Allergen-specific sublingual immunotherapy in the treatment of migraines: a prospective study.

BACKGROUND: Inflammation is a cardinal feature of migraines. A number of observations point to the possibility that an allergic component of a type I (IgE-mediated) nature may be involved in at least some migraineurs. Not only are migraines frequent among patients with allergic rhinitis but quite frequently the same medical approaches are beneficial in both diseases: anti-inflammatories, adrenergic tone modifiers, immune suppressants. The effect that immunotherapy for allergic rhinitis has upon migraines is studied. METHODS: Patients were recruited who suffered from typical migraines but were not treated with regular migraine controllers (beta blockers, antiepileptics, tricyclics, etc.). They underwent allergen-specific, sublingual immunotherapy with physician-formulated, individually-prepared airborne allergen extracts. Response to treatment was assessed with serum C-reactive protein level changes and symptom scores. Serum C-reactive protein (CRP), an acute phase reactant, was chosen as a marker because its usefulness has already been assessed in interictal migraine activity. RESULTS: Interictal serum CRP levels decline was observed in the course of sublingual immunotherapy. Concurrent improvement in symptom scores for both rhinitis and migraines was also observed. CONCLUSIONS: In patients with allergic rhinitis, migraine development and course may have a significant allergic component. Assessment of migraineurs for the possibility of coexisting allergic rhinitis is justified. Treatment of allergic rhinitis by immune response modifiers, such as immunotherapy, may have a place in the management of migraines for these patients.

Substrate specificity of an acylaminopeptidase that catalyzes the cleavage of the blocked amino termini of peptides.

An acylaminopeptidase purified from human red cells cleaves acetylated dipeptides in the decreasing order of acetyl-Ala, acetyl-Met, acetyl-Ser, acetyl-Gly and acetyl-Val. In addition, it was also found that the nature of the second amino-acid residue influenced the rate of cleavage of the blocked N-terminus: charged residues at the second position lead to reduced rates of cleavage. The possible use of this enzyme for structural studies on blocked peptide or protein substrates is evaluated.

Crystallization and preliminary X-ray studies of human erythrocyte acylpeptide hydrolase.

Crystals of acylpeptide hydrolase suitable for structure determination have been obtained. This enzyme removes the N-terminal formyl or acetyl group together with the first amino acid residue from N-terminal blocked peptides including bioactive peptides. One set of crystals, which diffract to 2.2 A, are in space group P2 with cell dimensions a = 118.6 A, b = 82.3 A, c = 182.1 A, beta = 91.6 degrees. The search for suitable heavy-atom derivatives is underway.

The ubiquitous cofactor NADH protects against substrate-induced inhibition of a pyridoxal enzyme.

In the usual reaction catalyzed by D-amino acid transaminase, cleavage of the alpha-H bond is followed by the reversible transfer of the alpha-NH2 to a keto acid cosubstrate in a two-step reaction mediated by the two vitamin B6 forms pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP). We report here a reaction not on the main pathway, i.e., beta-decarboxylation of D-aspartate to D-alanine, which occurs at 0.01% the rate of the major transaminase reaction. In this reaction, beta-C-C bond cleavage of the single substrate D-aspartate occurs rather than the usual alpha-bond cleavage in the transaminase reaction. The D-alanine produced from D-aspartate slowly inhibits both transaminase and decarboxylase activities, but NADH or NADPH instantaneously prevent D-aspartate turnover and D-alanine formation, thereby protecting the enzyme against inhibition. NADH has no effect on the enzyme spectrum itself in the absence of substrates, but it acts on the enzyme.D-aspartate complex with an apparent dissociation constant of 16 microM. Equivalent concentrations of NAD or thiols have no such effect. The suppression of beta-decarboxylase activity by NADH occurs concomitant with a reduction in the 415-nm absorbance due to the PLP form of the enzyme and an increase at 330 nm due to the PMP form of the enzyme. alpha-Ketoglutarate reverses the spectral changes caused by NADH and regenerates the active PLP form of the enzyme from the PMP form with an equilibrium constant of 10 microM. In addition to its known role in shuttling electrons in oxidation-reduction reactions, the niacin derivative NADH may also function by preventing aberrant damaging reactions for some enzyme-substrate intermediates. The D-aspartate-induced effect of NADH may indicate a slow transition between protein conformational studies if the reaction catalyzed is also slow.

