The hologenome concept of evolution: do mothers matter most?

The hologenome concept of evolution is discussed, with special emphasis placed upon the microbiome of women. The microbiome is dynamic, changing under different conditions, and differs between women and men. Genetic variation occurs not only in the host, but also in the microbiome by the acquisition of novel microbes, the amplification of specific microbes, and horizontal gene transfer. The majority of unique genes in human holobionts are found in microbiomes, and mothers are responsible for transferring most of these to their offspring during birth, breastfeeding, and physical contact. Thus, mothers are likely to be the primary providers of the majority of genetic information to offspring via mitochondria and the microbiome. TWEETABLE ABSTRACT: Microbiomes differ between women and men. Most genes in humans are in the microbiome. Mothers transfer most of these genes to offspring.

Structural interpretation of the 31P NMR chemical shifts in thiophosphate and phosphate: key effects due to spin-orbit and explicit solvent.

Structural interpretation of the 31P NMR shifts measured in O,O-diethyl thiophosphate (PT), 5,5-dimethyl-2-mercapto-1,3,2-dioxaphosphorinane 2-oxide (cPT), diethylphosphate (P) and 5,5-dimethyl-2-hydroxy-1,3,2-dioxaphosphinane 2-oxide (cP) was obtained by means of theoretical calculations including the effects of geometry, molecular dynamics, and solvent, relativistic effects and the effect of NMR reference. NMR calculations employed the B3LYP, BP86, BPW91, M06-2X, PBE0, MP2, and HF methods, the Iglo-n (n = II, III), cc-pVnZ (n = D, T, Q, 5), and pcS-n (n = 0, 1, 2, 3, 4) Gaussian-type basis sets and the Slater-type QZ4P atomic basis. Water solvent was described explicitly and/or implicitly. The effects due to molecular dynamics were calculated using molecular dynamics simulations with the GAFF force field and the TIP3P water molecules, and alternatively by means of the zero-point ro-vibrational averaging. Relativistic effects included the spin-orbit calculated within the two-component zero-order relativistic approximation and the effect with the four-component DFT method. Optimal geometries and large-amplitude dynamical motions within the "opened" PT and P molecules contrasted with notably different geometries and confined dynamical motions within the cPT and cP "closed" molecules. These structure-dynamical differences together with the different chemical structures of thiophosphate and phosphate due to a non-esterified sulphur or oxygen atom within the group considerably affected the magnitudes of 31P NMR shifts. The theoretical calculations enabled accurate and reliable structure-dynamical interpretation of the measured 31P NMR shifts. The effects due to explicit solvent and relativity turned out to be indispensable for obtaining accurate 31P NMR shifts particularly in the thiophosphates. Replacement of the non-esterified oxygen atom in the phosphate with sulphur makes NMR shielding of the phosphorus atom qualitatively different as compared to the NMR shielding of the phosphorus atom in phosphate, H3PO4 and PH3.

Distinct alkaline phosphatase in serum of patients with lymphatic leukemia and infectious mononucleosis.

A distinct alkaline phosphatase (phosphatase N) was demonstrated in the serum of patients with acute lymphatic leukemia, chronic lymphatic leukemia, and infectious mononucleosis. This enzyme closely resembles that extracted from the thymus of mice with lymphoma or lymphatic leukemia, both in its electro-phoretic mobility and its substrate specificity. The phosphatase N activity was related to the clinical state of patients with lymphatic leukemia and disappeared with recovery from infectious mnononucleosis.

Similarity of cruzin, an inhibitor of Trypanosoma cruzi neuraminidase, to high-density lipoprotein.

A specific inhibitor of the neuraminidase of the protozoan parasite Trypanosoma cruzi was isolated recently and named cruzin. It is now shown that cruzin is similar to high-density lipoprotein by amino acid homology, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by immunoblot analysis, and by isoelectric focusing. Cruzin purified by ion exchange chromatography and high-density lipoprotein isolated by density gradient ultracentrifugation inhibited Trypanosoma cruzi neuraminidase to the same extent. Cruzin or high-density lipoprotein restores to normal the decreased multiplication rate of Trypanosoma cruzi epimastigotes grown in a medium depleted of lipoproteins, suggesting that it may be important for survival of the parasite in nature.

