Biomedical subjects

G H Fisher

Publications and source records attributed to G H Fisher.

At least 19 recordsLinked to original sources

PubMed1996

Synergy between T cell Receptor and Fas (CD95/APO-1) signaling in mouse thymocyte death.

Administration of anti-TCR/CD3epsilon antibody in vivo or in thymic organ culture results in the apoptotic death of CD4+/CD8+ thymocytes. In contrast, purified thymocytes in suspension culture are resistant to TCR/CD3epsilon-induced apoptotic death. We show that induction of thymocyte death, in suspension culture, can be induced by the combination of TCR/CD3epsilon and Fas (CD95/Apo-1) signaling. No significant thymocyte death was observed after in vitro Fas cross-linking unless TCR/CD3epsilon was simultaneously co-cross-linked or metabolic inhibitors such as actinomycin D were added. Furthermore, TCR/CD3epsilon and Fas synergy did not operate through upregulation of Fas but by facilitation of the Fas-mediated death signal. Both TCRmid/lo/HSAhi/CD4+/CD8+ (double positive) and TCRhi/HSAlo/CD4+/CD8- or CD4-/CD8+ (single positive) thymocytes were susceptible to death induced by co-cross-linking of TCR/CD3epsilon and Fas. Our results reveal a signaling synergy between the Fas and TCR/CD3epsilon complex that has important implications for our understanding of in vivo vs in vitro models of thymocyte deletion.

Animals↗

PubMed1995

Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome.

Five unrelated children are described with a rare autoimmune lymphoproliferative syndrome (ALPS) characterized by massive nonmalignant lymphadenopathy, autoimmune phenomena, and expanded populations of TCR-CD3+CD4-CD8- lymphocytes. These findings, suggesting a genetic defect in the ability of T lymphocytes to respond to normal immunoregulatory mechanisms, prompted an evaluation of lymphocyte apoptosis. Each child had defective Fas-mediated T lymphocyte apoptosis associated with a unique, deleterious Fas gene mutation. One mutation appeared to cause a simple loss of function; however, four others had a dominant negative phenotype when coexpressed with normal Fas. Family studies demonstrated the inheritance of the mutant Fas alleles. The occurrence of Fas mutations together with abnormal T cell apoptosis in ALPS patients suggests an involvement of Fas in this recently recognized disorder of lymphocyte homeostasis and peripheral self-tolerance.

Antigens, Surface↗

PubMed1995

Free D-serine concentration in normal and Alzheimer human brain.

We have analyzed both free L- and D-serine in frontal cortex of normal and Alzheimer human brain by high-performance liquid chromatography (HPLC). There was no significant difference between the two brains. In normal brain, L- and D-serine concentrations were 666 +/- 222 and 66 +/- 41 nmol/g of wet tissue, respectively, and the ratio of D-isomer to L-isomer (D/L) was 0.099 +/- 0.031. In Alzheimer brain, the concentrations were 750 +/- 150 and 66 +/- 40 nmol/g, respectively, and the D/L ratio was 0.086 +/- 0.040. Thus, it was shown that the free D-serine concentration in the Alzheimer brain was comparable to that in the normal brain.

Aged↗

PubMed1995

Presence of D-aspartate and D-glutamate in tumor proteins.

Over 50 years ago Kögl and Erxleben reported that tumor proteins contain appreciable amounts of D-amino acids. This postulate remained highly controversial for several years, during which time several researchers either supported or refuted the hypothesis. We have analyzed several sets of tumors and normal control tissues for the presence of D-aspartate (D-Asp) and D-glutamate (D-Glu). Most tumors contain less D-Asp than the control tissues, whereas nearly half of the tumors contain 1.6 to 5.4 times more D-Glu than the controls. The tumors averaged 0.72% D-Asp and 0.61% D-Glu compared to 0.94% D-Asp and 0.35% D-Glu in the control tissues. However, within the limits of experimental error, there is no significant difference in the level of these D-amino acids between the tumors and normal tissues.

