Salivary lipid profiles of the leech (Hirudo medicinalis).

Saliva was collected from sixteen leeches (Hirudo medicinalis). The saliva was analyzed for its total lipid content (3.26 +/- 0.31 mg of total lipids per 100 mL saliva). The lipids were separated into polar and nonpolar by chromatographic techniques. The neutral fraction was approximately 2/3 of the total, and the polar fraction was approximately 1/3 of the total lipids. Thin-layer chromatography was used to obtain the individual profiles of the polar and nonpolar lipids. Of the identified lipids, phosphatidic acids and free fatty acids represented the largest percentage. These results suggest that the leech contains a unique lipid distribution, and that some of these components may be potent phospholipases and lipases that probably are present in its saliva for the purpose of preventing plugging or healing of the wound in the attacked organism.

Differential V region mutation of two transfected Ig genes and their interaction in cultured B cell lines.

We have established B cell culture systems in which transfected and stably integrated Ig constructs spontaneously undergo high rates of variable (V) region mutation. Mutation rates were determined using reversion analysis of an Ig V region nonsense codon (Vn). A construct (Vn/gamma2a) in which a Vn was associated with the gamma2a constant region and its intervening and immediate flanking sequences mutated at a high rate of 2.2 x 10(-4)/bp/generation in the NSO myeloma cell line. This same Vn, when associated with the mu constant region (Vn/mu), mutated at a 1000-fold lower rate in NSO. The Vn/gamma2a construct also mutated at high rates in the 18.81 pre-B and the S107 myeloma cell lines and at a low rate in the J558 myeloma cell line. In NSO, the presence of the gamma2a construct raised the mutation rate of the mu construct and the mu decreased the mutation rate of gamma2a. These results suggest that there is both positive and negative regulation of V region mutation and that different cell lines express different combinations and/or amounts of trans-acting factors that are involved in the mutational process.

Immunoglobulin variable region hypermutation in hybrids derived from a pre-B- and a myeloma cell line.

Somatic mutation of the variable (V) regions of immunoglobulin genes occurs in vivo at rates that have been estimated to be between 10(-3) and 10(-4) per bp per generation. To study this process in vitro, the 18.81 pre-B-cell line and hybrids derived by fusing 18.81 to the NSO myeloma fusion partner were transfected with a mu heavy-chain construct containing a nonsense mutation in the V region (Vn) or the constant region (Cn). Mutation was quantitated by reversion analysis using the ELISA spot assay to detect single cells secreting IgM. Fluctuation analysis revealed that V-region mutations spontaneously occurred in 18.81 cells at an average rate of 5.8 x 10(-6) per bp per cell generation and in selected 18.81-NSO hybrids at greatly increased rates of 1.6 x 10(-3) to 5.8 x 10(-4) per bp per generation. The Vn construct also reverted frequently in transgenic mice, indicating that it contained sufficient information to mutate at high rates both in vivo and in vitro. Sequence analysis of reverted genes revealed that reversion was due to point mutations. Since the rates and nature of the mutations that are occurring in these transfected genes are similar to those reported in vivo, it should be possible to use this system to identify the cis-acting sequences and trans-acting factors that are responsible for V-region somatic hypermutation.

A well-differentiated B-cell line is permissive for somatic mutation of a transfected immunoglobulin heavy-chain gene.

pSV2neo plasmids containing an IgM heavy-chain gene with nonsense mutations in either the variable (V) or the constant (C) region were transfected into four differentiated mouse plasma cell lines: S107 and the NSO fusion partner (myeloma cell lines) and 2C3 and 36.65 (hybridoma cell lines). The frequencies of reversion of the nonsense mutations in multiple independent transfectants were determined with the spot ELISA and rates of reversion were calculated by fluctuation analysis. Mutations in both V and C regions were confirmed by sequence analyses. In the S107 cell line, spontaneous point mutations occurred in the V region at a rate of approximately 5 x 10(-5)/bp per cell generation, > 400-fold higher than the rate of V-region mutation in the NSO cell line and considerably higher than the rates in 2C3 and 36.65 hybridoma cell lines. These studies suggest that S107 is a relatively permissive cell line in which V-region mutations can occur constitutively, even though it represents a late stage of B-cell differentiation. Further, the results show that the construct used contains sufficient information in its flanking and coding sequences to allow a relatively high rate of V-region mutation, at least in the S107 cell line.

