Identification and validation of differentially expressed genes from pig skeletal muscle.

Pig is an important animal for meat production; this is generally associated with characteristics determined prenatally during myogenesis. Expressed sequence tags (EST) can provide direct information on the transcriptome and indirect information on the relation between the genome and phenotype, giving information about differentially expressed genes (DEG). In this work, the identification and annotation of DEG from EST libraries of three pig breeds (Duroc, Large White and Local Breed Piau) were performed followed by real-time PCR analyses during pre- and postnatal stages (21, 40, 70 and 90 days of pregnancy and 107, 121 and 171 days postnatal) from commercial breed animals for analysis of genes expression levels. Therefore, 34 genes differentially expressed were identified, of which 21 grouped in a network related with muscle development. From this, the expression profile of 13 genes was measured, to confirm their relationship with myogenesis like ANKRD2, MYBPC1, NEB and MYL2. These genes showed a prenatal high expression in this study. Besides, novels candidates for muscle development (TP53 and DCTN1) were listed. These findings can contribute to better explaining gene function mechanism and are helpful in uncovering the pathways that mediate pre- and postnatal skeletal muscle development in vertebrates.

Qualidade de silagem de cultivares de brachiaria brizantha ensilado com diferentes níveis de farelo de milheto / Silage quality of brachiaria brizantha cultivars ensiled with different levels of millet meal

The silage quality of Brachiaria brizantha cultivars ensiled with different levels of millet meal was evaluated. The experimental design was a completely randomized with three replications in a factorial 3 x 4, with three cultivars of Brachiaria brizantha: marandu, xaraés, and piatã and four levels of millet meal 0, 5, 10, and 15 percent. The addition of millet meal improved the quality of B. brizantha silage. The inclusion of the additive at 15 percent provided the best nutritional values. The piatã silage had the lowest contents of neutral detergent fiber, acid detergent fiber, and lignin and the highest content of total digestible nutrients, being a better quality silage as compared to those of xaraés and marandu grasses.

Avaliou-se a qualidade de silagem de cultivares de Brachiaria brizantha ensilados com diferentes níveis de farelo de milheto. O delineamento experimental utilizado foi inteiramente ao acaso, com três repetições, em esquema fatorial 3 x 4, sendo, três cultivares de Brachiaria brizantha: marandu, xaraés e piatã e quatro níveis de farelo de milheto: 0, 5, 10 e 15 por cento. A adição de farelo de milheto melhorou a qualidade das silagens de cultivares de B. brizantha. A inclusão de 15 por cento do aditivo proporcionou os melhores valores nutritivos. A silagem de capim-piatã apresentou os menores teores de fibra em detergente neutro, de fibra em detergente ácido e de lignina, e o maior teor de nutrientes digestíveis totais, mostrando ser de melhor qualidade quando comparada com as silagens de capim-xaraés e capim-marandu.

Doses e fontes de nitrogênio na recuperação de pastagens do capim-marandu / Nitrogen doses and sources on pasture recuperation of grass marandu

Avaliaram-se o efeito de doses e fontes de nitrogênio na recuperação do capim-marandu, por um período de três anos, em pastagem estabelecida há mais de 10 anos, com baixa produção de forragem. O delineamento experimental foi em blocos completos ao acaso, com três repetições. Nas parcelas foi utilizado o fatorial 2 x 4, sendo duas fontes de nitrogênio (sulfato de amônio e uréia) e quatro doses de nitrogênio (0, 100, 200 e 300kg ha-1 ano-1). Nas subparcelas, foram alocados os três anos (2004, 2005 e 2006), referentes ao tempo de recuperação da pastagem. A aplicação de nitrogênio foi determinante para a recuperação do capim-marandu. A maior produção de massa seca foi observada no segundo ano e o maior teor de proteína bruta no terceiro ano de recuperação da pastagem. As maiores doses de nitrogênio promoveram acréscimos lineares na produção de massa seca e redução nos teores de fibra em detergente neutro e fibra em detergente ácido. O sulfato de amônio promoveu maior produção de massa seca do que a ureia, em todas as doses e anos avaliados.

