Beta-adrenergic control of trans-cutaneous evaporative cooling mechanisms in birds

Summary The decreasing effect of -adrenergic blockade on skin resistance to vapor diffusion and the onset of cutaneous water evaporation in the pigeon (Columba livia) was investigated. Oral administration of 1, 2.3 and 5 mg propranolol to pigeons (268±53 g) initiated intensive trans-cutaneous water evaporation (CWE) up to 29.1 mg H₂O·cm^–2·h^–1 in resting birds at 30°C air temperature (Ta), but had only a slight effect on CWE of birds exposed to 50 °C Ta.After 7 h of effective -adrenergic blockade (oral administration of 5 mg propranolol), skin and body temperature stabilized at 39.0±0.5 °C and 41.0±0.7 °C, compared to 40.2±0.8 °C and 41.9±0.6 °C in the control group, respectively. A slight hypothermia was accompanied by feather fluffing.Intradermal injection of 0.001, 0.01 and 0.12 mg propranolol also caused intensive CWE. Local -adrenergic blockade in relatively low blocker doses (0.001 and 0.01 mg propranolol) decreased skin resistance from a high value of 44.5 s·cm^–1 to about 6.0 s·cm^–1, and caused a sharp increase in CWE from a control value of about 4 to a high of 26.4 mg H₂O·cm^–2·h^–1 during the first two hours of exposure to 30°C Ta.The possible role of -adrenergic blockade in regulation of trans-cutaneous water evaporation of latent heat dissipation is discussed.     

Further photophysical and photochemical characterization of flavins associated with single-shelled vesicles

Summary In order to investigate whether the loop diuretic sensitive, sodium-chloride cotransport system described previously in shark rectal gland is in fact a sodium-potassium chloride cotransport system, plasma membrane vesicles were isolated from rectal glands ofSqualus acanthias and sodium and rubidium uptake were measured by a rapid filtration technique. In addition, the binding of N-methylfurosemide to the membranes was investigated. Sodium uptake into the vesicles in the presence of a 170mm KCl gradient was initially about five-fold higher than in the presence of a 170mm KNO₃ gradient. In the presence of chloride, sodium uptake was inhibited 56% by 0.4mm bumetanide and 40% by 0.8mm N-methylfurosemide. When potassium chloride was replaced by choline chloride or lithium chloride, sodium uptake decreased to the values observed in the presence of potassium nitrate. Replacement of potassium chloride by rubidium chloride, however, did not change sodium uptake. Initial rubidium uptake into the membrane vesicles was about 2.5-fold higher in the presence of a 170mm NaCl gradient than in the presence of a 170mm NaNO₃ gradient. The effect of chloride was completely abolished by 0.4mm bumetanide. Replacement of the sodium chloride gradient by a lithium chloride gradient decreased rubidium uptake by about 40%; replacement by a choline chloride gradient reduced the uptake even further. Rubidium uptake was also strongly inhibited by potassium. Sodium chloride dependence and bumetanide inhibition of rubidium flux were also found in tracer exchange experiments in the absence of salt gradients. The isolated plasma membranes bound³[H]-N-methylfurosemide in a dose-dependent manner. In Scatchard plots, one saturable component could be detected with an apparentK _D of 3.5×10^–6 m and a number of sitesn of 104 pmol/mg protein. At 0.8 m, N-methylfurosemide binding decreased 51% when sodium-free or low-potassium media were used. The same decrease was observed when the chloride concentration was increased from 200 to 600mm or when 1mm bumetanide or furosemide were added to the incubation medium. These studies indicate that the sodium-chloride cotransport system described previously in the rectal gland is in fact a sodium-potassium chloride cotransport system. It is postulated that this transport system plays an essential role in the secondary active chloride secretion of the rectal gland.     

