A Bicarbonate- and Weak Acid-permeable Chloride Conductance Controlled by Cytosolic Ca2+ and ATP in Rat Submandibular Acinar Cells

A Ca²⁺-activated Cl⁻ conductance in rat submandibular acinar cells was identified and characterized using whole-cell patch-clamp technique. When the cells were dialyzed with Cs-glutamate-rich pipette solutions containing 2 mm ATP and 1 μm free Ca²⁺ and bathed in N-methyl-d-glucamine chloride (NMDG-Cl) or Choline-Cl-rich solutions, they mainly exhibited slowly activating currents. Dialysis of the cells with pipette solutions containing 300 nm or less than 1 nm free Ca²⁺ strongly reduced the Cl⁻ currents, indicating the currents were Ca²⁺-dependent. Relaxation analysis of the ``on' currents of slowly activating currents suggested that the channels were voltage-dependent. The anion permeability sequence of the Cl⁻ channels was: NO⁻ ₃ (2.00) > I⁻ (1.85) ≥ Br⁻ (1.69) > Cl⁻ (1.00) > bicarbonate (0.77) ≥ acetate (0.70) > propionate (0.41) ≫ glutamate (0.09). When the ATP concentration in the pipette solutions was increased from 0 to 10 mm, the Ca²⁺-dependency of the Cl⁻ current amplitude shifted to lower free Ca²⁺ concentrations by about two orders of magnitude. Cells dialyzed with a pipette solution (pCa = 6) containing ATP-γS (2 mm) exhibited currents of similar magnitude to those observed with the solution containing ATP (2 mm). The addition of the calmodulin inhibitors trifluoperazine (100 μm) or calmidazolium (25 μm) to the bath solution and the inclusion of KN-62 (1 μm), a specific inhibitor of calmodulin kinase, or staurosporin (10 nm), an inhibitor of protein kinase C to the pipette solution had little, if any, effect on the Ca²⁺-activated Cl⁻ currents. This suggests that Ca²⁺/Calmodulin or calmodulin kinase II and protein kinase C are not involved in Ca²⁺-activated Cl⁻ currents. The outward Cl⁻ currents at +69 mV were inhibited by NPPB (100 μm), IAA-94 (100 μm), DIDS (0.03–1 mm), 9-AC (300 μm and 1 mm) and DPC (1 mm), whereas the inward currents at −101 mV were not. These results demonstrate the presence of a bicarbonate- and weak acid-permeable Cl⁻ conductance controlled by cytosolic Ca²⁺ and ATP levels in rat submandibular acinar cells. Received: 9 January 1996/Revised: 20 May 1996     

Change in floral nectar components from fresh to senescent flowers ofCapparis spinosa (Capparidaceae), a nocturnally flowering Mediterranean shrub

We studied the nectar characteristics in relation to flower age of the summer flowering Mediterranean shrubCapparis spinosa in three localities in Southern Greece. Anthesis was nocturnal. Nectar volume, concentration, and sucrose/hexose ratio varied with site, year, and between individual plants; amino acid concentration varied only with site. The sucrose/hexose ratio decreased considerably with flower age, while the glucose/fructose ratio remained constant (ca. 1), implying that nectar sucrose broke down in the course of anthesis. Sugar breakdown increased with water content of nectar. Amino acid concentration was strongly age-dependent: It was low in fresh flowers, relatively high in middle-aged ones (except aspartic acid that was extremely increased), and very high in senescent ones. We attribute the amino acid changes to phenomena related to flower senescence in the dark.     

Fatty acid double bond orientation alters interaction with L-cell fibroblasts

Relatively little is known of fatty acid specificity in cellular fatty acid uptake. In this study L-cells, a fibroblastic cell line with very low levels of endogenous cytosolic fatty acid binding protein, were used to examine the role of cis and trans unsaturation on fatty acid uptake. The fluorescent fatty acids, trans-parinaric acid and cis-parinaric acid, were used as analogs of straight-chain saturated, and kinked-chain unsaturated fatty acids, respectively, in order to evaluate the fatty acid specificity of the uptake system. Parinaric acid is poorly metabolizable; greater than 97% was unesterified while ³H-oleic acid was almost totally metabolized after 30 min uptake. Cis- and trans-parinaric acid uptake was saturable and dependent on the concentration of fatty acid. However, the initial rate and maximal amount of trans-parinaric acid taken up by the L-cells was greater than for cis-parinaric acid under the same conditions. The affinity of L-cell uptake for trans-parinaric acid (K_m = 0.12 uM) was 35-fold higher than that for cis-parinaric acid (K_m = 4.17 uM) . Based on competition studies with oleic and stearic acids, it was concluded that the cis- and trans-parinaric acid were taken up by the same L-cell fatty acid uptake system. The results suggest that the L-cell fatty acid uptake system has selectivity for straight chain rather than kinked chain unsaturated fatty acids.Abbreviations Cis-parinaric acid 9Z, 11E, 13E, 15Z-octatetraenoic acid - trans-parinaric acid 9E, I IE, 13E, 15E-octatetraenoic acid - EGTA ethylene glycol-bis(beta-amlno-ethyl ether) N,N,N,N-tetratacetic acid - BSA bovine serum albumin - PBS phosphate buffered saline     