Aspartic Acid↗

PubMed1994

D-aspartate and D-glutamate in microwaved versus conventionally heated milk.

OBJECTIVE: It has been reported that microwave heating of infant formulae can isomerize and racemize amino acids in the milk proteins, causing toxicity or affecting the nutritional value of the milk formulae. Therefore, we investigated whether microwave heating vs conventional heating would produce any D- enantiomers of aspartic acid (Asp) and glutamic acid (Glu) in milk. METHODS: Whole and skim milk samples were heated for 10 minutes in either a microwave oven at medium power or on a hot water bath at 80 degrees C. D-Asp and D-Glu were determined by high performance liquid chromatography. RESULTS: Unheated (control) samples were found to contain 0.40-0.45% D-Asp and D-Glu, inherent from the original pasteurizing process. Both conventional heating and microwave heating induce < 0.25% more racemization when compared to the control samples. CONCLUSION: Within experimental error, there is no significant difference in the levels of these D-amino acids between the conventionally heated and microwave heated milks, thus having no significant effect on the nutritional value of the milk proteins.

Animals↗

PubMed1993

Biological role of D-amino acid oxidase and D-aspartate oxidase. Effects of D-amino acids.

D-Amino acids administered to animals are absorbed by the intestine and transported through the blood-stream to solid tissues where they are oxidized in vivo by D-amino acid oxidase and D-aspartate oxidase to produce the same compounds they do in vitro; i.e. NH3, H2O2, and the keto acid corresponding to the amino acid ingested. In the liver and kidneys of the animals, an inverse relationship exists between the occurrence of D-amino acids and these oxidative enzymes. For example, younger animals have lower amounts of these oxidases and consequently higher concentrations of free D-amino acids compared to adult animals. If the ingested D-amino acids are not metabolized by these enzymes, they will accumulate in the tissues and may provoke serious damage, e.g. suppression of the synthesis of other essential enzymes and inhibition of the growth rate of the animals. A specific enzyme induction for these D-amino acid oxidases exists in young rats following ingestion of free D-amino acids by the mother. Specifically, when a mother rat ingests D-Ala or D-Asp during pregnancy and suckling, an increase in D-amino acid oxidase or D-aspartate oxidase is observed in the liver and kidneys of the baby rats. These results suggest that the in vivo biological role of these oxidases in animals is to act as detoxifying agents to metabolize D-amino acids which may have accumulated during aging.

Amino Acid Oxidoreductases↗

PubMed1992

Presence of D-alanine in proteins of normal and Alzheimer human brain.

This report constitutes the first demonstration of the presence of D-alanine in the proteins of the human nervous system. Proteins of the frontal lobe white and gray matter of human brains, both normal and Alzheimer subjects, contain D-alanine at concentrations between 0.50 and 1.28 mumol/g of wet tissue, 50-70-times lower than the concentration of L-alanine. Both white and gray matter of Alzheimer brains contain D-alanine 1.4-times higher than the respective regions of normal brains. The gray matter proteins of Alzheimer brains show a highly significant 8% decrease in total alanine content, when compared with normal brain gray matter proteins. Since Alzheimer's disease is exhibited by deterioration of the gray matter, the occurrence of elevated D-alanine levels in the gray matter of Alzheimer brains is a significant discovery and raises the question whether this enantiomer causes the degeneration of the gray matter proteins in Alzheimer's disease, or whether it is an effect of the disease.

Aged↗

PubMed1992

Quantification of D-aspartate in normal and Alzheimer brains.

Using a new procedure to hydrolyze proteins without provoking racemization of the amino acids and using enzymatic methods to determine D- and L-aspartate (Asp), we have quantified the content of protein-bound D-aspartate (both D-aspartic acid and D-asparagine) of human brain white and gray matter proteins from normal and Alzheimer subjects. The D-enantiomer is present in brain proteins at mean concentrations between 0.48 and 0.90 mumol/g of wet tissue, corresponding to concentrations 34-82 times lower than that of L-aspartate. The highest levels of D-aspartate were found in Alzheimer gray matter (0.60-0.90, mean 0.69 mumol/g of wet tissue). When expressed as the percentage of total (i.e. D- plus L-) aspartate, %D = [D/(D + L)] x 100, the Alzheimer brains show a significantly higher content of D-aspartate in both gray matter (2.08%) and white matter (1.80%) than in the corresponding tissues of normal brains (1.65% in gray, 1.58% in white).