Conditions for open-air outdoor culture of Dunaliella salina in southern Spain

The optimization of the operation, under the climatic conditions of southern Spain, of an experimental plant for -carotene production by Dunaliella has been pursued. The effects of mixing, culture depth, cell density and dilution cycles on -carotene and biomass productivity were studied under a semicontinuous culture regime in open tanks outdoors. Using 3 m²-surface containers, the highest productivity values, for both -carotene and biomass, were recorded with a flow rate of 0.55 m s^–1; 10 cm depth; 0.7 – 0.9 × 10⁶cell ml^–1, population density; and dilution cycles of two days. An average annual productivity of 1.65 g (dry wt) m^–2 d^–1 was estimated for Dunaliella biomass, being that for -carotene of about 0.1 g m^–2 d^–1. Under these optimized conditions, experiments have been carried out at the Cadiz Bay with 20 m²-surface tanks during a whole-year cycle. The results obtained have validated this location and the operating conditions established as being most appropriate for efficient mass production of -carotene rich D. salina.     

Permeation of divalent cations through α-latrotoxin channels in lipid bilayers: steady-state current-voltage relationships

Summary -Latrotoxin, a polypeptide neurotoxin known to cause massive release of transmitter from vertebrate nerve terminals, is thought to act by forming cation-selective channels in plasma membranes. This paper describes the steady-state current carried by Ca²⁺, Sr²⁺ and Ba²⁺ through pores of -LaTx molecules incorporated in artificial bilayer membranes made of neutral lipids. Even when the solutions separated by the membrane are identical, theI-V relations rectify strongly, the current being higher when the side to which the toxin is added is positive. The polarity of the rectification is consistent with the hypothesis that the mechanism of action of the toxin is, at least in part, that of promoting inwardly directed flow of cations, and thus, accumulation of Ca²⁺ and other ions in the intracellular spaces. The dependence of theI-V characteristics on voltage and Ca²⁺ concentration is well described by a one-site, one-ion model for a channel. Three parameters of the model are deduced: the binding constant of the site for Ca²⁺,K=1.5m ^–1 (orK=7m ^–1 when activities are used instead of concentrations); the electrical distance of the site from the toxin-containing solution, =0.3; the free energy difference between the two barrier peaks, F =0.26 kT. The values of the parameters deduced by studying the channel in the presence of Ca²⁺ give theoretical curves that also fit the data with Sr²⁺ and Ba²⁺, indicating a low level of discrimination among these three cations.     

Binding of chloride and a disulfonic stilbene transport inhibitor to red cell band 3

Summary The effect of chloride on 4,4-dibenzamido-2,2-disulfonic stilbene (DBDS) binding to band 3 in unsealed red cell ghost membranes was studied in buffer [NaCl (0 to 500mm) + Na citrate] at constant ionic strength (160 or 600mm). pH 7.4, 25°C. In the presence of chloride, DBDS binds to a single class of sites on band 3. At 160mm ionic strength, the dissociation constant of DBDS increases linearly with chloride concentration in the range [Cl]=450mm. The observed rate of DBDS binding to ghost membranes, as measured by fluorescence stopped-flow kinetic experiments, increases with chloride concentration at both 160 and 600mm ionic strength. The equilibrium and kinetic results have been incorporated into the following model of the DBDS-band 3 interaction: The equilibrium and rate constants of the model at 600mm ionic strength areK ₁=0.67±0.16 m,k ₂=1.6±0.7 sec^–1,k _–2=0.17±0.09 sec^–1,K ₁=6.3±1.7 m,k ₂=9±4 sec^–1 andk _–2=7±3 sec^–1. The apparent dissociation constants of chloride from band 3,K _Cl, are 40±4mm (160mm ionic strength) and 11±3mm (600mm ionic strength). Our results indicate that chloride and DBDS have distinct, interacting binding sites on band 3.     

