Absence Of Effector Research Articles

Antigen‐Specific Tolerance Induced by Autoimmunization with Photoinactivated Syngeneic Effector Cellsa

Development of a protocol that could invoke specific suppression of an undesired immune response, while sparing normal immune competence, would be of great clinical value. This report demonstrates that multiple infusions of splenocytes sensitized in vivo to sheep red blood cells (SRBC) and photoinactivated in vitro with 8-methoxypsoralen and ultraviolet A light can render a syngeneic recipient selectively unresponsive to subsequent challenge with this antigen. Mice treated in this fashion did not develop a T cell-mediated delayed type hypersensitivity (DTH) reaction to SRBC. In contrast, control mice exposed to nonimmune splenocytes pretreated in an identical manner developed a normal DTH response to SRBC, thereby demonstrating that drug and light in the absence of effector T cells were not suppressive. Inhibition of the DTH response was antigen specific, since animals rendered unresponsive to SRBC developed a normal DTH response to chicken red blood cells. Cell transfer experiments demonstrated that unprimed recipients of splenocytes from mice rendered unresponsive to SRBC could not mount a DTH reaction when challenged. Moreover, this procedure can also suppress established immunity to that antigen. The use of photoinactivated syngeneic antigen-reactive effector cells as immunosuppression agents suggests that this method may be clinically useful in inhibiting pathogenic antigen-specific immunologic reactions.

Activation of the Pseudomonas TOL plasmid upper pathway operon. Identification of binding sites for the positive regulator XylR and for integration host factor protein

Expression of the Pseudomonas putida TOL plasmid upper pathway operon requires a promoter that belongs to the -12/-24 class. Stimulation of transcription from this promoter is positively controlled by the effector-activated XylR protein and requires a form of RNA-polymerase holoenzyme containing the RpoN-encoded sigma factor, sigma 54. XylR-dependent stimulation of transcription from the Pseudomonas TOL upper pathway promoter was examined using deletions, insertions, and in vivo dimethyl sulfate footprinting. Two upstream activator sequences were identified in the -160 (UAS1) and -130 (UAS2) regions. Deletion of these two regions abolished transcription activation, although conservation of the UAS2 element alone allowed limited transcription stimulation. Separation of UAS1 from UAS2 by half a turn or a full turn significantly reduced XylR stimulation of transcription from the upper pathway operon promoter. An inverted repeated ATTTGN2CAAAT (where N is any nucleoside), which most likely represented the XylR recognition sequence, was identified. Binding of XylR was observed in vivo in the absence of effector, but changes in the binding pattern were induced in the presence of m-methylbenzyl alcohol, a XylR effector. In vivo footprinting analysis revealed that changes in the methylation pattern of G and T also occurred in the -50 to -90 region, which is probably occupied by integration host factor (IHF) protein. IHF was required for maximal expression from the TOL upper pathway operon promoter in Escherichia coli. Separation of the IHF site from UAS2 by a full helix turn did not significantly affect stimulation of transcription, which is consistent with this region playing a conformational role, rather than a regulatory one, in promoter function.

PH dependence of the kinetic properties of allosteric phosphofructokinase from Escherichia coli

The pH dependence of the activity of the allosteric phosphofructokinase from Escherichia coli has been studied in the pH range from 6 to 9, in the absence or presence of allosteric effectors. The sigmoidal cooperative saturation of phosphofructokinase by fructose 6-phosphate has been analyzed according to the Hill equation, and the following results have been obtained: (i) the apparent affinity for Fru-6P, as measured by the half-saturating concentration, [Fru-6P]0.5, does not change with pH; (ii) the cooperativity, as measured empirically by the Hill coefficient, nH, increases markedly with pH and reaches a value of 5.5-6 at pH 9; (iii) the catalytic rate constant, kcat, is controlled by the ionization of a critical group which has a pK of 7 in the absence of effector and must be deprotonated for phosphofructokinase to be active. The observation that pH affects both the cooperativity and the maximum velocity suggests that the catalytic efficiency of a given active site could be modified by the binding of fructose 6-phosphate to other remote sites. Finding values of the cooperativity coefficient larger than the number of substrate binding sites indicates that slow conformational changes may occur in phosphofructokinase. The cooperative saturation of phosphofructokinase by fructose 6-phosphate appears more complex than described by the classical concerted model at steady state and could involve two slowly interconverting states which differ in both their turnover rate constants and their affinities for fructose 6-phosphate. The presence of GDP shifts the pK of the critical group which controls kcat from 7 to 6.6.(ABSTRACT TRUNCATED AT 250 WORDS)

Covalent labeling of the acetylcholine receptor from Torpedo electric tissue with the channel blocker [3H]triphenylmethylphosphonium by ultraviolet irradiation.

