Spasmogenic and potentiating actions of some amino acids on the guinea-pig myometrium

Ectothiorhodospira shaposhnikovii, Chromatium minutissimum and Thiocapsa roseopersicina were grown in the dark under anaerobic conditions on media containing glucose or fructose and organic acids. Their cell contained the following enzymes of the fructose diphosphate pathway: phosphofructokinase, fructose diphosphate aldolase, and 3-phosphoglyceraldehyde dehydrogenase. The activity of fructose diphosphate aldolase was higher in the cells grown in the dark than in the cells grown in the light. The same enzymes of the tricarboxylic acid cycle were found in the cells cultivated in the dark on media containing organic acids as in the cells grown in the light, though the activity of some enzymes was lower. Only the activity of isocitrate lyase increased in the cells cultivated in the dark on a medium containing acetate.     

Antigen receptor-induced B lymphocyte apoptosis mediated via a protease of the caspase family

An extensive body of data, in a variety of systems, denoted the caspase family of proteases as a key player in the execution of programmed cell death. This family consists of cysteine proteases that cleave after asparagine-containing motifs. It is well established that the caspases are essential for the apoptosis mediated by Fas (CD95) and TNF receptor p55, molecules that contain the "death domain" in the cytoplasmic tail. However, little is known about the mechanisms underlying the antigen receptor-mediated cell death in B lymphocytes, a process instrumental in negative selection of potentially autoreactive B cells. Here, we investigated the involvement of caspases in cell death triggered via the antigen receptor in B lymphocytes (BCR) by using specific inhibitors. Initially, we used a well-established cell line, CH31, which is a model of B cell tolerance, to demonstrate that these proteases indeed participate in the BCR-induced apoptotic pathway. Next, we confirmed the physiological relevance of the caspase-mediated cell death pathway in splenic B cell populations isolated ex vivo that were induced to undergo apoptosis by extensive cross-linking of their BCR. Most interestingly, our data demonstrated that caspases regulate not only the nuclear DNA fragmentation, but also the surface membrane phosphatidylserine translocation as well as the degradation of a specific nuclear substrate. Taken together, this report supports the hypothesis that regulation of the caspase family is crucial in controlling the life/death decision in B lymphocytes mediated by the antigen receptor signal transduction.     

The role of excitatory amino acids in behavioral sensitization to psychomotor stimulants

Behavioral sensitization refers to the progressive augmentation of behavioral responses to psychomotor stimulants that develops during their repeated administration and persists even after long periods of withdrawal. It provides an animal model for the intensification of drug craving believed to underlie addiction in humans. Mechanistic similarities between sensitization and other forms of neuronal plasticity were first suggested on the basis of the ability of N-methyl-D-aspartate (NMDA) receptor antagonists to prevent the development of sensitization [Karler, R., Calder, L. D., Chaudhry, I. A. and Turkanis, S. A. (1989) Blockade of "reverse tolerance" to cocaine and amphetamine by MK-801. Life Sci., 45, 599-606]. This article will review the large number of subsequent studies addressing: (1) the roles of NMDA, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) and metabotropic glutamate receptors in the development and expression of behavioral sensitization, (2) excitatory amino acids (EAAs) and the role of conditioning in sensitization, (3) controversies regarding EAA involvement in behavioral sensitization based on studies with MK-801, (4) the effects of acute and repeated stimulant administration on EAA neurochemistry and EAA receptor expression, and (5) the neuroanatomy of EAA involvement in sensitization. To summarize, NMDA, AMPA metabotropic glutamate receptors all participate in the development of sensitization, while maintenance of the sensitized state involves alterations in neurochemical measures of EAA transmission as well as in the expression and sensitivity of AMPA and NMDA receptors. While behavioral sensitization likely involves complex neuronal circuits, with EAAs participating at several points within this circuitry, EAA projections originating in prefrontal cortex may play a particularly important role in the development of sensitization, perhaps via their regulatory effects on midbrain dopamine neurons. The review concludes by critically evaluating various hypotheses to account for EAA involvement in the development of behavioral sensitization, and considering the question of whether EAA receptors are involved in mediating the rewarding effects of psychomotor stimulants and sensitization of such rewarding effects.     