Angiotensin II,vasopressin and GTP[γ-S] inhibit inward-rectifying K+ channels in porcine cerebral capillary endothelial cells

Summary Cerebral capillaries from porcine brain were isolated. and endothelial cells were grown in primary culture. The whole-cell tight seal patch-clamp method was applied to freshly isolated single endothelial cells, and cells which were held in culture up to one week. With high K⁺ solution in the patch pipette and in the bath we observed inward-rectifying K⁺ currents, showing a time-dependent decay in part of the experiments. Ba²⁺ (1–10mm) in the bath blocked this current, whereas outside tetraethylammonium (10mm) decreased the peak current but increased the steady-state current. Addition of 1 m of angiotensin II or of arginine-vasopressin to the extracellular side caused a time-dependent inhibition of the inward-rectifying K⁺ current in part of the experiments. Addition of 100 m GTP[-S] to the patch pipette blocked the K⁺ inward rectifier. In cell-attached membrane patches two types of single inward-rectifying K⁺ channels were observed, with single channel conductances of 7 and 35 pS. Cell-attached patches were also obtained at the antiluminal membrane of intact isolated cerebral capillaries. Only one type of K⁺ channel withg=30 pS was recorded. In conclusion, inwardly rectifying K⁺ channels, which can be inhibited by extracellular angiotensin II and arginine-vasopressin, are present in cerebral capillary endothelial cells. The inhibition of this K⁺ conductance by GTP[-S] indicates that G-proteins are involved in channel regulation. It is suggested that angiotensin II and vasopressin regulate K⁺ transport across the blood-brain barrier, mediating their effects via G-proteins.     

Anion specificity of the jejunal folate carrier: Effects of reduced folate analogues on folate uptake and efflux

Summary We previously reported that³H-folate uptake by rabbit jejunal brush-border membrane (BBM) vesicles was markedly stimulated by an outwardly directed OH^– gradient (pH_in 7.7, pH_out 5.5), inhibited by anion exchange inhibitors (DIDS, SITS, furosemide), and saturable (folateK _m=0.19 m) suggesting carrier-mediated folate/OH^– exchange (or H⁺/folate cotransport). In the present study, the anion specificity of this transport process was examined. Under conditions of an outwardly directed OH^– gradient, DIDS-sensitive folate uptake wascis inhibited (>90%) by reduced folate analogues: dihydrofolate (IC₅₀=0.40 m), folinic acid (IC₅₀=0.50 m), 5-methyltetrahydrofolate (IC₅₀=0.53 m), and (+)amethopterin (IC₅₀=0.93 M). In contrast, 10 m (–)amethopterin had only a modest effect on folate uptake (18% inhibition) suggesting stereospecificity of the folate/OH^– exchanger. The nonpteridine compounds which are transported by the folate carrier in L1210 leukemic cells (phthalate, thiamine pyrophosphate, and PO ₄ ^–3 ) did not inhibit jejunal folate uptake. Furthermore, folate uptake was not inhibited by SO ₄ ^–2 (4mm) or oxalate (4mm) thereby distinguishing this carrier from the previously described intestinal SO ₄ ^–2 /OH^– and oxalate/Cl^– exchangers. After BBM vesicles were loaded with³H-folate, the initial velocity of³H-folate efflux was stimulated by unlabeled folate in the efflux medium. The transstimulation of³H-folate efflux by unlabeled folate was furosemide (or DIDS) inhibitable and temperature sensitive. Half-maximal stimulation of furosemide-sensitive³H-folate efflux was observed with 0.25±0.05 m unlabeled folate, a concentration similar to theK _m for folate uptake. These data suggest that folate-stimulated³H-folate efflux is mediated by the folate/OH^– exchanger. With the exception of (–) amethopterin, reduced folate analogues also transstimulated furosemide-sensitive³H-folate efflux in a concentration-dependent manner suggesting stereospecific transport of these analogues by the folate/OH^– exchanger. In summary, folate transport by the jejunal folate/OH^– exchanger demonstrates bothcis inhibition and transstimulation by reduced folate analogues, but not by other inorganic or organic anions suggesting bidirectional transport of folate and a high degree of anion specificity.     

