Macrophage membrane (MMs) camouflaged near-infrared (NIR) responsive bone defect area targeting nanocarrier delivery system (BTNDS) for rapid repair: promoting osteogenesis via phototherapy and modulating immunity.

Bone defects remain a significant challenge in clinical orthopedics, but no targeted medication can solve these problems. Inspired by inflammatory targeting properties of macrophages, inflammatory microenvironment of bone defects was exploited to develop a multifunctional nanocarrier capable of targeting bone defects and promoting bone regeneration. The avidin-modified black phosphorus nanosheets (BP-Avidin, BPAvi) were combined with biotin-modified Icaritin (ICT-Biotin, ICTBio) to synthesize Icaritin (ICT)-loaded black phosphorus nanosheets (BPICT). BPICT was then coated with macrophage membranes (MMs) to obtain MMs-camouflaged BPICT (M@BPICT). Herein, MMs allowed BPICT to target bone defects area, and BPICT accelerated the release of phosphate ions (PO43-) and ICT when exposed to NIR irradiation. PO43- recruited calcium ions (Ca2+) from the microenvironment to produce Ca3(PO4)2, and ICT increased the expression of osteogenesis-related proteins. Additionally, M@BPICT can decrease M1 polarization of macrophage and expression of pro-inflammatory factors to promote osteogenesis. According to the results, M@BPICT provided bone growth factor and bone repair material, modulated inflammatory microenvironment, and activated osteogenesis-related signaling pathways to promote bone regeneration. PTT could significantly enhance these effects. This strategy not only offers a solution to the challenging problem of drug-targeted delivery in bone defects but also expands the biomedical applications of MMs-camouflaged nanocarriers.

Effects of cecal oxytetracycline infusion, and dietary avidin and biotin supplementation on the biotin status of nongravid gilts.

The objective of this 49-d experiment was to test effects of cecal oxytetracycline (OTC) infusion, and dietary avidin and biotin supplementation on the biotin status of nongravid gilts. Twenty-eight crossbred gilts with an initial age of 160 d and BW of 120 kg were surgically fitted with a T-cannula in the terminal ileum, a cecal fistula, and an indwelling catheter in the anterior vena cava, and allotted to 7 dietary treatments. Treatments with the basal semipurified (SP) diet fed at 1.86 kg/d were: SP-1, negative control; SP-2, positive control with 270 µg of biotin/kg; SP-3, with spray-dried egg albumen (EA, 100 g/d) and OTC (2.56 g/d by cecal infusion); and SP-4, with EA, OTC, and 700 µg of biotin/kg. Treatments with the basal corn-soybean meal (CS) diet fed at 1.80 kg/d were: CS-1, negative control; CS-2, with EA and OTC; and CS-3, with EA, OTC, and 700 µg of biotin/kg. Response criteria were: fecal bacteria counts; plasma concentrations of biotin, glucose, and urea N (PUN); liver pyruvate carboxylase (PC) activity; kidney and epithelial tissue histology; ileal and fecal biotin concentrations; ileal and total tract N and energy utilization; daily gilt observation; and BW gain. Blood samples were collected every 7 d with serial samples collected on d 49. Total urine collections and fecal grab samples were made twice daily from d 44 to 49. Gilts were killed on d 50 and liver, kidney, and skin samples were collected. No gilts had symptoms of biotin deficiency. There were no treatment differences in BW gain, plasma glucose concentrations, liver PC activity, kidney and epithelial tissue histology, or fecal bacteria counts. Ileal and total tract N and energy digestibilities (%) did not differ among treatments within the same protein source, with greater (P ≤ 0.05) values for gilts on the SP treatments than the CS treatments. However, N retained/N absorbed and N retained/N intake (%) were less (P ≤ 0.05) and PUN concentrations were greater (P ≤ 0.05) for SP treatments with cecal OTC infusion. The overall fecal biotin concentration mean was 2.6-fold greater than the overall ileal biotin concentration mean. In conclusion, no gilts in the current experiment became biotin deficient because the biotin requirements were met primarily by microbial synthesis and absorption of biotin from the distal small intestine and large intestine, with corn and soybean meal contributing endogenous biotin. Therefore, supplementation of diets for gilts entering the breeding herd with 100% of the current NRC biotin requirement for sows is adequate.