Salty solutions: their effects on thermal set points in behavioral repertoires of albino rats.

Salt (sodium chloride) has been linked to increased blood pressure and a rise in core body temperature. The objective of this study was to investigate the role played by salt in altering behavioral thermoregulation in albino rats. Different doses of sodium chloride were administered (ip) prior to fixed-interval 2-min. schedules of microwave reinforcement in rats tested in a cold Skinner Box. Three Sprague-Dawley rats were conditioned to regulate their thermal environment with 5-sec. exposures of MW reinforcement in a repeated-measures reversal design. Friedman's non-parametric test showed significant differences among sodium chloride doses and physiologically normal saline. Post hoc sign tests showed that all doses of NaCl suppressed operant behavior for heat except 60 mg/kg. The hypothesis that sodium chloride lowers hypothalamic set point for heat was partially supported.

Acylpeptide hydrolase activity from erythrocytes.

Acylpeptide hydrolase, which cleaves the NH2-terminal acetylated or formylated amino acid from a blocked peptide, has been purified to apparent homogeneity from human erythrocytes. The enzyme catalyzes the hydrolysis of a diverse number of peptides and displays different pH optima for certain substrates in doing so. Zinc inhibits to the same extent the hydrolysis of both the most efficient and the least efficient substrates. This enzyme may play a pivotal role in the processing of polypeptide chains during biosynthesis.

Enzymic cleavage of the blocked amino terminal residues of peptides.

The substrate specificity of an enzyme that removes some N-terminal blocked amino acids from blocked peptides has been further explored with several naturally-occurring peptides. Chloride ion is an effective modulator of enzyme activity. Although the relative efficiency of the enzyme varies considerably with different peptide substrates, in each case there was significant although less than quantitative release of the N-terminal blocked amino acid. The possible application of this enzyme to structural studies on polypeptides is evaluated.

Generation of a new gamma delta T cell-specific monoclonal antibody (GD3.5). Biochemical comparisons of GD3.5 antigen with the previously described Workshop Cluster 1 (WC1) family.

In this report, we describe GD3.5, a new lineage-specific gamma delta T cell marker that is distinct from TCR and known Workshop Cluster 1 (WC1). FACS analysis indicated that GD3.5Ag is expressed on approximately 90% of the peripheral blood gamma delta T cell population and GD3.5 specifically stained gamma delta T cells and not alpha beta T cells, B cells, neutrophils, or monocytes. Also, a significant portion of the GD3.5-positive population was WC1-negative. Nonreducing Western blot analysis and immunoprecipitation experiments revealed a single 220- to 240-kDa glycoprotein recognized by GD3.5 compared with two WC1 bands at 200 kDa and 300 kDa recognized by the IL-A29 Ab. Cross-immunoprecipitation experiments demonstrated that GD3.5 could be immunoprecipitated from lysates cleared of IL-A29/WC1 complexes. Reciprocally, WC1 could be immunoprecipitated from lysates cleared of GD3.5Ab/GD3.5Ag complexes. Digestion of WC1 and GD3.5 Ag with V-8 protease resulted in digestion profiles that clearly distinguished the glycoproteins. Additionally, GD3.5 Ag and WC1 possess disparate sensitivity to PNGase F, O-sialoglycoprotease, and neuraminidase, indicating differences in N- and O-linked sugars and the presence of sialic acid residues. Both GD3.5 Ag and WC1 appeared to be sialomucin-like molecules that share similar O-sialoglycoprotein endopeptidase sensitivity with other cell surface molecules, such as PSGL-1. Lastly, GD3.5 Ag, but not WC1, was exquisitely sensitive to very low-dose chymotrypsin treatment. Therefore, our data suggest that GD3.5 Ag is a previously uncharacterized, lineage-specific gamma delta T cell Ag. Furthermore, we show that GD3.5 and WC1 are sialomucins, which provides important clues to their function.

Inactivation of bacterial D-amino acid transaminase by beta-chloro-D-alanine.