Changes in the lipid content of rat lymph after the ingestion of [14C] long-chain fatty acids.

Rats were orally administered separately one dose of three labelled long-chain fatty acids: [1-14C] oleic, [1-14C] palmitic or [1-14C] stearic. Samples of lymph were obtained from previously cannulated thoracic ducts and analyzed for the composition and content of labelled fatty acids. Most of the newly recovered and labelled fatty acids were qualitatively and quantitatively similar regardless of which 14C-fatty acid had been administered. Over 20% of the administered fat were interconverted in six hours. The results suggest that the mucosa of the small intestine, the first sites of fatty acid absorption, is also one of the sites of various metabolic processes such as beta oxidation and synthesis which are responsible for some of the changes observed. These processes indicated that by shortening or lengthening the fatty acid composition, the content of the lymph became approximately the same regardless of the precursor fatty acid. The intestinal mucosa became the first tissue to help maintain lipid homeostasis in the rat.

Hepatic lipid profiles in miniature pigs after alcohol feeding.

Changes in lipid profiles have not been reported for the known increases in total lipid content in livers of alcoholics. We have reported a lowering of the beta-oxidative capacity of alcoholic livers, and therefore would expect a lower turnover of fatty acids in these livers, and thus a change in lipid profile. The percentage composition of saturated and unsaturated fatty acids in the liver of alcohol-fed miniature pigs versus the controls, as well as the function of distance from the main hepatic vein, have both been determined in this study involving the feeding of ethanol for one year. Livers of alcohol-fed miniature pigs contained more total lipids than those of controls. Results also indicated significantly higher percentages of free fatty acids and triglycerides in the alcohol-fed miniature pigs, and also an increase in percentage total neutral lipids. The effect of distance from the main blood source (and therefore oxygenation) gave a fatty acid profile that showed an increase in the ratio of saturated to unsaturated fatty acids with increasing distance from the right hepatic vein. This change in ratio was independent of alcohol feeding.

Metabolism of inositol-1,4,5-trisphosphate in the taste organ of the channel catfish, Ictalurus punctatus.

1. The metabolism of inositol-1,4,5-trisphosphate was studied in the taste organ (barbel) of the channel catfish, Ictalurus punctatus. 2. Homogenates of epithelial barbel scrapings were incubated with [3H]-1,4,5-IP3, whose dephosphorylation or phosphorylation was assayed under first-order conditions by measuring the production of either [3H]-1,4-IP2 (representing the activity of IP3-5-phosphatase) or [3H]-1,3,4,5-IP4 (representing the activity of IP3-3-kinase). 3. Both enzymes were predominantly cytosolic, magnesium-dependent and maximally active at pH 6.4. For IP3-phosphatase, Km = 6 microM and Vmax = 10.5 nmol/min/mg. For IP3-kinase, Km = 0.23 microM and Vmax = 0.05 nmol/min/mg. 4. Neither enzyme was significantly affected by the presence of taste stimuli (amino acids), GTP gamma S, cAMP or phorbol esters. 5. In the presence of physiological levels of free calcium (0.05-12 microM) IP3-phosphatase was moderately activated whereas IP3-kinase was moderately inhibited. 6. IP3-phosphatase was moderately activated by Mn2+, unaffected by LiCl, and strongly inhibited by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, CuCl2, FeCl3 and ZnSO4 7. IP3-kinase was strongly activated by 2,3-diphosphoglycerate, Na-pyrophosphate, CdCl2, HgCl2, FeCl3 and LiCl and inhibited by ZnSO4 and Mn2+. 8. IP3-kinase was significantly activated in a calcium-dependent manner by exogenously-added phosphatidylcholine and sphingomyelin, and to a lesser extent by diacylglycerol. IP3-phosphatase was unaffected by exogenously-added lipids. 9. IP3-phosphatase may participate in taste transduction since calculations based on the first-order rate constant (6.9 sec-1) indicate that it is capable of dephosphorylating basal levels of IP3 with a half-life of 0.1 sec.

