Vitamin K5 Research Articles

Spermicide of Papaya Seed Oil and Compounds

This study aimed to determine the spermicidal activity of papaya seed oil (Carica papaya L) from local Bali varieties against Wistar rat spermatozoa by evaluating sperm quality and the correlation between the percentage of motility and viability spermatozoa to identify its active compounds. Papaya seed oil was obtained by extracting papaya seeds using n-hexane solvent. The study was designed as follows; group of spermatozoa Wistar rat were treated with NaCl 0.9% (control, P0), with Sodium carboxy methyl cellulose (NaCMC) 0.5% (P1), with n-hexane extract 0.1%(b/v) (P2), with n-hexane extract 0.3%(b/v) (P3), and with n-hexane extract 0.5%(b/v) (P4). Column chromatography was used to separate the n-hexane extract into two fractions, A and B. The spermicide test for fractions A and B followed the same design as the test for n-hexane extract but until the P7 group. The result showed that fraction B exhibited higher spermicidal activity, with 23.3% motility and 33.8% of viability sperm at 100 ppm. The identification of fraction B by LCMS showed at least 12 mixture compounds were venoterpine, vitamin K5, 5-phenyl-2-pyridinamine, harmalan, styrylquinoline, 1,2,3,4-tetrahydro-1-benzoyl-2-methyl-4-phenylamino-quinoline, calcitroic acid, siderol, N-benzyl-linoleamide, (3β)-3-(diisopropylamino)androst-5-en-17-one, pipereicosalidine, and 25-azacholesterol. Spermicidal activity may be a synergistic effect of several compounds that have a cytotoxic activity such as styrylquinoline, 1,2,3,4-tetrahydro-1-benzoyl-2-methyl-4-phenylamino-quinoline, siderol, and pipereicosalidine.

Vitamin K5 Is an Efficient Photosensitizer for Ultraviolet a Light Inactivation of Bacteria

Photodynamic inactivation of bacteria has been proven as an effective method to inactivate pathogenic bacteria. This study identified vitamin K5 (VK5) as an efficient photosensitizer for ultraviolet light A (UVA) induced bacterial inactivation.Six species of bacteria, including Bacillus cereus (vegetative form), Escherichia coli , Pseudomonas aeruginosa, Staphylococcus aureus , Staphylococcus epidermidis , Klebsiella pneumoniae , and two species of antibiotic-resistant bacteria, Pseudomonas aeruginosa and Staphylococcus aureus, were grown and mixed with VK5 and irradiated with UVA in phosphate buffered saline (PBS) in 6-well plates following our previous procedures (Xu & Vostal, 2014). A representative figure of bacterial reduction of VK5 and UVA irradiation treated E. coli , as determined by a colony forming unit (CFU) count, is shown in Figure 1. Increasing concentrations of VK5 from 0 to 1600 µmol l-1 with fixed UVA irradiation doses produce a concentration-depended reduction of bacteria. Similarly, a UVA dose response for bacterial reduction is evident with fixed VK5 concentrations. As shown in Table 1, UVA irradiation (5.8 J cm-2) with vitamin K5 (1600 µmol l-1) reduced the CFU of bacteria by three to seven logs. Antibiotic resistant bacteria (Pseudomonas A. and Staphylococcus A ) did not exhibit resistance to the bactericidal effects of UVA and vitamin K5 combination treatment.Efficacy of VK5 and UVA for bacterial reduction was also evident in plasma although higher concentrations of VK5 and UVA doses were required. VK5 (500 or 2000 µ mol l-1 ) in 35% or 50% bacteria spiked plasma diluted with Platelet Additive Solutions was exposed to UVA irradiation at 6 and 30 J cm-2. This treatment reduced Escherichia coli and Staphylococcus aureus CFU by up to seven logs as shown in Figures 2a and 2b, respectively.Reactive oxygen species, hydroxyl radicals (˙OH) and superoxide anion radicals (O2˙−), were found generated in vitamin K5 aqueous solution after UVA irradiation, suggesting these oxygen species may mediate the inactivation of the bacteria. The O2˙−was generated in UVA-irradiated-VK5 PBS solution. At a fixed UVA dose of 2.9 J cm-2, the generation of O2˙− coincided with an increase in VK5 concentration from 0 to 1000 µ mol l-1 (Figure 3a). The generation of O2˙− was further confirmed by carrying out quenching study with superoxide dismutase (SOD) as specific quenchers. The production of O2˙− was reduced as the SOD concentration was increased from 0 to 640 units ml-1 in a 500 µ mol l-1 VK5 solution irradiated with a UVA (2.9 J cm-2 ) (Figure 3b). VK5 and UVA light combination also caused the release of ˙OH as the VK5 concentrations increased from 0 to 1000 µ mol l-1 with a fixed UVA dose of 2.9 J cm-2 The generation of ˙OH was further confirmed by mannitol, a specific quencher for ˙OH. Less ˙OH was produced when mannitol concentration was increased from 0 to 1 mol l-1, in the presence of a fixed VK5 concentration of 500 µ mol l-1 and a UVA dose of 2.9 J cm-2.Vitamin K5 and UVA light treatment is an effective method for reduction of bacteria in solutions including plasma and may be an effective way to reduce transfusion transmitted septic reactions from contaminated plasma products. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Viral reduction of human blood by ultraviolet A-photosensitized vitamin K5.

