Interaction Between Nanoparticulate Anatase TiO<Subscript>2</Subscript> and Lactate Dehydrogenase

In order to study the mechanisms underlying the effects of TiO₂ nanoparticles on lactate dehydrogenase (LDH, EC1.1.1.27), Institute of Cancer Research region mice were injected with nanoparticulate anatase TiO₂ (5 nm) of various doses into the abdominal cavity daily for 14 days. We then examined LDH activity in vivo and in vitro and direct evident for interaction between nanoparticulate anatase TiO₂ and LDH using spectral methods. The results showed that nanoparticulate anatase TiO₂ could significantly activate LDH in vivo and in vitro; the kinetics constant (Km) and Vmax were 0.006 μM and 1,149 unit mg⁻¹ protein min⁻¹, respectively, at a low concentration of nanoparticulate anatase TiO₂, and 3.45 and 0.031 μM and 221 unit mg⁻¹ protein min⁻¹, respectively, at a high concentration of nanoparticulate anatase TiO₂. By fluorescence spectral assays, the nanoparticulate anatase TiO₂ was determined to be directly bound to LDH, and the binding constants of the binding site were 1.77 × 10⁸ L mol⁻¹ and 2.15 × 10⁷ L mol⁻¹, respectively, and the binding distance between nanoparticulate anatase TiO₂ and the Trp residue of LDH was 4.18 nm, and nanoparticulate anatase TiO₂ induced the protein unfolding. It was concluded that the binding of nanoparticulate anatase TiO₂ altered LDH structure and function.     

Identification and characterization of a novel xylanase derived from a rice straw degrading enrichment culture

A metagenomic library containing ca. 3.06 × 10⁸ bp insert DNA was constructed from a rice straw degrading enrichment culture. A xylanase gene, umxyn10A, was cloned by screening the library for xylanase activity. The encoded enzyme Umxyn10A showed 58% identity and 73% similarity with a xylanase from Thermobifida fusca YX. Sequence analyses showed that Umxyn10A contained a glycosyl hydrolase family 10 catalytic domain. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized biochemically. Recombinant Umxyn10A was highly active toward xylan. However, the purified enzyme could slightly hydrolyze β-1,3/4-glucan and β-1,3/6-glucan. Umxyn10A displayed maximal activity toward oat spelt xylan at a high temperature (75°C) and weak acidity (pH 6.5). The K _m and V _max of Umxyn10A toward oat spelt xylan were 3.2 mg ml⁻¹ and 0.22 mmol min⁻¹ mg⁻¹ and were 2.7 mg ml⁻¹ and 1.0 mmol min⁻¹ mg⁻¹ against birchwood xylan, respectively. Metal ions did not appear to be required for the catalytic activity of this enzyme. The enzyme Umxyn10A could efficiently hydrolyze birchwood xylan to release xylobiose as the major product and a negligible amount of xylose. The xylanase identified in this work may have potential application in producing xylobiose from xylan.     

The Application of Modified Flow-Through Cell Apparatus for the Assessment of Chlorhexidine Dihydrochloride Release from Lozenges Containing Sorbitol

The objective of this work was to apply a new apparatus for the assay of the drug release from lozenge tablet with a potential use in the treatment of oral candidosis and another conditions connected to microbial etiopathology in the oral cavity or as an antiplaque factor. Also, an approach to comparison of the applied method with the classical paddle apparatus method was performed. Tablets containing chlorhexidine dihydrochloride were formulated with granulated sorbitol of different grades (diameter of 110, 180, 480, and 650 μm, respectively), lactose, and magnesium stearate as excipients. Tablets were obtained through direct compression, and uniformity of weight, friability, breaking strength, disintegration, and release rate were evaluated. The disintegration times ranged between 10 and 21 min. In the next stage of the study, the release of chlorhexidine from lozenges prepared with granulated sorbitol grade 110 μm and different amounts of lactose and magnesium stearate was assessed. Two stages were observed during the release of chlorhexidine dihydrochloride from the lozenges, assayed by the classical paddle apparatus method II USP. In the first stage, release rates were between 2.6 × 10⁻² and 4.7 × 10⁻² min⁻¹, in the second stage between 1.7 × 10⁻³ and 7.7 × 10⁻³ min⁻¹. In the case of the in-house method, the release was near to first-order kinetics through the entire release experiment, with rate constants between 3.6 × 10⁻² and 6.6 × 10⁻² min⁻¹. The sorbitol granulate of granules with diameter 110 μm was found to be most suitable for the lozenges with chlorhexidine dihydrochloride and lactose. The in-house release method, proposed in this work, seems to be more realistic for the preliminary assessment of predicted drug concentrations in the oral cavity after the intake of a lozenge.     