The role of conserved amino acids in substrate binding and discrimination by photolyase

DNA photolyases catalyze the light-dependent repair of pyrimidine dimers in DNA. We have utilized chemical modification and site-directed mutagenesis to probe the interactions involved in substrate recognition by the yeast photolyase Phr1. Lys517 was protected from reductive methylation in the presence of substrate, but not in its absence, and the specific and nonspecific association constants for substrate binding by Phr1 (Lys517-->Ala) were decreased 10-fold. These results establish a role for Lys517 in substrate binding. Mutations at Arg507, Lys463, and Trp387 reduced both the overall affinity for substrate and substrate discrimination. Sites of altered interactions in ES complexes were identified by methylation and ethylation interference techniques. Interaction with the base immediately 3' to the dimer was altered in the Phr1(Lys517-->Ala). DNA complex, whereas interactions with the phosphate and base immediately 5' to the dimer were reduced when Phr1(Arg507-->Ala) bound substrate. Multiple interactions 5' and 3' to the dimer were perturbed in complexes containing Phr1(Trp387-->Ala) or Phr1(Lys463-->Ala). In addition the quantum yield for dimer photolysis by Phr1(Trp387-->Ala) was reduced 3-fold. The locations of these mutations establish that a portion of the DNA binding domain is comprised of residues in the highly conserved carboxyl-terminal half of the enzyme.     

The role of activin type I receptors in activin A-induced growth arrest and apoptosis in mouse B-cell hybridoma cells

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.     

Selective regulation of apoptosis: the cytotoxic lymphocyte serpin proteinase inhibitor 9 protects against granzyme B-mediated apoptosis without perturbing the Fas cell death pathway

Cytotoxic lymphocytes (CLs) induce caspase activation and apoptosis of target cells either through Fas activation or through release of granule cytotoxins, particularly granzyme B. CLs themselves resist granule-mediated apoptosis but are eventually cleared via Fas-mediated apoptosis. Here we show that the CL cytoplasmic serpin proteinase inhibitor 9 (PI-9) can protect transfected cells against apoptosis induced by either purified granzyme B and perforin or intact CLs. A PI-9 P1 mutant (Glu to Asp) is a 100-fold-less-efficient granzyme B inhibitor that no longer protects against granzyme B-mediated apoptosis. PI-9 is highly specific for granzyme B because it does not inhibit eight of the nine caspases tested or protect transfected cells against Fas-mediated apoptosis. In contrast, the P1(Asp) mutant is an effective caspase inhibitor that protects against Fas-mediated apoptosis. We propose that PI-9 shields CLs specifically against misdirected granzyme B to prevent autolysis or fratricide, but it does not interfere with homeostatic deletion via Fas-mediated apoptosis.     

Cerebral hypoxic and ischemic damage in newborn infants: cellular mechanisms and role of excitatory amino acids

Brain damage from hypoxia-ischemia plays a major role in neonatal mortality and morbidity. Different lesional mechanisms have been proposed. The most recent hypothesis involves the excitatory amino acids, especially glutamate. In this review, arguments in favour of a glutamate involvement in the lesional process are presented, with particular attention to the consequences of glutamate fixation on post synaptic receptors, especially N-methyl-D-aspartate receptors. Alterations of the intracellular concentration of calcium may also play a role in the pathogenesis of hypoxic-ischemic lesions. A better understanding of the metabolic processes could lead to new therapeutic possibilities.     

Differential role of calcium in tumour necrosis factor-mediated apoptosis and secretion of granulocyte-macrophage colony-stimulating factor in a T cell hybridoma

The authors investigated the dependence on extracellular and intracellular free Ca2+ in the induction of apoptosis and secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) by tumour necrosis factor (TNF) in a rat/mouse T cell hybridoma PC60 R55/R75, using the Ca2+ chelators EGTA and BAPTA/AM, respectively. TNF-induced apoptosis still occurred in the absence of free Ca2+, while GM-CSF production required the continuous presence of Ca2+. The latter was also true for GM-CSF production driven by interleukin 1 (IL-1). The dependence on Ca2+ in the induction of GM-CSF, but not of apoptosis, was further confirmed by the inhibition of TNF- or IL-1-induced cytokine production by cyclosporin A or FK506, drugs that block the Ca2+/calmodulin-dependent protein Ser/Thr phosphatase calcineurin. This differential requirement for Ca2+ illustrates the partial functional redundancy between TNF and IL-1, showing the activation of cytokine gene expression through a Ca(2+)-dependent activation of calcineurin, and a Ca(2+)-independent activation of apoptosis, exerted solely by TNF.     