Blood product transfusion can transmit viral pathogens. Pathogen reduction methods for blood products have been developed but, so far, are not available for whole blood. We evaluated if vitamin K5 (VK5) and ultraviolet A (UVA) irradiation could be used for virus inactivation in plasma and whole blood. Undiluted human plasma and whole blood diluted to 20% were spiked with high levels of vaccinia or Zika viruses. Infectious titers were measured by standard TCID50 assay before and after VK5/UVA treatments. Up to 3.6 log of vaccinia and 3.2 log of Zika were reduced in plasma by the combination of 500 μM VK5 and 3 J/cm2 UVA, and 3.1 log of vaccinia and 2.9 log of Zika were reduced in diluted human blood (20%) by the combination of 500 μM VK5 and 70 J/cm2 UVA. At end of whole blood treatment, hemolysis increased from 0.18% to 0.41% but remained below 1% hemolysis, which is acceptable to the Food and Drug Administration for red cell transfusion products. No significant alteration of biochemical parameters of red blood cells occurred with treatment. Our results provide proof of theconcept that a viral pathogen reduction method based on VK5/UVA may be developed for whole blood.

Shikonin, vitamin K3 and vitamin K5 inhibit multiple glycolytic enzymes in MCF-7 cells.

Glycolysis is the most important source of energy for the production of anabolic building blocks in cancer cells. Therefore, glycolytic enzymes are regarded as potential targets for cancer treatment. Previously, naphthaquinones, including shikonin, vitamin K3 and vitamin K5, have been proven to decrease the rate of glycolysis in cancer cells, which is partly due to suppressed pyruvate kinase activity. In the present study, enzymatic assays were performed using MCF-7 cell lysate in order to screen the profile of glycolytic enzymes in cancer cells inhibited by shikonin, vitamin K3 and vitamin K5, in addition to pyruvate kinase. Results revealed that hexokinase, phosphofructokinase-1, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase produced in the process of glycolysis were inhibited by shikonin, vitamin K3 and vitamin K5. The results indicated that shikonin, vitamin K3 and vitamin K5 are chemical inhibitors of glycolytic enzymes in cancer cells and have potential uses in translational medical applications.

Vitamin K5 is an efficient photosensitizer for ultraviolet A light inactivation of bacteria.

Photodynamic treatment combining light and a photosensitizer molecule can be an effective method to inactivate pathogenic bacteria. This study identified vitamin K5 as an efficient photosensitizer for ultraviolet light A (UVA)-induced bacterial inactivation. Six bacterial species, Bacillus cereus (vegetative form), Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and two species of antibiotic-resistant bacteria, Pseudomonas aeruginosa* and Staphylococcus aureus*, were suspended in aqueous solutions with or without vitamin K5 and exposed to UVA irradiation. UVA irradiation (5.8 J cm-2) with vitamin K5 (1600 μmol l-1) reduced the colony forming units (CFU) of these bacteria by three to seven logs. Antibiotic resistant bacteria were also susceptible to the bactericidal effects of UVA and vitamin K5 combination treatment. Inactivation of bacteria in human plasma required higher doses of UVA light and vitamin K5. UVA irradiation (30 J cm-2) with vitamin K5 (2000 μmol l-1) reduced E. coli and S. aureus spiked into human plasma by seven logs CFU/ml. Reactive oxygen species, such as superoxide anion radicals and hydroxyl radicals, were found to be generated in vitamin K5 aqueous solution after UVA irradiation, suggesting these oxygen species may mediate the inactivation of the bacteria.