Intestinal transport of sugars and amino acids in diabetic rats.

The specificity and mechanism of altered intestinal transport of diabetic rats was studied with an everted ring technique. Increased intracellular accumulation of amino acids, as well as galactose and 3-O-methylglucose, was demonstrated in diabetes. The greater accumulation by diabetic intestine could not be attributed to a direct effect of the agent used to induce diabetes or to an alteration in food consumption. Although the changes were related to the severity of diabetes and could be reversed with treatment with insulin, they could not be modified by addition of insulin in vitro. The changes could not be induced in control intestine either with hyperglycemia from glucose infusion or preincubation with glucose in vitro. Although the higher concentration gradients of amino acids, galactose, and 3-O-methylglucose could result from increased energy utilization by diabetic intestine, an alteration of cell membrane function, as well, is suggested by the demonstration with kinetic studies of increased influx with an increase in V(max).

Iodothyronine 5'-deiodinase in rat kidney microsomes. Kinetic behavior at low substrate concentrations.

The thiol-activated enzymatic outer-ring monodeiodination of iodothyronines by rat kidney microsomes at low (nanomolar) substrate concentrations shows an apparently sequential reaction mechanism and is further characterized by insensitivity to inhibition by dicoumarol, a moderate sensitivity to inhibition by propylthiouracil (Ki = 100 microM) and iopanoic acid (Ki = 0.9 mM), responsiveness to 5 mM glutathione (GSH), and a thermal activation profile that is concave downward with a Td of approximately 20 degrees C. In contrast, the activity at high (micromolar) substrate concentrations shows a ping-pong reaction mechanism, is inhibited by micromolar concentrations of propylthiouracil, iopanoic acid and dicoumarol, is unresponsive to 5 mM GSH, and shows a concave upward thermal activation profile. Analysis of the microsomal deiodinase reaction over a wide range of 3,3',5'-triiodothyronine (rT3) concentrations (0.1 nM to 10 microM) suggested the presence of two enzymatic activities, with apparent Michaelis constants (Km) of 0.5 microM and 2.5 nM. Lineweaver-Burk plots of reaction velocities at nanomolar substrate concentrations in presence of 100 microM propylthiouracil also revealed an operationally distinct enzymatic activity with Km's of 2.5 and 0.63 nM and maximum velocities (Vmax's) of 16 and 0.58 pmol/mg protein per h for rT3 and thyroxine (T4), respectively. These findings are consistent with the presence of a low Km iodothyronine 5'-deiodinase in rat kidney microsomes distinct from the well characterized high Km enzyme and suggest that at circulating levels of free T4 the postulated low Km enzyme could be physiologically important.

Inhibition of folate enzymes by sulfasalazine.

Sulfasalazine (salicylazosulfapyridine), an agent widely used for the treatment of ileitis and colitis, is also a competitive inhibitor of intestinal folate transport (1, 2). The mechanism of action of sulfasalazine remains uncertain. To further explore the mechanism of sulfasalazine action, the interaction of the drug with the folate recognition site was tested with three enzymes: dihydrofolate reductase, methylenetetrahydrofolate reductase, and serine transhydroxymethylase, each catalyzing a reaction involving a different folate derivative. Each of these enzymes was inhibited by sulfasalazine in the same concentration range as that previously observed to inhibit intestinal folate transport; the kinetic data are consistent with a competitive mode of inhibition. Specificity of inhibition was demonstrated by the finding that the reduction of the pteridine ring of pteroylheptaglutamic acid by dihydrofolate reductase was subject to inhibition, whereas the hydrolysis of the gamma-glutamyl peptide side chain by chicken pancreas conjugase was not affected. These results are interpreted to indicate that sulfasalazine interferes with a folate recognition site which is common to these enzymes and to the intestinal transport system. Sulfasalazine, therefore, has certain properties of an antifolate drug.

Rheumatoid cachexia: cytokine-driven hypermetabolism accompanying reduced body cell mass in chronic inflammation.