Growth factor protection against cytokine-induced apoptosis in neonatal rat islets of Langerhans: role of Fas

Treatment of neonatal rat islets of Langerhans with combined cytokines (interleukin-1beta 10(-10) M, tumour necrosis factor-alpha 10(-10) M, interferon-gamma 5 U/ml) led to extensive cell death, which was potentiated by Fas activation with the anti-Fas cytolytic antibody JO2. Pre-treatment with insulin (25 ng/ml) or insulin-like growth factor-1 (10(-8)M) gave only partial protection against cell killing, but prevented the Fas-mediated component. In the absence of cytokine treatment, Fas-mediated killing was not observed.     

Effect of some amino acids on the rectal irritation caused by sodium caprate in conscious rats

The effects of some amino acids such as L-glutamine (Gln), L-arginine (Arg) and L-methionine (Met) on rectal irritation caused by sodium caprate were studied in rats. Rectal irritation was assessed by the balloon method in fasting conscious rats. This method is based on measuring rectal contractions due to possible irritation caused by the presence of drugs and/or adjuvants in the rectum. Strong contractions were observed after rectal administration of an aqueous solution of 100 mM sodium caprate. However, the presence of Gln, Arg or Met (100 mM) in sodium caprate (100 mM) solution resulted in a significant decrease in the intensity of the rectal contraction caused by sodium caprate. The rectal absorption-promoting effect of sodium caprate on 6-carboxyfluorescein (6-CF) was examined following administration with amino acids in rats. The absorption of 6-CF was not influenced by the concurrent administration of amino acids. In addition, the rectal tissue interaction of sodium caprate, with or without Gln, was examined. The concentration of sodium caprate in rectal tissue was reduced by the presence of Gln.     

Mitogenic activities of amino acid substitution mutants of staphylococcal enterotoxin B in human and mouse lymphocyte cultures

Site-directed mutagenesis has been used to introduce amino acid substitutions at specific residues of the staphylococcal enterotoxin B (SEB) gene cloned from Staphylococcus aureus 10-275. The mitogenic activities of these derivatives were determined in two assay systems: (i) mouse spleen cells and (ii) a mixture of human peripheral blood mononuclear cells and lymphocytes. Substitution of either His-12, His-32, His-121, His-166, Lys-152, or Gly-205 did not significantly alter the mitogenic activity from that of the wild-type toxin in either proliferation assay. Substitution of either residue Asn-23, Phe-44, or Cys-93 reduced the mitogenicity of SEB by a degree that depended upon the assay system used. Similar to the results reported by others measuring toxin activation of mouse lymphoid cells, we found that substitutions of these three residues of SEB caused at least 800-fold reductions of mitogenic activity from that of the wild-type toxin. When tested for toxicity in vivo in D-galactosamine-treated mice, the reduced activities of these mutant toxins, however, were not as pronounced. In contrast, when tested in the human cell mitogenicity assay, these mutant toxins were active. Small alterations in activity (two- to fivefold reduction) were observable only at low concentrations. Our findings reveal the importance of using human lymphocytes in addition to the traditional mouse spleen cell assay when assessing biological activities of staphylococcal enterotoxins.     