Effects of vitamin K3 and K5 on proliferation, cytokine production, and regulatory T cell-frequency in human peripheral-blood mononuclear cells

AimsThe effects of vitamin K (VK) derivatives VK3 and VK5 on human immune cells have not been extensively investigated. We examined the effects of VK3 and VK5 on proliferation, apoptosis, cytokine production, and CD4+CD25+Foxp3+ regulatory T (Treg) cell-frequency in human peripheral blood mononuclear cells (PBMCs) activated by T cell mitogen in vitro. Main methodsAnti-proliferative effects of VK3 and VK5 on T-cell mitogen activated PBMCs were assessed by WST assay procedures. Apoptotic cells were determined as Annexin V positive/propidium iodide (PI) negative cells. Cytokine concentrations in the supernatant of the culture medium were measured with bead-array procedures followed by analysis with flow cytometry. The CD4+CD25+Foxp3+Treg cells in mitogen-activated PBMCs were stained with fluorescence-labeled specific antibodies followed by flow cytometry. Key findingsVK3 and VK5 suppressed the mitogen-activated proliferation of PBMCs significantly at 10–100μM (p<0.05). The data also suggest that VK3 and VK5 promote apoptosis in the mitogen-activated T cells. VK3 and VK5 significantly inhibited the production of tumor necrosis factor (TNF) α, interleukin (IL)-4, -6, and -10 from the activated PBMCs at 10–100μM (p<0.05). In contrast, VK3 and VK5 significantly increased Treg cell-frequency in the activated PBMCs at concentrations more than 10μM (p<0.001). SignificanceOur data suggest that VK3 and VK5 attenuate T cell mediated immunity by inhibiting the proliferative response and inducing apoptosis in activated T cells.

Innovations in Food Technology Special Issue

The development of novel processing techniques to obtain healthier and safer food products is one of the major challenges facing the food industry in the new century. On one hand, novel processes and products are driven by the stunning technological advances of this global era, to which food science and technology are not alien. Such advances include not only the design and application of new equipment and process lines, which constitute what has been called “emerging technologies”, but also the biotechnological and nanotechnological applications of such cutting-edge disciplines for food production and control. On the other hand, innovation in food technology flows in parallel with consumers’ demand for healthier and safer foods with improved quality and shelf life. As a consequence of these developments, and in an effort to give a response to such challenges, food technologists have paid special attention to the development and application of minimal processing strategies to a myriad of food products, thus avoiding the nutritional shortcomings that occur from the application of traditional preservation methods such as intense heat processing. Nevertheless, although the nutritional components of minimally processed foods may be more intact in products subjected to such light preservation methods, the presence of spoilage and pathogenic bacteria that are able to survive such minimal processing may incur novel risks. These risks require the development of robust and sensitive control methods to ensure not only the quality but also, and especially, the safety of such novel food products. In such an actively changing food technology environment, scientists working in this field are ready to find solutions from different approaches. Some of the most relevant solutions have been compiled in this “Innovations in Food Technology Special Issue”. Among the most promising novel technologies with a higher transfer potential from research to development and innovation, the application of pulsed electric fields and high-pressure processing are still at the top. Nevertheless, more specific and still less known techniques, such as hydrodynamic cavitation, are also gaining increasing importance for certain applications in water and food processing, while thermosonication and ultraviolet radiation are also gaining attention as food processing strategies. Sometimes, the combination of novel techniques and the obtention of added value food ingredients goes together, such is the case with the application of solvent-free strategies to the extraction of antioxidants that are relevant to the food industry. Novel food biopreservation techniques constitute another innovative field that deserves special attention. Active packaging techniques extend the possibility of keeping food quality at its best during storage, whereas the development of novel bacteriocins and other novel inhibitory agents, such as essential oils or the so-called vitamin K5, which are able to inhibit spoilage and pathogenic bacteria and fungi, represent valuable steps toward hurdle technologies for the production of minimally processed food products. The ever changing field of biotechnology also provides us with selected and engineered enzymes and starter cultures for novel food applications, such as microbial proteases and selected lactic acid bacteria for wine production, respectively. Novel technologies for cell immoJ. Barros Velazquez (*) Food Technology Laboratory, Department of Analytical Chemistry, Nutrition and Food Science School of Veterinary Sciences/College of Pharmacy University of Santiago de Compostela, 15782 Santiago de Compostela, Spain e-mail: jorge.barros@usc.es Food Bioprocess Technol (2011) 4:831–832 DOI 10.1007/s11947-011-0576-9