The cytokines IL-1 beta and TNF-alpha cause cachexia and hypermetabolism in animal models, but their role in human inflammation remains controversial. The relationship between in vitro cytokine production and metabolism was examined in 23 adults with RA and 23 healthy control subjects matched on age, sex, race, and weight. Body composition was measured by multicompartmental analysis of body cell mass, water, fat, and bone mass. Resting energy expenditure (REE) was measured by indirect calorimetry. Cytokine production by PBMC was measured by radioimmunoassay. Usual energy intake, physical activity, disability scores, medication use, and other confounders were also measured. Body cell mass was 13% lower (P < 0.00001), REE was 12% higher (P < 0.008), and physical activity was much lower (P < 0.001) in subjects with RA. Production of TNF-alpha was higher in RA than controls, both before and after stimulation with endotoxin (P < 0.05), while production of IL-1 beta was higher with endotoxin stimulation (P < 0.01). In multivariate analysis, cytokine production was directly associated with REE (P < 0.001) in patients but not in controls. While energy and protein intake were similar in the two groups and exceeded the Recommended Dietary Allowances, energy intake in subjects with RA was inversely associated with IL-1 beta production (P < 0.005). In this study we conclude that: loss of body cell mass is common in RA; cytokine production in RA is associated with altered energy metabolism and intake, despite a theoretically adequate diet; and TNF-alpha and IL-1 beta modulate energy metabolism and body composition in RA.

Multivitamin use and B vitamin status in a homebound elderly population.

OBJECTIVE: Homebound elderly are at increased risk for micronutrient deficiencies and nutritional status in this population has not been adequately described. There is evidence for beneficial effects of multivitamin use and a greater understanding of their nutritional contribution could identify behaviors that may help alleviate excess chronic disease. The purpose of this analysis is to investigate, in a racially diverse group of homebound elders, the association of multivitamin use with measures of plasma B vitamin concentrations. DESIGN: We examined the cross-sectional association between multivitamin use and plasma concentrations of B vitamins and homocysteine in 236 white and 182 black homebound elders (65-99y). Dietary intake was assessed and demographic and health information was ascertained. RESULTS: White and black elders had a high prevalence of dietary intakes below the Estimated Average Requirement for folate (38.1 and 40.7%), vitamin B6 (16.9 and 19.2%.), and vitamin B12 (3 and 3.9%) respectively. Multivitamin use was associated with higher mean plasma B vitamin concentrations in each group. In whites, multivitamin users had higher concentrations of vitamin B6 (64.6 vs. 32.4 nmol/L; p < 0.001), vitamin B12 (398 vs. 324 pmol/L;p < 0.001) and folate (39.4 vs. 30.4 nmol/L;p < 0.001). Black multivitamin users had higher concentrations of vitamin B6 (53.7 vs. 29.5 nmol/L; p < 0.001), B12 (427 vs. 372 pmol/L; p < 0.05) and folate (35.7 vs. 25.4 nmol/L; < 0.001) than non-users. CONCLUSIONS: Multivitamin supplementation was associated with higher mean plasma concentrations of vitamins B6, B12, and folate and lower prevalence of low plasma B vitamin status in a biracial homebound elderly.

B-Vitamin Therapy for Kidney Transplant Recipients Lowers Homocysteine and Improves Selective Cognitive Outcomes in the Randomized FAVORIT Ancillary Cognitive Trial.

