A sulfated galactan from the mucilaginous sheath of the red filamentous alga Chroodactylon ornatum (Stylonematophyceae,Rhodophyta)

The pseudofilamentous red alga Chroodactylon ornatum was grown in f/2 culture medium with the addition of 10, 34, and 100 μM (nominal concentration) cupric sulfate. The bioassays were terminated at two selected end points (days 4 and 12). Growth inhibition, changes in pigment composition, and oxidative stress indicators such as phenolic compounds and lipid peroxidation (dosed as thiobarbituric reactive substances) were observed in cultures with 34 and 100-μM cupric sulfate. Quinacrine (Atebrin) and chlorotetracycline fluorochromes showed abundant vacuoles of acidic content, related with mucilage secretion. Structural analyses by methylation, desulfation-methylation, alkaline treatment, and NMR spectroscopy revealed that the mucilaginous sheath of C. ornatum contains a sulfated galactan with a backbone of alternating 3-linked β-d-galactopyranose and 4-linked α-l-galactopyranose moieties, i.e., an agaran. The absence of 3,6-anhydrogalactose and of its precursor unit (α-galactose 6-sulfate) were confirmed. The highly sulfated polysaccharide contained these ester groups on the C-2 and C-4 hydroxyl groups of the 3-linked unit and on C-3 of the 4-linked units. The large proportion of sulfate esterification in this polysaccharide can be related to the extracellular biosorption of copper divalent cation (2.5?±?0.4 mg per gram of dry weight) and to copper tolerance in bioassays.     

Metabolic engineering of Escherichia coli for biosynthesis of d-galactonate

d-galactose is an attractive substrate for bioconversion. Herein, Escherichia coli was metabolically engineered to convert d-galactose into d-galactonate, a valuable compound in the polymer and cosmetic industries. d-galactonate productions by engineered E. coli strains were observed in shake flask cultivations containing 2 g L^?1 d-galactose. Engineered E. coli expressing gld coding for galactose dehydrogenase from Pseudomonas syringae was able to produce 0.17 g L^?1 d-galactonate. Inherent metabolic pathways for assimilating both d-galactose and d-galactonate were blocked to enhance the production of d-galactonate. This approach finally led to a 7.3-fold increase with d-galactonate concentration of 1.24 g L^?1 and yield of 62.0 %. Batch fermentation in 20 g L^?1 d-galactose of E. coli ?galK?dgoK mutant expressing the gld resulted in 17.6 g L^?1 of d-galactonate accumulation and highest yield of 88.1 %. Metabolic engineering strategy developed in this study could be useful for industrial production of d-galactonate.     

Seasonal variation of agar from <Emphasis Type="Italic">Gracilaria vermiculophylla,</Emphasis> effect of alkali treatment time,and stability of its Colagar

Gracilaria vermiculophylla, from Baja California Sur, Mexico, was studied in order to determine the seasonal variation of yield and quality of native and alkaline agar during 2007–2008. The highest alkaline agar yield was obtained in summer (17%) and the highest gel strength in spring (1,132 g cm⁻²). The highest melting temperature was 98°C (winter). The highest gelling temperature was 68°C (summer). The values obtained are within the range of the most important Gracilaria species harvested worldwide. During the agar extraction step, the best results were obtained after 30 min of alkali treatment with sodium hydroxide (7%), after which the quality decreased significantly. We produced Colagar from G. vermiculophylla which consists of the seaweeds treated with sodium hydroxide and dried. The yield and quality of the agar obtained from the Colagar shows stability in both yield and quality during 1 year of storage, suggesting that alkali treatment is a good method of avoiding agar hydrolysis during storage.     

Effect of alkali treatment time and extraction time on agar from <Emphasis Type="Italic">Gracilaria vermiculophylla</Emphasis>

The effects of alkali treatment time and extraction time of native agar and alkali treated agar obtained from Gracilaria vermiculophylla were studied. The response characteristics were mainly agar yield and gel strength. Alkali treatment was carried out at 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0 h. Agar yield and gel strength decreased with the increase in the time of the alkali treatment. The highest yield (15.3%) and highest gel strength (1,064 g cm⁻²) were obtained at 0.5 h, and therefore this time was used for the next experiment. The extraction of both native and alkali treated agars was carried out at 1.5, 2.0, 2.5, and 3.0 h. The best extraction time for alkali treated agar was 1.5 h, and for native agar 2.5 h. The alkali treated agar obtained with the different alkali treatment and extraction times showed higher melting (92.4–99.7°C) and gelling (35.7–39.6°C) temperatures. Native agar was lower in melting (60.2–64.1°C) and gelling (20.4–23.4°C) temperatures. The 3,6-anhydrogalactose content decreased with increasing alkali treatment time, with the opposite effect during the extraction of native and alkali treated agars.     