Preclinical pharmacology and safety of a novel avidin derivative for tissue-targeted delivery of radiolabelled biotin.

We recently described an oxidized avidin variant, named AvidinOX(®) , which is a product that chemically links to tissue proteins while maintaining the capacity to uptake intravenously administered biotin. Such product proved to be successful in targeting radionuclide therapy in a mouse model of inoperable breast cancer. Here, we show that the uptake of a single or multiple doses of biotin (up to five times), by the tissue-bound AvidinOX(®) , is stable for 2 weeks. Taking into account that oxidized avidin is the first chemically reactive protein to be proposed for clinical use, we evaluated its tolerability, immunogenicity and mutagenicity. Present in vitro data indicate that AvidinOX(®) (up to 10 µg/5 × 10(5) cells) does not affect cell viability or proliferation of PC3 human prostate cancer or 3T3 mouse fibroblast cell lines as well as primary mouse spleen cells. Safety pharmacology and toxicology studies were conducted using AvidinOX(®) up to the highest concentration compatible with its solubility (about 12 mg/mL), representing four times the product concentration intended for human use, and in the maximum administrable volume compatible with each study system. The intramuscular administration in rat and monkey induced a moderate to strong inflammatory response particularly after a second administration and consistently with the induction of an immune response. Interestingly, the intramuscular administration of AvidinOX(®) to rodents and monkeys exhibiting very high anti-avidin antibody titres was well tolerated with no systemic symptoms of any kind. Intravenous administration of AvidinOX(®) , performed to mimic an accidental injection of the dose intended for a local administration (15 µL of 3.3 mg/mL solution), showed significant localization of the product into the spleen not associated with uptake of the radiolabelled biotin intravenously injected after 24 hr, thus suggesting rapid inactivation. No mutagenic activity was induced by oxidized avidin in prokaryotic and eukaryotic cells. Overall, the present data indicate that AvidinOX(®) is well tolerated in rodents and non-human primates, thus supporting its clinical use within protocols of radionuclide therapy of inoperable tumour lesions.

The use of biotin-binding proteins for insect control.

Biotin-binding proteins (BBPs), expressed in transgenic plants, are insecticidal to a very wide range of insects. The expression levels required are generally low (approximately 100 ppm), and although higher than required for Bacillus thuringiensis (Bt) delta-endotoxins, BBPs are effective across a broader range of insect orders and other invertebrates than the Bt Cry proteins. Avidin and streptavidin, in particular, have been reported as causing death or severe growth reduction in at least 40 species of insects across five insect orders (Lepidoptera, Coleoptera, Orthoptera, Diptera, and leaf-eating Hymenoptera) and mites. In addition, due largely to its rapid dilution in ecosystems, no adverse impacts on nontarget microorganisms or invertebrates have been recorded. Because the target, biotin, cannot itself be modified to prevent it binding to BBPs and remain effective as a vitamin, the major avenue open to insects to develop resistance is unavailable. Two properties of the biotin-avidin complex make it highly suitable for use in transgenic plant crop protection strategies against a large range of insects; its extreme stability and its resistance to proteolysis. However, because the nutritional value of the plant could potentially be compromised in the absence of biotin supplementation, its use in nonfood crops such as fiber, forestry, and biofuel crops is seen as the most suitable initial focus for this technology.

Insecticidal activity of recombinant avidin produced in yeast.

An expression construct encoding chicken (Gallus gallus) avidin was assembled from amplified fragments of genomic DNA. Recombinant, functional avidin was produced in Pichia pastoris, with yields of up to 80 mg/l of culture supernatant. The recombinant avidin had similar insecticidal activity to egg white avidin when assayed against larvae of a lepidopteran crop pest, cabbage moth (Mamestra brassicae), causing >90% reduction in growth and 100% mortality when fed in optimised diets at levels of 1.5 microM and 15 microM (100 ppm and 1000 ppm wet weight of recombinant protein). The recombinant protein was also highly toxic to a hemipteran pest, the pea aphid (Acyrthosiphon pisum), when fed in liquid artificial diet, causing 100% mortality after 4 days when present at concentrations > or = 3.8 microM (0.25 mg/ml, 250 ppm). Mortality was dose-dependent, with an estimated LC(50) of 2.1 microM. Toxicity to A. pisum was prevented by biotin supplementation of diet. In contrast, avidin had no significant effects on the survival of cereal aphid (Sitobion avenae) at concentrations up to 30 microM in liquid diet. Analysis of genomic DNA showed that symbionts from both aphid species lack the ability to synthesise biotin de novo. Cereal aphids appear to be less sensitive to recombinant avidin in the diet through proteolysis of the ingested protein, which would allow recovery of bound biotin.

