Adoptive immunotherapy using immobilized anti-CD3 mAb-activated autologous lymphocytes: the strong cytotoxicity was supported by CD16+ cells which proliferated in prolonged cultures]

Inflammatory cytokines are able to facilitate the homing of transferred lymphocytes, tumor cell lysis through induction of adhesion molecules, also able to reduce tumor cell susceptibility to LAK cells by increasing tumor cell class I antigen. Investigation with 12 cell lines suggested that promotion of lysis by ICAM-1 was more responsible than protection by (allogeneic) class I Ags (Fig. 1). PBMC were cultured in anti-CD3 coated flasks with rIL-2. CD3+ cells dominated until day 7, decreased thereafter with CD4+. CD8+ and CD16+ increased (Fig. 2). Strong cytotoxicity obtained in some cultures correlated well with CD16+, contributing exclusively among several variables to the activity estimation in multiple regression analysis (Fig. 4). Among 6 cases, in which 2 or more cycles of transfer was done, 1 was prophylaxis of recurrence, in 2 of 3 advanced metastasis cases in which cells were transferred as BRM in the course of chemotherapy, survival of half a year was obtained in good QOL with suppressed disease and adequate level of PBL number. In 2 other cases, inflammation eliciting local treatments were combined. In the case 4, three large liver metastasis from colon cancer which resisted topical ethanol injection and chemotherapy, responded to the transfer with reduced lesions to 1/8 (Fig. 8). In the case 5, abdominal metastasis from colon cancer were removed, liver metastasis were injected of ethanol, and cells were transferred. Responses were obtained to immunotherapy in a certain degree, while never to any chemotherapy.     

Thyroid hormones selectively modulate human alcohol dehydrogenase isozyme catalyzed ethanol oxidation

Thyroid hormones are potent, instantaneous, and reversible inhibitors of ethanol oxidation catalyzed by isozymes of class I and II human alcohol dehydrogenase (ADH). None of the thyroid hormones inhibits class III ADH. At pH 7.40 the apparent Ki values vary between 55 and 110 microM for triiodothyronine, 35 and greater than 200 microM for thyroxine, and 10 and 23 microM for triiodothyroacetic acid. The inhibition is of a mixed type toward both NAD+ and ethanol. The binding of the thyroid hormone triiodothyronine to beta 1 gamma 1 ADH is mutually exclusive with 1,10-phenanthroline, 4-methylpyrazole, and testosterone, identifying a binding site(s) for the thyroid hormones, which overlap(s) both the 1,10-phenanthroline site near the active site zinc atom and the testosterone binding site, the latter being a regulatory site on the gamma-subunit-containing isozymes and distinct from their catalytic site. The inhibition by thyroid hormones may have implications for regulation of ADH catalysis of ethanol and alcohols in the intermediary metabolism of dopamine, norepinephrine, and serotonin and in steroid metabolism. In concert with other hormonal regulators, e.g., testosterone, the rate of ADH catalysis is capable of being fine tuned in accord with both substrate and modulator concentrations.     

The disposition of citric acid cycle intermediates by isolated rat heart mitochondria

The mechanism of depletion of tricarboxylic acid cycle intermediates by isolated rat heart mitochondria was studied using hydroxymalonate (an inhibitor of malic enzymes) and mercaptopicolinate (an inhibitor of phosphoenolpyruvate carboxykinase) as tools. Hydroxymalonate inhibited the respiration rate of isolated mitochondria in state 3 by 40% when 2 mM malate was the only external substrate, but no inhibition was found with 2 mM malate plus 0.5 mM pyruvate as substrates. In the prescence od bicarbonate, arsenite and ATP, propionate was converted to pyruvate and malate at the rates of 14.0 ± 2.9 and 2.8 ± 1.8 nmol/mg protein in 5 min, respectively. Under these conditions, 0.1 mM mercaptopicolinate did not affect this conversion, but 2 mM hydroxymalonate inhibited pyruvate formation completely and resulted in an accumulation of malate up to 13.2 ± 2.9 nmol/mg protein. No accumulation of phosphoenolpyruvate was found under any condition tested. It is concluded that malic enzymes but not phosphoenolpyruvate carboxykinase, are involved in conversion of propionate to pyruvate in isolated rat heart mitochondria.     

Local Conformation and Dynamics of Isoleucine in the Collagenase Cleavage Site Provide a Recognition Signal for Matrix Metalloproteinases

The mechanism by which enzymes recognize the “uniform” collagen triple helix is not well understood. Matrix metalloproteinases (MMPs) cleave collagen after the Gly residue of the triplet sequence Gly∼[Ile/Leu]-[Ala/Leu] at a single, unique, position along the peptide chain. Sequence analysis of types I-III collagen has revealed a 5-triplet sequence pattern in which the natural cleavage triplets are always flanked by a specific distribution of imino acids. NMR and MMP kinetic studies of a series of homotrimer peptides that model type III collagen have been performed to correlate conformation and dynamics at, and near, the cleavage site to collagenolytic activity. A peptide that models the natural cleavage site is significantly more active than a peptide that models a potential but non-cleavable site just 2-triplets away and NMR studies show clearly that the Ile in the leading chain of the cleavage peptide is more exposed to solvent and less locally stable than the Ile in the middle and lagging chains. We propose that the unique local instability of Ile at the cleavage site in part arises from the placement of the conserved Pro at the P₃ subsite. NMR studies of peptides with Pro substitutions indicate that the local dynamics of the three chains are directly modulated by their proximity to Pro. Correlation of peptide activity to NMR data shows that a single locally unstable chain at the cleavage site, rather than two or three labile chains, is more favorable for cleavage by MMP-1 and may be the determining factor for collagen recognition.     

