Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Retention of basic electrophysiologic properties by human sweat duct cells in primary culture

Summary The present investigation was undertaken to examine the usefulness of cultured human sweat duct cells for ion transport and related studies in the genetic disease, cystic fibrosis. Electrical properties of cultured duct (CD) cells were compared with electrical properties of microperfused duct (MPD) cells. The resting apical membrane potential (V _a ) of the CD cells was −26.4±0.9 mV,n=158 cells as compared to −24.3±0.6 mV,n=105 of MPD cells. The Na⁺−K⁺ pump inhibitor ouabain, when applied to the apical surface of the CD cells and basolateral surface of MPD cells, depolarized both CD cells (from −28.6±3.6 to −16.8±2.4 mV,n=5) and MPD cells (from −23.8±0.5 mV to −19.5±1.8 mV,n=6). The Na⁺ conductance inhibitor amiloride applied to the apical surface hyperpolarized the apical membrane potentials (V_a) of CD cells and MPD cells by −13.2±1.4 mV,n=43 and −34.3±3.1 mV,n=19), respectively, indicating the presence of amiloride sensitive Na⁺ channels in both groups of cells. However, the amiloride sensitivity of CD cells was dependent on the age of the culture. Cl⁻ substitution at the apical side by the impermeant anion gluconate depolarized the V _a of CD cells and MPD cells by 12.2±0.9 mV,n=32 and 37.9±4.3 mV,n=12, respectively. The effect of β-adrenergic agonist isoproterenol (IPR), was inconsistent. In CD cells, IPR either hyperpolarized (ΔV _a =−8.3±1.2mV,n=5) or depolarized (ΔV _a =8.2±2.3 mV,n=4) or had no effect,n=2. In contrast, most of the MPD cells did not respond to IPR, but three cells had a varied response to IPR. Our results suggest that CD cells, like MPD cells, retain significant Na⁺ and Cl⁻ conductances. CD cells seem to have developed a higher sensitivity to β-adrenergic stimulation in tissue culture as compared to MPD cells. This work was supported by grants from the National Institutes of Health, Bethesda, MD, DK26547, Getty Oil Co., the Gillette Co., Cystic Fibrosis Research Inc., and the U.S. National Cystic Fibrosis Foundation.     

Flow-Dependent Activation of Maxi K+ Channels in Apical Membrane of Rabbit Connecting Tubule

The Ca²⁺-activated maxi K⁺ channel was found in the apical membrane of everted rabbit connecting tubule (CNT) with a patch-clamp technique. The mean number of open channels (NP _o ) was markedly increased from 0.007 ± 0.004 to 0.189 ± 0.039 (n= 7) by stretching the patch membrane in a cell-attached configuration. This activation was suggested to be coupled with the stretch-activation of Ca²⁺-permeable cation channels, because the maxi K⁺ channel was not stretch-activated in both the cell-attached configuration using Ca²⁺-free pipette and in the inside-out one in the presence of 10 mm EGTA in the cytoplasmic side. The maxi K⁺ channel was completely blocked by extracellular 1 μm charybdotoxin (CTX), but was not by cytoplasmic 33 μm arachidonic acid (AA). On the other hand, the low-conductance K⁺ channel, which was also found in the same membrane, was completely inhibited by 11 μm AA, but not by 1 μm CTX. The apical K⁺ conductance in the CNT was estimated by the deflection of transepithelial voltage (ΔV _t ) when luminal K⁺ concentration was increased from 5 to 15 mEq. When the tubule was perfused with hydraulic pressure of 0.5 KPa, the ΔV _t was only −0.7 ± 0.4 mV. However, an increase in luminal fluid flow by increasing perfusion pressure to 1.5 KPa markedly enhanced ΔV _t to −9.4 ± 0.9 mV. Luminal application of 1 μm CTX reduced the ΔV _t to −1.3 ± 0.6 mV significantly in 6 tubules, whereas no significant change of ΔV _t was recorded by applying 33 μm AA into the lumen of 5 tubules (ΔV _t =−7.2 ± 0.5 mV in control vs.ΔV _t =−6.7 ± 0.6 mV in AA). These results suggest that the Ca²⁺-activated maxi K⁺ channel is responsible for flow-dependent K⁺ secretion by coupling with the stretch-activated Ca²⁺-permeable cation channel in the rabbit CNT. Received: 21 August 1997/Revised: 20 March 1998     