In vitro Growth Inhibition of Food-borne Pathogens and Food Spoilage Microorganism by Vitamin K5

The study investigates the effectiveness of a synthetic vitamin analog (vitamin K5) for the growth inhibition of a total of 14 bacterial pathogens, spoilage-inducing bacteria and three spoilage-inducing molds that were tested during this study. Bacterial growth inhibition by vitamin K5 and vitamin K5 combined with sodium chloride (NaCl) were tested by determining the Minimum Inhibitory Concentrations (MICs) using a broth microdilution method. Antifungal effects of vitamin K5 were determined by measuring the growth of three different spoilage molds on Malt agar plates containing different concentrations of vitamin K5. All bacterial strains assayed were inhibited by vitamin K5 in a range of 64-1,024 μg/ml. When vitamin K5 was combined with 3% NaCl, five out of the 14 bacterial strains assayed showed lower MICs than for vitamin K5 alone. When vitamin K5 was combined with 5% NaCl, nine of the 14 bacterial strains assayed displayed lower MICs than for vitamin K5 alone. With respect to inhibition of molds, 10 μg/ml vitamin K5 induced inhibitory effects against Aspergillus parasiticus NRRL 2999 and Penicillium expansum NRRL 6069, but not Aspergillus flavus NRRL 6540. In contrast, 30 or 50 μg/ml vitamin K5 showed inhibitory effects against the growth of all molds assayed; however, inhibitory effects against aflatoxin production by A. flavus NRRL 6540 were achieved only with 50 μg/ml. These data show that vitamin K5 is a useful antimicrobial agent that at low concentrations causes growth inhibition of a broad spectrum of bacterial pathogens and spoilage bacteria as well as certain molds.

Vitamins K2, K3 and K5 exert in vivo antitumor effects on hepatocellular carcinoma by regulating the expression of G1 phase-related cell cycle molecules

A number of studies have shown that various vitamins K, specifically vitamin K2, possessed antitumor activity on various types of rodent- and human-derived neoplastic cell lines. However, there are only a small number of reports demonstrating in vivo antitumor effects of vitamins K. Furthermore, the mechanism of antitumor effects of vitamins K still remains to be examined. In the present study, we examined the antitumor effects of vitamins K2, K3 and K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vivo. Furthermore, to examine the mechanism of antitumor actions of these vitamins K, mRNA expression levels of various G1 phase-related cell cycle molecules were evaluated by using a real-time reverse transcription-polymerase chain reaction (RT-PCR) method. HCC-bearing animals were produced by implanting PLC/PRF/5 cells subcutaneously into athymic nude mice, and drinking water containing vitamin K2, K3 or K5 was given to the animals. Treatments with vitamins K2, K3 and K5 were shown to markedly inhibit the growth of HCC tumors. To examine the mechanism of in vivo antitumor effects of vitamins K, total RNA was extracted from HCC tumors, and the expression of G1 phase-related cell cycle molecules was quantitatively examined. Real-time RT-PCR demonstrated that the expression of the cell cycle-driving molecule, cyclin-dependent kinase 4 (Cdk4), in HCC was significantly reduced by the treatments with vitamin K2, K3 and K5. Conversely, the expression of the cell cycle-suppressing molecules, Cdk inhibitor p16INK4a and retinoblastoma, in HCC was significantly enhanced by the treatments with vitamins K2, K3 and K5. These results indicate that vitamins K2, K3 and K5 exert antitumor effects on HCC by regulating the expression of G1 phase-related cell cycle molecules. These results also indicate that vitamins K2, K3 and K5 may be useful agents for the treatment of patients with HCC.