Phospholipid methyltransferase activity in pancreatic islets: activation by calcium

Pancreatic islet homogenates contain a Mg2+-requiring phospholipid methyltransferase activity, the activity of which was doubled by calcium (K0.5 less than 5 microM). Other divalent metal ions stimulated the activity from 11 to 35%, but zinc and strontium were inhibitory. Cyclic AMP had no effect on the enzyme activity and cyclic GMP inhibited it slightly. Calcium increased the Vmax of the enzyme without affecting its Km with respect to S-adenosylmethionine (6 microM). Chlorpromazine, trifluoperazine, and dibucaine inhibited the calcium-stimulatable activity without affecting the activity in the absence of calcium. Phosphatidylserine stimulated, and arachidonic acid and palmitic acid inhibited, the basal enzyme activity. The methylated products were found to be primarily mono- and dimethylphosphatidylethanolamine (30%) and phosphatidylcholine (43%) and an, as yet unidentified, nonpolar lipid fraction (27%), as judged by thin-layer chromatography. In the presence of calcium, incorporation of methyl groups into phosphatidylcholine, mono- and dimethylphosphatidylethanolamine, and nonpolar lipids was increased by 131, 60, and 46%, respectively. Based on the localization of the enzyme activity in the insulin secretory granule fraction, it is proposed that phospholipid methylation plays a role in coupling the stimulus to the initial events in insulin secretion, leading to the exocytosis of insulin.     

Role of a hisU gene in the control of stable RNA synthesis in Salmonella typhimurium

We have previously reported a fivefold reduction in expression of the ilvGEDA operon in a hisU mutant (hisU1820) originally isolated as a histidine regulatory mutant that exhibited derepressed (deattenuated) expression of the his operon. More recently, we have reported that a unitary explanation of the effect of this mutant on amino acid control is complicated by the observation of relaxed control of stable RNA synthesis during carbon/energy source downshifts. In the present study, we report the results of an analysis of the relaxation in control of RNA synthesis in relation to the accumulation of the guanosine polyphosphates, ppGpp and pppGpp. Unexpectedly, we observed that, despite the inability to restrict RNA accumulation upon carbon/energy downshifts, this mutant formed ppGpp at the normal rate. Further, the evidence clearly indicates that the defective control of RNA in this hisU mutant is not owing to an alteration in the spoT gene and that the relA-mediated RNA control is unaltered. However, relaxed RNA synthesis in hisU is suppressed by hyper-elevated levels of ppGpp; thus, an inverse correlation between RNA accumulation and ppGpp level during carbon/energy downshifts is still demonstrable in the hisU mutant. These data led us to the observation that the increased accumulation of stable RNA upon a carbon/energy downshift is apparently the consequence of a hisU-conferred increase in RNA stability.     

Choline Administration Elevates Brain Phosphorylcholine Concentrations

Abstract: The phosphorylcholine concentration of rat brain rises and falls in response to parallel changes in the concentration of circulating choline. A single oral dose of choline chloride (20 mmol/kg) elevated whole-brain concentrations of both choline and phosphorylcholine 5 h after administration; a greater proportion of exogenously administered choline was retained by the brain in its phosphorylated form than as the free arnine. Striatal phosphorylcholine concentrations were elevated within 2 h of choline administration and continued to be significantly greater than control values for up to 34 h after treatment. The response of striatal choline levels to exogenous choline was of shorter duration than that of phosphorylcholine and was correlated with a significant increase in striatal acetylcholine concentrations. The consumption of a choline-free diet for 7 days lowered both serum choline and striatal phosphorylcholine concentrations, but had no effect on striatal choline or acetylcholine. These results suggest that choline kinase is unsaturated by its substrate in vivo and may thus serve to modulate the response of brain choline concentrations to alterations in the supply of circulating choline.     

Mathematical model of the activation of prothrombin by factor X a and factor V t

A mathematical model of prothrombin activation is being proposed which includes the feedback mechanism of thrombin and the alteration of factor V by thrombin. This model is in good agreement with experimental data for the dependence of the rate of thrombin formation on the concentrations of factors V and X _a . In particular, it correctly predicts the existence and location of a maximum in both of these cases.     

Fractionation and purification of DNA-methylases from Shigella sonnei 47

Possible applications of various column chromatography techniques and isoelectrofocusing for the study of DNA-methylases of Shigella sonnei 47 cells were analyzed. A simple, rapid and convenient procedure based on the use of cation-exchange chromatography was developed for obtaining a highly active total preparation of methylases. Affinity chromatography on heparin-Sepharose was shown to be a promising approach for separating methylases according to their specificity towards nitrous bases. Isoelectrofocusing was used to identify in Shigella sonnei 47 cells six individual methylating enzymes differing in their pI values. Under the stipulation that Shigella sonnei 47 DNA-methylases show a tendency to aggregate in the course of fractionation, column chromatography is of little or no use in isolating and purifying individual methylating enzymes of the given strain. The advantages of the isoelectrofocusing technique and its utility in the study of different molecular forms of site-specific enzymes are discussed.     

Methylated poly(L-lysine): conformational effects and interactions with polynucleotides

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of epsilon-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally-induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO4)2. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from beta, to 50% alpha, to random coil at the maximum methylation. Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)n.(dAdT)n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)n.(dC)n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative psi-spectra are induced, which, in the case of (dGdC)n.(dGdC)n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration. It is suggested that the biological effects of methylation proteins may be evoke by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.