Finite element–based injury metrics for pulmonary contusion via concurrent model optimization

This study explores the relationship between impact severity and resulting pulmonary contusion (PC) for four impact conditions using a rat model of the injury. The force–deflection response from a Finite Element (FE) model of the lung was simultaneously matched to experimental data from distinct impacts via a genetic algorithm optimization. Sprague-Dawley rats underwent right-side thoracotomy prior to impact. Insults were applied directly to the lung via an instrumented piston. Five cohorts were tested: a sham group and four groups experiencing lung insults of varying degrees of severity. The values for impact velocity (V) and penetration depth (D) of the cohorts were Group 1, (V = 6.0 m · s⁻¹, D = 5.0 mm), Group 2, (V = 1.5 m · s⁻¹,D = 5.0 mm), Group 3, (V = 6 m · s⁻¹, D = 2.0 mm), and Group 4, (V = 1.5 m · s⁻¹, D = 2.0 mm). CT scans were acquired at 24 h, 48 h, and 1 week post-insult. Contusion volume was determined through segmentation. FE-based injury metrics for PC were determined at 24 h and 1 week post-impact, based on the observed volume of contusion and first principal strain. At 24 h post-impact, the volume of high radiopacity lung (HRL) was greatest for the severe impact group (mean HRL = 9.21 ± 4.89) and was significantly greater than all other cohorts but Group 3. The concurrent optimization matched simulated and observed impact energy within one standard deviation for Group 1 (energy = 3.88 ± 0.883 mJ, observed vs. 4.47 mJ, simulated) and Group 2 (energy = 1.46 ± 0.403 mJ, observed vs. 1.50 mJ, simulated) impacts. Statistically significant relationships between HRL and impact energy are presented. The FEA-based injury metrics at 24 h post-contusion are e_max·[(e)\dot]_max{\varepsilon_{\max}\cdot \dot {\varepsilon}_{\max}} exceeding 94.5 s⁻¹, ε _max exceeding 0.284 and [(e)\dot]_max{\dot{\varepsilon}_{\max}} exceeding 470 s⁻¹. Thresholds for injury to the lung still present at 1 week post-impact were also determined. They are e_max·[(e)\dot]_max{\varepsilon_{\max}\cdot \dot {\varepsilon}_{\max}} exceeding 149 s⁻¹, ε _max exceeding 0.343 and [(e)\dot]_max{\dot{\varepsilon}_{\max}} exceeding 573 s⁻¹. A mesh sensitivity study found that thresholds based on strain rate were more sensitive to changes to mesh density than the threshold based on strain only.     

Oxygen consumption and blood flow distribution in perfused skeletal muscle of chinook salmon

An isolated, perfused salmon tail preparation showed oxyconformance at low oxygen delivery rates. Addition of pig red blood cells to the perfusing solution at a haematocrit of 5 or 10% allowed the tail tissues to oxyregulate. Below ca. 60 ml O₂ kg⁻¹ h⁻¹ of oxygen delivery (DO₂), VO₂ was delivery dependent. Above this value additional oxygen delivery did not increase VO₂ of resting muscle above ca. 35 ml O₂ kg⁻¹ h⁻¹. Following electrical stimulation, VO₂ increased to ca. 65 ml O₂ kg⁻¹ h⁻¹, with a critical DO₂ of ca. 150 ml O₂ kg⁻¹ h⁻¹. Dorsal aortic pressure fell to 69% of the pre-stimulation value after 5 min of stimulation and to 54% after 10 min. Microspheres were used to determine blood flow distribution (BFD) to red (RM) and white muscle (WM) within the perfused myotome. Mass specific BFD ratio at rest was found to be 4.03 ± 0.49 (RM:WM). After 5 min of electrical stimulation the ratio did not change. Perfusion with saline containing the tetrazolium salt 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) revealed significantly more mitochondrial activity in RM. Formazan production from MTT was directly proportional to time of perfusion in both red and WM. The mitochondrial activity ratio (RM:WM) did not change over 90 min of perfusion.     