Omega-3 fatty acids and the peroxisome

The interactions between the omega-3 unsaturated fatty acids and peroxisomal function have been reviewed, in order to update and integrate knowledge in this area. Following a brief retrospective of the major clinical involvements of these fatty acids, the participation of the peroxisome in their metabolism has been appraised - the peroxisome being shown to exert a major influence on both the synthesis and degradation of the omega-3 fatty acids, with these effects flowing on to the widespread physiological implications of the derivative eicosanoids. Interactions between the omega-3 and omega-6 families of fatty acids have been discussed, as have the interdependent phenomena of peroxisome proliferation, membrane remodelling and cellular signalling. Amongst the signalling involvements covered were those of steroid hormone receptor superfamily, the phosphatidy1choline cycle, and the regulatory influences of oxygen free radicals. Comment has also been included on the separate biological roles of the individual omega-3 fatty acids, their influence on differential gene function, and on the molecular mechanisms of their pharmacological effects. It is concluded that the peroxisome is intimately involved in directing the metabolism and physiological influence of the omega-3 unsaturated fatty acids, and that this organelle merits much greater emphasis in future research aimed at unravelling the profound biological effects of these unique and multipotent compounds.     

Distribution and activity of microsomal NADPH-dependent monooxygenases and amino acid decarboxylases in cruciferous and non-cruciferous plants, and their relationship to foliar glucosinolate content

The NADPH-dependent conversion of amino acids to their aldoximes is an initial step in glucosinolate biosynthesis. A number of microsomal aldoxime-forming monooxygenase activities were detected in leaves from a variety of glucosi-nolate-containing species, whereas barley, bean and tobacco leaves did not contain any such activities. The substrates for these monooxygenases in each species largely correlated with the spectrum of glucosinolates found in that species. No activity was detected that metabolized homomethionine (supposed precursor of 2-propenylglucosinolate [sinigrin]), even in species where sinigrin was the major glucosinolate. In Sinapis species containing hydroxybenzylglucosinolate (sinalbin), activity with L-Tyr was detected, whereas Brassica species containing sinalbin had no such activity. However, these Brassicas did contain an L-Phe monooxygenase activity. Partial characterization of the monooxygenases indicated that in Brassica species, Nasturtium officinalis and Raphanus sativus these resembled the flavin-linked monooxygenases previously found in oilseed rape (Brassica napus) and Chinese cabbage (Brassica campestris). The L-Tyr-dependent activity in Sinapis species, and the L-Phe-dependent activity in Tropacolum majus, had characteristics of cytochrome P450-type enzymes. No similarity was found with any other known amino acid metabolizing enzymes (including decarboxylases, amino acid oxidases and diamine/polyamine oxidases).     

Co-purification,co-imniunoprecipitation,and coordinate expression of acetyl-coenzyme A carboxylase activity,biotin carboxylase,and biotin carboxyl carrier protein of higher plants

Acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) is a regulatory enzyme of fatty acid synthesis, and in some higher-plant plastids is a multi-subunit complex consisting of biotin carboxylase (BC), biotin-carboxyl carrier protein (BCCP), and carboxyl transferase (CT). We recently described a Nicotiana tabacum L. (tobacco) cDNA with a deduced amino acid sequence similar to that of prokaryotic BC. We here provide further biochemical and immunological evidence that this higher-plant polypeptide is an authentic BC component of ACCase. The BC protein co-purified with ACCase activity and with BCCP during gel permeation chromatography of Pisum sativum L. (pea) chloroplast proteins. Antibodies to the Ricinus communis L. (castor) BC co-precipitated ACCase activity and BCCP. During castor seed development, ACCase activity and the levels of BC and BCCP increased and subsequently decreased in parallel, indicating their coordinate regulation. The BC protein comprised about 0.8% of the soluble protein in developing castor seed, and less than 0.05% of the protein in young leaf or root. Polypeptides cross-reacting with antibodies to castor BC were detected in several dicotyledons and in the monocotyledons Hemerocallis fulva L. (day lily), Iris L., and Allium cepa L. (onion), but not in the Gramineae species Hordeum vulgare L. (barley) and Panicum virgatum L. (switchgrass). The castor endosperm and pea chloroplast ACCases were not significantly inhibited by long-chain acyl-acyl carrier protein, free fatty acids or acyl carrier protein. The BC polypeptide was detected throughout Brassica napus L. (rapeseed) embryo development, in contrast to the multi-functional ACCase isoenzyme which was only detected early in development. These results firmly establish the identity of the BC polypeptide in plants and provide insight into the structure, regulation and roles of higherplant ACCases.Abbreviations ACCase acetyl-CoA carboxylase - ACP acyl carrier protein - BC biotin carboxylase - BCCP biotin carboxyl carrier protein - CT carboxyl transferase - MF multi-functional - MS multi-subunit We thank our colleagues Nicki Engeseth and Vicki Eccleston for advice on fatty acid analysis and Sarah Hunter for providing the developing Iris seed. This work was supported in part by grant MCB 9406466 from NSF. Acknowledgement is also made to the Michigan Agriculture Experiment Station for its support of this research.     