Effects of mutations at threonine-654 on the insoluble glucan synthesized by Leuconostoc mesenteroides NRRL B-1118 glucansucrase

Twelve different amino acids were each substituted for threonine-654 in a cloned glucansucrase from Leuconostoc mesenteroides NRRL B-1118. Both the native and the cloned enzyme with threonine at position 654 produced a water-insoluble glucan containing approximately 44 mol% 1,3-disubstituted α-d-glucopyranosyl units and 29 mol% 1,6-disubstituted α-d-glucopyranosyl units. Several substitutions yielded an enzyme that produced an increased percentage of 1,3-disubstituted α-d-glucopyranosyl units, with corresponding decreases in 1,6-disubstituted α-d-glucopyranosyl units. Only one substitution, tyrosine, resulted in a significant increase in the percentage of 1,6-disubstituted α-d-glucopyranosyl units, with a concomitant increase in glucan yield. The mutated enzymes that produced the highest levels of 1,3-disubstituted α-d-glucopyranosyl units were also significantly activated by the addition of dextran, but glucan yields were also lower in these mutants.     

Characterization of a recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus that isomerizes l-fucose, d-arabinose, d-altrose, and l-galactose

A recombinant l-fucose isomerase from Caldicellulosiruptor saccharolyticus was purified as a single 68 kDa band with an activity of 76 U mg^?1. The molecular mass of the native enzyme was 204 kDa as a trimer. The maximum activity for l-fucose isomerization was at pH 7 and 75°C in the presence of 1 mM Mn²⁺. Its half-life at 70°C was 6.1 h. For aldose substrates, the enzyme displayed activity in decreasing order for l-fucose, with a k _cat of 11,910 min^?1 and a K _m of 140 mM, d-arabinose, d-altrose, and l-galactose. These aldoses were converted to the ketoses l-fuculose, d-ribulose, d-psicose, and l-tagatose, respectively, with 24, 24, 85, 55% conversion yields after 3 h.     

d-Tagatose production in the presence of borate by resting Lactococcus lactis cells harboring Bifidobacterium longum l-arabinose isomerase

Bifidobacterium longum NRRL B-41409 l-arabinose isomerase (l-AI) was overexpressed in Lactococcus lactis using a phosphate depletion inducible expression system. The resting L. lactis cells harboring the B. longum l-AI were used for production of d-tagatose from d-galactose in the presence of borate buffer. Multivariable analysis suggested that high pH, temperature and borate concentration favoured the conversion of d-galactose to d-tagatose. Almost quantitative conversion (92 %) was achieved at 20 g L^?1 substrate and at 37.5 °C after 5 days. The d-tagatose production rate of 185 g L^?1 day^?1 was obtained at 300 g L^?1 galactose, at 1.15 M borate, and at 41 °C during 10 days when the production medium was changed every 24 h. There was no significant loss in productivity during ten sequential 24 h batches. The initial d-tagatose production rate was 290 g L^?1 day^?1 under these conditions.     

Antioxidant, cytotoxic and hemolytic effects of sulfated galactans from edible red alga Hypnea musciformis

Polysaccharides, galactans, obtained from edible red seaweed Hypnea musciformis were characterized by molecular weight and infrared spectroscopy analysis and were evaluated for antioxidant activity in vitro and for their effects on cell viability. The main components were galactose and sulfate presenting low protein contamination. These sulfated galactans (F1.0) showed a polydisperse profile, and signs in infrared analysis were attributed to a sulfate ester S?=?O bond, the presence of a 3,6-anhydrogalactose C–O bond, nonsulfated β-d-galactose, and a C–O–SO₄ bond in galactose C4. The NMR analysis showed signals at about 95 and 92 attributed to anomeric carbon of 4-linked 3,6-anhydro-α-d-galactopyranose residue of κ-carrageenans and 4-linked 3,6-anhydro-α-d-galactopyranose2-sulfate of ι-carrageenans. Sulfated galactan F1.0 showed strong antioxidant activity under lipid peroxidation assay where F1.0 at 8 mg mL^?1 promoted 57.92% peroxidation inhibition and displayed the scavenging activity on hydroxyl radicals in a dose-dependent manner leading to 32.5% scavenging of these radicals when 5 mg mL^?1 of sulfated galactan F1.0 was used. The sulfated galactan fraction also exhibited strong inhibition on the H₂O₂-induced hemolysis model. Sulfated galactan F1.0 displayed low cytotoxic action in 3 T3 cells and moderate antitumoral action in HeLa cells. These results suggest that sulfated galactan F1.0 from H. musciformis has antioxidant potential, which is a great effect for a compound used as food and in the food industry.     