In vitro and in vivo antitumor effects of vitamin K5 on hepatocellular carcinoma

Although a number of studies have shown that vitamins K1, K2 and K3 exerted antitumor effects on various types of rodent- and human-derived neoplastic cell lines, it has not been examined whether or not vitamin K5 also possesses antitumor activity. In the present study, we examined the antitumor effects of vitamin K5 on PLC/PRF/5 human hepatocellular carcinoma (HCC) cells in vitro and in vivo. Furthermore, we examined the mechanisms of antitumor actions of vitamin K5 not only in vitro but also in vivo. Vitamin K5 was shown to suppress the proliferation of PLC/PRF/5 cells at a concentration of 30 microM. By a flow cytometric analysis, it was shown that although vitamin K5 did not induce apoptosis on PLC/PRF/5 cells, it did induce G1 arrest on PLC/PRF/5 cells. Subsequent in vivo study using subcutaneous HCC-bearing athymic nude mice demonstrated that vitamin K5 markedly suppressed the growth of HCC tumors. Although protein expression levels of cyclin D1 and p16INK4a cyclin-dependent kinase (Cdk) inhibitor in HCC tumors were not decreased by vitamin K5 treatment, those of Cdk4 were reduced significantly by the treatment. Taken collectively, vitamin K5 could induce potent antitumor effects on HCC not only in vitro but also in vivo, at least in part by inducing G1 arrest of cell cycle through downregulation of Cdk4 expression. The results demonstrated here indicate that vitamin K5 may be a useful agent for the treatment of patients with HCC.

Targeted delivery of drugs and proteins to the liver via receptor-mediated endocytosis

Targeting of drugs and proteins to the liver via the asialoglycoprotein receptor was investigated in mice. Carboxymethyl-dextran (CMD), carboxymethyl-amylose (CMA), and poly-l-glutamic acid (PLGA) were modified with 2-imino-2-methoxyethyl (IME)-thiogalactosides to obtain galactosylated derivatives as carriers of drugs with low-molecular weights. Proteins were targeted to the liver by direct attachment of galactose moieties. Pharmacokinetic analysis clearly showed that galactosylated derivatives were taken up by the liver depending on the molecular weight and configuration of macromolecules, the number of galactose residues, and the administered dose. Based on the obtained results, we attempted to selectively deliver vitamin K5, which acts as a coagulant in the liver. Galactosylated PLGA (Gal-PLGA) possessing 18 galactose residues was selected as a hepatotropic carrier since it was efficiently accumulated and gradually degraded in the liver after intravenous injection. The attachment of vitamin K5 did not alter the distribution properties of Gal-PLGA, and vitamin K5 was successfully delivered to the liver by the conjugation. The anti-hemorrhagic activity of the conjugate was assayed after intravenous injection in mice treated with warfarin. Vitamin K5 conjugated with Gal-PLGA showed coagulant activity at any periods studied after intravenous injection, while free vitamin K5 only showed the activity at 4 h after administration. These results indicate the usefulness of galactosylated macromolecules as hepatotropic carriers of drugs whose site of action is in the liver.

Insulin receptor and postbinding defects in KK mouse adipocytes and improvement by ciglitazone