The effects of nitrogen doses and sources were evaluated on pasture recuperation of grass marandu, in a three-year period. The pasture was established for more than ten years and it was presenting low herbage production being considered in moderate degradation phase. The experiment was arranged in a randomized complete block design with split-plots and three replications, in a 2x4 factorial, being two sources of N (ammonium sulphate and urea) and four doses of N (0, 100, 200, and 300kg ha-1 yr-1). The time of pasture evaluation was represented by the years 2004, 2005, and 2006. The highest dry matter production was observed in the second year and the highest crude protein in the third one. The highest nitrogen doses promoted linear increase on dry mass production and decrease in fiber concentration and in neutral and acid detergents. Ammonium sulphate resulted in higher dry mass production than urea in all doses applied and evaluated years.

Anti-inflammatory and anti-ulcerogenic properties of Stachytarpheta cayennensis (L.C. Rich) Vahl.

In the present work, the anti-inflammatory and gastroprotective properties of ethanolic extracts of Stachytarpheta cayennesis (L.C. Rich) Vahl (Verbenaceae) were assessed. Chromatographic analysis of the crude ethanolic extract, SC01, revealed high concentrations of the iridoid ipolamiide, whereas the SC02, the second ethanolic extract, presented the arylpropanoid verbacoside as a major constituent. The oral administration of SC01 (100 mg/kg) into Swiss mice failed to inhibit paw oedema and pleural exudation induced by carrageenan and zymosan, whereas SC02 (100 mg/kg, p.o.) inhibited oedema and protein extravasation in all instances. Both extracts inhibited total leukocyte accumulation into the pleural cavity 4 and 24h after the intrathoracic (i.t.) injection of carrageenan, due to the inhibition of neutrophil and mononuclear cell influx, whereas only SC02 was able to inhibit leukocyte mobilization induced by zymosan (100 microg/cavity, i.t.). SC02 inhibited LPS (250 ng/cavity)-induced total leukocyte, neutrophil and eosinophil accumulation in the pleural cavity, whereas SC01 selectively inhibited neutrophil influx. In addition, our data indicates that the extract SC02 presents an important anti-ulcerogenic activity, since it inhibited diclofenac-induced (100 mg/kg, p.o.) gastric ulcera. Overall, these data provide evidence for the anti-inflammatory and gastroprotective properties of Stachytarpheta cayennensis, supporting its use in folk medicine for such purposes.

Anti-allergic effects of natural tetranortriterpenoids isolated from Carapa guianensis Aublet on allergen-induced vascular permeability and hyperalgesia.

OBJECTIVE: We investigated the anti-allergic and analgesic properties of an oil and a derived fraction of tetranortriterpenoids (TNTP) obtained from the seeds of Carapa guianensis Aublet. MATERIALS AND METHODS: Pleurisy, paw and ear edema were induced in Swiss and C57/Bl10 mice mice, whereas thermal hyperalgesia was assessed in Wistar rats (n = 6-10 per group). Values of p < 0.05 were regarded as significant. RESULTS: C. guianensis oil (100 to 400 mg/kg, p.o.) and TNTP (12.5 to 100 mg/kg, p.o.) inhibited pleural exudation, paw and ear edema induced by ovalbumin (OVA) in sensitized mice. TNTP (12.5 to 100 mg/kg, p.o.) also inhibited paw edema induced by histamine, PAF and bradykinin. TNTP (100 mg/kg, p.o.) inhibited prostaglandin E(2) generation in the pleural cavity in response to antigenic challenge. Moreover, C. guianensis oil (100 to 400 mg/kg) and TNTP (12.5 to 100 mg/kg) decreased OVA- and histamine-induced hyperalgesia. CONCLUSION: Taken together, these findings demonstrate the anti-edematogenic and analgesic effects of C. guianensis oil, and points out TNTP as the responsible bioactive compounds.

Hepatic protein kinase C is not activated despite high intracellular 1,2-sn-diacylglycerol in obese Zucker rats.

High intracellular 1,2,-sn-diacylglycerol (DAG) usually activates protein kinase C (PKC). In choline-deficient Fischer 344 rats, we previously showed that fatty liver was associated with elevated hepatic DAG and sustained activation of PKC. Steatosis is a sequelae of many liver toxins, and we wanted to determine whether fatty liver is always associated with accumulation of DAG with activation of PKC. Obese Zucker rats had 11-fold more triacylglycerol in their livers and 2-fold more DAG in their hepatic plasma membrane than did lean control Zucker rats. However, this increased diacylglycerol was not associated with translocation or activation of PKC in hepatic plasma membrane (activity in obese rats was 897 pmol/mg protein X min(-1) vs. 780 pmol/mg protein X min(-1) in lean rats). No differences in PKC isoform expression were detected between obese and lean rats. In additional studies, we found that choline deficiency in the Zucker rat did not result in activation of PKC in liver, unlike our earlier observations in the choline deficient Fischer rat. This dissociation between fatty liver, DAG accumulation and PKC activation in Zucker rats supports previous reports of abnormalities in PKC signaling in this strain of rats.