l-Arabinose/d-galactose 1-dehydrogenase of Rhizobium leguminosarum bv. trifolii characterised and applied for bioconversion of l-arabinose to l-arabonate with Saccharomyces cerevisiae

Four potential dehydrogenases identified through literature and bioinformatic searches were tested for l-arabonate production from l-arabinose in the yeast Saccharomyces cerevisiae. The most efficient enzyme, annotated as a d-galactose 1-dehydrogenase from the pea root nodule bacterium Rhizobium leguminosarum bv. trifolii, was purified from S. cerevisiae as a homodimeric protein and characterised. We named the enzyme as a l-arabinose/d-galactose 1-dehydrogenase (EC 1.1.1.-), Rl AraDH. It belongs to the Gfo/Idh/MocA protein family, prefers NADP⁺ but uses also NAD⁺ as a cofactor, and showed highest catalytic efficiency (k _cat/K _m) towards l-arabinose, d-galactose and d-fucose. Based on nuclear magnetic resonance (NMR) and modelling studies, the enzyme prefers the α-pyranose form of l-arabinose, and the stable oxidation product detected is l-arabino-1,4-lactone which can, however, open slowly at neutral pH to a linear l-arabonate form. The pH optimum for the enzyme was pH 9, but use of a yeast-in-vivo-like buffer at pH 6.8 indicated that good catalytic efficiency could still be expected in vivo. Expression of the Rl AraDH dehydrogenase in S. cerevisiae, together with the galactose permease Gal2 for l-arabinose uptake, resulted in production of 18 g of l-arabonate per litre, at a rate of 248 mg of l-arabonate per litre per hour, with 86 % of the provided l-arabinose converted to l-arabonate. Expression of a lactonase-encoding gene from Caulobacter crescentus was not necessary for l-arabonate production in yeast.     

Function of aspartic acid residues in optimum pH control of l-arabinose isomerase from Lactobacillus fermentum

l-Arabinose isomerase (l-AI) catalyzes the isomerization of l-arabinose to l-ribulose and d-galactose to d-tagatose. Most reported l-AIs exhibit neutral or alkaline optimum pH, which is less beneficial than acidophilic ones in industrial d-tagatose production. Lactobacillus fermentum l-AI (LFAI) is a thermostable enzyme that can achieve a high conversion rate for d-galactose isomerization. However, its biocatalytic activity at acidic conditions can still be further improved. In this study, we report the single- and multiple-site mutagenesis on LFAI targeting three aspartic acid residues (D268, D269, and D299). Some of the lysine mutants, especially D268K/D269K/D299K, exhibited significant optimum pH shifts (from 6.5 to 5.0) and enhancement of pH stability (half-life time increased from 30 to 62 h at pH 6.0), which are more favorable for industrial applications. With the addition of borate, d-galactose was isomerized into d-tagatose by D268K/D269K/D299K at pH 5.0, resulting in a high conversion rate of 62 %. Based on the obtained 3.2-Å crystal structure of LFAI, the three aspartic acid residues were found to be distant from the active site and possibly did not participate in substrate catalysis. However, they were proven to possess similar optimum pH control ability in other l-AI, such as that derived from Escherichia coli. This study sheds light on the essential residues of l-AIs that can be modified for desired optimum pH and better pH stability, which are useful in d-tagatose bioproduction.     

NAD+-dependent xylitol dehydrogenase (xdhA) and l-arabitol-4-dehydrogenase (ladA) deletion mutants of Aspergillus oryzae for improved xylitol production

Xylitol dehydrogenase (XDHA) and l-arabitol dehydrogenase (LADA) are two key enzymes in xylan metabolism catalyzing the oxidation of xylitol to d-xylulose and arabitol to l-xylulose, respectively. In Aspergillus oryzae, XDHA and LADA are encoded by xdhA and ladA. We deleted xdhA and ladA and xdhA–ladA to generate mutants with decreased dehydrogenase activities and increased xylitol production. The mutants were constructed by homologous transformation into A. oryzae P4 (?pyrG) using pyrG as a selectable marker. The xylitol productivity of the mutants was measured using d-xylose as the sole carbohydrate source. xdhA, ladA, and the double-deletion mutant produced, respectively, 12.4 g xylitol/l with a yield of 0.24 g/g d-xylose, 12.4 g/l with a yield of 0.33 g/g d-xylose, and 8.6 g/l at a yield of 0.26 g/g d-xylose.     