Kinetics of lipase catalyzed isoamyl acetate synthesis at high hydrostatic pressure in hexane

Lipase-catalyzed synthesis of isoamyl acetate in hexane at 10–250 MPa at 80°C and 1–100 MPa at 40°C resulted in activation volumes of −12.9 ± 1.7 and −21.6 ± 2.9 cm³ mol⁻¹, respectively. Increasing pressure from 10 to 200 MPa resulted in approximately 10-fold increase in V _max at both 40 and 80°C. Pressure increased the K _m from 2.4 ± 0.004 to 38 ± 0.78 mM at 40°C. In contrast, at 80°C the pressure did not affect the K _m.     

Several Conserved Positively Charged Amino Acids in OATP1B1 are Involved in Binding or Translocation of Different Substrates

OATP1B1 and 1B3 are related transporters mediating uptake of numerous compounds into hepatocytes. A putative model of OATP1B3 with a “positive binding pocket” containing conserved positively charged amino acids was predicted (Meier-Abt et al. J Membr Biol 208:213–227, 2005). Based on this model, we tested the hypothesis that these positive amino acids are important for OATP1B1 function. We made mutants and measured surface expression and uptake of estradiol-17β-glucuronide, estrone-3-sulfate and bromosulfophthalein in HEK293 cells. Two of the mutants had low surface expression levels: R181K at 10% and R580A at 30% of wild-type OATP1B1. A lysine at position 580 (R580K) rescued the expression of R580A. Mutations of several amino acids resulted in substrate-dependent effects. The largest changes were seen for estradiol-17β-glucuronide, while estrone-3-sulfate and bromosulfophthalein transport were less affected. The wild-type OATP1B1 K _m value for estradiol-17β-glucuronide of 5.35 ± 0.54 μM was increased by R57A to 30.5 ± 3.64 μM and decreased by R580K to 0.52 ± 0.18 μM. For estrone-3-sulfate the wild-type high-affinity K _m value of 0.55 ± 0.12 μM was increased by K361R to 1.8 ± 0.47 μM and decreased by R580K to 0.1 ± 0.04 μM. In addition, R580K reduced the V _max values for all three substrates to <25% of wild-type OATP1B1. Mutations at intracellular K90, H92 and R93 mainly affected V _max values for estradiol-17β-glucuronide uptake. In conclusion, the conserved amino acids R57, K361 and R580 seem to be part of the substrate binding sites and/or translocation pathways in OATP1B1.     

The effectiveness of gadolinium MRI to improve target delineation for radiotherapy in hepatocellular carcinoma: A comparative study of rigid image registration techniques