Choline deficiency selects for resistance to p53-independent apoptosis and causes tumorigenic transformation of rat hepatocytes.

The mechanisms which drive initiated cells to progress to form carcinomas are poorly understood. CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, respond by inducing p53-independent apoptosis when acutely switched to medium containing low choline (16% apoptotic at 48 h in 5 microM choline) as compared with controls (1% apoptotic at 48 h in 70 microM choline). The rate of apoptosis was inversely correlated with cellular phosphatidylcholine content. Choline deficiency (CD)-induced apoptosis is probably mediated by TGFbeta1 and reactive oxygen species, since immunoneutralization of TGFbeta1 in the medium or treatment with N-acetylcysteine (an antioxidant) or addition of neocuproine (a transition metal chelator) prevented CD-induced apoptosis. CWSV-1 hepatocytes could be gradually adapted to survive in 5 microM choline. CD-adapted cells had increased membrane phosphatidylcholine concentrations (compared with acute CD cells). Adapted cells acquired relative resistance to CD-induced apoptosis (7% of adapted cells compared with 19% of non-adapted cells were apoptotic at 48 h in 5 microM choline). They also became relatively resistant to another p53-independent form of apoptosis (TGFbeta1-induced). CD-adapted hepatocytes developed increased capability for anchorage-independent growth and formed tumors when transplanted into nude mice; passage-matched control hepatocytes did not possess these properties. Cell transformation was dependent on exposure to the selective pressure of CD apoptosis, as we observed that when CD apoptosis was inhibited with an antioxidant during adaptation, cells did not become anchorage independent. Acquisition by p53-deficient cells of resistance to p53-independent inducers of apoptosis (CD, TGFbeta1 and reactive oxygen species) may leave cells without another important apoptotic defensive barrier and may be responsible for the progression of initiated cells to frank carcinomas.

Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency.

Choline-deficiency causes liver cells to die by apoptosis, and it has not been clear whether the effects of choline-deficiency are mediated by methyl-deficiency or by lack of choline moieties. SV40 immortalized CWSV-1 hepatocytes were cultivated in media that were choline-sufficient, choline-deficient, choline-deficient with methyl-donors (betaine or methionine), or choline-deficient with extra folate/vitamin B12. Choline-deficient CWSV-1 hepatocytes were not methyl-deficient as they had increased intracellular S-adenosylmethionine concentrations (132% of control; P < 0.01). Despite increased phosphatidylcholine synthesis via sequential methylation of phosphatidylethanol-amine, choline-deficient hepatocytes had significantly decreased (P < 0.01) intracellular concentrations of choline (20% of control), phosphocholine (6% of control), glycerophosphocholine (15% of control), and phosphatidylcholine (55% of control). Methyl-supplementation in choline-deficiency enhanced intracellular methyl-group availability, but did not correct choline-deficiency induced abnormalities in either choline metabolite or phospholipid content in hepatocytes. Methyl-supplemented, choline-deficient cells died by apoptosis. In a rat study, 2 weeks of a choline deficient diet supplemented with betaine did not prevent the occurrence of fatty liver and the increased DNA strand breakage induced by choline-deficiency. Though dietary supplementation with betaine restored hepatic betaine concentration and increased hepatic S-adenosylmethionine/S-adenosylhomocysteine ratio, it did not correct depleted choline (15% of control), phosphocholine (6% control), or phosphatidylcholine (48% of control) concentrations in deficient livers. These data show that decreased intracellular choline and/or choline metabolite concentrations, and not methyl deficiency, are associated with apoptotic death of hepatocytes.

Choline deficiency induces apoptosis in SV40-immortalized CWSV-1 rat hepatocytes in culture.

