Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.     

DNA fingerprinting of red clover (Trifolium pratense L.) with Jeffrey's probes: detection of somaclonal variation and other applications

Summary DNA fingerprints generated by the Jeffreys' probes, 33.6 and 33.15, indicated the presence of minisatellite-like sequences in the red clover genome. The fingerprints generated by probe 33.6 gave less background and fewer but better defined bands than those obtained with probe 33.15. Assay of a regenerative somaclonal variant (F49R) by DNA fingerprinting with probe 33.6 detected mutation that was unlinked to the regenerative trait. The fingerprints obtained under the applied conditions also demonstrated genetic stability of consecutive generations of the regenerants in tissue culture. DNA fingerprints of F₁ plants revealed that each polymorphic band was inherited from either one or the other parent. Both probes distinguished individual-specific genotypes in seven cultivars of red clover. Greater variability in DNA fingerprints was detected between (V=0.899) than within (0.417≤V≤0.548) cultivars.     

Tissue distribution of bupivacaine enantiomers in sheep

rac-Bupivacaine HCl was infused intravenously to constant arterial blood drug concentrations in sheep using a regimen of 4 mg/min for 15 min followed by 1 mg/min to 24 h. At 24 h, arterial blood was sampled, the animal was killed with a bolus of KCl solution, then rapidly dissected and samples were obtained from heart, brain, lung, kidney, liver, muscle, fat, gut, and rumen. Tissue:blood distribution coefficients for (+)-(R)-bupivacaine exceeded those of (?)-(S)-bupivacaine (P < 0.05) for heart, brain, lung, fat, gut, and rumen by an overall mean of 43%. Blood:plasma distribution coefficients of (?)-(S)-bupivacaine exceeded those of (+)-(R)-bupivacaine by a mean of 29% and this offset the tissue:blood distribution coefficients so that the previously significant enantioselective differences disappeared. It is concluded that although enantioselectivity of bupivacame distribution is shown by the measured tissue:blood distribution coefficients, it is not shown when tissue:plasma water distribution coefficients are calculated, suggesting that there is no intrinsic difference between the bupivacaine enantiomers in tissue affinity. Sheep given fatal intravenous bolus doses of rac-bupivacaine had significantly greater concentrations of (+)-(R)-bupivacaine than (?)-(S)-bupivacaine in brain (P = 0.028) and ventricle (P = 0.036); these could augment the greater myocardial toxicity of this enantiomer found in vitro. © 1993 Wiley-Liss, Inc.     

EFFECTS OF THE PHYSICAL ENVIRONMENT ON SOME LIPOPROTEIN LAYER SYSTEMS AND OBSERVATIONS ON THEIR MORPHOGENESIS

Lipoprotein membrane systems such as chloroplasts and the endoplasmic reticulum exhibit a generalized swelling response. The initial effect is an increase in interlamellar spacing, but as swelling proceeds, the membranes are transformed into closed thin-walled spherical vesicles. Available evidence suggests that morphogenesis of the endoplasmic reticulum of Nitella and the lamellar system of the Zea chloroplasts involves fusion of small spherical vesicles to yield closed double membrane structures, which subsequently undergo further differentiation. It is suggested that the vesicles comprise a convenient "micellar" form by which lipides may be transported within the cell from the sites of lipide synthesis to regions of lamellar growth. The characteristic formation of vesicles in swelling and the apparent fusion of vesicles in morphogenesis appear to represent two aspects of a fundamental plasticity of lipoprotein layer systems.     

Suckling behaviour in the brown long-eared bat (Plecotus auritus)

Thirty-two adult female brown long-eard bats were taken into captivity. Eight individuals gave birth to single young in captivity (known mother-young pairs), 10 were lactating when captured (putative mother-young pairs), and the remaining 14 bats were non-reproductive. Bats were maintained in five groups consisting of females from sigle(n=3) or mixed (n=2) wild roosts. All bats were housed in outdoor, free-flight enclosures and fed mainly on free-flying noctuid moths. Bats were individually were determined daily (n=152) for a single gruup of bats containing four known mother-young pairs and five non-reproductive bats. The probability of a being attached to the nipple declined from 100% of records at 1-5 days of age to 5% of records at 36-40 days of age. Females were always found suckling their own young. Suckling associations were determined using infra-red sensitive video-recordings of bat behaviour within the roost box. For both known (n=8) and putative mother-young pairs (n=10), there were no records of young attached to lactating females other than their own mothers (from the same or different wild roosts).     

