft atherosclerosis has been noted. Immunosuppression has included a three-drug protocol of cyclosporine, azathioprine, and prednisone. A steroid taper to alternate day steroids or off completely by 6 months has been the goal and has been accomplished in 6 of 12 infants. Heart-lung and lung transplantation has been performed in 5 infants. One infant in each group died: 1 infant secondary to airway complications and sepsis and another due to pulmonary sepsis. A pulmonary lobe from a larger and older donor was transplanted into a 4-week-old infant as a single-lung transplant with good outcome. The 3 surviving infants are well 24, 18, and 2 months after transplantation. Obliterative bronchiolitis has not been clinically apparent in this group. These data support the clinical efficacy of heart, heart-lung, and lung transplantation in the first year of life.

Actuarial Analysis↗
PubMed1992

Spectrum of injury produced in the duodenum by perfusion with luminal acid in the rat.

The dose and time dependence of duodenal mucosal injury by luminal acid perfusion was studied. Saline, 0.01, 0.05, 0.15, and 0.3N HCl, were perfused through the proximal duodena of rats for 5, 15, or 30 minutes and then harvested for histological examination. In a second set of studies, after a 30-minute perfusion, duodena were harvested either immediately or 2, 4, 8, or 24 hours after the perfusion to study the recovery from injury. Acid disappearance (acid delivered minus acid recovered) was measured in all groups. Duodena were examined grossly, then fixed, stained, and scored histologically. Whereas no gross mucosal injury was noted, there was graded histological injury proportional to acid concentration. Injury occurred early in the perfusion and changed little with increased perfusion durations. The initial injury lead to an acid disappearance rate that was proportional to acid concentration and, therefore, the degree of injury. After the initial injury occurred, the rate of acid neutralization was unchanged by increased duration of acid perfusion. This acid neutralization protected against further injury despite the continued presence of acid. Recovery from injury was complete with physiological (0.01 and 0.05N HCl) but not pharmacological (0.15 and 0.3N HCl) concentrations of acid. It is concluded that acid-induced duodenal injury occurs within 5 minutes of exposure, is proportional to the acid concentration, and results in acid neutralization that protects against extension of the injury with continued acid exposure.

Animals↗
PubMed1992

Cholecystokinin receptor antagonist MK-329 blocks intestinal fat-induced inhibition of meal-stimulated gastric acid secretion.

MK-329, a selective type A cholecystokinin (CCK) receptor antagonist, was given to dogs to test the hypothesis that CCK is one of the principal physiological enterogastrones mediating fat-induced decreases in gastric acid secretion. Gastric acid secretion in response to 300 mL 8% peptone meals was measured by intragastric titration to pH 5.5 in six awake dogs with chronic gastric, duodenal, and jejunal fistulas. Gastric emptying was measured by a dye-dilution technique. During the last hour of peptone stimulation, the intestine was perfused with either control solution or 20% lipid (Intralipid; Kabi Vitrum, Alamedo, CA) intraduodenally or intrajejunally. Compared with control perfusions, mean gastric acid outputs were decreased significantly after lipid perfusion of the duodenum (47% of control) and jejunum (24% of control). Similarly, mean gastric emptying rates were significantly less after lipid perfusion of the duodenum (56%) and jejunum (26%). Oral pretreatment with MK-329 (1 mg/kg) significantly reversed the inhibition of gastric acid output caused by lipid perfusion of the duodenum and jejunum, but fat-induced inhibition of gastric emptying was not significantly affected. These studies provide evidence for an important inhibitory role for CCK as an enterogastrone but do not implicate CCK as being important in fat-induced delayed gastric emptying of a liquid meal in dogs.

Animals↗
PubMed1992

Molecular mechanism of contractile dysfunction in cardiac allograft rejection.

Alterations in the beta-adrenergic receptor adenylyl cyclase pathway are well known in heart failure. To determine if an alteration in this pathway occurs during the reversible phase of cardiac allograft rejection, we used a rat heterotopic heart transplant model. Lewis rats received either isografts or Lewis Brown Norway allografts. Cardiac grafts and native hearts were explanted 4, 5, or 6 days later. Receptor-mediated modulation of adenylyl cyclase activity was investigated using isoproterenol, forskolin, and the muscarinic and adenosine receptor agonists carbachol and R-N6-(C2-phenyl-isopropyl)-adenosine (R-PIA), respectively. Allografts demonstrated evidence of histological rejection and a significantly impaired response to forskolin and isoproterenol on all days: [table: see text] (% increase in cAMP in response to forskolin or isoproterenol +/- standard error. All results P less than 0.03 except Day 4 forskolin and Day 5 isoproterenol.) No significant difference was noted between isografts and allografts stimulated with carbachol and R-PIA. These data suggest that a primary alteration in adenylyl cyclase activity may be a component of the molecular basis of reversible contractile dysfunction in cardiac allograft rejection.