Immortalized CWSV-1 rat hepatocytes, in which p53 protein is inactivated by SV40 large T antigen, had increased numbers of cells with strand breaks in genomic DNA (terminal dUTP end labeling) when grown in 0 Micron choline (67-73% of cells) than when grown in 70 Micron choline (2-3% of cells). Internucleosomal fragmentation of DNA (DNA ladders) was detected in cells grown with 5 Micron and 0 Micron choline for 72h. Cells treated with 0 or 5 Micron choline for 72h detached from the substrate in high numbers (58% of choline deficient cells vs. 1.4% of choline sufficient cells detached) exhibited a high incidence of apoptosis (apoptotic bodies were seen in 55-75% of cells; 67-73% had DNA strand breaks), and an absence of mitosis and proliferating cell nuclear antigen (PCNA) expression. Cells undergoing DNA fragmentation had functioning mitochondria. At 24h, cells grown in 0 or 5 Micron choline synthesize DNA more rapidly than those grown in 70 Micron choline. By 72h, the cells grown in 0 or 5 Micron choline were forming DNA much more slowly than control cells (assessed by thymidine incorporation, PCNA expression, and mitotic index). Western blot analysis showed that p53 in the nucleus of cells was detected in direct association with SV40 T-antigen, and was therefore likely to be inactive. We conclude that choline deficiency kills CWSV-1 hepatocytes in culture by inducing apoptosis via what may be a p53-independent process, and that this process begins in viable cells before they detach from the culture dish.

Pregnancy and lactation are associated with diminished concentrations of choline and its metabolites in rat liver.

Choline is an important nutrient that is actively transported from mother to fetus across the placenta and from mother to infant across the mammary gland. Thus, pregnancy and lactation are times when dietary requirements for choline may be increased. Pregnant rats eating AIN-76A diet (with and without choline) for 6 d (d 12-18 gestation) were compared with nonmated female and male rats eating the same diets. Similarly, lactating rats were compared with nonmated female rats, both groups eating these same diets for 25 d (gestation d 12-postpartum d 15). We measured choline and choline metabolites in livers on the last day of feeding. Nonmated female rats, eating the control diet, had higher hepatic choline metabolites concentrations than did male rats (choline, 98%; betaine, 96%; and phosphorylcholine, 55% higher), pregnant rats (phosphorylcholine, 47%; and betaine, 42% higher) or lactating rats (phosphorylcholine, 49%; phosphatidylcholine, 37%; and betaine, 273% higher). We found that nonmated females eating a choline deficient diet had only a modest diminution (33%) of the labile choline metabolite PCho in liver, compared with similar rats eating a control diet. When compared with similar rats fed a choline-adequate diet, pregnant rats fed a choline-deficient diet had significantly great diminution of hepatic phosphorylcholine (83% lower) than did nonmated females. Liver phosphorylcholine was only 12% lower than in controls in nonmated females fed the deficient diet for the same 25-d period. Lactating rats were the most sensitive to choline deficiency, with liver phosphorylcholine 88% lower than in similar rats fed control diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of prolonged (1 year) choline deficiency and subsequent re-feeding of choline on 1,2-sn-diradylglycerol, fatty acids and protein kinase C in rat liver.

Rats fed a choline-deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. They also develop fatty livers, because choline is needed for hepatic secretion of lipoproteins. We have previously reported that 1,2-sn-diradylglycerol accumulates in the livers of rats fed a choline-deficient diet for 1-27 weeks, and that protein kinase C activity in the hepatic plasma membrane is elevated during that time (da Costa et al., J. Biol. Chem., 268, 2100-2105, 1993). In the present study, we examined the changes that occur in rat liver at 52 weeks of choline deficiency and determined whether these changes were reversible when choline was returned to the diet of the deficient animals for 1 or 16 weeks. At 52 weeks, non-tumor liver samples from the experimental animals had increased 1,2-sn-diradylglycerol concentrations in the lipid droplets compared with control animals. Plasma membrane 1,2-sn-diradylglycerol levels in the liver did not differ between the two groups, but an age-related increase in membrane 1,2-sn-diradylglycerol concentrations was observed. Unsaturated free fatty acids, another activator of protein kinase C, accumulated in the deficient livers. Protein kinase C activity associated with the plasma membrane remained significantly elevated at 52 weeks in deficient livers. Hepatic foci expressing gamma-glutamyltranspeptidase were detected only in the deficient rats (0.83% of liver volume) and 15% of these rats had hepatocellular carcinoma at 1 year on the diet. At 53 weeks (1 week after choline was returned to the deficient group), 1,2-sn-diradylglycerol concentrations in the lipid droplets and hepatic free fatty acids had dropped to control levels. By 68 weeks (16 weeks of re-feeding choline), the membrane protein kinase C activity had returned to normal. At this time, 14% of the experimental animals had hepatocellular carcinoma. We suggest that choline deficiency altered the protein kinase C-mediated signal transduction within liver and this contributed to hepatic carcinogenesis in these animals.

