Biosynthesis and properties of an extracellular metalloprotease from the Antarctic marine bacterium Sphingomonas paucimobilis

An extracellular protease from the marine bacterium Sphingomonas paucimobilis, strain 116, isolated from the stomach of Antarctic krill, Euphausia superba Dana, was purified and characterized. The excretion of protease was maximal at temperatures from 5 to 10°C, i.e. below the temperature optimum for the strain growth (15°C). The highly purified enzyme was a metalloprotease [sensivity to ethylenediaminetetraacetic acid (EDTA)] and showed maximal activity against proteins at 20–30°C and pH 6.5–7.0, and towards N-benzoyl-tyrosine ethyl ester (BzTyrOEt) at pH 8.0. At 0°C the enzyme retained as much as 47% of maximal activity in hydrolysis of urea denatured haemoglobin (Hb) (at pH 7.0), and at −5 and −10°C, 37 and 30%, respectively. The metalloprotease was stable up to 30°C for 15 min and up to 20°C for 60 min. These results indicate that the proteinase from S. paucimobilis 116 is a cold-adapted enzyme.     

Some properties of levansucrase of Bacillus natto stabilized with periodate oxidized yeast glucomannan

It was observed that levansucrase from Bacillus natto became unstable and was easily inactivated when the salts were removed from the enzyme solution, while the enzyme was stable for long time in a buffered saline. After modification with periodate oxidized yeast glucomannan, the enzyme increased thermal stability up to 45°C, in which it conserved more than 90% of its activity after 15 min treatement. The optimum temperature was also shifted from 40°C in the case of original enzyme to 50°C for the modified enzyme after 10 min reaction time. The half-life time increased significantly from 9 min to 55 min at 50°C, however it increased from 30 min and 22 min respectively at 40°C and 45°C to more than 1 h at the same temperature. The content of carbohydrates of modified enzyme was 25% that increases the molecular weight from 57 KDa to 80 KDa. The products from sucrose by the modified enzyme were the same as the case using original enzyme. Namely, the products confirmed were levan and 3 kestoses (6-, 1-, and neo-kestose).     

Microwave irradiation increases recovery of neuropeptides from brain tissues

The effect of focused high energy microwave treatment (MW) on brain concentrations and molecular forms of substance P, neurokinin A, neuropeptide Y, neurotensin, galanin and calcitonin gene-related peptide was investigated. Groups of rats were treated as follows: 1) MW, storage for 60 min at 22°C, 2) Decapitation, storage for 60 min at 22°C, 3) Decapitation, storage for 60 min at 22°C, MW treatment, 4) MW, decapitation, storage for 2 min at 22°C and 5) Decapitation, storage for 2 min at 22°C. Peptide concentrations were in all instances highest in the MW sacrificed groups. MW increased the concentration of intact peptides by rapid inhibition of peptidase activity and increase in peptide solubility/extractability.     

Investigation of the transport of intact glutathione in human and rat type II pneumocytes