Liver lipid profiles of adults taking therapeutic doses of aspirin.

The distributions of lipids of hepatic specimens obtained at autopsy from 7 adult patients who had been taking large amounts of aspirin for arthritis were compared to 7 control samples obtained from livers of autopsied adults without prior liver disease. The total neutral lipid levels of control livers were approximately one-third lower than those observed for livers of patients on aspirin. In addition, the phospholipid content of control specimens was significantly greater than that of livers from adult patients that had been on a high dose of aspirin for a long time. Examination of individual lipid classes showed that the concentrations of free fatty acids, triacylglycerols, and mono- and diacylglycerols were highest in livers of patients with aspirin exposure, and that all phospholipids were diminished. Phosphatidylcholines and phosphatidylethanolamines showed the greatest decrease. These results suggest that the livers of patients taking large amounts of aspirin may accumulate fatty acids and neutral lipids due to an impairment in the oxidation of fatty acids by hepatocytes. The data obtained also suggest that needle biopsy of the liver with measurement of distribution of hepatic lipids, perhaps together with histopathologic examination, may provide useful diagnostic information.

I-E expression does not by itself influence growth of or T cell unresponsiveness to SJL lymphomas.

The nature of the antigen on SJL lymphoma (reticulum cell sarcomas, RCS) cells that is strongly stimulatory to syngeneic CD4+ T cells is still elusive. Previously, we showed that the response to RCS of T cells from F1 hybrids of SJL by strains expressing I-Ak,d and/or I-Ek,d was much lower than that of T cells from SJL mice or from F1 hybrids of SJL by H2b- or H2s-bearing strains. We now show that removal of CD8+T cells from the responding cell population of (SJL x BALB/c)F1 or (SJL x A.TL)F1 mice does not enhance their responses, suggesting that the negative effect of H2k,d is not due to suppressor cells. Moreover, repeated injections of RCS cells into such F1 mice also fail to enhance the response, suggesting that these mice lack responder cells. T cells from I-E alpha transgenic (C57BL x SJL)F1 mice backcrossed to SJL respond to RCS as do T cells from I-E alpha- littermates or SJL mice. Similarly, I-E alpha+ SJL mice support RCS growth in vivo to the same (LN + spleen)/body weight ratio as do I-E- littermates. Thus, while I-E appears to have a negative influence on T cell responsiveness and RCS growth in F1 mice, it does not have such an effect when present, by itself, on a SJL background. The role of V beta 17 a+ T cells in the response of SJL T cells to RCS was also examined, because such cells are known to be responsive to I-E. The responses of V beta 17a(+)-depleted (0.3% V beta 17 a+) and V beta 17 a(+)-enriched (25.3% V beta 17a+) SJL T cell populations to RCS were examined by limiting dilution. We found the incidence of responding cells to be slightly higher in the depleted (0.016%) than in the (0.006%) enriched population. Furthermore, lymph node blast cell populations responding to RCS do not exhibit a higher percentage of cells staining for V beta 17a than do blast cells responding to Con A or unstimulated lymph node cells.

Lipid composition and biosynthesis in the gingiva of the domestic cat.