Choline and hepatocarcinogenesis in the rat.

Rats fed a choline deficient diet develop foci of enzyme-altered hepatocytes with subsequent formation of hepatic tumors. This is the only nutritional deficiency that, in itself, causes cancer. We suggested that carcinogenesis is triggered, in part, because of abnormalities in cell signals which regulate cell proliferation and cell death. Because choline deficient rats develop fatty liver (choline is needed for hepatic secretion of certain lipoproteins), we examined whether an important lipid second messenger involved in proliferative signaling, 1,2-sn-diacylglycerol, accumulated in liver and resulted in the prolonged activation of protein kinase C. We observed that 1,2-sn-diacylglycerol accumulated in the plasma membrane from the non-tumor portion of livers of rats fed a choline deficient diet, and that unsaturated free fatty acids, another activator of protein kinase C, also accumulated in deficient livers. Protein kinase C in the hepatic plasma membrane and nucleus of choline deficient rats was elevated for months; this is the only model system which exhibits such prolonged activation of protein kinase C. Premalignant, abnormal hepatic foci were detected only in the deficient rats, and 15% of deficient rats (none of the controls) had hepatocellular carcinoma at 1 year on the diet. In rats, an early event in choline deficiency is an increase in the rate of cell death. In liver from choline deficient rats, we observed an increase in the numbers of liver cells with fragmented DNA (characteristic of programmed cell death; apoptosis). We used a cell culture model (immortalized rat hepatocytes) to study the effects of choline deficiency on apoptosis. Liver cells grown in a choline deficient medium became depleted of choline, accumulated triacylglycerol and 1,2-sn-diacylglycerol, and had increased DNA fragmentation and other morphologic and biochemical changes associated with apoptosis. This model has great potential as a tool for studying the underlying link between choline deficiency and the regulation of the balance between cell proliferation and cell death. We suggest that choline deficiency altered the cell proliferation signals mediated by protein kinase C within liver, and altered cell apoptosis. These changes in cell signaling may be the triggering events which result in hepatic carcinogenesis.

Severe folate deficiency causes secondary depletion of choline and phosphocholine in rat liver.

It has previously been shown that choline deficiency causes depletion of hepatic folate concentration in rats. Two separate experiments were undertaken to investigate the converse phenomenon: whether folate deficiency would lead to depletion of hepatic choline. In Experiment 1, severe folate deficiency was induced in rats by feeding an amino acid-defined diet containing (per kg diet) 1.4 g choline, 0 mg folate and 10 g succinylsulfathiazole. Control rats were fed the same diet containing 8 mg folate/kg. After 4 wk, plasma and hepatic folate concentrations were significantly depleted in the severely folate-deficient rats compared with controls (P < 0.001), and hepatic choline and phosphocholine concentrations were 65 and 80% lower, respectively (P < 0.001). In Experiment 2, moderate folate deficiency was induced in rats by feeding the same diet as described above, but with the succinylsulfathiazole omitted. After 24 wk, significant systemic folate deficiency was present in the moderately folate-deficient rats compared with controls (P < 0.001). A modest reduction (36%, P = 0.087) in hepatic choline concentration was observed in the moderately folate-deficient rats compared with controls. No significant differences in hepatic phosphocholine concentrations were detected between the two groups. These results indicate that severe folate deficiency causes secondary hepatic choline deficiency in rats.

Accumulation of 1,2-sn-diradylglycerol with increased membrane-associated protein kinase C may be the mechanism for spontaneous hepatocarcinogenesis in choline-deficient rats.