The aim of the study was to investigate whether there is transmembrane transport of intact glutathione ([³H]-GSH, 0.1 μCi) in rat and human type II pneumocytes (T2P), and if this transport might be dependent on the redox state of the extracellular fluid. The T2P were pretreated with acivicin (250 μM) to inhibit γ-glutamyl-transferase activity and with L-buthionine-[SR]-sulfoximine (1 mM) to inhibit intracellular GSH synthesis. After 48 h in culture, initial GSH influx rate was 0.70 ± 0.20 nmol/min/mg protein (37°C) and 0.35 ± 0.04 nmol/min/mg protein (4°C) during the first 5 min in rat T2P. In human T2P, the initial GSH influx rate was 0.36 ± 0.30 nmol/min/mg protein (37°C) and 0.32 ± 0.06 nmol/min/mg protein (4°C) during the first 10 min. Thereafter no further influx was found. The influx of 1 mM GSH in freshly isolated rat and human T2P in suspension was 2.3 ± 0.3 and 1.2 ± 0.3 nmol/mg protein after 15 min at 37°C, and 2.8 ± 0.2 and 1.0 ± 0.3 nmol/mg protein at 4°C, respectively. When GSH influx was studied at different concentrations between 0 and 40 mM, a linear increase without saturation or difference between 37°C and 4°C was found. Preexposure to ouabain had no effect on GSH influx. Efflux of GSH was stimulated and influx inhibited by preexposure of the cells to reduced thiols, while disulphides inhibited efflux and favoured inward uptake. Thus, in human and rat T2P a GSH-carrier exists which operates as an effluxer. At GSH concentrations in the physiological range no uptake is seen, but some uptake can be observed at GSH concentrations above normal physiological levels. The uptake appears to be energy-independent and non-saturable. Efflux of GSH is stimulated and influx inhibited by reduced thiols, while disulphides inhibit the efflux and favour inward uptake. GSH uptake in T2P thus may depend on concentration gradients and driving forces, such as the redox state of the extracellular fluid.     

Effects of some thermal, chemical and mechanical treatments on lipase activity in Shammi goat milk

The effects of heating (20, 37 or 50 °C), cooling (5 °C), pasteurisation (71 °C for 15 s), boiling (100 °C), agitation (5 or 10 min), pH (acid or alkaline), and addition of chemicals such as silver and lead nitrates, copper sulphate and sodium chloride on lipase activity in Shammi goat milk were studied. There were non-significant differences (P < 0.01) in chemical composition between Shammi goat milk and Arabi cow milk. Lipase activity in Shammi goat milk was non-significantly (P < 0.01) lower than in Arabi cow milk. Lipase activity in milk of Shammi goats and Arabi cows was reduced when the milk was subjected to heating, cooling, pasteurisation, boiling, or when chemicals or acid was added, whereas in agitated and alkaline milk, the lipase activity was increased. The increase following agitation was greater after 10 min than 5 min. It can be concluded that heating, pasteurising, boiling, cooling, addition of certain chemicals and acidity are means by which lipase activity in milk can be reduced.     

Enhancement of albumin secretion by short-term hypothermic incubation of primary rat hepatocytes

A feasibility of hypothermic incubation of hepatocytes as a means of enhancing liver-specific activity was investigated to obtain preferable hepatocytes for a bioartificial liver (BAL) system. Freshly isolated rat hepatocytes were incubated at hypothermic temperatures from 10 to 33 °C for several days, and subsequently cultured at normothermic temperature of 37 °C to evaluate cell viability and albumin secretion activity. The cell viability was decreased by 3-day hypothermic incubations at 10 and 20 °C, while it was maintained even after 3-day hypothermic incubations between 25 and 33 °C. The activity of albumin secretion gradually decreased with prolonging the period of hypothermic incubation at 25 °C. Enhancement of albumin secretion activity was observed in the hypothermic incubations at 30 and 33 °C. The maximum activation of albumin secretion was obtained when hypothermic incubation was performed for 3 days at 30 °C, where the activity increased to 145% of the original activity. The hypothermic incubation at 30 °C also reduced the required time to be the peak of the activity of albumin secretion in the normothermic culture. It was considered that the hypothermic incubation at 30 °C would be effective as a method for pretreatment of isolated hepatocytes for a BAL system.     

Characteristics of galactomannanase for degrading konjac gel

Galactomannanase (Glmnase) is an enzyme product derived from Aspergillus niger. The activity of Glmnase degrading (hydrolyzing) the konjac gel were investigated. Significant loss in the enzyme activity was found when the temperature above 60 °C. Similar observations were obtained when the reaction pH above 5. Further increase in the pH value resulted in entirely loss of enzyme activity at the alkaline pH region (pH 8.0 and above). The optimal hydrolyzing temperature and pH were at 60 °C and 5.0, respectively. For the stability test, the purified Glmnase increased its thermostability up to 70 °C at pH 5.0, but it retained only about 60% activity after 60 min incubation at this temperature and its activity became zero after 20 min incubation at 80 °C. The Glmnase was stable at the pH range from 3.0 to 7.0 at room temperature and retained at least 80% activity for 60 min. For the storage temperature test, the lyophilized Glmnase still conserved about 90% activity during 7 days at 30 °C, and was higher than about 80% at 4 °C. The K_m and V_max, were 0.018 mg/ml konjac powder and 0.20 mg/ml reducing sugar per min, respectively.     