Periodontal disease in the domestic cat may assume debilitating and serious consequences; however, little is known of the biochemical composition or metabolism of feline gingiva in health or disease. In this report the chemical composition and metabolism of gingival lipids from inflamed an non-inflamed sites is presented and compared to other species with naturally occurring periodontitis. The neutral and phospholipid composition of feline gingiva was found to be distinct from that of porcine and human. As a measure of de novo lipid synthesis, the total incorporation of 14C-acetate into fractionated lipid components was determined and revealed an approximate 2 to 3 fold decrease in inflamed versus non-inflamed gingiva. The decrease in 14C-acetate incorporation appeared to result from a 2-fold increase in free acetate pools in inflamed compared to non-inflamed gingival samples, since total lipase and phospholipase activity were comparable in inflamed and non-inflamed gingiva and total lipid composition was not changed between inflamed and non-inflamed sites. These data are similar to those reported for periodontally involved human gingival tissue and suggest a common effect of periodontal inflammation on lipid metabolism in both species.

A probable defect in the beta-oxidation of lipids in rats fed alcohol for 6 months.

Studies on fatty acid oxidation were made in rats fed for 6 months on a liquid diet containing 15% total calories as ethanol. After 6 months, a marked diminution was observed in the in vivo production of 14CO2 from labeled palmitate and octanoate in the ethanol-fed animals compared to their pair-fed isocaloric controls not receiving alcohol. Similar results were obtained in 14CO2 formation from 14C-fatty acids using liver mitochondria from these ethanol-fed rats after 6 months. The effect on octanoate beta-oxidation was larger than that for palmitate. Addition of purified acyl CoA dehydrogenase complexes and additional electron transfer flavoprotein complex to the mitochondria suggested that the ethanol-fed animals could have been deficient in the medium-chain acyl CoA-dehydrogenase complex.

Germinal center cells are a major IL-5-responsive B cell population in peripheral lymph nodes engaged in the immune response.

Germinal center formation and the development of B cell memory in lymphoid tissue is a T cell-dependent process. The specific B cell-T cell interactions, and/or cytokines, resulting in germinal center cell growth have not yet been identified. Germinal center B cells were separated from other lymph node (LN) B cells by panning on peanut agglutinin (PNA)-coated dishes. Resulting fractions enriched for PNA+ (germinal center) B cells, and the PNA- (other) LN B cells from immune SJL mice were assayed for proliferation in the presence of cytokines. PNA+ and PNA- B cells responded equally to IL-4 in the anti-mu co-stimulator assay. In contrast, PNA+ B cells responded to murine (r)IL-5 or human B cell growth factor in the dextran sulfate (DxS) co-stimulator assay, to a much greater degree than did PNA- B cells. The same results were obtained with PNA+ and PNA- cells from LAF1 mice. Unfractionated LN B cells from nonimmunized SJL or BALB/c mice did not respond to IL-5 with or without DxS. B cell populations from BALB/c mice such as from spleen and peritoneal cavity, which are known to be high in Ly-1+B cells, responded to IL-5 alone, and more dramatically, to IL-5 as a co-stimulator with DxS. Such populations of cells from SJL mice, which are known to contain low numbers of Ly-1+B cells, responded markedly less. These results are consistent with those of others which show that in nonimmunized mice, Ly-1+B cells are a major IL-5 responsive subpopulation. IL-1 enhanced the proliferation of PNA+ cells in response to rIL-5 and had no effect on PNA- cells. IL-4 and IL-5 did not enhance each other's effects as co-stimulators of proliferation. In contrast to PNA+ B cells from immune LN, B cells activated by Escherichia coli endotoxin exhibited no responses to rIL-5. The present results indicate that in immune LN, PNA+, germinal center B cells constitute a prominent IL-5-responsive population.

Lipid metabolic interrelationships and phospholipase activity in gustatory epithelium of Ictalurus punctatus in vitro.