Study ofO-sialylation of glycoproteins in C6 glioma cells treated with retinoic acid

When treated with retinoic acidin vivo, C6 glioma cells show an enhancement of CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase activity. A 300kDa glycoprotein was detected by lectin affinoblotting in retinoic acid-treated C6 cells which stained weakly or not at all in control cells. Comparative studies with different lectins demonstrated that this glycoprotein contains 2,3 Neu5Ac Gal-GalNAc O-glycan moieties. Cultures in the presence of an inhibitor of O-glycan synthesis (N-acetylgalactosaminide -O-benzyl) demonstrated that enhancement of staining of the 300 kDa glycoprotein was not due to the increase of the 2,3 sialyltransferase but to thede novo synthesis of the polypeptide chain of this glycoprotein.Abbreviations RA retinoic acid - Neu5Ac N-acetylneuraminic acid - CMP-Neu5Ac cytidine 5 monophosphosialate - 2,3 ST CMP-Neu5Ac:Gal 1–3 GalNAc-R -2,3 sialyltransferase - GalNAc-O-benzyl N-acetylgalactosaminide -O-benzyl - Gal1-3GalNAc-O-benzyl Galactosyl 1-3N-acetylgalactosaminide -O-benzyl - TBS Tris-HCl buffer 50mm pH 7.5 containing NaCl 0.15m and Tween 20 0.05% - B1 buffer TBS containing MgCl₂ 1mm, MnCl₂ 1mm and CaCl₂ 1mm     

Mode of uptake of sterol by Arthrobacter simplex

The mechanism of uptake of water-insoluble -sitosterol by a newly isolated strain of Arthrobacter simplex SS-7 was studied. The production of an extracellular sterol-pseudosolubilizing protein during growth of A. simplex on -sitosterol was demonstrated by isolating the factor from the cell-free supernatant and its subsequent purification by Sephadex G-150 column chromatography. The M _r of the purified sterol-pseudosolubilizing protein determined by SDS–PAGE was 19kDa. The rate of sterol pseudosolubilization (5.2×10^–3g l^–1h^–1) could not adequately account for the rate of sterol uptake (72×10^–3g l^–1h^–1) and the specific growth rate (56×10^–3 h^–1). However in the unfavourable growth condition, when the cells were treated with sodium azide at the level of 30–60% of MIC, the sterol pseudosolubilization accounted for nearly 74% of the total growth containing 96% free cells. Cellular adherence to substrate particles was found to play an active role in the normal growth of the strain on -sitosterol. Unlike sodium acetate-grown cells, whose surface activity was negligible (60mNm^–1), the sterol-grown cells had strong surface activity (40mNm^–1). The high lipid content and long chain fatty acids in the cell-wall of -sitosterol-grown cells probably contribute to the high sterol adherence activity of the cells.     

The sodium concentration of the lateral intercellular spaces of MDCK cells: A microspectrofluorimetric study

MDCK cell monolayers grown on glass coverslips were used to examine the Na⁺ concentration in individual lateral intercellular spaces (LIS) by video fluorescence microscopy. The LIS was filled with the Na⁺-sensitive fluorescent dye SBFO by incubation of the monolayers for 75–90 min with 250 m of the membrane impermeant form of the dye. After dye loading, the monolayers were perfused at 37°C with solutions buffered with HEPES or bicarbonate/CO₂ containing 142 mm Na⁺. Ratios of the fluorescence images after sequential excitation with 340 nm and 380 nm light were performed and in situ calibration of LIS Na⁺ was accomplished after blocking the Na⁺ pump with 5 × 10^–4 m ouabain. Measurements of Na⁺ along the basolateral-to-apical axis of the LIS at 1.0 or 1.5 m intervals did not reveal a Na⁺ gradient when the perfusate was either HEPES or bicarbonate/CO₂ solutions. In bicarbonate solutions, the mean Na⁺ concentration (mm) was 157.2 ± 2.3, 15 mm higher than the bath Na⁺ concentration. In HEPES solutions, however, the Na⁺ concentration was not different from the bath concentration (142.7 ± 3.1 mm). The time course of Na⁺ changes in LIS was investigated by rapidly switching the perfusate from 142 to 80 mm Na⁺ and measuring the Na⁺ changes at one focal plane.We would like to thank P.H. Tran and C. Gibson for their technical and computational assistance as well as Dr. B.-E. Persson (University of Uppsala, Sweden) for his contribution in the early phases of the study.     