A d-psicose 3-epimerase with neutral pH optimum from Clostridium bolteae for d-psicose production: cloning, expression, purification, and characterization

d-Tagatose 3-epimerase family enzymes can efficiently catalyze the epimerization of free keto-sugars, which could be used for d-psicose production from d-fructose. In previous studies, all optimum pH values of these enzymes were found to be alkaline. In this study, a d-psicose 3-epimerase (DPEase) with neutral pH optimum from Clostridium bolteae (ATCC BAA-613) was identified and characterized. The gene encoding the recombinant DPEase was cloned and expressed in Escherichia coli. In order to characterize the catalytic properties, the recombinant DPEase was purified to electrophoretic homogeneity using nickel-affinity chromatography. Ethylenediaminetetraacetic acid was shown to inhibit the enzyme activity completely; therefore, the enzyme was identified as a metalloprotein that exhibited the highest activity in the presence of Co²⁺. Although the DPEase demonstrated the most activity at a pH ranging from 6.5 to 7.5, it exhibited optimal activity at pH 7.0. The optimal temperature for the recombinant DPEase was 55 °C, and the half-life was 156 min at 55 °C. Using d-psicose as the substrate, the apparent K _m, k _cat, and catalytic efficiency (k _cat/K _m) were 27.4 mM, 49 s^?1, and 1.78 s^?1 mM^?1, respectively. Under the optimal conditions, the equilibrium ratio of d-fructose to d-psicose was 69:31. For high production of d-psicose, 216 g/L d-psicose could be produced with 28.8 % turnover yield at pH 6.5 and 55 °C. The recombinant DPEase exhibited weak-acid stability and thermostability and had a high affinity and turnover for the substrate d-fructose, indicating that the enzyme was a potential d-psicose producer for industrial production.     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Agar properties of two species of Gracilariaceae from the Gulf of California,Mexico

Agar properties of two potentially commercial important seaweeds from the Gulf of California were studied. Maximum yield in Gracilaria vermiculophylla (45.7%) occurred during the summer months, coinciding with high water temperatures (31°C) whereas minimum yields (11.6%) were obtained during the coldest months of the year when populations of this species diminish in the bay. Gracilariopsis longissima showed two yield peaks, one in spring and another in fall, before the maximum and minimum seawater temperatures. Gel strength in native agar from the two species was low (<22.5 g cm⁻²) for most of the year. G. vermiculophylla native agar showed a slight increase in gel strength from June to August, which were the hottest months. Maximum value was 85 g cm⁻¹ in August. Maximum gel strength in G. longissima was observed in October (91 g cm⁻¹), and an unusual native agar with no detectable gel strength was observed in March and April samples. Gelling temperatures range from 27.7 to 36.5°C in G. vermiculophyla and from 26.6 to 34.9°C in G. longissima, meanwhile melting points were 73.9 – 53.5°C and 75.5 – 56.6°C, respectively. Sulfate content was high, 6.3–13.9% in G. vermiculophylla and 1.9–11.9% in G. longissima, and on the other hand 3,6 anhydrogalactose content was low 12.1–26.7% and 9.1–23%, respectively compared to other species. Results obtained showed that mean native agar yields of Gracilaria vermiculophylla and Gracilariopsis longissima from the Gulf of California are comparable to other tropical Gracilaria. However, the low gel strength, high sulfate content and low 3,6 anhydrogalactose content observed in the native agar extracted from these species make this an agaroid, thus alternative methods of extraction should be used to evaluate the possibility of commercial utilization of both species.     