To achieve consistent target delineation in radiotherapy for hepatocellular carcinoma (HCC), image registration between simulation CT and diagnostic MRI was explored.Twenty patients with advanced HCC were included. The median interval between MRI and CT was 11 days. CT was obtained with shallow free breathing and MRI at exhale phase. On each CT and MRI, the liver and the gross target volume (GTV) were drawn. A rigid image registration was taken according to point information of vascular bifurcation (Method[A]) and pixel information of volume of interest only including the periphery of the liver (Method[B]) and manually drawn liver (Method[C]). In nine cases with an indefinite GTV on CT, a virtual sphere was generated at the epicenter of the GTV. The GTV from CT (V_GTV[CT]) and MRI (V_GTV[MR]) and the expanded GTV from MRI (V_+GTV[MR]) considering geometrical registration error were defined. The underestimation (uncovered V[CT] by V[MR]) and the overestimation (excessive V[MR] by V[CT]) were calculated. Through a paired T-test, the difference between image registration techniques was analyzed.For method[A], the underestimation rates of V_GTV[MR] and V_+GTV[MR] were 16.4 ± 8.9% and 3.2 ± 3.7%, and the overestimation rates were 16.6 ± 8.7% and 28.4 ± 10.3%, respectively. For V_GTV[MR] and V_+GTV[MR], the underestimation rates and overestimation rates of method[A] were better than method[C]. The underestimation rates and overestimation rates of the V_GTV[MR] were better in method[B] than method[C]. By image registration and additional margin, about 97% of HCC could be covered. Method[A] or method[B] could be recommended according to physician preference.     

Uptake kinetics and storage capacity of dissolved inorganic phosphorus and corresponding dissolved inorganic nitrate uptake in Saccharina latissima and Laminaria digitata (Phaeophyceae)

Uptake rates of dissolved inorganic phosphorus and dissolved inorganic nitrogen under unsaturated and saturated conditions were studied in young sporophytes of the seaweeds Saccharina latissima and Laminaria digitata (Phaeophyceae) using a “pulse‐and‐chase” assay under fully controlled laboratory conditions. In a subsequent second “pulse‐and‐chase” assay, internal storage capacity (ISC) was calculated based on V_M and the parameter for photosynthetic efficiency F_v/F_m. Sporophytes of S. latissima showed a V_S of 0.80 ± 0.03 μmol · cm^?2 · d^?1 and a V_M of 0.30 ± 0.09 μmol · cm^?2 · d^?1 for dissolved inorganic phosphate (DIP), whereas V_S for DIN was 11.26 ± 0.56 μmol · cm^?2 · d^?1 and V_M was 3.94 ± 0.67 μmol · cm^?2 · d^?1. In L. digitata, uptake kinetics for DIP and DIN were substantially lower: V_S for DIP did not exceed 0.38 ± 0.03 μmol · cm^?2 · d^?1 while V_M for DIP was 0.22 ± 0.01 μmol · cm^?2 · d^?1. V_S for DIN was 3.92 ± 0.08 μmol · cm^?2 · d^?1 and the V_M for DIN was 1.81 ± 0.38 μmol · cm^?2 · d^?1. Accordingly, S. latissima exhibited a larger ISC for DIP (27 μmol · cm^?2) than L. digitata (10 μmol · cm^?2), and was able to maintain high growth rates for a longer period under limiting DIP conditions. Our standardized data add to the physiological understanding of S. latissima and L. digitata, thus helping to identify potential locations for their cultivation. This could further contribute to the development and modification of applications in a bio‐based economy, for example, in evaluating the potential for bioremediation in integrated multitrophic aquacultures that produce biomass simultaneously for use in the food, feed, and energy industries.     

Using light as a lure is an efficient predatory strategy in <Emphasis Type="Italic">Arachnocampa flava,</Emphasis> an Australian glowworm

Trap-building, sit-and-wait predators such as spiders, flies and antlions tend to have low standard metabolic rates (SMRs) but potentially high metabolic costs of trap construction. Members of the genus Arachnocampa (glowworms) use an unusual predatory strategy: larvae bioluminesce to lure positively phototropic insects into their adhesive webs. We investigated the metabolic costs associated with bioluminescence and web maintenance in larval Arachnocampa flava. The mean rate of CO₂ production ([(V)\dot] \dot{V}CO₂) during continuous bioluminescence was 4.38 μl h⁻¹ ± 0.78 (SEM). The mean [(V)\dot] \dot{V}CO₂ of inactive, non-bioluminescing larvae was 3.49 ± 0.35 μl h⁻¹. The mean [(V)\dot] \dot{V}CO₂ during web maintenance when not bioluminescencing was 8.95 ± 1.78 μl h⁻¹, a value significantly lower than that measured during trap construction by other predatory arthropods. These results indicate that bioluminescence itself is not energetically expensive, in accordance with our prediction that a high cost of bioluminescence would render the Arachnocampa sit-and-lure predatory strategy inefficient. In laboratory experiments, both elevated feeding rates and daily web removal caused an increase in bioluminescent output. Thus, larvae increase their investment in light output when food is plentiful or when stressed through having to rebuild their webs. As light production is efficient and the cost of web maintenance is relatively low, the energetic returns associated with continuing to glow may outweigh the costs of continuing to attract prey.     