Identification of a GDP-Fuc:Galβ1–3GalNAc-R (Fuc to Gal)α1–2 fucosyltransferase and a GDP-Fuc:Galβ1–4GlcNAc (Fuc to GlcNAc)α1–3 fucosyltransferase in connective tissue of the snailLymnaea stagnalis

Connective tissue of the freshwater pulmonateLymnaea stagnalis was shown to contain fucosyltransferase activity capable of transferring fucose from GDP-Fuc in 1–2 linkage to terminal Gal of type 3 (Gal1–3GalNAc) acceptors, and in 1–3 linkage to GlcNAc of type 2 (Gal1–4GlcNAc) acceptors. The 1–2 fucosyltransferase was active with Gal1–3GalNAc1-OCH₂CH=CH₂ (K _m=12 mM,V _max=1.3 mU ml^–1) and Gal1–3GalNAc (K _m=20 mM,V _max=2.1 mU ml^–1), whereas the 1–3 fucosyltransferase was active with Gal1–4GlcNAc (K _m=23 mM,V _max=1.1 mU ml^–1). The products formed from Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4GlcNAc were purified by high performance liquid chromatography, and identified by 500 MHz¹H-NMR spectroscopy and methylation analysis to be Fuc1–2Gal1–3GalNAc1-OCH₂CH=CH₂ and Gal1–4(Fuc1–3)GlcNAc, respectively. Competition experiments suggest that the two fucosyltransferase activities are due to two distinct enzymes.Abbreviations 2Fuc-T 1–2 fucosyltransferase - 3Fuc-T 1–3 fucosyltransferase - MeO-3Man 3-O-methyl-D-mannose - MeO-3Gal 3-O-methyl-D-galactose     

Apparent loss of calcium-activated potassium current in internally perfused snail neurons is due to accumulation of free intracellular calcium

Summary Internal perfusion ofHelix neurons with a solution containing potassium aspartate, MgCl₂, ATP, and HEPES causes the calcium-activated potassium current (I _K(Ca)) evoked by depolarizing voltage steps to decrease with time. When internal free Ca⁺⁺ is strongly buffered to 10^–7 m by including 0.5mm EGTA and 0.225mm CaCl₂ in the internal solution,I _K(Ca) remains constant for up to 3 hours of perfusion. In cells whereI _K(Ca) is small at the start of perfusion, perfusion with the strongly buffered 10^–7 m free Ca⁺⁺ solution produces increases inI _K(Ca) which ultimately saturate. In cells perfused with solutions buffered to 10^–6 m free Ca⁺⁺,I _K(Ca) is low and does not change with perfusion. These results lead us to conclude thatI _K(Ca) is stable in perfusedHelix neurons and that the apparent loss ofI _K(Ca) seen initially with perfusion is due to accumulation of cytoplasmic calcium. Since the calcium current (I _Ca) provides the Ca⁺⁺ which activatesI _K(Ca) during a depolarizing pulse,I _Ca is also stable in perfused cells when free intracellular Ca⁺⁺ is buffered.Perfusion with 1 m calmodulin (CaM) produces no effect onI _K(Ca) with either 10^–7 or 10^–6 m free internal calcium. Inhibiting endogenous CaM by including 50 m trifluoperazine (TFP) in both the bath and the internal perfusion solution also produces no effect onI _K(Ca) with 10^–7 m free internal calciu. It is concluded that CaM plays no role inI _K(Ca) activation.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

Enzymatic synthesis of lactic acid derivatives with emulsifying properties

Novozym 435 (50 g l^–1) catalyzed the synthesis of n-octyl--d-glucopyranoside lactate by transesterification between n-octyl--d-glucopyranoside (30 g l^–1) and ethyl lactate (100 g l^–1) in acetone. In 12 h, a molar yield of 87% n-octyl -d-glucopiranoside lactate was obtained at a overall conversion of 90%.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