Adenylyl Cyclases↗
PubMed1992

Cross-species reactivity of the anti-idiotype anti-OKT3 cascade between mice and humans.

The administration of murine mAb specific for the CD3 epsilon subunit of the TCR complex (OKT3) has been demonstrated to engender in humans an anti-OKT3 idiotypic cascade. This study used murine-derived anti-OKT3 (Ab2) as a bioreagent to determine whether this Ab2 and polyclonal anti-(anti-OKT3) (Ab3) generated in some human kidney transplant patients are idiotypically connected. Two anti-OKT3 mAbs G-880 (IgG1) and M-12 (IgM) were derived by immunizing BALB/c mice with the OKT3-secreting hybridoma. The two mAbs exhibited specificity for OKT3 F(ab)'2 idiotypic determinants. Both mAbs were tested for their ability to inhibit OKT3 induced mitogenesis and to block FITC-OKT3 binding to cell surface CD3 epsilon chain. The M-12 mAb inhibited OKT3-induced mitogenesis and blocked (approximately 60%) the binding of OKT3 to peripheral blood (PBL) T-cell CD3 epsilon chain in flow cytometry. In contrast, the G-880 mAb did not inhibit mitogenesis and only weakly blocked OKT3 binding to CD3 epsilon chain (approximately 12%). Sera of kidney transplant recipients who received OKT3 antirejection therapy and who developed antiidiotypic anti-OKT3 antibodies could be divided into two subgroups exhibiting anti-OKT3 activity: (a) those who had similar specificity as M-12 and failed to enhance the M-12 inhibition of OKT3 binding to PBL T-cell CD3 epsilon chain when added as a third component (n = 3), and (b) those with anti-OKT3 antibodies with idiotype specificity dissimilar to M-12 and who were able to increase the (maximum 60%) inhibition obtained with M-12 in the OKT3 to T-cell CD3-binding assay (n = 4). From these observations, we conclude that M-12 had the characteristics of an Ab2 beta and G-880 that of an Ab2 alpha. Additionally, there was an idiotypic connectivity of mouse-derived M-12 anti-OKT3 (Ab2) and OKT3-engendered human polyclonal anti-(anti-OKT3) (Ab3), in that three of seven patients examined had human serum IgG antibodies that specifically recognized M-12 idiotypic determinants as demonstrated in ELISA.

Animals↗
PubMed1992

Kynurenine and probenecid inhibit pentylenetetrazol- and NMDLA-induced seizures and increase kynurenic acid concentrations in the brain.

Kynurenine is a direct precursor of kynurenic acid, the only known endogenous antagonist of excitatory amino acid receptors in the brain. Kynurenine administered intraperitoneally (150, 450, 900 mg/kg) 2 h before pentylenetetrazol injection dose-dependently increased the time to seizures, the time to death and the survivorship of mice. Kynurenine dose-dependently increased the time to seizures and the time to death in mice with NMDLA-induced seizures. Kynurenine, 900 mg/kg, was equally efficacious to diazepam, 2 mg/kg. Probenecid dose-dependently increased the time to seizures, the time to death and the survivorship of mice with pentylenetetrazol-induced seizures. Probenecid had no significant effects on NMDLA-induced seizures, although the time to death was prolonged in the NMDLA 500 mg/kg group. Probenecid potentiated the effects of kynurenine in these tests. Both probenecid and kynurenine significantly increased kynurenine and kynurenic acid concentrations in mouse cerebral cortex and striatum. These findings suggest that kynurenine (metabolized to kynurenic acid) has anticonvulsant effects, and probenecid potentiates these effects in mice.

Analysis of Variance↗
PubMed1992

Doxorubicin in advanced breast cancer: influence of schedule on response, survival and quality of life.

The influence of scheduling of doxorubicin on response, survival and quality of life was assessed in a randomised trial in patients with advanced breast cancer, none of whom had previously received cytotoxic chemotherapy for advanced disease. 28 patients received 75 mg/m2 doxorubicin every 3 weeks for four courses (arm 1) and 31 patients received 25 mg/m2 weekly for 12 courses (arm 2). Response rates and median time to progression were similar in the two arms and median survival was 8 months in both arms. However, amongst patients receiving treatment every 3 weeks, psychological distress measured using the Rotterdam symptom checklist fell significantly over the course; no such change was observed in those treated weekly. Physical symptoms related to cancer improved during treatment similarly for both groups.

Adult↗