The role of in vitro-induced lymphocyte apoptosis in feline immunodeficiency virus infection: correlation with different markers of disease progression

Human immunodeficiency virus infection is characterized by a progressive decline in the number of peripheral blood CD4(+) T lymphocytes, which finally leads to AIDS. This T-cell decline correlates with the degree of in vitro-induced lymphocyte apoptosis. However, such a correlation has not yet been described in feline AIDS, caused by feline immunodeficiency virus (FIV) infection. We therefore investigated the intensity of in vitro-induced apoptosis in peripheral blood lymphocytes from cats experimentally infected with a Swiss isolate of FIV for 1 year and for 6 years and from a number of long-term FIV-infected cats which were coinfected with feline leukemia virus. Purified peripheral blood lymphocytes were either cultured overnight under nonstimulating conditions or stimulated with phytohemagglutinin and interleukin-2 for 60 h. Under stimulating conditions, the isolates from the infected cats showed significantly higher relative counts of apoptotic cells than did those from noninfected controls (1-year-infected cats, P = 0.01; 6-year-infected cats, P = 0.006). The frequency of in vitro-induced apoptosis was inversely correlated with the CD4(+) cell count (P = 0. 002), bright CD8(+) cell count (P = 0.009), and CD4/CD8 ratio (P = 0. 01) and directly correlated with the percentage of bright major histocompatibility complex class II-positive peripheral blood lymphocytes (P = 0.004). However, we found no correlation between in vitro-induced apoptosis and the viral load in serum samples. Coinfection with feline leukemia virus enhanced the degree of in vitro-induced apoptosis compared with that in FIV monoinfected cats. We concluded that the degree of in vitro-induced apoptosis was closely related to FIV-mediated T-cell depletion and lymphocyte activation and could be used as an additional marker for disease progression in FIV infection.     

p-Chlorotetrafluorophenyl esters of N-protected amino acids

p-Chlorotetrafluorophenyl (Tfc) esters of protected amino acids and peptides are more reactive than are the well known pentafluorophenyl (Pfp) esters. Two reagents, p-chlorotetrafluorophenyltrifluoroacetate (Tfc-OTfa) and di-(p-chlorotetrafluorophenyl)carbonate (di-Tfc-carbonate), can be used for their syntheses, thereby avoiding use of the allergic dicyclohexylcarbodiimide. This is especially important for bulk preparations. Many Fmoc- and Boc-amino acid-OTfc esters have been synthesized and characterized. The hexadecameric tandem repeat H-(AlaAlaLysPro)4-OH was synthesized using di-Tfc-carbonate for the preparation of Tfc-esters.     

Brain metabolism of branched-chain amino acids

Domestic swine (n=12 in each group) were randomized to daily treatment with the thromboxane A2 (TXA2) receptor antagonist BMS-180291 (group I), aspirin (group II), or no drug (group III) prior to prosthetic carotid graft implantation. Platelet and arterial wall receptor density were measured by equilibrium binding using 125I-BOP. At 6 weeks, means (s.e.m.) platelet receptor density (pmol/mg) had increased in groups I (3.3(0.6) versus 1.8(0.3); P<0.05) and II (2.6(0.6) versus 1.7(0.2); P<0.05), but not in group III (1.3(0.3) versus 1.2(0.2)). Aortic membrane TXA2 receptor density (fmol/mg) was significantly greater (P<0.05) in groups I (150(50)) and II (68(10)) compared with group III (39(6)). Chronic exposure to a TXA2 receptor antagonist or aspirin is associated with increased platelet and aortic receptor density in pigs.     

Kinetic study of apo B100 containing lipoprotein metabolism using amino acids labeled with stable isotopes: methodological aspects

Kinetic disturbances of lipoprotein metabolism are important to know for a better understanding of lipid diseases or effects of drugs. These kinetic aspects were previously studied with radioactive tracers. The ethical concerns related to these tracers can be now overcome at a reasonable cost with the new development of small bench top mass spectrometers and the increased production of stable isotope tracers. In this review, we will discuss some methodological aspects related to stable isotope tracers and the analysis of the data with non-compartmental or compartmental models.     

Protection of mice against lethal coxsackievirus B3 infection by using DNA immunization

Vaccination with DNA and recombinant vaccinia viruses (rec.VV) has been studied with the coxsackievirus B3 (CVB3) model system. Plasmids encoding all structural proteins of CVB3, when injected intramuscularly, induced only low levels of virus-specific antibodies. However, DNA vaccination with the major structural protein VP1 protected 72.2% of mice from lethal challenge, whereas VP1 expressed by rec.VV was much less efficient.     