Effect of Mg2+ on the Structure and Function of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

Mg²⁺ in various concentrations was added to purified Rubisco in vitro to gain insight into the mechanism of molecular interactions between Mg²⁺ and Rubisco. The enzyme activity assays showed that the reaction between Rubisco and Mg²⁺ was two order, which means that the enhancement of Rubisco activity was accelerated by low concentration of Mg²⁺ and slowed by high concentration of Mg²⁺. The kinetics constant (K _m) and V _max was 1.91 μM and 1.13 μmol CO₂ mg⁻¹ protein∙min⁻¹, respectively, at a low concentration of Mg²⁺, and 3.45 μM and 0.32 μmol CO₂∙mg⁻¹ protein∙min⁻¹, respectively, at a high concentration of Mg²⁺. By UV absorption and fluorescence spectroscopy assays, the Mg²⁺ was determined to be directly bound to Rubisco; the binding site of Mg²⁺ to Rubisco was 0.275, the binding constants (K _A) of the binding site were 6.33 × 10⁴ and 5.5 × 10⁴ l·mol⁻¹. Based on the analysis of the circular dichroism (CD) spectra, it was concluded that the binding of Mg²⁺ did not alter the secondary structure of Rubisco, suggesting that the observed enhancement of Rubisco carboxylase activity was caused by a subtle structural change in the active site through the formation of the complex with Mg²⁺.     

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K _d values of 2.6 ± 0.1 μM for HBA and 1.9 ± 0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min⁻¹ and 61 ± 1 min⁻¹, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω−1- and ω−2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω−2-hydroxynonyloxybenzoic acid and ω−2,ω−4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω−2- and 0.5 mM ω−1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min⁻¹, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min⁻¹.     

Production of β-galactosidase from thermophilic fungus Rhizomucor sp

A thermostable β-galactosidase was produced extracellularly by a thermophilic Rhizomucor sp, with maximum enzyme activity (0.21 U mg⁻¹) after 4 days under submerged fermentation condition (SmF). Solid state fermentation (SSF) resulted in a nine-fold increase in enzyme activity (2.04 U mg⁻¹). The temperature range for production of the enzyme was 38–55°C with maximum activity at 45°C. The optimum pH and temperature for the partially purified enzyme was 4.5 and 60°C, respectively. The enzyme retained its original activity on incubation at 60°C up to 1 h. Divalent cations like Co²⁺, Mn²⁺, Fe²⁺ and Zn²⁺ had strong inhibitory effects on the enzyme activity. The K _m and V _max for p-nitrophenyl-β- _D-galactopyranoside and o-nitrophenyl-β - _D-galactopyranoside were 0.39 mM, 0.785 mM and 232.1 mmol min⁻¹ mg⁻¹ respectively. The K _m and V _max for the natural substrate lactose were 66.66 μM and 0.20 μ mol min⁻¹ mg⁻¹. Received 10 March 1997/ Accepted in revised form 17 July 1997     

Effect of intense interval workouts on running economy using three recovery durations