Development of idraparinux and idrabiotaparinux for anticoagulant therapy.

Idraparinux is an analogue of fondaparinux binding with high affinity to antithrombin. It was designed for weekly, rather than daily, administration, with an exceptionally long half-life. One potential problem with small heparin-like fragments of this type is the difficulty of neutralising excessive activity in the case of side-effects or overdose. The efficacy of idraparinux was was proven in clinical studies with patients suffering from venous thromboembolism (VTE) or atrial fibrillation. Due to major bleeding events during treatment for more than six months the development of idraparinux was stopped. Idrabiotaparinux has an attached biotin moiety at the non-reducing end unit, which allows its neutralisation with avidin, an egg-derived protein with low antigenicity. This compound is currently investigated in clinical trials for prevention of recurrent VTE in patients with acute pulmonary embolism. The future of idrabiotaparinux depends also on the safety and efficacy of avidin.

Molecular events contributing to cell death in malignant human hematopoietic cells elicited by an IgG3-avidin fusion protein targeting the transferrin receptor.

We have previously reported that an anti-human transferrin receptor IgG3-avidin fusion protein (anti-hTfR IgG3-Av) inhibits the proliferation of an erythroleukemia-cell line. We have now found that anti-hTfR IgG3-Av also inhibits the proliferation of additional human malignant B and plasma cells. Anti-hTfR IgG3-Av induces internalization and rapid degradation of the TfR. These events can be reproduced in cells treated with anti-hTfR IgG3 cross-linked with a secondary Ab, suggesting that they result from increased TfR cross-linking. Confocal microscopy of cells treated with anti-hTfR IgG3-Av shows that the TfR is directed to an intracellular compartment expressing the lysosomal marker LAMP-1. The degradation of TfR is partially blocked by cysteine protease inhibitors. Furthermore, cells treated with anti-hTfR IgG3-Av exhibit mitochondrial depolarization and activation of caspases 9, 8, and 3. The mitochondrial damage and cell death can be prevented by iron supplementation, but cannot be fully blocked by a pan-caspase inhibitor. These results suggest that anti-hTfR IgG3-Av induces lethal iron deprivation, but the resulting cell death does not solely depend on caspase activation. This report provides insights into the mechanism of cell death induced by anti-TfR Abs such as anti-hTfR IgG3-Av, a molecule that may be useful in the treatment of B-cell malignancies such as multiple myeloma.

Insecticidal activity of avidin combined with genetically engineered and traditional host plant resistance against Colorado potato beetle (Coleoptera: Chrysomelidae) larvae.

Colorado potato beetle, Leptinotarsa decemlineata (Say), is a destructive pest of potato, Solanum tuberosum (L.), in North America. It is renowned for adapting to insecticides. With the arsenal of effective insecticides decreasing, it is important to consider alternative forms of control. Biotin is an essential coenzyme for insect growth and development. Avidin is a protein found in chicken egg that sequesters biotin and has shown insecticidal properties against a range of insect. We assessed the effectiveness of avidin against the Colorado potato beetle neonates in a no-choice detached leaf bioassay at 0, 17, 34, 51, 102, and 204 microg avidin/ml over 12 d. The LC50 was 136 microg avidin/ml (108-188 95% CL). The combined effects of avidin (136 microg avidin/ml) with Bt-Cry3A or leptines were evaluated with neonates and third instars over 12 and 6 d, respectively. Three potato lines were used: susceptible line, a line engineered to express Cry3A from Bacillus thuringiensis, and a line expressing the natural resistance factor leptines. The addition of avidin at the LC50 concentration significantly reduced consumption by neonates, but it did not affect consumption by third instars feeding on the susceptible line and the leptine line. Survival of neonates feeding on the susceptible line with avidin was significantly reduced compared with the susceptible line. Survival of third instars on the Bt-Cry3A with avidin was significantly reduced after 3 d compared with survival on the Bt-Cry3A, suggesting the addition of avidin may increase susceptibility to Bt-Cry3A.

