Effects of sodium salicylate on epithelial cells of the rectal mucosa of the rat: A light and electron microscopic study

Using a ligation method, rat rectal epithelium was exposed to 2% sodium salicylate, and light and electron microscopic methods were used to assay for: 1) permeability of the epithelium to a marker dye, trypan blue, and 2) damage expressed in terms of disruption of the epithelial surface. Rectal mucosa was exposed to salicylate at pH 4.8, 7.0, and 9.0, and the effects of pretreatment with phlorizin were also studied. Results indicated that 2% sodium salicylate does very little damage to rectal epithelial cells at pH 7.0 while enhancing their permiability to trypan blue, an effect that is reversed upon washing out the sodium salicylate. The major cellular change induced by salicylate was a reduction in the length or distribution of glycocalyx filaments on microvilli of epithelial cells. It was also noted that pretreatment with phlorizin counteracted some of the effects of salicylate treatment.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. I. Establishment of the experimental conditions for the termination of immunological tolerance to human gamma globulin.

In order to study the role of hapten-reactive helper T cells in the induction of autoimmunity in mice, an attempt was made to establish an experimental model for the development of hapten-reactive helper T cells and the termination of immunological tolerance against heterologous proteins. Spleen cells taken from mice which were immunized with hapten-isologous protein conjugates (PAB-MGG) demonstrated helper activity for the anti-DNP antibody response of DNP-primed B cells responding to DNP and PAB-conjugated protein, but spleen cells from hapten-heterologous protein conjugate (PAB-HGG)-primed mice could not respond to PAB-determinant. Thus, hapten-reactive helper T cells can develop in mice by the immunization with hapten-isologous protein conjugate, but not with hapten-heterologous protein conjugate. However, spleen cells from mice which had been rendered tolerant by treatment with 2.5 or 0.2 mg of DHGG and then immunized with PAB-HGG could demonstrate helper activity responding to PAB-determinant. This helper activity was PAB-specific, because these spleen cells did not demonstrate helper activity if PAB-determinant was omitted in the primary and the secondary antigen. This helper activity was abrogated by the treatment of spleen cells with anti-θ serum and complement. Thus, hapten-reactive helper T cells were successfully induced by the challenge with hapten-heterologous protein conjugate in carrier-protein tolerant mice. When mice were treated with 2.5 or 0.2 mg of DHGG, no anti-HGG antibody response was induced by the challenge with HGG or PAB-HGG. However, the termination of HGG-tolerance was demonstrated only when the mice were preimmunized with PAB-MGG to raise PAB-rcactive helper T cells, treated with 0.2 mg of DHGG, and then challenged with PAB-HGG. This termination of immunological tolerance was not observed when the mice were preimmunized with PAB-BαA to raise PAB-specific B cells and anti-PAB antibody, or when the mice were treated with 2.5 mg of DHGG. Thus, if HGG-specific B cells remain intact in mice such as treated with low dose of DHGG, these B cells can be activated by some bypass mechanisms in the presence of PAB-reactive helper T cells through the PAB-determinant even in the absence of HGG-reactive helper T cells. These data clearly showed the role of hapten-reactive helper T cells in the termination of immunological tolerance and provide experimental supports to the hypothesis on the termination mechanism proposed by Weigle. The cellular mechanism for the development of hapten-reactive helper T cells in tolerant animals and the cellular mechanism of autoantibody production were discussed on the basis of T-B cell collaboration.     

H+ stoichiometry of sites 1 + 2 of the respiratory chain of normal and tumor mitochondria

The mechanistic stoichiometry for vectorial H+ ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on cyanide-inhibited mitochondria from rat liver, rat heart, and Ehrlich ascites tumor cells, and on rat liver mitoplasts with ferricyanide or ferricytochrome c as electron acceptors. K+ (+ valinomycin) and Ca2+ were employed as permeant cations. Three different methods were employed. In the first, known pulses of ferricyanide were added, and the total H+ ejected was determined with a glass electrode. Such measurements gave H+/2e-values exceeding 7.0 for both normal and tumor mitochondria with beta-hydroxybutyrate and other NAD-linked substrates; uptake of Ca2+ was also measured and gave the expected q+/2e-ratios. The second type of measurement was initiated by addition of ferricytochrome c to rat liver mitoplasts, with H+ ejection monitored with the glass electrode and ferricytochrome c reduction by dual-wavelength spectrophotometry; the H+/2e-ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing ferrocytochrome c in situ with a small amount of ferricyanide. H+/2e-ratios for rat liver mitoplasts oxidizing beta-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H+/2e-ejection ratios significantly exceeding 7.0, which correlated closely with H+/2e-measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.     