Formation of nesquehonite and other minerals as a consequence of biofilm dehydration

A microbial biofilm community was established over 971 days within gravel in an aquarium so as to model biofouling of an aquifer. When the water was allowed to evaporate slowly, white crystalline deposits, containing several carbonate and sulphate minerals including nesquehonite (MgCO₃.3H2O), were seen at the highest points on the surface of the biofouled gravel. No such deposits occurred in regions lacking biofilms. These crystals appeared to originate from evaporation of dissolved salts which had migrated through the biofilm. Surfaceadherent microbial biofilms may conceivably provide a conduit for solute transport in porous media such as soils and aquifers.     

Biomass and feeding activity of phagotrophic mixotrophs in the northwestern Black Sea during the summer 1995

Biomass and activities of planktonicmicroorganisms (bacteria, nanoplankton andmicroplankton) were measured in the northwestern BlackSea during summer 1995. The method based on theuptake of fluorescently labeled prey was chosen todetermine the ingestion rate of bacteria andnanoplankton by phagotrophic microorganisms. Thismethod revealed the presence of mixotrophic organismssuch as ’plastid-retaining ciliates‘ in the wholecoastal area. Mixotrophic ciliates were dominated bymicro-sized forms and maximum biomasses were recorded inthe water masses characterised by low nutrientconcentrations but high food particle concentrations. Mixotrophic nanoflagellates were absentand mixotrophic dinoflagellates were observed at onestation only. Mixotrophic ciliates were shown to ingestpreferably bacteria while mixotrophic dinoflagellateswere grazing almost exclusively on nanoflagellates.Although the biomass of mixotrophic organisms weresignificantly lower than those of aplastidic protozoa,their feeding activity contributed to 14 and 24% ofthe ingestion of bacteria and nanoplankton, respectively.This is due to the high specificingestion rate of mixotrophic micro-sized ciliates anddinoflagellates, which were two and three times higher,respectively, than the specific ingestion rate ofbacteria and nanoplankton by aplastidic protozoa. Thissuggests a significant contribution of phagotrophicmixotrophs to the microbial network of thenorthwestern Black Sea. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characteristics of triplex-directed photoadduct formation by psoralen-linked oligodeoxynucleotides.

A triplex-forming oligopyrimidine has been attached at its 5'-end to a photoreactive psoralen derivative and used to target a sequence which forms part of the coding region of the human aromatase gene. The 20 base pair sequence is not a perfect triplex target since it contains three pyrimidine interruptions within the purine-rich strand. Despite this, we have detected triplex-directed photoadduct formation at pH 7.0 between the psoralen-linked oligonucleotide and a 30mer duplex representing the aromatase target. Photoadduct formation was found to be sensitive to pH, temperature, cation concentration and the base composition of the third strand. By varying the base sequence of the target duplex around the psoralen intercalation site, we have characterised the site and mode of psoralen intercalation. The attached psoralen has been found to intercalate at the triplex-duplex junction with a strong preference for one orientation. We have shown that the psoralen will bind at the junction even when there is a preferred TpA step at an adjacent site. We have also compared the binding affinity and photoreactivity of oligodeoxyribonucleotides linked to two different psoralen derivatives and found differences in the rate of crosslinking and the extent of crosslink formation. Finally, we have examined oligodeoxyribonucleotides which are attached to psoralen by polymethylene linkers of different lengths.     

Fructose 2,6-bisphosphate, sugar phosphates and adenine nucleotides in the regulation of glucose metabolism in the lactating rat mammary gland

Fructose 2,6-bisphosphate is present in the rat mammary gland, rising from a value of 1.4 nmol/g in pregnancy to 4.3 nmol/g tissue at 14 days lactation; the equivalent values calculated/ml intracellular water are 5.2 and 11.6 nmol, respectively. The tissue content of fructose 6-phosphate, fructose 1,6-bisphosphate, ATP and phosphoenolpyruvate remain relatively constant in the transition from pregnancy to the height of lactation. The changes in AMP, cyclic AMP, and citrate content of the mammary gland during lactation are such as to promote an increase in fructose 2,6-bisphosphate formation and flux through phosphofructokinase.