Pretargeted adjuvant radioimmunotherapy with yttrium-90-biotin in malignant glioma patients: a pilot study.

In a previous study we applied a three-step avidin-biotin pretargeting approach to target 90Y-biotin to the tumour in patients with recurrent high grade glioma. The encouraging results obtained in this phase I-II study prompted us to apply the same approach in an adjuvant setting, to evaluate (i) time to relapse and (ii) overall survival. We enrolled 37 high grade glioma patients, 17 with grade III glioma and 20 with glioblastoma, in a controlled open non-randomized study. All patients received surgery and radiotherapy and were disease-free by neuroradiological examinations. Nineteen patients (treated) received adjuvant treatment with radioimmunotherapy. In the treated glioblastoma patients, median disease-free interval was 28 months (range=9-59); median survival was 33.5 months and one patient is still without evidence of disease. All 12 control glioblastoma patients died after a median survival from diagnosis of 8 months. In the treated grade III glioma patients median disease-free interval was 56 months (range=15-60) and survival cannot be calculated as only two, within this group, died. Three-step radioimmunotherapy promises to have an important role as adjuvant treatment in high grade gliomas, particularly in glioblastoma where it impedes progression, prolonging time to relapse and overall survival. A further randomized trial is justified.

Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis.

Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.

Serum concentrations of bisnorbiotin and biotin sulfoxide increase during both acute and chronic biotin supplementation.

In addition to the pharmacokinetic interest, serum concentrations of biotin and biotin metabolites are important because biotin in serum might interfere with assays that use avidin-biotin detection systems. With acute and chronic oral administration of biotin the serum concentration of biotin increases. Because of limited specificity of bioassays or avidin-binding assays used in previous studies, the proportion of the increase attributable to biotin metabolites (if any) remains unknown. To address these questions 15 adults consumed 1,200 microg biotin daily for 14 days. Blood samples were obtained before biotin ingestion and at 3 hours after biotin ingestion on the first day ("acute supplementation") and the fourteenth day ("chronic supplementation"). Biotin, bisnorbiotin, and biotin sulfoxide were measured with a chemically specific high-pressure liquid chromatography/avidin-binding assay. Serum concentrations of biotin, bisnorbiotin, and biotin sulfoxide increased approximately fiftyfold with acute supplementation of biotin; each increased further with chronic supplementation. With acute supplementation the proportion of the total attributable to metabolites did not decrease significantly, suggesting that pathways for biotin catabolism are not easily saturated. With chronic supplementation the proportion of the total attributable to metabolites did not increase significantly, suggesting that biotin catabolism was not substantially induced. We conclude that on a mole basis the contribution of biotin metabolites is important, and we provide an estimate of the biotin and biotin metabolite concentration that might be encountered in individuals who self-select large biotin supplements.

Induction of histamine release from non-immunized guinea pigs: a possible involvement of lectin-like factor(s) in pollinosis.

To investigate nonimmune pathogenic functions of pollens, vascular permeability enhancement (VPE) activity of pollen extracts was examined using guinea pigs nonimmunized against pollens. Ryegrass, ragweed, mesquite and almond, but not common cattail and sumac, induced VPE which was inhibited primarily by an anti-histamine drug. Ryegrass pollen VPE activity was extracted more at pH 7.3 than at pH 6.5 or 8.0 and the maximal activity was extracted in 30 min. Interestingly, more than 60% of the maximal activity was extracted in 5 min. The maximal VPE activity had a dose-dependency similar to histamine (3 x 10(-5) M) but lasted longer than the histamine activity. The VPE activity was inhibited by oligomannose-glycosylated ovalbumin or avidin, as well as the oligosaccharides but not by the deglycosylated proteins. These results indicate that some pollens contain lectin-like, histamine-releasing factor(s), which may be involved in part in pollinosis, by inducing mast cell degranulation through a nonimmune mechanism and resulting in allergy-like symptoms.

Complete inactivation of target mRNA by biotinylated antisense oligodeoxynucleotide-avidin conjugates.