The role of hapten-reactive T lymphocytes in the induction of autoimmunity in mice. II. Termination of self-tolerance to erythrocytes by immunization with hapten-isologous erythrocytes.

When mice which had been primed with hapten-isologous protein conjugate (PAG-MGG) were challenged with PAB-conjugated isologous mouse erythrocytes (MRBC), they developed Coombs positivity and anemia. However, when mice primed with hapten-heterologous protein conjugate (PAG-HGG) were challenged with PAB-MRBC, neither Coombs positivity nor anemia developed.Since it was demonstrated that PAB-reactive helper T cells were generated by immunization with PAB-MGG but not with PAB-HGG, PAB-reactive helper T cells were considered to play a very crucial role in the induction of autoantibody. These results, as a model for autoantibody production in mice, were discussed on the basis of cellular cooperation mediated by a hapten-mechanism, and are consistent with the hypothesis proposed by Weigle for the mechanism of termination of self-tolerance.     

Regulation of glycolysis in muscle. The role of ammonium and synergism among the positive effectors of phosphofructokinase

It is observed that the decrease in the energy charge, increase in P_i, NH₄⁺, and fructose-6-phosphate observed in stimulated frog muscle act synergistically in increasing the activity of rabbit muscle phosphofructokinase 300-fold over its activity observed at the concentrations of above effector substrates found in the muscle at rest.The activity of phosphofructokinase at various concentrations of Mg²⁺ and various fixed concentrations of NH₄⁺, at levels of energy charge and P_i corresponding to the resting and stimulated muscle were also studied.These results suggest that variations in the concentrations of effectors of phosphofructokinase resulting from contraction of muscle are responsible for the increase in the activity of enzyme in stimulated muscle and that this activation may not necessarily be geared to the contractile process itself as postulated by Karpatkin el al.     

Stimulation of the hexose-monophosphate pentose pathway by delta 1-pyrroline-5-carboxylic acid in human fibroblasts.

L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.     

Synthesis, surface deposition, and secretion of immunoglobulins by Abelson virus-transformed lymphosarcoma cell lines.

Three Abelson virus-transformed lymphoma cell lines were established in tissue culture and the immunoglobulin biosynthesis by these cell lines was studied. Two of the cell lines (ABLS-1 and ABLS-5) were found to synthesize monomeric IgM molecules which were deposited in the cell membrane, probably to serve as an antigen receptor. The third cell line (ABLS-8) was found to synthesize membrane-associated IgM as well as cellular IgG molecules. In addition, these cell lines were found to synthesize a protein of 35,000 molecular weight which is also membrane-associated and which has the capability to bind the immunoglobulin (MAID). It is speculated that this protein might play a role in adapting the receptor immunoglobulin molecule to the hydrophobic environment of the cell membrane. The kinetics of amino acid incorporation into immunoglobulins by these cell lines show that they produce immunoglobulins at a rate which is two orders of magnitude smaller than plasmacytoma cells (MOPC 104E). These results suggest that Abelson virus transforms thymus-independent lymphocytes in various stages of maturation and these lymphocytes might be of B cell origin. The T lymphoma (P1798) used as a control cell line was found occasionally to produce minute amounts of immunoglobulin.     

Effect of the arginine analog,canavanine, on the synthesis and secretion of procollagen

Canavanine was shown to competitively inhibit the activation of arginine when tested with tRNA and synthetases prepared from whole chick embryos. The canavanine has no effect when tested with other amino acids. The K_m for arginine was 2.5 μm and the K_i for canavanine was 35 μm. When fibroblasts from embryonic chick tendons were incubated with [³H]arginine and increasing concentrations of canavanine, there was a progressive decrease in the incorporation of [³H]arginine so that at 3 mm the incorporation into nondialyzable protein was only 14% of the control. A much smaller decrease in the incorporation of other radioactive amino acids was observed. Amino acid analysis of proteins isolated from cells incubated with canavanine showed conclusively that the analog was incorporated. When the cells were incubated with [¹⁴C]proline or [³H]glycine and 3 mm canavanine, the labeled procollagen containing the canavanine was secreted more slowly than normal and accumulated intracellularly. The retained procollagen chains were normally hydroxylated, disulfide linked, and triple helical. However, slab gel electrophoresis in sodium dodecyl sulfate demonstrated that they migrated with a lower mobility than control procollagen chains. We postulate that incorporation of canavanine inhibits normal proteolytic processing of signal sequences resulting in delayed secretion of the procollagen.     