Backbone and side-chain 1H, 15N and 13C assignment of apo- and imipenem-acylated l,d-transpeptidase from Bacillus subtilis

The d,d-transpeptidase activity of Penicillin Binding Proteins (PBPs) is essential to maintain cell wall integrity. PBPs catalyze the final step of the peptidoglycan synthesis by forming 4 → 3 cross-links between two peptide stems. Recently, a novel β-lactam resistance mechanism involving l,d-transpeptidases has been identified in Enterococcus faecium and Mycobacterium tuberculosis. In this resistance pathway, the classical 4 → 3 cross-links are replaced by 3 → 3 cross-links, whose formation are catalyzed by the l,d-transpeptidases. To date, only one class of the entire β-lactam family, the carbapenems, is able to inhibit the l,d-transpeptidase activity. Nevertheless, the specificity of this inactivation is still not understood. Hence, the study of this new transpeptidase family is of considerable interest in order to understand the mechanism of the l,d-transpeptidases inhibition by carbapenems. In this context, we present herein the backbone and side-chain ¹H, ¹⁵N and ¹³C NMR assignment of the l,d-transpeptidase from Bacillus subtilis (Ldt_Bs) in the apo and in the acylated form with a carbapenem, the imipenem.     

Reactions upstream of glycerate-1,3-bisphosphate drive Corynebacterium glutamicum d-lactate productivity under oxygen deprivation

We previously demonstrated the simplicity of oxygen-deprived Corynebacterium glutamicum to produce d-lactate, a primary building block of next-generation biodegradable plastics, at very high optical purity by introducing heterologous D-ldhA gene from Lactobacillus delbrueckii. Here, we independently evaluated the effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production in C. glutamicum. We consequently show that while the reactions catalyzed by glucokinase (GLK), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phosphofructokinase (PFK), triosephosphate isomerase (TPI), and bisphosphate aldolase had positive effects on d-lactate productivity by increasing 98, 39, 15, 13, and 10 %, respectively, in 10 h reactions in minimal salts medium, the reaction catalyzed by pyruvate kinase had large negative effect by decreasing 70 %. The other glycolytic enzymes did not affect d-lactate productivity when each of encoding genes was overexpressed. It is noteworthy that all reactions associated with positive effects are located upstream of glycerate-1,3-bisphosphate in the glycolytic pathway. The d-lactate yield also increased by especially overexpressing TPI encoding gene up to 94.5 %. Interestingly, overexpression of PFK encoding gene reduced the yield of succinate, one of the main by-products of d-lactate production, by 52 %, whereas overexpression of GAPDH encoding gene increased succinate yield by 26 %. Overexpression of GLK encoding gene markedly increased the yield of dihydroxyacetone and glycerol by 10- and 5.8-fold in exchange with decreasing the d-lactate yield. The effect of overexpressing glycolytic genes was also evaluated in 80 h long-term reactions. The variety of effects of overexpressing each of genes encoding the ten glycolytic enzymes on d-lactate production is discussed.     

Vegetation and fire history with their implication for climatic change and fire events since the last deglacial in the Aso Valley, central Kyushu, southwestern Japan: new pollen and charcoal data

To better understand the response of forest vegetation to climate and fire regimes with reference to human activities over the last deglacial period in the Aso Caldera, central Kyushu, southwestern Japan, a 33.9 m long sediment core was examined in order to reconstruct the vegetational and fire history using pollen and charcoal analyses. The results show that a cool temperate broad-leaved deciduous forest, dominated by Quercus (deciduous oaks) with Carpinus and Fagus, prevailed in the Aso Valley from ca. 14.6 ka cal. b.p., indicating warming since the last glacial period. The landscape was presumably covered by a mosaic of deciduous Quercus forests and terrestrial Artemisia communities. Around 12.8–11.7 ka cal. b.p., Quercus dominated the forest and fires occurred frequently. Co-expansion of distinctive Ulmus–Zelkova and Celtis–Aphananthe forests coupled with a progressive retreat of Quercus in the early Holocene could reflect a strengthening of the East Asian summer monsoon under mild and humid climate conditions. Around 8 ka cal. b.p., significant increases in Cyclobalanopsis (evergreen oaks), Castanopsis/Castanea and Podocarpus indicate a further warming, in particular an increased winter temperature. Warm temperate lucidophyllous forests, dominated by Cyclobalanopsis, flourished after 7.3 ka cal. b.p., probably corresponding to the “Holocene Climatic Optimum” interval. Progressive expansion of Quercus at the expense of Cyclobalanopsis began around 6.4 ka cal. b.p. and paralleled an increase in charcoal until ca. 4.8 ka cal. b.p.; this could be evidence of fire disturbance induced by the early-middle Jomon people. The disturbed evergreen forest experienced a temporary recovery but then opened again from 3.6 ka cal. b.p. due to extensive fire deforestation, as suggested by the high charcoal levels during this time. Human exploitation and buckwheat (Fagopyrum) agriculture may have contributed to the opening of the forest, which allowed secondary forests (primarily Pinus and Quercus) and herbaceous communities (mainly Poaceae) to spread. These results are discussed in comparison with other high-resolution pollen data from western Japan to better elucidate the vegetation and fire history over the last deglacial in the Aso Caldera.     