Alterations of the insulin receptor and of glucose metabolism were examined in adipocytes from genetically diabetic KK mice. Compared with those from control C57BL/6 mice, adipocytes from KK mice showed weaker insulin binding and were less sensitive and less responsive to insulin with respect to 3-O-methylglucose uptake and [1-14C]glucose oxidation. However, insulin had no difference in effect between the two groups of mice with respect to [6-14C]glucose oxidation, glyceride-glycerol synthesis, or fatty acid synthesis from [1-14C]- and [6-14C]glucose. [1-14C]Glucose oxidation of KK cells was also insensitive to insulin mimics such as vitamin K5 and H2O2. When adipose tissues were precultured with insulin or insulin mimics for 24 h, adipocytes from C57BL/6 mice showed decreased insulin binding, but KK cells did not. When administered orally to KK mice for 2 weeks, ciglitazone made adipocytes more sensitive to insulin, more responsive to insulin with respect to glucose uptake and oxidation, and more capable of binding insulin. Impairment of the downregulation at the insulin receptor caused by exposure to insulin or insulin mimics was normalized by ciglitazone treatment in KK cells. In conclusion, KK cells are insulin-resistant due to defects of the insulin receptor and postbinding system in the glucose uptake and pentose pathways. In addition, regulation of the insulin receptor seems to be closely related to the postbinding system.

Induction of Flowering in Lemna paucicostata by Dicoumarol and 4-Hydroxycoumarin

Dicoumarol, an antagonist of vitamin K, not only promoted the flower-inducing activity of vitamin K5, but also induced flowering in Lemna paucicostata when added alone to the medium. The flower-inducing activity of dicoumarol was comparable to that of benzoic acid and could be greatly intensified by simultaneous application of benzyladenine as was the case with benzoic acid. Warfarin, another antagonist of vitamin K, did not induce flowering. 4-Hydroxycoumarin, a component of dicoumarol, was also active, but coumarin and 7-hydroxycoumarin were not. Dicoumarol had only a slight flower-inducing activity for strains 441 and 6746 under continuous light, but had a strong flower-promoting effect under a near critical photoperiod. That is, the effect of dicoumarol was daylength-dependent.

Differential effects of insulin, antibodies against rat adipocyte plasma membranes, and other agents that mimic insulin action in rat adipocytes

The effects of insulin and several insulin-mimetic agents on rat adipocyte D-glucose metabolism were studied in an effort to determine if any of the insulin-mimetic agents could be used to define the mechanism of insulin action. Antibodies against rat adipocyte plasma membranes have been characterized as having insulin-mimetic effects on glucose transport and these effects may be caused by divalent clustering of cell surface antigens. In contrast to insulin, antimembrane antibodies had little stimulatory effect on D-glucose conversion to lipids in isolated rat adipocytes, under conditions where both reagents stimulated D-glucose oxidation. Among other insulin-mimetic agents tested, the reagents hydrogen peroxide and concanavalin A most closely resembled insulin in their ability to increase both [ 14C]-CO 2 and [ 14C]-lipid formation from [ 14C]D-glucose in rat adipocytes. Vitamin K5 and diamide had the unusual effect of inhibiting [1- 14C]D-glucose conversion to [ 14C]-lipids at a concentration that gave maximal stimulation of glucose oxidation by rat adipocytes. Analysis of the lipid components into which glucose derivatives were incorporated revealed that insulin increased D-glucose incorporation into both nonesterified fatty acids and triglycerides and H 2O 2 and concanavalin A had similar effects. These findings argue against the possibility that insulin and the antimembrane antibodies or the insulin-mimetic agents other than H 2O 2 and concanavalin A share the same mechanism of action.

Inhibition of down regulation by chloroquine in cultured lymphocytes (RPMI-1788 line)

We studied the effect of chronic exposure to insulin on insulin receptors of cultured lymphocytes (RPMI-1788 line). The cells treated with insulin (2 micrograms/ml) at 37 degrees C for 12 h, showed a 36.5% decrease in the number of binding sites. Solubilized extract from the cells treated with insulin showed a 35.9% decrease of binding capacity, suggesting that insulin exposure induced the loss of total (cell surface and intracellular) receptors. Insulin-induced loss of receptors was blocked by chloroquine, suggesting that receptor loss is mediated by a chloroquine sensitive pathway. Bacitracin, which inhibited the insulin degradation, had no effect on insulin-induced receptor loss in this cell line. We also found that vitamin K5, one of the insulin mimickers, induced a 31.5% loss of insulin receptors. Therefore, the post-receptor process appeared to mediate down regulation. In cultured lymphocytes, insulin exposure caused a significant loss of total receptors, suggesting that insulin-induced receptor loss may be due to receptor degradation. Insulin-induced receptor loss is mediated by a chloroquine sensitive pathway, and is related to the post-binding process stimulated by vitamin K5.