State-Dependent Inhibition of Inactivation-Deficient CaV1.2 and CaV2.3 Channels By Mibefradil

The structural determinants of mibefradil inhibition were analyzed using wild-type and inactivation-modified Ca_V1.2 (α1C) and Ca_V2.3 (α1E) channels. Mibefradil inhibition of peak Ba²⁺ currents was dose- and voltage-dependent. An increase of holding potentials from −80 to −100 mV significantly shifted dose-response curves toward higher mibefradil concentrations, namely from a concentration of 108 ± 21 μm (n= 7) to 288 ± 17 μm (n= 3) for inhibition of half of the Ca_v1.2 currents (IC ₅₀) and from IC ₅₀= 8 ± 2 μm (n= 9) to 33 ± 7 μm (n= 4) for Ca_V2.3 currents. In the presence of mibefradil, Ca_V1.2 and Ca_V2.3 experienced significant use-dependent inhibition (0.1 to 1 Hz) and slower recovery from inactivation suggesting mibefradil could promote transition(s) to an absorbing inactivated state. In order to investigate the relationship between inactivation and drug sensitivity, mibefradil inhibition was studied in inactivation-altered Ca_V1.2 and Ca_V2.3 mutants. Mibefradil significantly delayed the onset of channel recovery from inactivation in CEEE (Repeat I + part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel), in EC(AID)EEE (part of the I–II linker from Ca_V1.2 in the Ca_V2.3 host channel) as well as in Ca_V1.2 E462R, and Ca_V2.3 R378E (point mutation in the β-subunit binding motif) channels. Mibefradil inhibited the faster inactivating chimera EC(IS1-6)EEE with an IC ₅₀= 7 ± 1 μm (n= 3), whereas the slower inactivating chimeras EC(AID)EEE and CEEE were, respectively, inhibited with IC ₅₀= 41 ± 5 μm (n= 4) and IC ₅₀= 68 ± 9 μm (n= 5). Dose-response curves were superimposable for the faster EC(IS1-6)EEE and Ca_V2.3, whereas intermediate-inactivating channel kinetics (CEEE, Ca_V1.2 E462R, and Ca_V1.2 E462K) were inhibited by similar concentrations of mibefradil with IC ₅₀≈ 55–75 μm. The slower Ca_V1.2 wild-type and Ca_V1.2 Q473K channels responded to higher doses of mibefradil with IC ₅₀≈ 100–120 μm. Mibefradil was also found to significantly speed up the inactivation kinetics of slower channels (Ca_V1.2, CEEE) with little effect on the inactivation kinetics of faster-inactivating channels (Ca_V2.3). A open-channel block model for mibefradil interaction with high-voltage-activated Ca²⁺ channels is discussed and shown to qualitatively account for our observations. Hence, our data agree reasonably well with a ``receptor guarded mechanism' where fast inactivation kinetics efficiently trap mibefradil into the channel. Received: 14 March 2001/Revised: 25 June 2001     

Heterologous expression and characterization of a proxidomal ascorbate peroxidase from <Emphasis Type="Italic">Populus tomentosa</Emphasis>

The present study reported for the first time, cloning, expression and characteristics of a Proxidomal APX gene (PpAPX) from Populus tomentosa. The PpAPX gene encodes a protein of 287 amino acid residues with a calculated molecular mass of 31.58 kDa. The over-expressed recombinant PpAPX protein showed high activity towards the substrates ascorbate aicd (ASA) and H₂O₂. At fixed ASA concentrations, the K _m and V _max values were 0.12 ± 0.01 mM and 23.4 ± 4.2 mmol/min mg for H₂O₂. And at fixed H₂O₂ concentrations, the K _m and V _max values were 0.53 ± 0.04 mM and 20.0 ± 2.3 mmol/min mg for ASA.     