pH Modulation of the kinetics of rabbit jejunal,brush-border folate transport

Summary In jejunal brush-border membrane vesicles, an out-wardly directed OH^– gradient (in>out) stimulates DIDS-sensitive, saturable folate (F) uptake (Schron, C.M., 1985).J. Clin. Invest. 76:2030–2033), suggesting carrier-mediated folate: OH^– exchange (or phenomenologically indistiguishable H⁺: folate cotransport). In the present study, the precise role of pH in the transport process was elucidated by examinin F uptake at varying pH. For pH gradients of identical magnitude, F uptake (0.1 M) was geater at lower (pH_int/pH_ext:5.5/4.5) compared with higher (6.5/5.5) pH ranges. In the absence of a pH gradient, internal Ftrans stimulated DIDS-sensitive³H-folate uptake only at pH6.0. Since setepwise increments ininternal pH (4.57.5; pH_ext=4.5) stimulated F uptake, an inhibitory effect of higherinternal pH was excluded. In contrast, with increasing external pH(4.356.5; pH_int=7.8), a 50-fold decrement in F uptake was observed (H⁺ K _m =12.8±1.2m). Hill plots of these data suggest involvement of at least one H⁺ (OH^–) at high pH (divalent F^–2 predominates). Since an inside-negative electrical potential did not affect F uptake at either pH_ext 4.55 or 5.8, transport of F^– and F^–2 is electroneutral. Kinetic parameters for F^– and F^–2 were calculated from uptake data at pH_ext 4.55 and 5.0. Comparision of predictedvs. experimentally determined kinetic parameters at pH_ext 5.8 (K _m =1.33vs. 1.70 m;V _max=12.8vs. 58.0 pmol/mg prot min) suggest that increasing external pH lowers theV _max, but does not affect thatK _m, for carrier-mediated F transport. These data are consistent with similarK _i's for sulfasalazine (competitive inhibitor) at pH_ext 5.35 and 5.8 (64.7 and 58.5 m, respectively). In summary, the jejunal F carrier mediates electroneutral transport of mono- and divalen F and is sensitive to extermal pH with a H⁺ K _m (or OH^– IC₅₀) corresponding to pH 4.89. External pH affects theV _max, but not theK _m for carriermediated F uptake suggesting a reaction mechanism involving a ternary complex between the outward-facing conformation of the carrier and the transported ions (F and either OH^– or H⁺) rather than competitive binding that is mutually exclusive.     

Swelling-activated Chloride and Potassium Conductance in Primary Cultures of Mouse Proximal Tubules. Implication of KCNE1 Protein

Volume-sensitive chloride and potassium currents were studied, using the whole-cell clamp technique, in cultured wild-type mouse proximal convoluted tubule (PCT) epithelial cells and compared with those measured in PCT cells from null mutant kcne1 –/– mice. In wild-type PCT cells in primary culture, a Cl^– conductance activated by cell swelling was identified. The initial current exhibited an outwardly rectifying current-voltage (I-V) relationship, whereas steady-state current showed decay at depolarized membrane potentials. The ion selectivity was I^– > Br^– > Cl^– >> gluconate. This conductance was sensitive to 1 mM 4,4-Diisothiocyanostilbene-2,2-disulfonic acid (DIDS), 0.1 mM 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and 1 mM diphenylamine-2-carboxylate (DPC). Osmotic stress also activated K⁺ currents. These currents are time-independent, activated at depolarized potentials, and inhibited by 0.5 mM quinidine, 5 mM barium, and 10 µM clofilium but are insensitive to 1 mM tetraethylammonium (TEA), 10 nM charybdotoxin (CTX), and 10 µM 293B. In contrast, the null mutation of kcne1 completely impaired volume-sensitive chloride and potassium currents in PCT. The transitory transfection of kcne1 restores both Cl^– and K⁺ swelling-activated currents, confirming the implication of KCNE1 protein in the cell-volume regulation in PCT cells in primary cultures.     