Alzheimer Disease↗

PubMed1992

Altered aspartate in Alzheimer neurofibrillary tangles.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo post-translational modification by racemization to D-aspartate, or by isomerization to the L-isoaspartyl form in which the peptide chain links through the beta carboxyl group of the residue. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a significantly greater number of these modified aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

Aged↗

PubMed1992

Racemized D-aspartate in Alzheimer neurofibrillary tangles.

Normal protein-bound L-aspartyl/L-asparaginyl residues may undergo posttranslational modification by racemization to D-aspartate. Based on preliminary results reported here, proteins associated with Alzheimer neurofibrillary tangle preparations contain a greater number of these racemized D-aspartyl residues than the unaffected proteins from the surrounding gray matter or in comparable preparations from normal brains.

Alzheimer Disease↗

PubMed1991

Free D-aspartate and D-alanine in normal and Alzheimer brain.

In this report we present evidence for the presence of free D-aspartic acid (D-Asp) and D-alanine (D-Ala) in the white and gray matter of normal human brains and brains of individuals with Alzheimer's disease. D-Asp occurs at about the same concentration in the gray matter of both normal (18.6 nmol/g) and Alzheimer (14.8 nmol/g) brains, whereas in white matter its concentration is more than two times higher in normal than Alzheimer brains (22.4 and 10.5 nmol/g, respectively). D-Ala occurs in white matter at approximately the same concentration in both normal and Alzheimer brains (12.3 and 13.8 nmol/g, respectively), whereas in Alzheimer gray matter the D-Ala concentration is more than twice that found in normal gray matter (20.8 and 9.5 nmol/g, respectively). However, when the results are expressed as a percentage of D-amino acid/D+L, only small differences occur in all tissues examined.

Adult↗

PubMed1990

T lymphocytes can recognize determinants unique to neuropeptides of guinea pig myelin basic protein containing a single D-isomer amino acid substitution.

The present studies were undertaken to examine how the substitution of racemized forms of selected amino acids in synthetic peptides of guinea pig myelin basic protein (GPMBP) would alter the host's immunological ability to recognize such molecules. Using peptides from the 69-84 sequence of GPMBP containing a D-serine at position 70 or 75 (69-84[D-ser70 or D-ser75]) or D-aspartate at position 82 (69-84[D-asp82]), the findings demonstrated that the position of the diastereomer substitution on these neuropeptides was critical with respect to the ability of the immune system to recognize the molecule. Thus substitution of D-asp at position 82 or D-ser at position 75 abrogated the ability of these peptides to induce experimental autoimmune encephalitis and proliferation of host T cells. In contrast, a peptide containing a D-ser70 residue was capable of inducing clinical disease in rats, as well as stimulating T lymphocytes from 69-84-(D-ser70)-injected animals. Moreover, although this D-peptide was shown to share at least some determinant(s) with the 69-84 peptide, the use of 69-84(D-ser70)-stimulated cell lines demonstrated that some epitope(s) unique to this molecule could stimulate CD4+ syngeneic T cells.

Animals↗

PubMed1987

D-aspartate in human brain.

The presence of the biologically uncommon D-aspartic acid (D-aspartate) in human brain white matter has been previously reported. The earlier study has now been expanded to include D/L-aspartate ratios from 67 normal brains. The data show that the D-aspartate content increases rapidly from 1 year to approximately 35 years of age, levels off in middle age, and then appears to decrease somewhat. The D-aspartate content in gray matter remains at a consistently low level (half of that found in white matter) throughout the human life span. Within the limitations of current analytical methods, there was no detectable difference in D/L-aspartate ratios in white and gray matter of brains with Alzheimer's disease and several other pathologies when compared with brains of normal subjects. However, the presence of a significant D-aspartate level in white matter during the adult life span may lead to changes in protein configuration related to dysfunctions associated with the aging brain.