Biotinylation of phosphodiester oligodeoxynucleotides (PO-ODN) allows for conjugation to avidin-based transcellular delivery systems. In addition, biotinylation of PO-ODN at the 3'-terminus provides complete protection against serum 3'-exonuclease degradation. The present study was undertaken to determine if antisense 3'-biotinylated PO-ODN-avidin constructs are able to recognize and inactivate the target mRNA through RNase H-mediated degradation. A 21-mer antisense PO-ODN complementary to the tat gene encompassing nucleotides 5402-5422 of the HIV-1 genome was synthesized with biotin conjugated to the 3'-terminus (bio-tat). Gel mobility assays using [5'-32P]-labeled bio-tat ODN and avidin showed that the bio-tat ODN was fully monobiotinylated. Aliquots of [32P]-labeled sense or antisense tat RNA (337 and 351 nucleotides, respectively) were prepared from transcription plasmids and were preincubated with an excess of bio-tat ODN with or without avidin constructs and digested with RNase H. Products were resolved with sequencing gel and analyzed by autoradiography. Complete conversion to predicted RNA fragments resulting from RNase H digestion of the RNA-ODN duplex (53 and 263 nucleotides) was observed when [32P]-tat sense RNA was incubated with antisense bio-tat ODN or conjugated to avidin or an avidin-cationized human serum albumin (cHSA) complex. Conversely, no degradation of [32P]-tat-antisense RNA was observed after incubation with antisense bio-tat ODN and RNase H. In addition, the avidin-cHSA complex significantly increased (84-fold) the uptake of [32P]-internally labeled bio-tat ODN and its stability against cellular nuclease degradation in peripheral blood lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Complete protection of antisense oligonucleotides against serum nuclease degradation by an avidin-biotin system.

It has been recently demonstrated that a complex of avidin, a cationic protein, and a monobiotinylated antisense oligonucleotide for the GLUT1 glucose transporter mRNA is taken up by cells in vitro and by organs in vivo via absorptive-mediated endocytosis. In the present study, a GLUT1 biotinylated oligonucleotide-avidin construct showing complete protection against serum 3'-exonuclease-mediated degradation is described. 21-mer antisense oligonucleotides complementary to nucleotides 162-182 and 161-181 of the bovine GLUT1 glucose transporter mRNA were synthesized with a 6-aminodeoxyuridine at positions 3 and 20, respectively, biotinylated with NHS- or NHS-XX-biotin to yield near 5'- or near 3'-biotinylated oligonucleotide (bio-DNA), and 5'- and 3'-end radiolabeled. Serum induced a rapid degradation of unprotected (no avidin) [5'-32P]-5'-bio-DNA (> 95% at 30 min). Avidin partially protected this construct (approximately 31% of intact 21-mer oligo remained at 1 h). Similar results were obtained with the [3'-32P]-5'-bio-DNA; however, no degradation products of varying size were observed, confirming that the degradation is mediated primarily by a 3'-exonuclease. Incubation of the [5'-32P]-3'-bio-DNA with serum showed a rapid conversion to the 20- and 19-mer forms (t1/2 approximately 13 min). Conversely, avidin totally protected this construct against the serum 3'-exonuclease. In conclusion, avidin fully protects antisense oligonucleotides biotinylated at the near 3'-terminus against serum 3'-exonuclease degradation, and this property may be useful for avidin-mediated drug delivery of oligonucleotides to tissues in vivo or to cultured cells in vitro.

Primary cultures of astrocytes from rat as a model for biotin deficiency in nervous tissue.

The activities and biotin-dependence of the three mitochondrial biotin-dependent carboxylases: pyruvate carboxylase, propionyl CoA carboxylase, and beta-methylcrotonyl CoA carboxylase of primary culture of astrocytes have been examined. An increase of the three mitochondrial carboxylase activities was observed during cell growth, as was the case for developing rat brain. Mitochondrial carboxylase activities from 3-wk-old primary cultures of astrocytes were higher than those in the neonatal rat brain. When astrocytes were grown in a 10% serum-enriched medium supplemented with avidin to bind biotin, the mitochondrial carboxylase activities were reduced to 15% of control value. Consistent with these results, after 3 wk in culture, the 3-hydroxyisovaleric acid concentration in the growth medium was tenfold higher than the controls. In this culture condition, cellular growth and the nonbiotin-dependent enzyme, glutamine synthetase, were not modified with respect to control. Primary cultures from newborn rat brain hemispheres are suggested as an experimental approach to the study of biotin deficiency in nervous tissue.