BACKGROUND: Objectives: Elevated plasma total homocysteine (tHcy) is associated with increased risk of cardiovascular disease, stroke and dementia. Results of clinical trials using B-vitamins to reduce the cognitive risks attributed to tHcy have been inconsistent. The high prevalence of both hyperhomocysteinemia and cognitive impairment among kidney transplant recipients makes them an important population in which to evaluate the effect of lowering homocysteine on cognitive function. We therefore evaluated whether B-vitamin therapy to lower tHcy would prevent cognitive-decline in a cohort of stable kidney transplant recipients. DESIGN: The study was a longitudinal ancillary of the FAVORIT trial, a randomized, placebo-controlled multi-site trial of high-dose B vitamins to reduce cardiovascular and cerebrovascular events in clinically stable kidney transplant recipients with elevated tHcy. PARTICIPANTS: 584 participants from 18 sites across North America. INTERVENTION: The intervention consisted of a daily multivitamin containing high-doses of folate (5.0 mg), vitamin B12 (1.0 mg) and vitamin B6 (50 mg). The placebo consisted of a daily multi-vitamin containing no folate and recommended daily allowances of vitamins B12 and B6 (0 mg folate; 2.0 µg vitamin B12; 1.4 mg vitamin B6). MEASUREMENTS: Annual neuropsychological assessment for up to 5 years (mean 3.3 years) using a standardized test battery. Efficacy was analyzed on an intention-to-treat basis using end-of-trial data. Subgroup analyses included stratification for baseline plasma B-vitamin and tHcy concentrations. RESULTS: At baseline, cognitive impairment was common with 61% of participants falling more than one standard deviation below published norms for at least one cognitive test. Fewer than 1% of participants had insufficient plasma folate < 5 ng/ml or vitamin B12 < 148 pmol/L. However, 44.6% had plasma B6 concentrations < 30 nmol/L. At follow-up, processing speed and memory scores were modestly but significantly better in the B-vitamin supplement group than in controls (p≤0.05). There was no interaction between baseline tHcy, B-vitamin status and treatment on the cognitive outcomes. CONCLUSIONS: High-dose B-vitamin supplementation provided modest cognitive benefit for kidney transplant recipients with elevated baseline tHcy. Since nearly all participants were folate and vitamin B12 sufficient at baseline, the potential cognitive benefits of folate and B12 supplementation in individuals with poor B-vitamin status remains to be determined.

-CH2- lengthening of the internucleotide linkage in the ApA dimer can improve its conformational compatibility with its natural polynucleotide counterpart.

The complete family of ApA phosphonate analogues with the internucleotide linkage elongated by insertion of a -CH2- group was prepared and the hybridisation and structural properties of its members in interaction with polyuridylic acid were investigated using an original 2D Raman approach. Except for the conformationally restricted A(CH)pA(2'3'endo-5') modification, all of the isopolar, non-isosteric analogues form triplex-like complexes with poly(rU) at room temperature, in which two polymer strands are bound by Watson-Crick and Hoogsteen bonds to a central pseudostrand consisting of a 'chain' of A-dimers. For all of these dimers, the overall conformation of the triplexes was found to be similar according to their extracted Raman spectra. A simple semi-empirical model was introduced to explain the observed dependency of the efficiency of triplex formation on the adenine concentration. Apparently, for most of the modifications studied, the creation of a stable complex at room temperature requires the formation of a central pseudostrand, consisting of several adenine dimers. Molecular dynamics calculations were finally performed to interpret the differences in 'cooperative' behaviour between the different dimers studied. The results indicate that the exceptional properties of the Ap(CH2)A(3'-5') dimer could be caused by the 3D conformational compatibility of this modified linkage with the second (Hoogsteen) poly(rU) strand.

Magnetic resonance imaging in the evaluation of temporomandibular joint disc displacement--a review of 144 cases.

One hundred and forty four patients underwent magnetic resonance imaging (MRI) for evaluation of suspected internal derangement (ID) of the temporomandibular joint (TMJ). All scans were performed on a state-of-the-art scanner by highly experienced technologists and evaluated by a single Head and Neck/Maxillofacial radiologist. Seventy-nine percent of patients were female and 21% male. Age distribution of the cases was bi-modal with first peak at 20-30 years of age and second peak at 50-60 years of age. Of the 82.5% of cases with disc displacement, 59.5% demonstrated reduction with opening and 40.5% did not reduce. Anterior disc displacement is common (44%) and sideways displacement rare (4%). Antero-lateral displacement was the second commonest type of displacement (29%) probably related to the weakness of the lateral disc attachment.

Transformation of C127 mouse fibroblasts by human papillomavirus 16.