The lipophilic cation [3H]triphenylmethylphosphonium, frequently used as a voltage sensor in membrane systems, binds reversibly to a site different from the acetylcholine binding site. This is concluded from the different pH dependences of the binding of these two ligands. Furthermore [3H]triphenylmethylphosphonium, previously identified as a channel blocker, can be covalently incorporated into acetylcholine receptor-rich membranes from Torpedo electric tissue by UV irradiation of the receptor-ligand complex. In the absence of effector, predominantly the alpha-polypeptide chains (Mr 40000) of the receptor protein are labeled by the radioactive ligand. The agonist carbamoylcholine strongly stimulates the labeling, but it directs the label predominantly to the delta- and beta-polypeptide chains. The antagonist D-tubocurarine and the virtually irreversible competitive antagonist alpha-bungarotoxin have qualitatively the same effect as the agonist carbamoylcholine. Significant differences were obtained with receptor-rich membranes prepared from Torpedo marmorata and Torpedo californica: No agonist- or antagonist-stimulated reaction was observed with the latter. The results are interpreted as an indication of a rearrangement of the receptor's quaternary structure caused by cholinergic effector binding preceding discrimination between agonists and antagonists.

Effect of Polysaccharides from Ganoderma applanatum on Immune Responses

AbstractPrior intraperitoneal (i.p.) or oral administration of the polysaccharide preparation from a kind of mushroom, Ganoderma applanatum (Pers.) Pat. of Basidiomycetes, exerted an enhancing effect on the induction of delayed hypersensitivity (DH) to protein antigen as measured by the footpad reaction (FPR), and expanded the size of T cell memory for the IgG antibody response. One of the active principles was partially purified and found to be associated with a polysaccharide‐rich fraction. The induction of DH was enhanced by treatment with an appropriate dose of the mushroom extract, whereas increasing the dose resulted in almost complete loss of the enhancing activity.The mechanism for the enhancing effect of the mushroom extract on the induction of DH was explored by the adoptive cell transfer technique. Although an i.p. injection of methylated bacterial α‐amylase (M‐BαA) in incomplete Freund's adjuvant (IFA) has been found to generate in the spleen the antigen‐specific suppressor T cells capable of inhibiting the induction of DH 5 days after immunization, the same treatment of mice given prior injections of the mushroom extract did not raise the suppressor cell activity, but transfer of these spleen cells (6 × 107) into syngeneic recipient mice which had been primed with a subcutaneous (s.c.) injection of M‐BαA in complete Freund's adjuvant (CFA) resulted in substantial amplification of the expression of DH. The absence of effector T cells for DH in the transferred spleen cells was confirmed by the failure to transfer DH into cyclophosphamide (CY)‐treated mice with the amplifying cells. The amplifying activity was antigen‐nonspecific and mediated by cells sensitive to treatment with anti‐θ antiserum plus complement. Therefore, the nonspecific enhancing effect of the mushroom extract could not be explained by the possibility that pretreatment with the extract eliminated the antigen‐specific suppressor T cells. Other adoptive cell transfer experiments revealed that nylon wool‐passed cells from mice unprimed but treated with the mushroom extract were able to exert an enhancing activity on the expression of effector T cells in DH. The results indicate that the treatment with an appropriate dose of the extract enhances the induction of DH by activation of the nonspecific amplifier T cells.

Reaction mechanism of L-glutamate dehydrogenase. Transient complexes in the oxidative deamination of L-glutamate catalyzed by NAD(P)-dependent L-glutamate dehydrogenase.

Characteristic optical properties of the reduced nicotinamide group of the coenzyme NADPH (absorbance, fluorescence, and circular dichroism) have enabled us to describe the sequential formation of two transients during the oxidative deamination of l-glutamste catalyzed by l-glutamate dehydrogenase in the presence of NADP. The first transient detected, called complex I, is characterized by a reduced nicotinamide absorption maximum at 333 nm and a very low specific NADPH fluorescence (Fs∼ 0.2). This complex is identified as the E · NADPH · 2-oxoglutarate complex which is known to possess very similar optical properties. The second transient, called complex II, is characterized and identified as the E · NADPH ·l-glutamate complex by the detection of an unique negative ellipticity during the steady-state phases. Using certain kinetic arguments, the formation of complex II (i.e., binding of l-glutamate to E · NADPH) is shown to be rate-limited by the dissociation of 2-oxoglutarate from complex II (i.e., E · NADPH · 2-oxo-glutarate). The conclusion is also drawn that the complex II accumulates prior to the liberation of the free product NADPH during the presteady-states phases. The rate-limiting step of the overall reaction is the dissociation of NADPH from the complex II which attains a, steady-state concentration level corresponding to 80% of the total enzyme concentration under our experimental conditions. The influence of the known catalytic effectors GTP and ADP on the accumulation of these transients has also been studied. The inhibitor GTP, under these same conditions, allows the accumulation of complex I, although its rate of formation is slower (2-fold), but thereafter blocks the reaction (>90%) by inhibiting the liberation of the products, especially that of 2-oxoglutarate. There is no formation of complex II which confirms the fact that 2-oxoglutarate is retained under the complex E · NADPH · 2-oxoglutarate form. The complex I accumulates to the same maximal concentration (∼ 50%, total available enzyme sites) as the reaction in the absence of effector. It is not known whether the extremely slow increase in absorbance which follows is due to a slow binding of the substrate (2-oxoglutarate) to the remaining sites and/or to a very slow liberation of NADPH permitting the enzyme turnover. The activator ADP modulates the accumulation of these two transients in a different fashion. The complex I is also formed more slowly (2-fold) but it then decomposes more quickly (2-oxoglutarate liberated twice as fast). Its maximal accumulation is thus lower than in the reaction in the absence of ADP (∼ 30%). The formation of complex II however does occur and accumulates to about 80% of the enzyme concentration. Here again the enzyme turnover is thought to be governed by the release of NADPH from complex II, which is about 2–3 times faster than the reaction without effector. This fact suffices to explain the activation (2–3 times) observed by classical steady-state kinetics.