Effects of elevated water temperature on the critical swim speeds of yearling rainbow trout Salmo gairdneri

Effects of elevated water temperature on the critical swim speeds of rainbow trout, Salmo gairdneri, were investigated. Trout acclimated to 10°C were exposed to 10, 15, 20 and 20°C while swimming and at rest. Initial swim speed of 20 cms⁻¹ was increased in 10 cms⁻¹ increments every 20 min until the fish fatigued. Critical swim speeds were calculated in absolute values (cms⁻¹) and relative performance values (body lengths s⁻¹). Critical swim speeds were similar at 10, 15 and 25°C. Swimming performance was significantly decreased at 25°C. Performance measured as critical swim speed was unaffected by temperature elevations up to 10°C above acclimation temperature of 10°C.     

Cheek skin temperature and thermal resistance in active and inactive individuals during exposure to cold wind

1. The present study examined the effect of the thermal state of the body (as reflected by rectal temperature) on cheek skin temperature and thermal resistance in active and inactive subjects.

2. Active subjects were exposed to a 30 min conditioning period (CP) (0 °C air with a 2 m/s wind), followed immediately by a 30 min experimental period (EP) (0 °C with a 5 m/s wind). Inactive subjects were exposed to a 30 min CP (22 °C air with no wind), followed immediately by a 45 min EP (0 °C air with a 4.5 m/s wind). The CP period was used to establish a core temperature difference between the active and inactive subjects prior to the start of EP. The 0 °C exposure was replaced with a −10 °C ambient air exposure and the experiment was repeated on a separate day. Subjects were comfortably dressed for each ambient condition.

3. Cheek skin temperature was not significantly higher in active subjects when compared to inactive subjects, but thermal resistance was higher in active subjects.

4. Cheek skin temperature and thermal resistance both decreased as ambient temperature decreased from 0 to −10 °C. The lower cheek thermal resistance at −10 °C may have been due to a greater cheek blood flow as a result of cold-induced vasodilation.

Extraction of defatted rice bran by subcritical water treatment

Defatted rice bran was treated with subcritical water in the temperature range of 180–280 °C for 5 min using 117 mL and 9 mL vessels to produce the extracts. The total sugar and protein contents and radical scavenging activity of the extracts were then estimated for both vessels. The total sugar concentration of ca. 0.3 g/L-extract was the highest for the extracts at 200 °C, and it significantly decreased at the higher temperatures. The protein concentration and radical scavenging activity were higher at the higher temperatures. Extraction was also done at 200 °C and 260 °C for various times using the small vessel. The total sugar concentration decreased with the increasing extraction time, while the protein concentration and radical scavenging activity only slightly depended on the extraction time. The extracts at 200 °C or lower temperatures using the large vessel possessed the emulsifying and emulsion-stabilizing activities. The HPLC analysis of the extract at 260 °C for 5 min using the small vessel indicated that it contained both hydrophilic and hydrophobic substances. The hydrophilic fraction of the extract mainly contained low-molecular-mass substances.     

Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Isolation of a thermostable uricase-producing bacterium and study on its enzyme production conditions

A uricase-producing bacterium was isolated from soil with a medium containing uric acid as the only carbon source. Based on its morphological and physiological characteristics, as well as 16S rDNA sequence and phylogenetic tree analysis, this new isolate belong to the genus Microbacterium. After heat treatment at 70 °C for 30 min, the uricase retained about 100% of the initial activity. The enzyme activity remained largely unchanged when it was stored in borate buffer at pH 8.5 at 37 °C for 40 days. The effects of different factors on the enzyme production were studied. Maize milk was the best C and N resources, and the uric acid showed to be an inducer for uricase production. When the strain was cultured at 30 °C at pH 7.5 for 30–36 h, the uricase activity peaked at 1.0 U/ml.     