The purposes of this study were to determine whether running economy (RE) is adversely affected following intense interval bouts of 10 × 400-m running, and whether there is an interaction effect between RE and recovery duration during the workouts. Twelve highly trained male endurance athletes [maximal oxygen consumption; V˙O_{2 max} =72.5 (4.3) ml·kg⁻¹·min⁻¹; mean (SD)] performed three interval running workouts of 10 × 400 m with a minimum of 4 days between runs. Recovery duration between the repetitions was randomly assigned at 60, 120 or 180 s. The velocity for each 400-m run was determined from a treadmill V˙O_{2 max} test. The average running velocity was 357.9 (9.0) m · min⁻¹. Following the workout, the rating of perceived exertion (RPE) increased significantly (P < 0.01) as recovery duration between the 400-m repetitions decreased (14.4, 16.1, and 17.7 at 180s, 120s, and 60 s recovery, respectively). Prior to and following each workout, RE was measured at speeds of 200 and 268 m · min⁻¹. Changes in RE from pre- to post-workout, as well as heart rate (HR) and respiratory exchange ratio (R) were similar for the three recovery conditions. When averaged across conditions, oxygen consumption (V˙O₂) increased significantly (P < 0.01) from pre- to post-test (from 38.5 to 40.5 ml · kg⁻¹ · min⁻¹ at 200 m · min⁻¹, and from 53.1 to 54.5 ml · kg⁻¹ · min⁻¹ at 268 m · min⁻¹, respectively). HR increased (from 124 to 138, and from 151 to 157 beats · min⁻¹ respectively) and R decreased (from 0.90 to 0.78, and from 0.93 to 0.89, respectively) at 200 and 268 m · min⁻¹, respectively (P < 0.01). This study showed that RE can be perturbed after a high-intensity interval workout and that the changes in V˙O₂, HR and R were independent of the recovery duration between the repetitions. Accepted: 23 June 1997     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Characterization of Xyn10A, a highly active xylanase from the human gut bacterium Bacteroides xylanisolvens XB1A

A xylanase gene xyn10A was isolated from the human gut bacterium Bacteroides xylanisolvens XB1A and the gene product was characterized. Xyn10A is a 40-kDa xylanase composed of a glycoside hydrolase family 10 catalytic domain with a signal peptide. A recombinant His-tagged Xyn10A was produced in Escherichia coli and purified. It was active on oat spelt and birchwood xylans and on wheat arabinoxylans. It cleaved xylotetraose, xylopentaose, and xylohexaose but not xylobiose, clearly indicating that Xyn10A is a xylanase. Surprisingly, it showed a low activity against carboxymethylcellulose but no activity at all against aryl-cellobioside and cellooligosaccharides. The enzyme exhibited K _m and V _max of 1.6 mg ml⁻¹ and 118 μmol min⁻¹ mg⁻¹ on oat spelt xylan, and its optimal temperature and pH for activity were 37°C and pH 6.0, respectively. Its catalytic properties (k _cat/K _m = 3,300 ml mg⁻¹ min⁻¹) suggested that Xyn10A is one of the most active GH10 xylanase described to date. Phylogenetic analyses showed that Xyn10A was closely related to other GH10 xylanases from human Bacteroides. The xyn10A gene was expressed in B. xylanisolvens XB1A cultured with glucose, xylose or xylans, and the protein was associated with the cells. Xyn10A is the first family 10 xylanase characterized from B. xylanisolvens XB1A.     

The viability to a wall shear stress and propagation of <Emphasis Type="Italic">Bifidobacterium longum</Emphasis> in the intensive membrane bioreactor

Bifidobacterium longum grew at 65 L pilot scale of the membrane bioreactor (MBR), externally fitted with ceramic membrane (0.7 m²). Cell mass at the MBR reached 22.18 g L⁻¹ as dry cell weight in 12 h, which is 8.44 times higher than cell mass attained at the vial culture. The growth rate in the vial culture was μ = 0.385 h⁻ and at the batch culture was μ = 1.13 h⁻ in the exponential period and μ = 0.31 h⁻¹ in the stationary period. In the fed-batch mode was μ = 1.102 h⁻¹ for 6 h with inoculation and declined to μ = 0.456 h⁻¹ with feeding of feed medium. The growth rate at the MBR was μ = 0.134 h⁻¹. The number of viable cells was 6.01 × 10¹² cfu L⁻¹ at the batch culture, but increased to 1.15 × 10¹⁴ cfu L⁻¹ at the MBR culture. The specific growth rate of viable cell number (colony-forming units per liter, per hour) improved by 6.01 times from the batch to the MBR culture. The wall shear stress mainly generated by the pump, and the membrane incorporated into the MBR was controlled during the cultivation at the MBR. The viability of B. longum declined to under 10% in the first 2 weeks of the 4-week stability test (40°C) as B. longum was exposed to over wall shear stress 713 Pa, but the viability improved to 30–40% in wall shear stress of 260 Pa or STR culture. The loss in the cell viability can be saved by managing with wall shear stress during the cultivation at the MBR.     