Inhibition by estrogen of autofeedback regulation of prolactin secretion

The effect of estradiol-17 beta (E2) on autofeedback regulation of prolactin (PRL) secretion was tested in ovariectomized rats after s.c. implantation of an (E2)-containing or empty silastic capsule, followed by i.v. injection of bovine PRL (b-PRL) or bovine serum albumin (BSA; 500 micrograms/100 g B.W.). Implantation of an E2 capsule (day 0), 2.5 mm or 5.0 mm in length, produced plasma E2 concentrations of 79 +/- 6 (9) and 140 +/- 8 pg/ml (8), respectively. Assay of PRL in plasma samples collected at 1 h intervals between 1100-1800 h on days 3, 4 and 5, after E2 capsule implantation showed a daily afternoon PRL surge. Empty capsule-treated rats did not show any afternoon PRL surge. Injection of b-PRL, but not BSA, at 1200 h on day 3 reduced basal PRL release both on days 3 and 4 in empty capsule-treated rats. In ovariectomized rats treated with a smaller E2 capsule (2.5 mm), b-PRL injection at 1200 h on day 3 reduced the amplitude of the afternoon surge of PRL and the total amount of PRL released on day 4. b-PRL, however, was ineffective in reducing PRL release in rats bearing the large E2 capsule (5.0 mm). These results suggest that high E2 levels in the blood can block the negative feedback action of PRL on PRL release.     

A radiochemical assay for N5-formyltetrahydrofolic acid (citrovorum factor) in serum and urine.

N⁵-Methyltetrahydrofolate, but not N⁵-formyltetrahydrofolate, can be measured in biological fluids by ligand-binding radioassay. Therefore, in order to measure N⁵-formyltetrahydrofolate by radioassay, it is chemically converted to N⁵-methyltetrahydrofolate by acidification followed by reduction with borohydride. By this method, 70–113% of N⁵-formyltetrahydrofolate added to serum and urine was recovered. The plasma clearance of the mixture of diastereoisomer of N⁵-formyltetrahydrofolate (Leucovorin) following intravenous administration to two normal subjects was rapid for the first 30 min, but then plateaued and cleared very slowly over the next 90 min, most probably because of the accumulation of the inactive isomer which was slowly excreted in the urine during this time period.     

Avian fatty acid synthetase: Evidence for short-term regulation of activity

Liver biopsies were performed on starved chicks at 0 and 4 h after refeeding a fat-free diet. Fatty acid synthetase activity increased after refeeding, and administration of cycloheximide did not prevent the rise of enzyme activity. Incorporation of [carboxyl-¹⁴C]leucine into fatty acid synthetase was measured in enzyme purified from the livers of starved chicks, starved-refed (4 h) chicks, and starved-refed chicks injected with cycloheximide. The data suggest that the synthesis of enzyme protein was inhibited in starved and cycloheximide-treated refed chicks in comparison with refed chicks. Liver cytosol from fed or starved chicks was filtered through centrifuge ultrafiltration membranes and the residues were suspended in the same or opposite filtrates. Fatty acid synthetase activity in residues from starved chicks was stimulated when suspended in filtrates from fed chicks. The evidence is consistent with the hypothesis that a portion of the fatty acid synthetase in the liver of starved chicks is present as an inactive form which can be activated upon refeeding.     

Neural and hormonal control of Brunner's gland secretion

For many years it has been speculated that the physiological function of Brunner's glands was to secrete mucus to protect the proximal duodenum from the corrosive effects of acidified gastric juice. However the control of Brunner's gland secretion remains an enigma. Some evidence exists which indicates both cholinergic and adrenergic innervation of these glands, but current consensus weighs heavily in favor of a hormonal stimulus for glandular secretion. This is based in part on evidence obtained from denervated Brunner's gland pouches following a feeding stimulus. A number of hormones and hormone-like substances have been investigated as possible mediators in this secretory response, however, no specificity was ever demonstrated. The inability to pinpoint a given substance as a common mediator can be attributed to the fact that most active agents employed also affect duodenal motility. We present evidence that Brunner's gland secretion can be observed to be a diphasic response. The initial, transient response is always observed in the presence of increased duodenal motility. The sustained response does not require duodenal motility and is probably hormonally mediated.     