Finger temperatures during military field training at 0 to −29 °C

1. Skin and rectal temperatures were recorded continuously in 70 measurements during typical tasks of infantry and artillery training at 0 to −29 °C. The duration of the measurements varied from 55 min to 9.5 h.

2. The distribution of finger skin temperatures was quite similar at ambient temperature ranges 0 to −10 °C and −10 to −20 °C, while at −20 to −30 °C the finger temperatures were clearly lower.

3. At different ambient temperature ranges, 20–69% of finger temperatures were low enough to cause cold thermal sensations.

4. Sensation of cold was experienced at a finger temperature of 11.6±3.7 °C (mean±SD).     

Enzyme stabilization using synthetic compensatory solutes

The protective effect of the synthetic compensatory solutes, dimethylthetin (CAS 4727-41-7) and homodeanol betaine (N, N-dimethyl-N-(2-hydroxyethyl)-N-(2 carboxyethyl) ammonium inner salt, CAS 6249-53-2), on two enzymes: lactate dehydrogenase (LDH from rabbit muscle) and a microbial lipase, was compared with that of glycine betaine, trehalose and sorbitol. When the enzyme plus 1 M solute were heated for 10 min at temperatures between 35-75°C, the temperature at which 50% of enzyme activity was lost increased most in the presence of trehalose (7.9° for LDH, 11.6° for lipase) and homodeanol betaine (10.7° for LDH, 11.0° for lipase). With both enzymes, more activity was retained at extreme temperatures in the presence of homodeanol betaine than with trehalose. Glycine betaine, dimethylthetin and sorbitol were less effective. Enzyme plus 1 M stabilizer solutions were frozen at -30°C and freeze-dried for 24 h. Trehalose was the most effective stabilizer of lactate dehydrogenase, and homodeanol betaine of lipase, during freeze-drying.     

'P' solubilization potential of plant growth promoting Pseudomonas mutants at low temperature

Pseudomonas fluorescens strain GRS₁, PRS₉ and their cold tolerant mutants were examined for their tricalcium phosphate (TCP) solubilizing activity in NBRIP (broth) media at 10°C and 25°C. Invariably, all the cold tolerant mutants of GRS₁ and PRS₉ were found more efficient than their respective wild type counterparts for ‘P’ solubilization activity at 10°C as compared to 25°C. ‘P’ solubilization potential of CRM was found maximum among all the strains followed by CRPF₆ and CRPF₄. To the best of out knowledge, this is the first report regarding low temperature ‘P’ solubilization activity.     

Studies on the optimal cooling rate for freezing human diploid fibroblasts

Ampoules containing each 1 ml of Dulbecco's Modified Eagle Medium with 16.6% fetal calf serum and 10% dimethylsulfoxide were insulated in various ways and placed into different cooling boxes. The resulting cooling velocities of the medium ranged from about 0.7 to 102 °C/min. As revealed by cellular attachment in recovery cultures, human diploid fibroblasts cooled at about 1.5 to 4.5 °C/min to − 78 °C prior to storage in liquid nitrogen showed an optimal survival of about 60%. Survival was about 25% at cooling rates of 0.7 and 19 °C/min, respectively. The optimal cooling rate was achieved by insulating the freezing ampoules with 1 to 3 closed vessels and placing them into a dry ice chest, or into a dry ice/ethanol bath.     