We have compared the ability of cloned DNAs of HPV16, a human papillomavirus associated with cervical carcinoma, and BPV1, a papillomavirus inducing skin lesions in cattle, to transform murine C127 cells. Unlike BPV1, HPV16 DNA failed to induce foci when C127 cells were transfected and maintained as monolayers; HPV16-transformed C127 cells could only be detected after cotransfection with HPV16 and pSV2neo DNA, selection for resistance to G418, and assay of pooled selectants for colony growth in agar. HPV16 and BPV1 C127 cells differed in terms of the size and morphology of their colonies in agar, but not in their colony-forming efficiencies. In addition, the tumors they induced in nude mice were clearly histologically distinct, with the HPV16 C127 tumors considerably more anaplastic. The HPV16 C127 cells contained viral DNA at high copy numbers integrated at random sites in the C127 genome, while the BPV1 C127, as expected, contained episomal BPV1 DNA molecules. The high complexity of the integrated HPV16 DNA was maintained in the pooled cells grown through extended passage in vitro, in clonal lines derived from single agar colonies, in nude mouse tumors induced by the cells, and in a nude mouse tumor-derived cell line, indicating the stability of the HPV16 sequences in the cells. HPV16 transcripts in the transformed C127 cells were present in three size classes (1.5, 2, and 4 kilobases) on Northern blots. The different transformed phenotypes in the same cell line induced by two structurally similar, yet distinct viruses imply differences in the underlying transforming mechanisms and possible different virus-host cell molecular interactions.

Identification of a novel variant form of fibroblast growth factor receptor 3 (FGFR3 IIIb) in human colonic epithelium.

Although several tyrosine kinase-type growth factor receptors have been demonstrated in human colonic epithelial cells, the full spectrum of growth factor receptors has not been identified. Low stringency screening of a complementary DNA library prepared from the human colon cancer-derived cell line HT-29 with a probe containing the tyrosine kinase domain of human c-src kinase led to the identification and isolation of a clone containing a receptor class tyrosine kinase. This putative receptor was found to be identical to the human fibroblast growth factor receptor 3 (FGFR3) except for a region of 150 nucleotides (50 amino acids) encoding the presumptive ligand-binding domain, where it exhibited only 32% homology with the previously described FGFR3. The variant domain corresponded precisely to the splicing junctions of the exon encoding the carboxyl terminal half of the third immunoglobulin-like domain, suggesting that two forms of FGFR3 result from splicing of alternate exons in a manner similar to that previously found for FGFR1 and FGFR2. By prior convention, the previously reported from of FGFR3 was designated IIIc due to its high degree of homology with the IIIc domain of FGFR1 (83% homology) and the IIIc domain of FGFR2 (81% homology). However, the ligand-binding domain of FGFR3 found in the HT-29 cell line was more highly divergent from all previously reported FGFR immunoglobulin-like domain IIIs than any other two members of this receptor family. Therefore, we propose to designate the newly reported form as the FGFR3 IIIb variant. Genomic polymerase chain reaction confirmed that the IIIb-containing exon occupies a position 5' relative to the IIIc-containing exon within the FGFR3 gene. Northern blot analysis using a probe encompassing sequences unique to the FGFR3 IIIb mRNA confirmed the expression of a 4.4-kilobase transcript in two colon cancer-derived cell lines as well as normal human colonic mucosa. Using a technique combining reverse transcriptase polymerase chain reaction with restriction endonuclease digestion, cell lines, primary cells, and tissues were assessed for IIIb and IIIc transcripts; expression of the IIIb variant was associated with an epithelial lineage, while the IIIc variant was expressed predominantly in nonepithelial cells and tissues.

Folate deficiency enhances the development of colonic neoplasia in dimethylhydrazine-treated rats.

In patients with ulcerative colitis, epidemiological work has suggested an association between low folate status and an increased risk of colonic neoplasia. The aim of the present study was to determine if experimental folate deficiency increases the likelihood of developing neoplasia in rats treated with the carcinogen dimethylhydrazine. Weanling male Sprague-Dawley rats were fed with an amino acid-defined diet containing either 8 or 0 mg/kg folic acid. After 5 weeks of defined diet, weekly s.c. injections of dimethylhydrazine (20 mg/kg) were administered to both groups. Serum, whole blood, liver, and colonic folate concentrations at the time of sacrifice were significantly lower in folate-depleted animals (P less than 0.001). There were significant differences in the incidence of colonic neoplasia between the two groups after 20 weeks of dimethylhydrazine exposure: folate-deficient rats had a greater incidence of dysplasia (6 of 7 versus 2 of 7 animals; P less than 0.05) and carcinoma (6 of 7 versus 1 of 7 animals; P less than 0.01). Furthermore, a significantly greater proportion of folate-replete rats than folate-deficient rats were free of neoplastic lesions (5 of 7 versus 0 of 7 animals; P less than 0.05). These results suggest that, in this animal model, folate deficiency increases the risk of malignancy when there is an underlying predisposition to colorectal cancer.