Induction of tumour-suppressor phosphoprotein p53 in the apoptosis of cultured rat cerebellar neurones triggered by excitatory amino acids

We have isolated the collagenase/gelatinase activity of fibronectin from a bovine lens capsule hydrolysate, using heparin-agarose, gelatin-agarose, immunopurification with polyclonal antibodies directed against bovine plasma fibronectin, and immunopurification with a monoclonal antibody directed against the extra-domain A of cellular fibronectin. The expression of collagenase/gelatinase activity by the purified fibronectin fragment was dependent on the incubation time at 37 degrees C and the addition of gelatin to the purified sample. Under these conditions, the purified fibronectin fragment exhibited collagenase/gelatinase activity, as measured by means of gelatin zymography and the intramolecularly quenched fluorogenic substrate of collagenases (7-methoxycoumarin-4-yl)-acetylprolylleucylglycylleucyl-[3-(2,4-di nitrophenyl)-L-2,3-diaminopropionyl]-alanylarginylamide. This activity was due to proteins of 47 kDa and 37 kDa, as indicated by the gelatin-zymography pattern. When the processing was analyzed, by means of SDS/PAGE under reducing conditions, purified starting material of 66 kDa and 55 kDa was observed, and molecular masses of 45, 30 and 27 kDa were found for the processed samples. Under these conditions, the processing was more significant when a substrate, i.e the fluorogenic peptide or gelatin, was added to the processing mixture. An inhibition-profile study showed a zinc-dependent collagenase activity. Using the 45-kDa chymotryptic fragment from human plasma fibronectin, which contains the collagen-binding site, the same results were obtained. These results allow us to define a thiol-dependent zinc metalloproteinase expressed after limited proteolysis of both basement membrane and plasma fibronectins. This proteinase contains a collagen-binding domain, a zinc-binding sequence, and a cysteine involved in catalysis. This enzyme is a member of the thimet family of zinc metalloproteinases.     

Production of amino acids by yogurt bacteria

The dynamics of free amino acid production by the selected strains Streptococcus thermophilus 13a and Lactobacillus bulgaricus 2-11 were studied in pure and mixed cultivations during yogurt starter culture manufacture. L. bulgaricus 2-11 showed the highest activity for producing free amino acids with high individual concentrations over the first hour of growth (50% of the total amount). By the end of milk's full coagulation (4.5 h), 70% of the total amount of amino acids was released. S. thermophilus 13a showed poor proteolytic properties and consumed up to 70% of the free amino acids produced by L. bulgaricus 2-11 in the process of coagulation of milk with the mixed culture.     

Utilization of ileal digestible amino acids by growing pigs: methionine

An experiment was conducted to determine the utilization of ileal digestible methionine by growing pigs. Three methionine-deficient diets (0.09 g ileal digestible methionine/MJ digestible energy (DE)) were formulated using cottonseed meal, meat-and-bone meal and soya-bean meal respectively as the only source of methionine in the diet. An additional three diets were formulated with supplements of methionine to confirm that methionine was limiting in the first three diets. The growth performance and retention of methionine by pigs given the six diets over the 20-45 kg growth phase was then determined. Growth rates (g/d) of pigs given the three diets formulated to 0.09 g ileal digestible methionine/MJ DE were significantly different (P < 0.01): cottonseed meal 411, meat-and-bone meal 442, soya-bean meal 496 (SED 24.6). The response of pigs to the addition of methionine confirmed that methionine was limiting in these diets. Crude protein (N x 6.25) deposited by the pigs (g/d) was significantly higher (P < 0.05) for those given soya-bean meal (61) and meat-and-bone meal (57) relative to cottonseed meal (47; SED 3.3). The proportion of ileal digestible methionine retained by pigs given the three protein concentrates was: cottonseed meal 0.39, meat-and-bone meal 0.45, soya-bean meal 0.47 (SED 0.019). These results indicate that values for the ileal digestibility of methionine in protein concentrates do not reflect the proportion of methionine that can be utilized by the pig. It appears that, with heat-processed meals, a considerable proportion of the methionine is absorbed in a form(s) that is (are) inefficiently utilized.