Relationship between maximal oxygen uptake on different ergometers, lean arm volume and strength in paraplegic subjects

The present experiment was designed to study the importance of strength and muscle mass as factors limiting maximal oxygen uptake (V˙O_{2 max} ) in wheelchair subjects. Thirteen paraplegic subjects [mean age 29.8 (8.7) years] were studied during continuous incremental exercises until exhaustion on an arm-cranking ergometer (AC), a wheelchair ergometer (WE) and motor-driven treadmill (TM). Lean arm volume (LAV) was estimated using an anthropometric method based upon the measurement of various circumferences of the arm and forearm. Maximal strength (MVF) was measured while pushing on the rim of the wheelchair for three positions of the hand on the rim (−30°, 0° and +30°). The results indicate that paraplegic subjects reached a similar V˙O_{2 max} [1.23 (0.34) l · min⁻¹, 1.25 (0.38) l · min⁻¹, 1.22 (0.18) l · min⁻¹ for AC, TM and WE, respectively] and V˙O_{2 max} /body mass [19.7 (5.2) ml · min⁻¹ · kg⁻¹, 19.5 (6.14) ml · min⁻¹ · kg⁻¹, 19.18 (4.27) ml · min⁻¹ · kg⁻¹ for AC, TM and WE, respectively on the three ergometers. Maximal heart rate f _{c max} during the last minute of AC (173 (17) beats · min⁻¹], TM [168 (14) beats · min⁻¹], and WE [165 (16) beats · min⁻¹], were correlated, but f _{c max} was significantly higher for AC than for TM (P<0.03). There were significant correlations between MVF and LAV (P<0.001) and between the MVF data obtained at different angles of the hand on the rim [311.9 (90.1) N, 313.2 (81.2) N, 257.1 (71) N, at −30°, 0° and +30°, respectively]. There was no correlation between V˙O_{2 max} and LAV or MVF. The relatively low values of f _{c max} suggest that V˙O_{2 max} was, at least in part, limited by local aerobic factors instead of central cardiovascular factors. On the other hand, the lack of a significant correlation between V˙O_{2 max} and MVF or muscle mass was not in favour of muscle strength being the main factor limiting V˙O_{2 max} in our subjects. Accepted: 31 January 1997     

Temperature dependence of habituation of the initial responses to cold-water immersion

The initial responses to cold-water immersion, evoked by stimulation of peripheral cold receptors, include tachycardia, a reflex inspiratory gasp and uncontrollable hyperventilation. When immersed naked, the maximum responses are initiated in water at 10°C, with smaller responses being observed following immersion in water at 15°C. Habituation of the initial responses can be achieved following repeated immersions, but the specificity of this response with regard to water temperature is not known. Thirteen healthy male volunteers were divided into a control (C) group (n = 5) and a habituation (H) group (n = 8). Each subject undertook two 3-min head-out immersions in water at 10°C wearing swimming trunks. These immersions took place at a corresponding time of day with 4 days separating the two immersions. In the intervening period the C group were not exposed to cold water, while the H group undertook another six, 3-min, head-out immersions in water at 15°C. Respiratory rate (f _R), inspiratory minute volume (V˙ _I) and heart rate (f _H) were measured continuously throughout each immersion. Following repeated immersions in water at 15°C, the f _R, V˙ _I and f _H responses of the H group over the first 30 s of immersion were reduced (P < 0.01) from 33.3 breaths · min⁻¹, 50.5 l · min⁻¹ and 114 beats · min⁻¹ respectively, to 19.8 breaths · min⁻¹, 26.4 l · min⁻¹ and 98 beats · min⁻¹, respectively. In water at 10°C these responses were reduced (P < 0.01) from 47.3 breaths · min⁻¹, 67.6 l · min⁻¹ and 128 beats · min⁻¹ to 24.0 breaths · min⁻¹, 29.5 l · min⁻¹ and 109 beats · min⁻¹, respectively over a corresponding period of immersion. Similar reductions were observed during the last 2.5 min of immersions. The initial responses of the C group were unchanged. It is concluded that habituation of the cold shock response can be achieved by immersion in warmer water than that for which protection is required. This suggests that repeated submaximal stimulation of the cutaneous cold receptors is sufficient to attenuate the responses to more maximal stimulation. Accepted: 6 February 1998     