The catfish, Ictalurus punctatus, is an important model for studying the biochemical mechanisms of taste at the peripheral level. The type, amount and metabolic activity of the lipids within this tissue play important roles in taste transduction by forming the matrix in which the receptors for taste stimuli are imbedded and by acting as precursors to second messengers. The metabolic interconversions that occur among the lipids on the taste organ (barbels) of this animal are reported here. When sodium [32P]phosphate was incubated with minced pieces of epithelium from the taste organ of I. punctatus, phospholipids became labeled. Maximal incorporation occurred near 20 min for lysophosphatidylcholines (LPC), phosphatidylcholines (PC) and phosphatidylinositols (PI). The phosphatidylethanolamines (PE) and phosphatidylserines (PS) became labeled more slowly. The label in LPC and PC declined from 20 min to 120 min, while that of the other fractions increased or was stable over the 20-120 min time period. Upon addition of 1,2-di-[1'-14C]palmitoyl-sn-glycero-3-phosphocholine to the medium, 14C was found within minutes in all of the phospholipids assayed. The amount of label incorporated increased with time, with maximum labeling for all phospholipids occurring at 15 min. However, 14C appeared predominantly first (by 5 min) in a neutral lipid fraction (fraction AG, consisting of free fatty acids, mono- and diglycerides, triglycerides and methyl esters), then declined rapidly as the phospholipids gradually incorporated more label. Within minutes of addition of 1-[1'-14C]palmitoyl-sn-glycero-3-phosphocholine (lysophosphatidylcholine) the 14C-label was detected in the neutral lipid fraction AG, then in the PC fraction, and later in the other phospholipids.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in whole body lipid composition in a murine model of insulin-dependent diabetes mellitus.

To assess the effect of experimentally induced insulin-dependent diabetes mellitus (IDDM) on total body lipid composition, homogenates of neonatal (0-day) and 6-day Sprague-Dawley rat pups treated on day 0 with 65 mg/kg body weight of streptozotocin (STZ) or citrate buffer alone were compared using thin-layer and gas-liquid chromatographic techniques. STZ-treated littermates in a parallel study were markedly hyperglycemic, hypoinsulinemic and attained only 50% of the gain in weight of citrate-treated controls. Although both groups were similar in protein to body weight ratios, STZ-treated pups exhibited 60% of the total lipid content of citrate-treated littermates when compared by weight. The decrease in total lipid content in the STZ-treated group resulted specifically from decreased neutral and not phospholipid content, although a small increase in phosphatidic acid and sphigomyelin was observed in this group. The changes in relative whole body lipids with short-term, high-dose STZ parallel those reported in human IDDM.

Lipid characterization and 14C-acetate metabolism in catfish taste epithelium.

The catfish, Ictalurus punctatus is an important model system for the study of the biochemical mechanisms of taste reception. A detailed lipid analysis of epithelial tissue from the taste organ (barbel) of the catfish has been performed. Polar lipids account for 62 +/- 1% of the total, neutrals for 38 +/- 1%. Phosphatidyl-cholines, serines and ethanolamines are the major constituents of the polar fraction. Plasmalogen concentration is high relative to that of non-neural tissues. [14C]-Acetate is incorporated into cell lipid fractions after incubation of barbel tissue at 37 degrees C for 60 min. Percentage amounts of most lipids change with time during this in vitro incubation. The phospholipids are the most metabolically active fractions. This work yields information for continuing reconstitution experiments and indicates that the taste epithelium of this important model system is a metabolically active tissue capable of supporting lipid turnover/synthesis.

Incorporation of 140-lanthanum into bones, teeth and hydroxyapatite.

The incorporation of lanthanum in the form of 140-lanthanum onto the surface of teeth, bone and synthetic hydroxyapatite was investigated. A small amount of lanthanum was taken up by the surface of all of the materials studied regardless of their origin. The depth of penetration into bone and teeth was dependent upon lanthanum concentration and time of incubation and, in these experiments, ranged from an estimated 5 to 15 microns. An exchange of lanthanum for calcium in the apatite matrix may be responsible for increased resistance of the hard tissues to acid dissolution. The effects of pH, temperature, time and concentration of the lanthanum solutions on this incorporation were investigated. Possible clinical uses of this effect are discussed.

Effect of 2 ppm ozone exposure on rat lung lipid fatty acids.