Liver and intestinal fatty acid binding proteins in control and TGFβ1 gene targeted deficient mice

The effect of transforming growth factor beta-1 (TGF1) expression on fatty acid binding proteins was examined in control and two strains of gene targeted TGF1-deficient mice. Homozygous TGF1-deficient 129 × CF-1, expressing multifocal inflammatory syndrome, had 25% less liver fatty acid binding protein (L-FABP) when compared to control mice. The decrease in L-FABP expression was not due to multifocal inflammatory syndrome since homozygous TGF1-deficient/immunodeficient C3H mice on a SLID background had 36% lower liver L-FABP than controls. This effect was developmentally related and specific to liver, but not the proximal intestine, where L-FABP is also expressed. Finally, the proximal intestine also expresses intestinal-FABP (1-FABP) which decreased 3-fold in the TGF1-deficient/immunodeficient C3H mice only. Thus, TGF1 appears to regulate the expression of L-FABP and I-FABP in the liver and the proximal intestine, respectively.Abbreviations L-FABP liver fatty acid binding protein - I-FABP intestinal fatty acid binding protein - TGF1 transforming growth factor beta-1 - TNF- tumor necrosis factor- - MIP- macrophage inflammatory protein- - PMSF phenylmethyl sulfonyl fluoride - PBS phosphate buffered saline     

Carbohydrate and amino acid metabolism during batch culture of a human lymphoblastoid cell line,BTSN6

This work presents data on the carbohydrate and amino acid metabolism of a lymphoblastoid cell line producing an IgG1 antibody. In static culture, it was observed that lactate levels were significantly lowered when the cells were cultured on galactose as a carbon source. The use of carbohydrate substitution may be useful in lowering lactate levels, if it is established that this component is toxic to the cells. In addition, carbohydrate substitution may be used to modify glycosylation patterns and hence pharmacokinetic properties of glycoproteins.The amino acids glutamine and tryptophan were shown to be limiting in batch culture on this medium (DR, a 1:1 mixture of DMEM and RPMI, with 4mM glutamine). Amino acids produced included alanine, proline and glutamate. Serine was consumed to exhaustion, which was followed by a depletion of extracellular glycine. Amino acid metabolism, specific antibody productivity and specific growth rate were shown to be functions of the inoculation density in stirred flask culture. The results have implications for the design of media for both low and high density antibody manufacture by these cell lines.     

FLIGHT ACTIVITY AND FATTY ACID UTILIZATION IN MYTHIMNA SEPARATA (WALKER) MOTHS*

Abstract The fatty acid (FA) compositions for total lipids from fat body, hemolymph and flight muscle of the armyworm moths, Mythirnna separata, at rest and after tethered flight for 1 h were determined by GC and GC-MS. The composition in these tissues comprises myristic acid (1%-2%), palmitic acid (more than 35%1, palmitoleic acid (9%-11%), stearic acid (less than 1%), oleic acid (about 32%), linoleic acid (12%-17%) and linolenic acid (3%-6%). After flight, FA level in the fat body, compared to that at rest, shows a significant decline at about 20 μg/mg tissue.h^-1; the concentration of FAs in hemolymph rises evidently, but change of FA content in flight muscle appears to be small. From the changes of proportional composition of FAs in fat body, hemolymph and flight muscle, it is found that the FAs selectively utilized for flight in flight muscle are predominantly the palmitic acid and oleic acid.     

腐殖酸对小麦抗旱性的生理效应

在干旱气候条件下,喷施腐殖酸钠（ＨＡ）、黄腐酸钠（ＦＡ）可降低土壤水分损耗,提高小麦叶片持水能力,叶片细胞超氧物歧化酶（ＳＯＤ）、过氧化氢酶（ＣＡＴ）活性明显提高,丙二醛（ＭＤＡ）含量和电解质渗出率明显降低,可减缓叶绿素降解,增强光合速率和光合产物积累,延缓植株衰老,小麦抗旱性增强,千粒重增加．