Regulation of glucose-6-phosphate dehydrogenase by reversible phosphorylation in liver of a freeze tolerant frog

Glucose-6-phosphate dehydrogenase (G6PDH) and the pentose phosphate pathway play a key role in reductive biosynthesis and antioxidant defense, while diverting glucose from other cellular functions. G6PDH was isolated from liver of the wood frog, Rana sylvatica, a freeze tolerant species that uses glucose as a cryoprotectant. Analysis of kinetic parameters (K _m and V _max) of G6PDH showed a significant increase in K _m G6P (from 98.2 ± 3.8 to 121 ± 5.3 μM) and K _m NADP⁺ (from 65.5 ± 2.3 to 89.1 ± 4.8 μM) in frogs following freezing exposure, indicating lower affinity for G6PDH substrates in this state. Subsequent analyses indicated that differential phosphorylation of G6PDH between the two states was responsible for the altered kinetic properties. Thus, two differentially charged forms of G6PDH were resolved by DEAE ion-exchange chromatography and, compared with controls, the proportion of G6PDH activity in peak I decreased and in peak II increased in liver from frozen frogs. G6PDH in peak I had a K _m G6P of 94.1 ± 1.1 μM and K _m NADP⁺ of 61.2 ± 3.5 μM, whereas Peak II G6PDH showed higher values (K _m G6P was 172 ± 4.3 μM, K _m NADP⁺ was 98.2 ± 3.3 μM). G6PDH from each peak was incubated with ions and second messengers to stimulate the actions of protein kinases with results indicating that G6PDH can be phosphorylated by protein kinase G, protein kinase C, AMP-activated protein kinase, or calmodulin-dependent protein kinase. The data indicate that in control frogs, G6PDH is in a high phosphate form and displays a high substrate affinity, whereas in frozen frogs G6PDH is less phosphorylated, with lower substrate affinity.     

Genetic polymorphism of <Emphasis Type="Italic">CYP2C19</Emphasis> in a Jordanian population: influence of allele frequencies of <Emphasis Type="Italic">CYP2C19*1</Emphasis> and <Emphasis Type="Italic">CYP2C19*2</Emphasis> on the pharmacokinetic profile of lansoprazole

To relate the pharmacokinetics of orally administered lansoprazole in healthy adult Jordanian men with CYP2C19 polymorphisms and to determine the percentage of CYP2C19 polymorphism in Jordanian population and the allelic frequency of CYP2C19*2 and CYP2C19*3. A total of 78 healthy Jordanian volunteers were included in this study from three different bioequivalence studies, one of these studies which included 26 volunteers was done on lansoprazole. Genotyping for CYP2C19*1, CYP2C19*2, CYP2C19*3 was done for all 78 volunteers, the data of genotyping of all subjects used for screening the frequency of different genotypes and the allelic frequency of different polymorphisms in healthy Jordanian men, the pharmacokinetics and genotyping data for the study of lansoprazole was matched and compared to investigate presence of statistical differences in pharmacokinetic parameters. In Jordanian subjects, the allele frequencies of the CYP2C19*2 and CYP2C19*3 mutation were 0.16 and 0, respectively. The concentration–time curves in the two groups [homozygote extensive metabolizer (homEM, n = 19) and heterozygote extensive metabolizer (homEM, n = 7)] groups were fitted to a non-compartment model. In the homEM and in the hetEM groups, the main kinetic parameters were as follows: T_max (2.1875 ± 0.777) and (2.54 ± 1.87) h, C_max (697.875 ± 335) and (833.58 ± 436.26) mg/l, t_1/2 (1.3 ± 0.43) and (2.38 ± 1.64) h, AUC_(0→∞) were (1,684.9 ± 888) and (3,609.8 ± 318) mg h l⁻¹, respectively. The Jordanian population showed similarities in CYP2C19 allele and genotype distribution pattern with Caucasians and Africans. CYP2C19 allele and poor metabolizer (PM) genotype frequencies in the Jordanian population are distinct from populations’ from East Asia such as Japanese and Koreans. Although lower pharmacokinetic parameters were found in homEM compared to hetEM but there was no significant difference between the two groups (P < 0.05).     