Solubilization of a phospholipid-requiring enzyme, iodothyronine 5'-deiodinase, from rat kidney membranes.

Enzymic activities catalyzing the reductive 5'-deiodination of thyroxine and 3,3',5'-triiodothyronine were solubilized from rat kidney microsomes by treatment with 0.2% deoxycholate. Deoxycholate reversibly inhibited the enzyme(s); removal of detergent restored activity and resulted in the formation of enzymatically active aggregates with a buoyant density of 1.17 g/ml resembling that of membranes. Fractionation of the solubilized membrane components in the presence of 0.2% deoxycholate by either gel filtration or sucrose gradient centrifugation inactivated the enzyme(s) and activity could be restored by the addition of partially purified soybean phospholipids; this allowed some of the physical properties of the enzyme(s) to be determined. 5'-Deiodinating activity of both thyroxine and 3,3',5'-triiodothyronine was associated with protein(s) with S20,W of 3.5 S, Stokes' radius of 32 A, and a calculated molecular weight of 49 900. A partial specific volume of 0.74 cm3/g was calculated from sedimentation in 2H2O and H2O sucrose gradients. Phospholipid reactivation of lipid-depleted enzyme preparations was concentration-dependent, with near maximal restoration when sufficient phospholipid was added to restore the phospholipid:protein ratio to that of thyroxine and 3,3',5'-triiodothyronine could not be resolved by sedimentation or molecular sieving and showed similar behavior toward deoxycholate solubilization and phospholipid reconstitution.

Effect of anions on folate binding by isolated brush border membranes from rat kidney.

The characteristics of folate binding by brush border membranes from rat kidney homogenates were investigated. At pH 7.4, binding of [3',5',9-3H]-pteroylglutamic acid to membranes containing endogenous folate is inhibited by anions, with chloride being most effective followed by bromide, thiocyanate, iodide, phosphate and sulfate. A maximum inhibition of 70-75% is attained at a concentration of 0.1 M chloride and an incubation time of 30 min. The inhibition diminishes with increased incubation time and at 24 h is negligible. The binding of [3',5',9-3H]pteroylglutamic acid to brush border membranes stripped of endogenous folate by acid treatment is not inhibited by anions. Anion sensitivity can be restored to these treated membranes by reconstitution with membrane-derived folate, particularly 5-methyltetrahydropteroyl-glutamic acid, or by preincubation with synthetic 5-methyltetrahydropteroyl-glutamic acid. Inhibition of [3',5',9-3H]pteroylglutamic acid binding by anions in membranes with endogenous folate is best explained by an anion-induced stabilization of endogenous folate-binding protein complex resulting in a decreased rate of exchange with exogenous [3',5',9-3H]pteroylglutamic acid.

Carrier affinity as a mechanism for the pH-dependence of folate transport in the small intestine.

A mildly acidic pH in the lumen of the small intestine markedly enhances the transport of folate. This study investigated the relationship between pH and the affinity between folic acid and the apical membrane transporter using brush border membrane vesicles from rat jejunum and differentiated monolayer cultures of the colon carcinoma cell line, CaCo-2. Uptake studies with BBMV were conducted at folic acid concentrations of 0.1 to 50 mumol/l, conditions which were suitable for analyzing uptake data based on the Michaelis-Menten equation modified to include a nonsaturable component. These analyses yielded apparent Km values of 0.6 and 12.3 microM at pH 5.5 and pH 7.4, respectively (P less than 0.05). Values for Vmax were lower at pH 5.5 than at pH 7.4 (0.8 vs. 1.6 pmol/mg protein per 10 s, P less than 0.05). The studies with CaCo-2 cells employed folic acid concentrations of 0.1 to 5 mumol/l. Under these conditions the apparent Km for folic uptake was lowest at pH 6.0, where the Km was 0.7 mumol/l. The apparent Km increased sharply as a neutral pH was approached; reaching a value of 13.9 mumol/l at pH 7.1. These data suggest that the prominent pH effect on intestinal folate transport is, in part, explained by an increased affinity of the folate substrate for its membrane transporter.