Choline deficiency, via deprivation of labile methyl groups, is associated with a greatly increased incidence of hepatocarcinoma in experimental animals. This dietary deficiency also causes fatty liver, because choline is needed for hepatic secretion of lipoproteins. We hypothesized that fatty liver might be associated with the accumulation of 1,2-sn-diradylglycerol and subsequent activation of protein kinase C. Several lines of evidence indicate that cancers might develop secondary to abnormalities in protein kinase C-mediated signal transduction. We observed that rats fed a choline-deficient diet for 1, 6, or 27 weeks had increased hepatic concentrations of 1,2-diradylglycerol. At 1 and 6 weeks, hepatic plasma membrane from choline-deficient rats had increased concentrations of 1,2-sn-diacylglycerol and 1-alkyl, 2-acylglycerol, with the latter accounting for 20-26% of membrane 1,2-sn-diradylglycerol (as compared with only 2-5% in controls). Protein kinase C activity was increased in hepatic plasma membrane at 1 week of choline deficiency. By Western blotting there was an increase in the amount of protein kinase C zeta and a decrease in the amount of protein kinase C delta in liver at 1 week. By 6 weeks of choline deficiency, hepatic plasma membrane and cytosolic protein kinase C (PKC) activities were increased significantly, with increased amounts of hepatic plasma membrane protein kinase C alpha, and delta detected by Western blotting. Glycogen synthase activity in liver was diminished after 1 week of choline deficiency; this enzyme is inhibited by PKC-mediated phosphorylation. We suggest that choline deficiency perturbed PKC-mediated transmembrane signaling within liver and that this contributed to the development of hepatic cancer in these animals.

Effects of amiodarone on elastin biosynthesis in primary hamster lung cell cultures.

Amiodarone is a Class III antiarrhythmic agent that has been implicated as a cause of human pulmonary fibrosis. Pulmonary fibrosis is associated with increased levels of connective tissue proteins such as collagen and elastin. The purpose of this investigation was to determine whether elastin synthesis would be altered by in vitro amiodarone administration. Primary hamster lung cell cultures were utilized. Cultures were treated with 2, 10, and 20 micrograms/ml amiodarone. Following treatment, elastin synthesis was monitored by a biochemical tracer assay based on the presence of the cross-linking amino acids: desmosine/isodesmosine. These cross-links are found only in elastin. Addition of [14C] lysine to cultures results in uptake of the radiolabel into the cross-links. Cross-links were isolated and identified using chromatography and electrophoresis. At all doses of amiodarone, elastin synthesis was seen to increase above control levels. Light and electron microscopy confirmed the presence of an extracellular matrix. The morphologic studies also revealed the presence of cytoplasmic inclusion bodies and vacuoles that are often associated with cationic, amphiphilic drugs such as amiodarone.

Choline, an essential nutrient for humans.

Choline is required to make essential membrane phospholipids. It is a precursor for the biosynthesis of the neurotransmitter acetylcholine and also is an important source of labile methyl groups. Mammals fed a choline-deficient diet develop liver dysfunction; however, choline is not considered an essential nutrient in humans. Healthy male volunteers were hospitalized and fed a semisynthetic diet devoid of choline supplemented with 500 mg/day choline for 1 wk. Subjects were randomly divided into two groups, one that continued to receive choline (control), and the other that received no choline (deficient) for three additional wk. During the 5th wk of the study all subjects received choline. The semisynthetic diet contained adequate, but no excess, methionine. In the choline-deficient group, plasma choline and phosphatidylcholine concentrations decreased an average of 30% during the 3-wk period when a choline-deficient diet was ingested; plasma and erthrocyte phosphatidylcholine decreased 15%; no such changes occurred in the control group. In the choline-deficient group, serum alanine aminotransferase activity increased steadily from a mean of 0.42 mukat/liter to a mean of 0.62 mukat/liter during the 3-wk period when a choline-deficient diet was ingested; no such change occurred in the control group. Other tests of liver and renal function were unchanged in both groups during the study. Serum cholesterol decreased an average of 15% in the deficient group and did not change in the control group. Healthy humans consuming a choline-deficient diet for 3 wk had depleted stores of choline in tissues and developed signs of incipient liver dysfunction. Our observations support the conclusion and choline is an essential nutrient for humans when excess methionine and folate are not available in the diet.

Small subunit contacts in ribulose-1,5-bisphosphate carboxylase.

The arrangement of subunits of ribulosebisphosphate carboxylase in solution has been studied by exposing the enzyme to the cross-linking agents tetranitromethane, dimethyl suberimidate, and dimethyl adipimidate, and the cleavable cross-linking agent, methyl 4-mercaptobutyrimidate followed by gel electrophoresis in the presence of dodecyl sulfate. All these agents caused the formation of dimers of the enzyme's small subunit, independently of protein concentration. In addition, trimers and tetramers of small subunit were detected in the mercaptobutyrimidate-treated enzyme. The data show that small subunits are closely paired in the native enzyme and may be in layers of four, or a ring of eight.