Enhanced cellular uptake of biotinylated antisense oligonucleotide or peptide mediated by avidin, a cationic protein.

The cellular uptake of a model antisense oligonucleotide complementary to 21 bases of the bovine GLUT-1 glucose transporter mRNA and a model vasopressin peptide that were biotinylated, was markedly stimulated by the presence of avidin, a cationic protein. Conversely, the bacteria homologue of avidin, streptavidin, which is a slightly acidic protein, did not facilitate cellular uptake. The avidin-mediated uptake of biotinylated derivatives was competitively inhibited by another cationic protein, protamine, with a Ki of 5 micrograms/ml; was saturable, temperature- and time-dependent; and was associated with endocytosis. The use of the avidin-biotin system provides a new approach to increasing the cellular uptake of antisense oligonucleotides or peptides.

Pyruvate carboxylase as a model for oligosubstituted enzyme-ligand conjugates in homogeneous enzyme immunoassays.

Theoretical models suggest that the detection capabilities of homogeneous enzyme immunoassays can be improved by the use of oligosubstituted enzyme-ligand conjugates rather than the traditionally used multisubstituted ones. The natural form of pyruvate carboxylase contains four covalently bound biotins (one per subunit) and it can be considered as an oligosubstituted enzyme-biotin conjugate. The enzyme is nearly completely inhibited in the presence of the natural binder for biotin, avidin. When the enzyme is incubated with avidin and free biotin, a competition occurs between the free biotin and the prosthetic group of the enzyme for the avidin. Steep dose-response curves are obtained by relating the observed inhibition to the free biotin concentration. By variation of the amount of avidin or enzyme in the assay, the detection limits of the system can be altered allowing for sensitive determinations over a wide range of biotin concentrations. Such data from real sample analysis of several vitamin supplements are reported.

Effect of biotin on alkaloid production during submerged cultivation of Claviceps sp. strain SD-58.

Addition of biotin to culture medium NL-406 significantly increased alkaloid yield during submerged cultivation of Claviceps sp. strain SD-58. Alkaloid yield was further enhanced by incorporating leucine in biotin-supplemented culture medium. Increased alkaloid production was associated with an increase in the lipid content of cells and in the number of chlamydospores. Biotin deficiency caused a reduction in alkaloid yield and a parallel decrease in lipid content and chlamydospore numbers.

The biotin status of weanling pigs fed semipurified diets as evaluated by plasma and hepatic parameters.

Twenty-four pigs were weaned at four weeks of age and fed either spray dried egg albumen (DA) or autoclaved DA (ADA) as the only protein source were used to evaluate plasma and hepatic parameters as indicators of biotin status. Pigs were fed one of four semipurified diets during the 42-day study: DA basal, DA + 1.1 g sulfamethazine/day (DA + S), ADA basal or ADA + .5 mg biotin/day (ADA + H). Autoclaving DA at 121 degrees C for 30 minutes reduced both the biotin binding properties of the avidin fraction and the trypsin inhibitor activity of DA. Pigs fed ADA and ADA + H had greater (P less than .05) liver pyruvate carboxylase (EC 6.4.1.1) activity and DNA concentrations, lower (P less than .05) plasma urea nitrogen levels and greater and more efficient (P less than .05) weight gain than either DA or DA + S fed pigs. Biotin supplementation further enhanced (P less than .05) liver DNA concentration but had no effect on the liver levels of RNA or total protein. Plasma levels of glucose and free fatty acids or blood levels of lactate and pyruvate were not influenced by the addition of biotin to the ADA basal diet.

Stimulation of lymphocytes by concanavalin A. Temperature-dependent effect of fatty acid replacements.

The membrane fatty acyl composition of lymphocytes was altered by growth in lipid-depleted serum containing fatty acid supplements, as well as avidin to block endogenous synthesis of fatty acids. Under these growth conditions over 50% of the total fatty acid in membrane phospholipid were derived from the added fatty acid. Enrichment of lymphocyte membranes with oleate (cis C18:1) or elaidate (trans C18:1) shifted the optimum temperature for mitogenic stimulation by concanavalin A as measured by [3H]thymidine incorporation. These results suggest that the fluidity of the membrane lipid phase plays a role in the process of lymphocyte stimulation by lectins.