Anti-apoptosis effect of polysaccharide isolated from the seeds of Cuscuta chinensis Lam on cardiomyocytes in aging rats

To investigate the mechanism of apoptosis in myocardial cells of aging rats induced by d-galactose and to study the effect of the Polysaccharide isolated from the seeds of Cuscuta chinensis Lam (PCCL) on apoptosis of cardiomyocytes and its corresponding machinasim in aging rat model. Fifty male SD rats were randomly divided into 5 groups. Normal control group (NC). d-galactose (100 mg·kg^?1d^?1 for 56 day) indued aging group (MC), d-galactose plus 100 mg kg^?1 d^?1 PCCL group (ML), d-galactose plus 200 mg kg^?1 d^?1 PCCL group (MM), and d-galactose plus 400 mg kg^?1 d^?1 PCCL group (MH). Same volume of solution (water, or PCCL aqueous solution) was given by gavage for 56 days. Then the hearts were collected and apoptosis parameters were evaluated. Caspase-3 and Cyt c were determined by fluorescence spectrometer, the apoptosis rate was assessed by AnnexinV-FITC method by Flow-Cytometry, [Ca²⁺]i and [Ca²⁺]i overloaded by KCL were observed by laser scanning confocal microscopy (LSCM); Bcl-2 and Bax were examined by immunohistochemistry. The content of Cyt C, [Ca²⁺]i of cardiomyocytes, the activity of Caspase-3, Bax expression level in d-galactose induced aging group were higher than NC (p < 0.05). The ratio of Bcl-2/Bax was decreased in d-galactose induced aging group compared to NC. On the other hand, the content of Cyt C, [Ca²⁺]i of cardiomyocytes, the activity of Caspase-3 and apoptosis rate, as well as Bax expression level in all three PCCL groups were decreased compared to galactose induced group (p < 0.05). Bcl-2/Bax ratio was increased in all PCCL groups compared to galactose induced aging group. PCCL could decrease the apoptosis of cardiomyocytes by the mitochondria apoptosis pathway.     

K+-dependent 3H-d-glucose transport by hepatopancreatic brush border membrane vesicles of a marine shrimp

The effects of sodium, potassium, sugar inhibitors, and membrane potential on ³H-d-glucose uptake by hepatopancreatic epithelial brush border membrane vesicles (BBMV) of the Atlantic marine shrimp, Litopenaeus setiferus, were investigated. Brush border membrane vesicles were prepared using a MgCl₂/EGTA precipitation method and uptake experiments were conducted using a high speed filtration technique. ³H-d-Glucose uptake was stimulated by both sodium and potassium and these transport rates were almost doubled in the presence of an inside-negative-induced membrane potential. Kinetics of ³H-d-glucose influx were hyperbolic functions of both external Na⁺ or K⁺, and an induced membrane potential increased influx J _max and lowered K_m in both salts. ³H-d-Glucose influx versus [glucose] in both Na⁺ or K⁺ media also displayed Michaelis–Menten properties that were only slightly affected by induced membrane potential. Phloridzin was a poor inhibitor of 0.5 mM ³H-d-glucose influx, requiring at least 5 mM in NaCl and 10 mM in KCl to significantly reduce hexose transport. Several sugars (d-galactose, α-methyl-d-gluco-pyranoside, unlabeled d-glucose, d-fructose, and d-mannose) were used at 75 mM as potential inhibitors of 0.1 mM ³H-d-glucose influx. Only unlabeled d-glucose, d-fructose, and d-mannose significantly (p < 0.05) reduced labeled glucose transport. An additional experiment using increasing concentrations of d-mannose (0, 10, 25, 75, and 100 mM) showed this hexose to be an effective inhibitor of 0.1 mM ³H-d-glucose uptake at concentrations of 75 mM and higher. As a whole these results suggest that ³H-d-glucose transport by hepatopancreatic BBMV occurs by a carrier system that is able to use both Na⁺ and K⁺ as drivers, is enhanced by membrane potential, is relatively refractory to phloridzin, and is only inhibited by itself, d-fructose, and d-mannose. These properties are similar to those exhibited by the mammalian SLC5A9/SGLT4 transporter, suggesting that an invertebrate analogue of this protein may occur in shrimp.