Based on in vitro studies, the initial damage to lung cells by ozone exposure is believed to result in part from the breakdown of lipid polyunsaturated fatty acids to aldehydes, ozonides, and peroxides. The present study measured lipid breakdown products in lungs isolated from rats pretreated with [1-14C]acetate 12 h before exposure for 4 h to either air or 2 ppm ozone. Lipid fatty acid breakdown was indicated by a 112% increase in thiobarbituric acid-reactive substances on ozone exposure and by changes in chemical and radioactive measurements of mono- and dicarboxylic acids formed by treatment of lipid fractions with hydrogen peroxide. Ozone exposure resulted in a 63% increase in recovery of short-chain fatty acids accounted for by increased recoveries of malonic acid by 37%, hexanoic acid by 47%, nonanoic acid by 118%, and azelaic acid by 107%. Recovery of glutaric acid was enhanced 15-fold by ozone exposure. Although decreases in tissue arachidonic acid could not be detected, oleic acid was significantly decreased by 36%. Recovery of radiolabel as short-chain fatty acids was increased by 65% on ozone exposure and was mainly accounted for by enhanced labeling of nonanoic and glutaric acid fractions. The failure to observe significant increases in 14C recovery in the other fractions suggested ozone-induced breakdown of unlabeled fatty acids. These results demonstrate the cleavage of unsaturated fatty acid double bonds following in vivo exposure of lungs to ozone. Breakdown of arachidonic and oleic acids was specifically identified by increased recoveries of glutaric and nonanoic acids, respectively.

Tissue uptake and intracellular distribution of 140-lanthanum after oral intake by the rat.

The ability of lanthanum to stabilize hydroxyapatite against acid dissolution is well known. It is possible to use lanthanum to experimentally alter hard tissues in vivo and in vitro. It was, therefore, of interest to determine the tissue distribution of lanthanum following oral ingestion of a LaCl3 solution. Oral intake of 140-lanthanum (as LaCl3 in drinking water) in adult rats over a 3-d period was voluntary and amounted to 0.27 mmol LaCl3 per animal per day. The teeth sowed increases in 140-lanthanum uptake with time. Distribution of 140-lanthanum within the incisors of animals drinking the LaCl3 solution showed that the highest specific activity of 140-lanthanum was associated with the outer layer of the tooth (that portion exposed to the oral environment). The soft tissues, such as lung, kidney, and liver, maintained a constant 140-lanthanum concentration after the first day of intake. The intracellular distribution of 140-lanthanum was measured in liver, with the soluble fraction showing the highest content. No histological changes were observed in the rat tissues after 3 d of oral intake (0.27 mmol/d) of lanthanum.

Altered Na+-K+-ATPase, cell Na+ and lipid profiles in canine arterial wall with chronic cigarette smoking.

1. We evaluated the influence of cigarette smoking on arterial wall membranes, using Na+-K+-ATPase activity, free cholesterol (FC) and phospholipid (PL) contents as indices of membrane structural and functional integrity. 2. Segments of aorta, carotid and femoral arteries were obtained from normal dogs (controls) and dogs subjected to chronic cigarette smoking for 2 yr (12 cigarettes a day). 3. Na+-K+-ATPase activity was assessed in segments of carotid and femoral arteries using a ouabain-sensitive 86Rb uptake procedure for intact tissues. 4. Free cholesterol and phospholipids were separated, identified, and quantitated from extracts of aortic samples by means of two dimensional thin-layer chromatography. 5. Na+-K+-ATPase activity was reduced in the smoker group in both carotid and femoral arteries. This reduced enzyme activity was accompanied by a rise in cell Na+ levels at both arterial sites. 6. Aortic FC was elevated and the PL profile was altered in the smoker group; as a result, phosphatidylcholine was reduced, whereas lysophosphatidylcholine, phosphatidic acid, and cardiolipin were elevated. 7. Phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingolipid levels were unchanged. In addition, the FC/PL ratio was increased in the smokers. 8. Taken together, the changes in Na+-K+-ATPase activity, FC/PL ratio and phospholipid profiles observed are consistent with the hypothesis that chronic cigarette smoking causes a reorganization of the phospholipid bilayer in the smooth-muscle cell membrane of the arterial wall.