Multiple acid phosphatases in avian pectoral muscle--the postmicrosomal supernatant acid phosphatase is elevated in avian dystrophic muscle

There are at least three forms of acid phosphatase in avian pectoralis muscle differing in molecular weight, subcellular location, and response to various substrates and inhibitors. These enzymes are separated by differential sedimentation into postmicrosomal supernatant, lysosomal, and microsomal activities with apparent molecular weights in Triton X-100 of 68,000, 198,000, and 365,000, respectively. All of the enzymes show acid pH optima (pH approximately 5), but the postmicrosomal supernatant form is distinctly different from the other two forms in its resistance to most common phosphatase inhibitors and in its reduced activity against several organic phosphates. Quantitation of these three forms of acid phosphatase in normal and dystrophic avian pectoralis muscle shows that the postmicrosomal supernatant form is significantly elevated in dystrophic muscle; at 33 days ex ovo, 84% of the increased acid phosphatase activity in dystrophic muscle can be attributed to the postmicrosomal supernatant form. The microsomal form is only slightly elevated; the level of the lysosomal form is not altered.     

Structural and functional role of leucine residues in proteins

Circular dichroism and potentiometric titration studies of leucine random copolymers in aqueous solutions, as well as a comparison of the conformational stability in poly-α-amino acids, indicate that leucine may possibly be the amino acid with the highest propensity for forming α-helical structures. This suggests that leucine might be found most frequently in the helical regions of proteins. A survey was made on 15 different proteins containing 2473 residues with known sequence and conformation determined by X-ray crystallography: carboxy-peptidase A, α-chymotrypsin, cytochrome b₅, elastase, ferricytochrome c, α- and β-hemoglobin, insulin, lysozyme, myogen, myoglobin, papain, ribonuclease A, staphylococcal nuclease, and subtilisin BPN′. It was found that 888 residues in these proteins are in helices, and 422 of them reside in the internal turns of helical regions. While Glu, Ala, Leu and His were found to be present with the highest percentages in helical regions, Leu was clearly the most abundant residue in the inner helical cores of proteins. Polar residues are found preferentially at the helix-coil boundary regions; Asp and Glu at the N-terminal and His, Lys and Arg at the C-terminal helical ends. These findings agree with Ptitsyn's (1969) analysis on seven proteins containing 1132 residues. A more comprehensive analysis in the present survey showed that Ile, Met and Val occur with the greatest frequency in the β-regions of proteins. Leu was also found as the strongest structure-forming residue in proteins (total helical and β-regions). The functional-structural role of leucine was established by showing that it occurs most frequently among residues surrounding the heme in five of the heme proteins. In addition, the greater abundance of leucine as neighbors to active-site residues in enzymes provides strong evidence that hydrophobic residues create a non-aqueous environment, aiding the polar residues in substrate binding and enzymic catalysis. Examples of conservative and non-conservative mutations of leucine in heme proteins are given to illustrate the structure—function relation of proteins, and explain why most leucine residues in the insulin, hemoglobin, and cytochrome c homologs are invariant. Finally, the strong helical-forming power of leucine, as demonstrated experimentally in synthetic copolypeptides and its high occurrence in the inner helical cores of proteins, suggests that it could have a major role as nucleation centers in the folding and evolution of large protein molecules.     

A possible role for guanosine 3′,5′-monophosphate in the stimulus-secretion coupling in exocrine pancreas

Carbamylcholine, caerulein and cholecystokinin octapeptide rapidly increased the cyclic GMP concentration and amylase secretion in isolated guinea pig pancreatic slices. The cyclic GMP concentration was increased eight-fold over the basal concentration in 30 s, with concomitant increase in the rate of amylase secretion. The tissue concentration of cyclic GMP then rapidly declined to a plateau value of approx. 16% of the peak level within 10 min and was maintained at that concentration for the duration of the experiment. We have shown earlier (Kapoor, C.L. and Krishna, G. (1977) Science 196, 1003–1005) that the decrease of tissue cyclic GMP was due mainly to the secretion of cyclic GMP into the medium. The cyclic AMP concentration in the tissue was not changed, nor was it secreted into the medium.There was a correlation between the concentration response to various agents for the increase in cyclic GMP concentration and amylase secretion in pancreatic slices. Carbamylcholine increased both the cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 1.5 μM concentration. Caerulein and cholecystokinin octapeptide were 5000 times more potent than carbamylcholine in increasing cyclic GMP concentration and amylase secretion; the half-maximal effect was achieved at 0.3 nM concentration. Atropine, which completely inhibited the increase in cyclic GMP and amylase secretion induced by carbamylcholine, did not block the effects of caerulein or cholecystokinin octapeptide. These results suggest that various secretagogues induced amylase secretion by increasing the cyclic GMP concentration, but the mechanism by which cyclic GMP caused amylase secretion remains to be elucidated.     