Carp expresses fast skeletal myosin isoforms with altered motor functions and structural stabilities to compensate for changes in environmental temperature

1. 1. Myosin and its subfragment-1 (Sl) from carp acclimated to 10°C showed higher actin-activated Mg²⁺-ATPase activity and lower thermostability than their counterparts from carp acclimated to 30°C. Accordingly, filament velocity for the 10°C-acclimated carp myosin was higher at any measuring temperatures from 3 to 23°C than that for the 30°C-acclimated carp myosin.

2. 2. Three types of cDNA clones encoding myosin heavy chains were isolated from thermally acclimated carp. The 10 and 30°C types were predominating in carp acclimated to 10 and 30°C, respectively, whereas the intermediate type was found as a minor component in the 10°C-acclimated carp with an intermediate feature in both DNA nucleotide and deduced amino acid sequences between those of the 10 and 30°C types.

3. 3. The three types of myosin rod all showed a typical coiled-coil structure of -helices. DSC scans demonstrated that myosin rod prepared from carp acclimated to 10°C had a lower thermostability than that from carp acclimated to 30°C, showing that low thermostability in cold-acclimated carp myosin prevails over the entire molecule.

4. 4. cDNA clones encoding myosin alkali light chains were isolated from thermally acclimated carp. Northern blot analysis showed that the ratios of LC3/LC1 mRNAs were significantly higher (3.92) in the 30°C- than 10°C-acclimated (3.10) carp.

     

Thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) from Lenzites betulinus

A thermally stable and hydrogen peroxide tolerant manganese peroxidase (MnP) was purified from the culture medium of Lenzites betulinus by ion exchange chromatography, gel filtration and isoelectric focusing chromatography. The MnP purified from L. betulinus (L-MnP) has a molecular mass of 40 kDa and its isoelectric point was determined to be 6.2. The first 19 amino acids at the N-terminal end of the L-MnP sequence were found to exhibit 74% identity with those of a Phlebia radiata MnP. L-MnP was proved to have the highest hydrogen peroxide tolerance among MnPs reported so far. It retained more than 60% of the initial activity after thermal treatment at 60°C for 60 min, and also retained more than 60% of the initial activity after exposure to 10 mM hydrogen peroxide for 5 min at 37°C.     

Thermostable hydantoinase from a hyperthermophilic archaeon, Methanococcus jannaschii

A hyperthermophilic hydantoinase from Methanococcus jannaschii with an optimum growth at 85°C was cloned and expressed in E. coli. The recombinant hydantoinase was purified by affinity and anion-exchange chromatography and determined to be homotetrameric protein by gel filtration chromatography. The best substrate for the hydantoinase was D,L-5-hydroxyhydantoin, which has the specific activity of 183.4 U/mg. The optimum pH and temperature for the hydantoinase activity was 8.0 and 80°C, respectively. The half-life of the hydantoinase was measured to be 100 min at 90°C in the buffer containing 500 mM KCl. Manganese ions were the most effective for the hydantoinase activity. Stereospecificity was determined to be L-specific for the 5-hydroxymethylhydantoin and 5-methylhydantoin by chiral TLC. The activity yields as well as the operational stabilities of the thermostable M. jannaschii hydantoinase could be significantly improved by immobilization method.     

An Aceto-Osmium Method for Demonstrating Nematodes in Citrus Roots

Difficulties are encountered in observing nematodes in citrus feeder roots because of the presence of suberin and other unsaturated compounds. To obviate these difficulties, infected citrus roots, either fresh or preserved, are immersed in a covered jar for 2 hr at 52° C in a solution composed of distilled water, 16 parts; 10% acetic acid, 10 parts; and 2% aqueous osmium tetroxide, 2 parts. The stained, blackened roots are washed in running water for at least 1 hr and then bleached in 10-30% hydrogen peroxide at 32°C for a few seconds until the color of the roots lightens perceptibly. After several washings in water to stop the oxidation reaction, roots are dehydrated in 70, 95, and absolute ethanol held at 52 °C for 30 min at each concentration. After dehydration, roots are cleared in methyl salicylate at 52°C. Examination for nematodes in most cases, can be made after 30 min.