Adolescent↗

PubMed1986

D-aspartic acid in purified myelin and myelin basic protein.

The presence of the biologically uncommon D-isomer of aspartic acid in the white matter of human brains has been reported previously from this laboratory (1). We now report that the level of D-aspartate in human brains is higher in purified myelin than in white matter and is even higher in the myelin basic protein fraction. There also appears to be a difference in the level of D-aspartate found in human brain as compared to bovine brain, possibly a species or age-related difference.

Adult↗

PubMed1986

Synthetic inhibitors of carboxypeptidase N.

Further to explore the functions of carboxypeptidase N (CPN) in vivo, we undertook two studies to find CPN inhibitors of high potency and relatively long duration of action. In each study we examined for inhibition of hydrolysis of [3H]benzoyl-Ala-Arg using pure bovine serum CPN or human serum. In the first such study we synthesized a series of acyl amino acids and acyl di - and tripeptides containing arginine, lysine or both. All proved to be weak inhibitors (Ki = 10(-3) to 10(-4) M). N alpha-carbamoyl-Arg was the strongest: Ki = 3.5 X 10(-5) M. In the second study we prepared S-acyl (thio ester) derivatives of the highly potent CPN inhibitor 2-mercaptomethyl-3-guanidinoethylthiopropionic acid (2-MGP), as certain S-acyl groups markedly increase the duration of captopril, another mercapto-containing compound. Acetyl-, Boc-phenylalanyl-, phenylalanyl-, benzoyl-alanyl-, alanyl-, and Boc-alanyl-2-MGP retained the high potency of 2-MGP in vitro. Although Ala-2-MGP exerted maximum effects in vivo, like those of 2-MGP, the duration of action of Ala-2-MGP was slightly shorter than that of 2-MGP. These results indicate that the mercapto group of 2-MGP can be taken up in some forms of thioester linkage and still remain virtually the full potency of 2-MGP itself. Thus, it appears that a free mercapto function is not essential for the action of 2-MGP.

Amino Acids↗

PubMed1985

Analysis of problems encountered in the determination of amino acid enantiomeric ratios by gas chromatography.

A previously described procedure for determining the enantiomeric ratios of amino acids has produced inconsistent results when determining relatively low (less than or equal to 0.110) D/L ratios. The method involves synthesis of diastereomeric N-trifluoroacetyl-L-prolyl-D/L-amino acid ester dipeptides which are resolved by gas chromatography (GC). We have found that triethylamine, which is added to maintain a basic pH during the coupling reaction, racemizes the chiral reagent N-trifluoroacetyl-L-prolyl chloride (TPC). Coupling of partially racemized TPC to D/L-amino acid esters results in the formation of four dipeptides (two pairs of enantiomers) instead of the expected two diastereomeric dipeptides. The enantiomeric dipeptides coelute on an achiral GC column, resulting in erroneous D/L ratios. More accurate D/L ratios are obtained by preparing the volatile N-trifluoroacetyl-D/L-amino acid isopropyl ester derivative which can be separated into its enantiomers on a chiral GC column such as the Chirasil-Val III (registered trademark of Applied Science Laboratories).

Amino Acids↗

PubMed1983

Accumulation of D-aspartic acid with age in the human brain.

An age-related accumulation of D-aspartic acid was detected in the white matter of ten normal brains from individuals aged 30 to 80 years. Gray matter showed no systematic increase in D-aspartic acid. The rate constant for D-aspartate formation in the brain is equal to the predicted value calculated for 37 degrees C. Accumulation of the uncommon D-aspartate isomer in myelinated white matter implies that there is little or no turnover of this tissue, and this may have a bearing on dysfunction of the aging brain or on other diseases of myelin.

Adult↗

PubMed1983

Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists.

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.

Animals↗