Circulatory function at sub-zero temperature: venous responses to catecholamines and angiotensin II in the Antarctic fish <Emphasis Type="Italic">Pagothenia borchgrevinki</Emphasis>

Catecholamines increase arterial pressure by increasing cardiac output (Q) and stroke volume (V _s), while angiotensin II (ang II) also increases vascular resistance (R _sys) in the Antarctic fish Pagothenia borchgrevinki. Adrenaline, phenylephrine and ang II (Asn¹, Val⁵) were injected into P. borchgrevinki. Cardiovascular variables, including central venous pressure (P _cv) and mean circulatory filling pressure (P _mcf; an index of venous capacitance), were recorded to investigate if venous vasoconstriction can explain the increased V _s and Q and the arterial pressor response in this species. Routine P _cv and P _mcf were 0.11 ± 0.01 and 0.18 ± 0.02 kPa, respectively. All of the drugs caused moderate increases in P _cv and P _mcf and the responses were attenuated after α-adrenergic blockade with prazosin. Although dorsal aortic pressure (P _da) also increased in response to all agonists, the mechanisms differed. Adrenaline caused sustained increases in V _s and Q, while R _sys only rose transiently. Ang II had a slower effect than adrenaline and increased both R _sys and Q, while phenylephrine only increased R _sys. This study demonstrates that P _cv is positive and controlled by an α-adrenergic mechanism in P. borchgrevinki. However, given the relatively small venous response to adrenaline it seems more likely that the increases in V _s and Q from this agonist are due to direct effects on the heart.     

Transport of sodium and chloride across earthworm skin in vitro

We present a new invertebrate model for the study of epithelial sodium transport in tight epithelia, the earthworm integument. Dissected segments of earthworm integument were mounted in modified Ussing chambers and perfused with either pond water (PW) or earthworm ringer solution (ERS) on the apical side. In order to investigate ion transport under near-in vivo physiological conditions, measurements were performed under current-clamp conditions by monitoring the transepithelial potential (V _T), as well as the transepithelial resistance (R _T). These were recorded continuously and the virtual short circuit current (I _SC) was calculated. The integument has a high transepithelial resistance (R _T=9,037±502 Ω cm² for PW, n=24, and 11,055±1,320 Ω cm² for ERS, n=32). V _T was −3.7±2.2 mV for PW (n=24) and −1.5±1.0 mV for ERS (n=32), and I _SC was −0.57±0.30 μA/cm² for PW (n=24) and −0.44±0.24 μA/cm² for ERS (n=32). Only under PW, but not under ERS conditions, was there a pronounced inhibition of I _SC by low doses of amiloride or its analogues phenamil and benzamil. The resistance of the paracellular pathway was found to be very high. The terrestrial oligochaete Lumbricus seems especially adapted to the environmental conditions because it has an ultra-tight integument and a very fast up- and down-regulation of apical Na⁺ channels.     