Biodegradation of propanol and isopropanol by a mixed microbial consortium

The aerobic biodegradation of high concentrations of 1-propanol and 2-propanol (IPA) by a mixed microbial consortium was investigated. Solvent concentrations were one order of magnitude greater than any previously reported in the literature. The consortium utilized these solvents as their sole carbon source to a maximum cell density of 2.4 × 10⁹ cells ml⁻¹. Enrichment experiments with propanol or IPA as carbon sources were carried out in batch culture and maximum specific growth rates (μ_max) calculated. At 20 °C, μ _max values were calculated to be 0.0305 h⁻¹ and 0.1093 h⁻¹ on 1% (v/v) IPA and 1-propanol, respectively. Growth on propanol and IPA was carried out between temperatures of 10 °C and 45 °C. Temperature shock responses by the microbial consortium at temperatures above 45 °C were demonstrated by considerable cell flocculation. An increase in propanol substrate concentration from 1% (v/v) to 2% (v/v) decreased the μ _max from 0.1093 h⁻¹ to 0.0715 h⁻¹. Maximum achievable biodegradation rates of propanol and IPA were 6.11 × 10⁻³% (v/v) h⁻¹ and 2.72 × 10⁻³% (v/v) h⁻¹, respectively. Generation of acetone during IPA biodegradation commenced at 264 h and reached a maximum concentration of 0.4% (v/v). The results demonstrate the potential of mixed microbial consortia in the bioremediation of solvent-containing waste streams. Received: 14 December 1999 / Received revision: 3 April 2000 / Accepted: 7 April 2000     

An altered camelid-like single domain anti-idiotypic antibody fragment of HM-1 killer toxin: acts as an effective antifungal agent

Phage-display and competitive panning elution leads to the identification of minimum-sized antigen binders together with conventional antibodies from a mouse cDNA library constructed from HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT). Antigen-specific altered camelid-like single-domain heavy chain antibody (scFv K2) and a conventional antibody (scFv K1) have been isolated against the idiotypic antigen nmAb-KT. The objectives of the study were to examine (1) their properties as compared to conventional antibodies and also (2) their antifungal activity against different pathogenic and non-pathogenic fungal species. The alternative small antigen-binder, i.e., the single-domain heavy chain antibody, was originated from a conventional mouse scFv phage library through somatic hyper-mutation while selection against antigen. This single-domain antibody fragment was well expressed in bacteria and specifically bound with the idiotypic antigen nmAb-KT and had a high stability and solubility. Experimental data showed that the binding affinity for this single-domain antibody was 272-fold higher (K _d = 1.07 × 10⁻¹⁰ M) and antifungal activity was three- to fivefold more efficient (IC₅₀ = 0.46 × 10⁻⁶ to 1.17 × 10⁻⁶ M) than that for the conventional antibody (K _d = 2.91 × 10⁻⁸ M and IC₅₀ = 2.14 × 10⁻⁶ to 3.78 × 10⁻⁶ M). The derived single-domain antibody might be an ideal scaffold for anti-idiotypic antibody therapy and the development of smaller peptides or peptide mimetic drugs due to their less complex antigen-binding site. We expect that such single-domain synthetic antibodies will find their way into a number of biotechnological or medical applications.     

Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> exchange activity in the alkaliphile halotolerant cyanobacterium <Emphasis Type="Italic">Aphanothece halophytica</Emphasis>

The activity of Na⁺/H⁺ exchanger to remove toxic Na⁺ is important for growth of organisms under high salinity. In this study, the halotolerant cyanobacterium Aphanothece halophytica was shown to possess Na⁺/H⁺ exchange activity since exogenously added Na⁺ could dissipate a pre-formed pH gradient, and decrease extracellular pH. Kinetic analysis yielded apparent K _m (Na⁺) and V _max of 20.7 ± 3.1 mM and 3,333 ± 370 nmol H⁺ min⁻¹ mg⁻¹, respectively. For cells grown under salt-stress condition, the apparent K _m (Na⁺) and V _max was 18.3 ± 3.5 mM and 3,703 ± 350 nmol H⁺ min⁻¹ mg⁻¹, respectively. Three cations with decreasing efficiency namely Li⁺, Ca²⁺, and K⁺ were also able to dissipate pH gradient. Only marginal exchange activity was observed for Mg²⁺. The exchange activity was strongly inhibited by Na⁺-gradient dissipators, monensin, and sodium ionophore as well as by CCCP, a protonophore. A. halophytica showed high Na⁺/H⁺ exchange activity at neutral and alkaline pH up to pH 10. Cells grown at pH 7.6 under high salinity exhibited higher Na⁺/H⁺ exchange activity than those grown under low salinity during 15 days of growth suggesting a role of Na⁺/H⁺ exchanger for salt tolerance in A. halophytica. Cells grown at alkaline pH of 9.0 also exhibited a progressive increase of Na⁺/H⁺ exchange activity during 15 days of growth.     

Cardiorespiratory responses to underwater treadmill walking in healthy females

This study compared the cardiorespiratory responses of eight healthy women (mean age 30.25 years) to submaximal exercise on land (LTm) and water treadmills (WTm) in chest-deep water (Aquaciser). In addition, the effects of two different water temperatures were examined (28 and 36°C). Each exercise test consisted of three consecutive 5-min bouts at 3.5, 4.5 and 5.5 km · h⁻¹. Oxygen consumption (V˙O₂) and heart rate (HR), measured using open-circuit spirometry and telemetry, respectively, increased linearly with increasing speed both in water and on land. At 3.5 km · h⁻¹ V˙O₂ was similar across procedures [χ = 0.6 (0.05) l · min⁻¹]. At 4.5 and 5.5 km · h⁻¹ V˙O₂ was significantly higher in water than on land, but there was no temperature effect (WTm: 0.9 and 1.4, respectively; LTm: 0.8 and 0.9 l · min⁻¹, respectively). HR was significantly higher in WTm at 36°C compared to WTm at 28°C at all speeds, and compared to LTm at 4.5 and 5.5 km · h⁻¹ (P ≤ 0.003). The HR-V˙O₂ relationship showed that at a V˙O₂ of 0.9 l · min⁻¹, HR was higher in water at 36°C (115 beats · min⁻¹) than either on land (100 beats · min⁻¹) or in water at 28°C (99 beats · min⁻¹). The Borg scale of perceived exertion showed that walking in water at 4.5 and 5.5 km · h⁻¹ was significantly harder than on land (WTm: 11.4 and 14, respectively; LTm: 9.9 and 11, respectively; P ≤ 0.001). These cardiorespiratory changes occurred despite a slower cadence in water (the mean difference at all speeds was 27 steps/min). Thus, walking in chest-deep water yields higher energy costs than walking at similar speeds on land. This data has implications for therapists working in hydrotherapy pools. Accepted: 3 September 1997     