Purified D-amino acid transaminase from Bacillus sphaericus catalyzes an alpha,beta elimination from the D isomer of beta-chloroalanine to yield equivalent amounts of pyruvate, chloride, and ammonia; the L isomer of chloroalanine is not a substrate for this transaminase. During the beta elimination there is a synchronous loss in enzyme activity; the Kinact for beta-chloroalanine was estimated to be about 10 micrometers. The alpha-aminoacrylate-Schiff base intermediate formed after beta elimination of chloride ion is probably the key intermediate that partitions between one inactivation event for every 1500 turnovers. In the presence of D-alanine and alpha-ketoglutarate, which are good substrates for the transaminase activity of this enzyme, beta-chloroalanine is a potent, competitive inhibitor (K1 = 10 micrometers) with D-alanine and a weak, uncompetitive inhibitor with alpha-ketoglutarate.

Determination of free D-amino acids with a bacterial transaminase: their depletion leads to inhibition of bacterial growth.

A general procedure is described to determine the common free D-amino acids except D-proline in mixtures that also contain L-amino acids. The system employs exogenous pure bacterial D-amino acid transaminase coupled with 2-oxohexanoate, which accepts the amino group from D-amino acids to form D-norleucine. This amino acid is readily quantified by amino acid analysis since it elutes in a position not occupied by any of the common amino acids. Formation of norleucine denotes the presence of some D-amino acid(s) whose identity can be established by a corresponding decrease in the susceptible amino acid(s) after treatment. The utility of the procedure is demonstrated by determination of the amounts of free D-alanine and free D-glutamate in extracts of Escherichia coli JM-103 grown on minimal medium; D-alanine was the major D-amino acid. By the same principle, 2-oxohexanoate through coupling with endogenous bacterial D-amino acid transaminase is shown to be capable of inhibiting the growth of E. coli by depleting it of the D-alanine and D-glutamate.

Inhibition of bacterial growth by beta-chloro-D-alanine.

The D- and L-isomers of beta-chloroalanine inhibit the growth of Diplococcus pneumoniae, Streptococcus pyogenes, Bacillus subtilis, and Escherichia coli. With pneumococcus the inhibition by beta-chloro-D-alanine is completely prevented by either D-alanine or D-alanyl-D-alanine, while L-alanine is not effective in preventing the inhibition. The inhibition of growth by beta-chloro-L-alanine is not affected by D-alanine and is only partially prevented by high concentrations of L-alanine. The intracellular free alanine in untreated E. coli and B. subtilis is about 95% in the D-configuration while the free intracellular alanine in both organisms after treatment with beta-chloro-D-alanine is predominantly the L-isomer. These results suggested that the beta-chloroamino acid inactivates alanine racemase (EC 5.1.1.1). Indeed, when extracts of E. coli or B. subtilis were treated with beta-chloro-D-alanine, the activities of alanine racemase and of D-glutamate-D-alanine transaminase were found to be 90-95% inhibited. Studies with mice have shown that beta-chloro-D-alanine is an effective antibacterial agent in vivo againt D. pneumoniae, S. pyogenes, and E. coli.

Human acylpeptide hydrolase. Studies on its thiol groups and mechanism of action.

The presence of a cysteine residue(s) near the active site of acylpeptide hydrolase was suggested by inactivation of the enzyme with sulfhydryl-modifying agents and by the substantial protection against inactivation afforded by the competitive inhibitor acetylmethionine. 5,5'-dithiobis-(2-nitrobenzoate) titrations of the native and the denatured enzyme together with analysis for cysteic acid after performic acid oxidation showed that the enzyme contained 12 free SH groups and three disulfide bonds/monomer. Chemical modification with radiolabeled iodoacetamide led to the labeling of Cys-30 and Cys-64 suggesting that one or both of these Cys residues are close to the active site. Modification of one or both of them probably inhibits the enzyme either because of a distortion of the active site or because the adducts present a barrier to the efficient diffusion of substrates into and products out of the active site. Studies on the mechanism of action of acylpeptide hydrolase have employed p-nitrophenyl-N-propyl carbamate as a potent active site-directed inhibitor. Enzyme inactivation, which follows pseudo first-order kinetics, is diminished by the competitive inhibitor acetylmethionine. The inhibited enzyme slowly regains activity at a rate that is increased in the presence of the nucleophile hydroxylamine. A general mechanism involving an acyl-enzyme intermediate is supported by evidence for the formation of acetyl-alanyl hydroxamate during hydrolysis of acetyl-alanine p-nitroanilide in the presence of hydroxylamine. The effect on Vmax and Km during this reaction indicate that hydrolysis of the acyl-enzyme intermediate is rate-limiting.