Biochemical characterization of glyceraldehyde-3-phosphate dehydrogenase from <Emphasis Type="Italic">Thermococcus kodakarensis</Emphasis> KOD1

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an essential role in glycolysis by catalyzing the conversion of d-glyceraldehyde 3-phosphate (d-G3P) to 1,3-diphosphoglycerate using NAD⁺ as a cofactor. In this report, the GAPDH gene from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (GAPDH-tk) was cloned and the protein was purified to homogeneity. GAPDH-tk exists as a homotetramer with a native molecular mass of 145 kDa; the subunit molecular mass was 37 kDa. GAPDH-tk is a thermostable protein with a half-life of 5 h at 80–90°C. The apparent K _m values for NAD⁺ and d-G3P were 77.8 ± 7.5 μM and 49.3 ± 3.0 μM, respectively, with V _max values of 45.1 ± 0.8 U/mg and 59.6 ± 1.3 U/mg, respectively. Transmission electron microscopy (TEM) and image processing confirmed that GAPDH-tk has a tetrameric structure. Interestingly, GAPDH-tk migrates as high molecular mass forms (~232 kDa and ~669 kDa) in response to oxidative stress.     

Influences of forested watershed conditions on fluctuations in stream water temperature with special reference to watershed area and forest type

In order to gain insight into the effect of watershed conditions on fluctuations in stream water temperature, we statistically analyzed water temperature data for 1 year, using root mean square (Rms) and harmonic (A Amplitude, φ delay time) methods. The average values of delay time (days) between air and water temperatures (T _a and T _w) of small (< 0.5 ha), medium (0.5–100 ha) and large (> 100 ha) watersheds were 4.53 ± 0.82 days, 11.83 ± 3.88 days and 4.45 ± 1.52 days, respectively. Fluctuations in stream water temperature expressed by Rms (Rms T _w/Rms T _a) and harmonic methods (A −T _w/A −T _a) in the medium-sized watersheds with moderate slope gradients were 0.37 ± 0.09 and 0.56 ± 0.14, respectively. These values increased in the larger watersheds with low slope gradients, including five large rivers covered by various landscapes, with their averages of 0.53 ± 0.09 and 0.78 ± 0.09, respectively, indicating the influences of solar radiation and heat transfer processes. In the smaller watersheds with high slope gradients, these values were 0.73 ± 0.02 and 0.87 ± 0.03, respectively, suggesting that shorter passage time affected water temperatures. With respect to forest type, these values at badly managed hinoki forest watersheds (0.45 ± 0.04 and 0.73 ± 0.07) were larger than those at broadleaf forest (0.34 ± 0.04 and 0.51 ± 0.12) and well-managed hinoki forest (0.33 ± 0.04 and 0.51 ± 0.07) watersheds, indicating different proportions of flow paths.     

Transport of hemicellulose monomers in the xylose-fermenting yeastCandida shehatae

Summary Cells ofCandida shehatae repressed by growth in glucose- or D-xylose-medium produced a facilitated diffusion system that transported glucose (K _s±2 mM,V _max±2.3 mmoles g⁻¹ h⁻¹),d-xylose (K _s±125 mM,V _max±22.5 mmoles g⁻¹ h⁻¹) and D-mannose, but neither D-galactose norl-arabinose. Cells derepressed by starvation formed several sugar-proton symports. One proton symport accumulated 3-0-methylglucose about 400-fold and transported glucose (K _s±0.12 mM,V _max ± 3.2 mmoles g⁻¹ h⁻¹) andd-mannose, a second proton symport transportedd-xylose (K _s± 1.0 mM,V _max 1.4 mmoles g⁻¹ h⁻¹) andd-galactose, whilel-arabinose apparently used a third proton symport. The stoicheiometry was one proton for each molecule of glucose or D-xylose transported. Substrates of one sugar proton symport inhibited non-competitively the transport of substrates of the other symports. Starvation, while inducing the sugar-proton symports, silenced the facilitated diffusion system with respect to glucose transport but not with respect to the transport of D-xylose, facilitated diffusion functioning simultaneously with thed-xylose-proton symport.