Inhibition of insulin-stimulated xylose uptake in rat soleus muscle by cycloheximide.

Cycloheximide (20-200 mg/l) did not affect basal D-[U-14C]xylose uptake by rat soleus muscle (2.4 +/- 0.2 mumol . g-1 . h-1). However, the stimulatory effect of insulin on sugar transport was progressively reduced from 375% above basal in control muscles to 170% in muscles exposed to 200 mg cycloheximide/l but above this concentration cycloheximide inhibited basal xylose uptake without further effect on the incremental effect of insulin. Cycloheximide affected the insulin dose-response curve both by depressing insulin sensitivity and by reducing the maximum stimulatory effect of the hormone. In contrast to the inhibition of insulin action, which increased progressively over the range 20-200 mg cycloheximide/l, muscle protein synthesis was inhibited maximally at a concentration of 10 mg/l. Cycloheximide also inhibited the insulinomimetic effects of anoxia, 2:4-dinitrophenol, salicylate, cooling, hydrogen peroxide, diamide, vitamin K5, hyperosmolarity and EDTA, but did not affect concanavalin A-stimulated xylose uptake. It is concluded that cycloheximide inhibits insulin-stimulated sugar transport at some late post-receptor step, and that this effect of cycloheximide is not secondary to the inhibition of protein synthesis.

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.

Cellular basis of insulin resistance in chronic uremia.

To define the cellular alterations responsible for insulin resistance during uremia, we studied insulin action in adipocytes isolated from rats 2 wk after 75% partial nephrectomy. Insulin binding to fat cells and purified liver plasma membranes prepared from uremic rats was unaltered. In contrast, hexose transport was significantly decreased, with and without insulin, in the fat cells from the uremic animals. The concentration of insulin that elicited half-maximal response was not altered. Glucose utilization was reduced in the absence or presence of insulin by partial nephrectomy. The stimulation of hexose transport and glucose metabolism by the insulin mimickers, hydrogen peroxide and vitamin K5, were also inhibited. Hexose transport activity in adipocytes obtained from uremic rats was no longer decreased when pieces of fat tissue were cultured for 20 h. Finally, hexose transport was reduced in the cells isolated from normal adipose tissue that was preincubated for 3 h with uremic serum, but insulin binding was not different than control. Thus, insulin resistance associated with uremia may be primarily accounted for by altered postreceptor events that appear to result from a circulating factor(s).

Effect of vitamins other than vitamin C on viruses: virus-inactivating activity of vitamin K5.

Effect of vitamins other than vitamin C on viruses: virus-inactivating activity of vitamin K5.

Inhibition of insulin-stimulated xylose uptake in denervated rat soleus muscle: a post-receptor effect

Insulin (100 U/l) stimulated xylose uptake in rat soleus muscle from a basal value of 2.3 +/- 0.5 to 11.6 +/- 2.1 mumol . g-1 . h-1. Denervation (section of the sciatic nerve) markedly reduced the stimulatory action of insulin (basal 1.3 +/- 0.4 mumol . g-1 . h-1; insulin-stimulated 4.5 +/- 0.6 mumol . g-1 . h-1). This effect appeared 3 days after denervation and was maximal after 5 days. Denervation also affected the insulin dose response curve; there was no effect of insulin in denervated muscle until the concentration exceeded 1 U/l. Denervation inhibited insulin-stimulated alpha-aminoisobutyrate uptake by 77% but did not affect 125I-insulin binding or glucose-independent activation of glycogen synthase by insulin. There was no effect of denervation on the insulinomimetic effects of concanavalin A, hydrogen peroxide, vitamin K5, anoxia, 2:4-dinitrophenol, cooling, hyperosmolarity or EDTA, but the effect of diamide was inhibited. It is concluded [1] that denervation inhibits insulin-stimulated sugar transport at some early post-receptor step, and [2] that the mechanism whereby insulin activates glycogen synthase is different from the activation of the membrane transport of sugars and amino acids.