The interaction of protein kinase C and other specific cytoplasmic proteins with phospholipid bilayers.

The role of lipid composition in the interaction of purified protein kinase C with large unilamellar vesicles was determined by the extent of photolabelling of the enzyme with 5-[125I]iodonaphthalene-I-azide. The protein kinase C was only slightly labelled when exposed to phosphatidylcholine (PC) liposomes. The addition of phorbol 12-myristate 13-acetate (PMA) or of diacylglycerol to the PC liposomes enhanced significantly the labelling of the protein kinase C at low calcium concentrations. A further enhancement in the photolabelling of the protein kinase C was observed in liposomes containing 2% phosphatidylserine (PS). At low calcium concentrations, the binding of the enzyme to these liposomes increased in the presence of added PMA or diacylglycerol. Raising the levels of PS beyond 2% in the liposomes did not enhance the binding of the protein kinase C. However, when the enzymatic activity of the protein kinase C was measured using basic histones as substrates, maximum phosphorylation was obtained in liposomes with a PC to PS ratio of 1. The fact that the translocation of the protein kinase C from solution to the surface of the liposomes could be monitored by its labelling with 5-iodonaphthalene 1-azide prompted us to determine whether other cytoplasmic proteins might share this property. The interaction of cytoplasmic proteins from HeLa cells with PC liposomes gave trace labelling irrespective of whether calcium was added. When the HeLa cell cytoplasmic proteins were allowed to interact with liposomes containing PS, selective 5-iodonaphthalene-1-azide photolabelling was observed in distinct proteins. Addition of calcium and of PMA or diacylglycerol modified the labelling of some but not all of these proteins. These results suggest that the methodology developed might serve to identify proteins that move to the membrane during stimulation of cells by phorbol esters or by growth factors which induce the generation of diacylglycerol. These results also suggest a role for the phospholipid composition of the plasma membrane (or any intracellular membrane) in the modulation of the activation processes of specific phospholipid-dependent proteins, in particular protein kinase C.

Effect of 1-oleoyl-2-acetylglycerol and other lipids on phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in cultured type II pneumocytes.

We previously reported that addition of phosphatidylglycerol to the culture medium stimulates phosphatidylcholine synthesis and cholinephosphate cytidylyltransferase activity in type II pneumocytes. In view of the known biological effects of diacylglycerols and since phosphatidylglycerol could be metabolized to diacylglycerol, we now examined the effects of diacylglycerols on the same parameters. The rate of choline incorporation into phosphatidylcholine was increased 30-60% by 10 microM phosphatidylglycerol, diolein, mixed diacylglycerols and 1-oleoyl-2-acetylglycerol (OAG). The effects of phosphatidylglycerol and OAG were not additive, suggesting a similar mechanism of action. The diacylglycerols and phosphatidylglycerol increased the activity of cholinephosphate cytidylyltransferase in type II cell sonicates by 35-50%, but had no effect on the activities of choline kinase, cholinephosphotransferase or 1-acylglycerophosphocholine acyltransferase. Again, the effects of OAG and phosphatidylglycerol on cytidylyltransferase were not additive. It is known that addition of lipids to the assay mixture increases the activity of cholinephosphate cytidylyltransferase in vitro and inclusion of the above lipids (1.1 mM) in the in vitro assay mixture increased cytidylyltransferase activity in type II cell sonicates. In addition, the stimulatory effects of OAG and of diolein, as well as of phosphatidylglycerol as reported previously, in the culture medium on cytidylyltransferase activity in type II cells were diminished or abolished when the assay was carried out in the presence of sufficient amounts of the same lipids to stimulate maximally the activity in vitro. These data show that lipids in the culture medium stimulate phosphatidylcholine biosynthesis in type II cells by direct activation of cholinephosphate cytidylyltransferase.