Causes of Parkinson’s disease: genetics of <Emphasis Type="Italic">DJ-1</Emphasis>

The identification of Mendelian mutations in rare forms of familial Parkinsons disease (PD) have provided significant insights into the molecular pathogenesis of this common complex disorder. DJ-1 is the third of four genes known to be definitively causal in familial PD, the three others being -synuclein, parkin and the recently identified PINK1. Mutations in the DJ-1 gene were identified in two European families, a Dutch kindred harbouring a large homozygous genomic deletion encompassing exons 1–5 of the gene and an Italian kindred with a homozygous L166P missense mutation. The clinical phenotype of the two families was similar to that of parkin cases. Age of onset was in the mid-thirties with good responsiveness to l-dopa and slow disease progression. Focal dystonias and blepharospasm were also evident as were behavioural disturbances early in the course of the disease. To date, there are no studies of pathological material from known DJ-1 patients. It therefore remains to be determined whether these patients form Lewy bodies and/or Lewy neurites, the eosinophilic fibrillary inclusions that contain predominantly -synuclein and that are the pathological hallmark of PD.     

Biochemical and genetic factors in the heat inactivation of murine β-glucuronidase

The enzymes coded for by two alleles at the glucuronidase structural locus (Gus) were compared in their response to pH, buffering anion, buffer molarity, ionic strength, and temperature. The heat-labile Gus^h gene product responded in a qualitatively similar but quantitatively reduced manner compared to the relatively heat-stable Gus ^b gene product. In all buffers tested, the enzyme was most heat stable at pH 5.0. Ranking of the various buffer anions tested, according to increasing heat stabilization, was water acetate phosphate < citrate. Varying the molarity of the buffers from 0.01 to 0.6 m at pH 5.0 revealed further differences among the buffers. Increasing ionic strength exerted a destabilizing force on the protein. The half-life of the enzyme decreased by as much as a hundredfold between 71 and 75 C. The Gus^h/Gus^h genotype also results in decreased activity levels in all tissues, reportedly because of decreased synthesis. The heat inactivation curves of Gus^b/Gus^h heterozygotes were incompatible with any theoretical curve based on the assumption that the Gus^b and Gus^h chromosomes in the heterozygote behave in a manner similar to that seen in the homozygotes.This research was supported by a Basil O'Connor Starter Research Grant from the National Foundation—March of Dimes (R. J. M.) and by a grant from The Jane Coffin Childs Memorial Fund for Medical Research (K. H.).Fellow of The Jane Coffin Childs Memorial Fund for Medical Research.     

Effects induced by mono- and divalent cations on protein regions responsible for thermal adaptation in β-glycosidase from<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis>

The perturbation induced by mono- and divalent cations on the thermophilicity and thermostability of Solfolobus solfataricus -glycosidase, a hyperthermophilic tetrameric enzyme, has been investigated by spectroscopic and computational simulation methods to ascertain the Hofmeister effects on two strategic protein regions identified previously. Specifically, (1) an extra segment (83–124), present only in the sequence of hyperthermophilic glycosidases and recognized as an important thermostability determinant for the enzyme structure; and (2) a restricted area of the subunit interface responsible for the quaternary structure maintenance. Mono- and divalent cations inhibit to a different extent the -glycosidase activity, whose kinetic constants show an apparent competitive inhibition of the catalytic process that reflects the Hofmeister order. The thermostability is also affected by the nature and charge of the cations, reaching maximal effects for the case of Mg²⁺. Fourier transform infrared spectroscopy has revealed very small changes in the protein secondary structure in the presence of the investigated cations at 20 °C, while large effects on the protein melting temperatures are observed. Computational analysis of the enzyme structure has identified negative patches on the accessible surface of the two identified regions. Following the Hofmeister series, cations weaken the existing electrostatic network that links the extra segment to the remaining protein matrix. In particular, the perturbing action of cations could involve the ionic pair interactions E107–R245 and E109–R185, thus leading to a local destructuring of the extra segment as a possible starting event for thermal destabilization. A detailed investigation of the electrostatic network at the A–C intermolecular interface of Sgly after energy minimization suggests that cations could cause a strong attenuation of the ion pair interactions E474–K72 and D473–R402, with consequent partial dissociation of the tetrameric structure.Abbreviations Amide I amide I band in a ²H₂O medium - EM energy minimization - ONPG o-nitrophenyl--d-galactopyranoside - Sgly Escherichia coli expressed Sulfolobus solfataricus -glycosidase