Ribonucleoside Diphosphate Reductase Induced by Bacteriophage T4: II. ALLOSTERIC REGULATION OF SUBSTRATE SPECIFICITY AND CATALYTIC ACTIVITY

Abstract Ribonucleoside diphosphate reductase induced in Escherichia coli by infection with bacteriophage T4 catalyzes the reduction of the 5'-diphosphates of cytidine, uridine, guanosine, and adenosine (CDP, UDP, GDP, and ADP). The specificity of the enzyme for the substrates is governed by different nucleoside triphosphates which act both by changing the maximal velocity of the reaction and the apparent Km for the substrates. Deoxyadenosine triphosphate (dATP) and ATP are the prime effectors for the reduction of the pyrimidine ribonucleotides CDP and UDP, deoxythymidine triphosphate (dTTP) is the prime effector for the reduction of GDP, and dGTP is the prime effector for the reduction of ADP. None of the nucleotides is a general inhibitor. Substrate saturation curves are hyperbolic both in the absence and presence of effector. In the presence of the prime effector the maximal velocity with each of the four substrates is of the same magnitude. The Km values for CDP, GDP, and ADP are about 0.3 mm in the absence of effector and about 0.05 mm in the presence of a prime effector. The corresponding Km values for UDP are 1.2 mm and 0.1 mm, respectively. It is suggested that the nucleoside triphosphates act by allosteric mechanisms. Competition experiments indicate that the enzyme contains a single allosteric site and a single catalytic site. The allosteric properties of T4 ribonucleotide reductase are in many ways similar to those of the E. coli enzyme. A main difference, however, concerns the effect of dATP. With the T4 enzyme this nucleotide is a prime effector for the reduction of pyrimidine ribonucleotides while it is an inhibitor for the reduction of all ribonucleotides with the E. coli enzyme.

Activation by bicarbonate, orthophosphate, and sulfate of rat liver microsomal glucose dehydrogenase

1. 1. The effects of the anions bicarbonate, orthophosphate, and sulfate on rat liver microsomal glucose dehydrogenase (β- d-glucose:NAD(P) + oxidoreductase, EC 1.1.1.47) have been studied in detail. 2. 2. Significant activations of glucose dehydrogenase activity were produced by all these anions in the range from 2.5 to 50 mM. 30 mM sulfate, orthophosphate, and bicarbonate, respectively, effected increases in activity levels to approximately 550, 400, and 300% of control values. 3. 3. Activations were noted at all of a variety of H + concentrations between pH 7 and 10. The pH optimum, 9.2 in the absence of effector, was shifted slightly towards neutrality in the presence of increasing concentrations of the various anions. 4. 4. Kinetic studies revealed that K m values for glucose and for nucleotide substrate were independent of second substrate concentrations. The various anions all effected increases in the v max value and decreases in K m values for both glucose and NAD. 5. 5. Relatively small but significant inhibitions of glucose 6-P dehydrogenase activity also catalyzed by this multifunctional enzyme were produced by the various anions, while ATP was a potent inhibitor. 6. 6. It is suggested on the basis of similarities of the effects noted with the two multifunctional enzymes that the mechanism of anion action previously proposed by H orne et al. 3 ( Biochemistry, 9 (1970) 610) for yeast glucose-6-P dehydrogenase also obtains, with a slight modification, for the mammalian microsomal glucose dehydrogenase. 7. 7. Although the high K m value for glucose (0.33–1.25 M) may contraindicate a physiological role for glucose dehydrogenase activity, the observation that in the presence of physiological levels of anionic activators glucose dehydrogenase activity with 0.5 M d-glucose may approach or exceed the level of total hepatic cellular glucose-6-P dehydrogenase activity (microsomal plus cytosolic) raises the possibility that, as previously has been suggested by M etzger et al. 38 a specialized, compartmentalized biological role for this hepatic activity is possible.