Comparison of E-selectin-binding glycoprotein ligands on human lymphocytes, neutrophils, and bovine gamma delta T cells.

We compared E-selectin-binding cell surface ligands on bovine gamma delta T cells and human leukocytes using an E-selectin/Ig chimera. The chimera worked well in flow cytometric studies and showed that bovine gamma delta T cells were the only lymphocyte population in newborn animals that bound E-selectin chimera. Furthermore, the chimera blocked gamma delta T cell rolling on E-selectin. Chimera reacted with four potential glycoprotein ligands of 180, 200, 250, and 300 kDa in Western blot analysis of gamma delta T cell detergent lysates, and it specifically precipitated at least two of these E-selectin ligands (200 and 250 kDa) from lysates of cell surface biotinylated gamma delta T cells. Preclearing bovine gamma delta lysates of GD3.5 Ag and workshop cluster 1 did not abrogate E-selectin ligand precipitation, suggesting that these surface markers do not represent E-selectin ligands. Human neutrophils possessed three E-selectin-binding ligands of approximately 80 to 90, 130, and 230 kDa, while human lymphocytes variably possessed three ligands of 120, approximately 220 to 240, and 260 kDa. Cross-precipitation experiments confirmed the results of others that neutrophil L-selectin serves as the 80 to 90-kDa E-selectin ligand. The human lymphocyte approximately 220 to 240-kDa and 260-kDa ligands may be analogous to the bovine gamma delta T cell molecules, whereas the 120-kDa is unique to human cells. The identities of the human and bovine lymphocyte E-selectin ligands are unknown. Finally, E-selectin ligand-1 apparently may have a minimal role, if any, in lymphocyte/E-selectin interactions, since a polyclonal anti-E-selectin ligand-1 serum stained a minimal number of cutaneous lymphocyte-associated Ag-positive human lymphocytes and bovine gamma delta T cells.

Blood pressure effects of lower abdominal aortic surgery with particular reference to the use of morphine and droperidol in modifying the responses.

Hypertensive responses during operations on the abdominal aorta are common. We have shown that hypertensive changes occur before cross-clamping of the aorta. We have also shown that droperidol modifies the hypertensive changes in operations on the lower aorta. Although further studies are required, our preliminary results show a higher rise of serum epinephrine and norepinephrine levels in one patient receiving morphine, as compared to one patient receiving the same dose per unit body weight of morphine plus droperidol. With maintenance of adequate pre-operative hydration and intra-operative fluid and blood replacement, declamping hypotension was transient in both groups of our patients.

Inhibition of deoxyhemoglobin S polymerization by glyceraldehyde.

Glyceraldehyde reacts with hemoglobin S in the intact erythrocyte to reduce the degree of polymerization, thereby inhibiting sickling of the erythrocyte. Only five of the 24 amino groups per alpha beta dimer react with glyceraldehyde; the adducts are present as ketoamine structures, formed by Amadori rearrangement of the initial Schiff base adducts on the protein. The reactive amino groups are the epsilon-amino group of Lys-16 of the alpha-chain, and the alpha-amino group of Val-1 as well as the epsilon-amino groups Lys-82, Lys-59, and Lys-120 of the beta-chain. Hybrid tetramers were prepared with the modification only on Lys-16 of the alpha-chain or on the reactive lysine residues of the beta-chain. The former derivative gels at a much higher hemoglobin concentration (23 g/dl) than either the latter derivative (16 g/dl) or unmodified deoxyhemoglobin S (15 g/dl). Thus, the modification at Lys-16 of the alpha-chain is a major factor in the inhibition of sickling by glyceraldehyde.