Pairing of inosine with adenosine in codon position two in the translation of polyinosinic acid

Poly (I) codes for valine even more than for glycine. Since the ratio is constant over a wide range of Mg²⁺ concentrations the ambiguity appears to be inherent in the translation of I-I-I, rather than induced by the relatively high Mg²⁺ concentration (or the streptomycin) required with this messenger. These findings indicate that I in codon position 2 can pair readily not only with C (complementing glycine codon GG_⁵) but also with A (complementing valine codon GU_).I in anticodon position 3 is also known to pair with A (as well as with U and C). Crick's model for this pair would require a large increase in interstrand distance, rather than simply “wobble”. This stretch would be avoided in an alternative model, suggested by Sakore &; Sobell (1969), in which A would rotate from the usual anti to the syn conformation. Since a large stretch would seem difficult to accommodate in the less flexible middle codon position the present findings suggest that the rotation model for I·A pairing merits further exploration.     

12-O-tetradecanoylphorbol-13-acetate stimulates release of arachidonic acid, prostaglandin E2 and prostaglandin F2 alpha from TPA nonproliferative variants of 3T3 cells

The Proteinase Inhibitor Inducing Factor, PIIF, a pectic polysaccharide that induces synthesis and accumulation of proteinase inhibitor proteins in tomato and potato leaves, is an effective elicitor of the phytoalexin pisatin in pea pod tissues. The levels of pisatin induced by PIIF, and the time course of elicitation, are similar to those induced by chitosans, β-1,4 glucosamine polymers, which are potent elicitors of pisatin in pea pods. Similarly, the chitosans, found in both insect and fungal cell walls, are the most potent inducers yet found of proteinase inhibitor accumulation in excised tomato cotyledons. The similarity in the induction of synthesis of proteinase inhibitors in tomato cotyledons and of pisatin in pea pods by pectic polysaccharides and chitosans suggests that the two polysaccharide types may be triggering a similar fundamental system present in pea and tomato plants that regulates the expression of genes for natural protection systems.     

Detachment variants of Chinese hamster cells : Hyaluronic acid as a modulator of cell detachment

Variants of the Chinese hamster cell line CHO have been isolated and characterized with respect to attachment and trypsin- or EGTA-mediated detachment kinetics, cell morphologies, and the complex carbohydrates (labeled with [³H]glucosamine) of the cell surface. The variant which was more readily detached from the substratum exhibited a more rounded cell shape and had three times more label as hyaluronic acid on the cell surface than the parental cell. The slowly detaching variant had a morphology similar to the parental cell but only half the radioactivity ascribable to hyaluronic acid. Endogenous levels of cAMP were unaltered in the variants. Exogenous dbcAMP caused the cells to elongate and flatten but did not alter the characteristic detachment kinetics. The role of hyaluronic acid as a modulator of the cell substratum interface is discussed.     

Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Huntington's disease: regional alteration in muscarinic cholinergic receptor binding in human brain.

Huntington's Disease, an autosomal dominant neurological disorder, is characterized by diffuse neuronal degeneration particularly in the basal ganglia and cerebral cortex. The purpose of this study was to examine various discrete regions of choreic and control brains for alterations in muscarinic cholinergic receptor binding and choline acetyltransferase (ChAc) activity. Nine postmortem brains, three from patients with Huntington's Disease and six controls, were dissected into 17 discrete regions. Each regional homogenate was assayed for muscarinic receptor concentration by measuring specific membrane binding of [³H]-QNB, a potent muscarinic antagonist which selectively labels brain muscarinic receptors. Aliquots from each brain region were also assayed for ChAc activity. Of significance was the marked reduction in specific [³H]-QNB receptor binding in the caudate nucleus, putamen and globus pallidus of choreic brain while no significant alterations were detected in other brain regions. Significant decreases in ChAc activity were found in the caudate nucleus, putamen, and globus pallidus with no alterations in ChAc activity in the rest of the brain regions examined. The tissues were chosen such that protein levels were similar in both choreic and normal brain samples. The apparent reduction in the number of muscarinic cholinergic receptors in the choreic brains suggests that treatment with cholinomimetic drugs might be beneficial in Huntington's Disease.