A Novel β-Agarase with High pH Stability from Marine Agarivorans sp. LQ48

A novel endo-type β-agarase gene, agaA, was cloned from a newly isolated marine bacterium, Agarivorans sp. LQ48. It encodes a protein of 457 amino acids with a calculated molecular mass of 51.2 kDa. The deduced protein contains a typical N-terminal signal peptide of 25 amino acid residues, followed by a catalytic module, which is homologous to that of glycoside hydrolase family 16. A sequence similar to a carbohydrate-binding module is found in the C-terminal region of the enzyme. The overall amino acid sequence shares a highest identity of 73% with the sequence of beta-agarase AgaB from Pseudoalteromonas sp. strain CY24. The mature agarase was highly expressed extracellularly in Escherichia coli. At pH 7.0 and 40°C, the purified recombinant AgaA had a high specific activity of 349.3 μmol min⁻¹ mg⁻¹, a K _m of 3.9 mg ml⁻¹, and a V _max of 909.1 μmol min⁻¹ mg⁻¹ for agarose. The recombinant enzyme hydrolyzed the β-1,4-glycosidic linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main products. Enzyme activity analysis revealed that the optimal temperature and pH of the recombinant AgaA were 40°C and 7.0, respectively. Notably, AgaA still retained more than 95% activity after incubation at pH 3.0–11.0 for 1 h, a characteristic much different from other agarases reported. It is the first agarase identified to have so wide a pH range stability. This favorable property could make AgaA to be attractive to the food, cosmetic, and medical industrial applications.     

The development of an isokinetic squat device: reliability and relationship to functional performance

The aims of the present study were: (1) to assess aerobic metabolism in paraplegic (P) athletes (spinal lesion level, T4–L3) by means of peak oxygen uptake (V˙O_2peak) and ventilatory threshold (VT), and (2) to determine the nature of exercise limitation in these athletes by means of cardioventilatory responses at peak exercise. Eight P athletes underwent conventional spirographic measurements and then performed an incremental wheelchair exercise on an adapted treadmill. Ventilatory data were collected every minute using an automated metabolic system: ventilation (l · min⁻¹), oxygen uptake (V˙O₂, l · min⁻¹, ml · min⁻¹ · kg⁻¹), carbon dioxide production (V˙CO₂, ml · min⁻¹), respiratory exchange ratio, breathing frequency and tidal volume. Heart rate (HR, beats · min⁻¹) was collected with the aid of a standard electrocardiogram. V˙O_2peak was determined using conventional criteria. VT was determined by the breakpoint in the V˙CO₂−V˙O₂ relationship, and is expressed as the absolute VT (V˙O₂, ml · min⁻¹ · kg⁻¹) and relative VT (percentage of V˙O_2peak). Spirometric values and cardioventilatory responses at rest and at peak exercise allowed the measurement of ventilatory reserve (VR), heart rate reserve (HRr), heart rate response (HRR), and O₂ pulse (O₂ P). Results showed a V˙O_2peak value of 40.6 (2.5) ml · min⁻¹ · kg⁻¹, an absolute VT detected at 23.1 (1.5) ml · min⁻¹ · kg⁻¹ V˙O₂ and a relative VT at 56.4 (2.2)% V˙O_2peak. HRr [15.8 (3.2) beats · min⁻¹], HRR [48.6 (4.3) beat · l⁻¹], and O₂ P [0.23 (0.02) ml · kg⁻¹ · beat⁻¹] were normal, whereas VR at peak exercise [42.7 (2.4)%] was increased. As wheelchair exercise excluded the use of an able-bodied (AB) control group, we compared our V˙O_2peak and VT results with those for other P subjects and AB controls reported in the literature, and we compared our cardioventilatory responses with those for respiratory and cardiac patients. The low V˙O_2peak values obtained compared with subject values obtained during an arm-crank exercise may be due to a reduced active muscle mass. Absolute VT was somewhat comparable to that of AB subjects, mainly due to the similar muscle mass involved in wheelchair and arm-crank exercise by P and AB subjects, respectively. The increased VR, as reported in patients with chronic heart failure, suggested that P athletes exhibited cardiac limitation at peak exercise, and this contributed to the lower V˙O_2peak measured in these subjects. Accepted: 22 April 1997     

Production of hexanoic acid from d-galactitol by a newly isolated Clostridium sp. BS-1

In a study screening anaerobic microbes utilizing d-galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with d-galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294^T, with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from d-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from d-galactitol and d-glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O.