Induction of oviposition by injection of male-derived extracts in two Callosobruchus species

In some insect species, certain substances in the seminal fluid of males induce egg production and laying in females. We determined the effects of male-derived substances on female oviposition behaviour in two Callosobruchus species, C. chinensis and C. maculatus. Aqueous extracts of the accessory gland; testis; and seminal vesicle, including the ejaculatory duct, were prepared. The injection of these extracts into abdomen of females induced oviposition in both species. Oviposition was induced by the testis and seminal vesicle extracts in C. chinensis and by the accessory gland extracts in C. maculatus. The extracts were separated into three fractions by ultrafiltration: fractions I, molecular weight (MW) <3 kDa; fraction II, 3-14 kDa; and fraction III, >14 kDa. Fraction III induced oviposition in both species. These results suggest that in these two species, the substances that induce oviposition have similar MW but are present in different organs. Oviposition was induced by high-MW (>14 kDa) substances in the testis and seminal vesicle in C. chinensis, and by high-MW substances in accessory gland in C. maculatus. Here, we have discussed the relationship between oviposition and the abovementioned male-derived substances.     

Preparation and characterization of molecular weight fractions of glycosaminoglycan from sea cucumber Thelenata ananas using free radical depolymerization

A glycosaminoglycan from sea cucumber Thelenata anana (THG) was isolated as a polymer of molecular weight of around 70 kDa. Its low molecular weight derivatives were first prepared by free radical depolymerization with hydrogen peroxide in the presence of copper(II) ion. The parameters of the process were investigated by a high-performance gel permeation chromatography. Analyses of chemical composition and molecular weight distribution indicated that the fragmentation of the main-chain of THG occurred randomly, obeyed pseudo first-order kinetics, and produced species with rather narrow and unimodal distribution of molar mass. The characterization of different molecular weight fractions was investigated by using viscometry and atomic force microscopy (AFM). Analysis of molecular weight and intrinsic viscosity in terms of the known theories for unperturbed wormlike cylinder yielded 1201 ± 110 nm⁻¹, 15.3 ± 1.5 nm, and 1.5 ± 0.3 nm for molar mass per unit contour length M_L, persistence length q, and diameter d, respectively. The M_L and d values were approximately consistent with those observed by AFM. The present data suggest that THG may dissolve in 0.1 M aqueous NaCl as single-stranded helical chains.     

Comparative binding of Cry1Ab and Cry1F Bacillus thuringiensis toxins to brush border membrane proteins from Ostrinia nubilalis, Ostrinia furnacalis and Diatraea saccharalis (Lepidoptera: Crambidae) midgut tissue

The European (Ostrinia nubilalis Hübner) and Asian corn borers (Ostrinia furnacalis Guenée) are closely related and display similar sensitivity to Cry1 toxins. In this study, we compared the binding patterns of Cry1Ab and Cry1F toxins between both Ostrinia spp., as well as the expression of putative cadherin- and aminopeptidase-N (APN)-like protein receptors. Additionally, cDNA sequences of these putative toxin receptors from both Ostrinia species were compared. Ligand blots for both species indicated a similar binding pattern for Cry1Ab with the strongest immunoreactive band at 260 kDa in both species. In addition, similar expression of the putative cadherin- and APN-like protein receptors were observed at 260 and 135 kDa, respectively. A high degree of similarity (98% amino acid sequence identity) of cDNA sequences for both putative receptor sequences was observed. The Cry1F ligand blot revealed that O. furnacalis and O. nubilalis BBMV exhibited slightly different binding patterns, with strong binding to putative proteins at 150 and 140 kDa, respectively. Both proteins appeared to also bind Cry1Ab, although the signal intensity was much reduced with Cry1Ab. O. furnacalis showed an additional but weaker band at 210 kDa relative to the 150 kDa band. Diatraea saccharalis (Fabricius), which was used as an outgroup species, exhibited different binding patterns than either Ostrinia species, with both Cry1Ab and Cry1F toxins binding to a 210 kDa protein. These results support the previous experiments indicating that O. nubilalis and O. furnacalis share similar patterns of susceptibility to Cry toxins.     

On the molecular mass of the extracellular hemoglobin of Glossoscolex paulistus: Analytical ultracentrifugation reexamination

The giant extracellular hemoglobin of Glossoscolex paulistus (HbGp) is constituted by subunits containing heme groups with molecular masses (M) in the range of 15 to 19 kDa, monomers of 16 kDa (d), and trimers of 51 to 52 kDa (abc) linked by nonheme structures named linkers of 24 to 32 kDa (L). HbGp is homologous to Lumbricus terrestris hemoglobin (HbLt). Several reports propose M of HbLt in the range of 3.6 to 4.4 MDa. Based on subunits M determined by mass spectrometry and assuming HbGp stoichiometry of 12(abcd)₃L₃ (Vinogradov model) plus 144 heme groups, a value of M for HbGp oligomer of 3560 kDa can be predicted. This value is nearly 500 kDa higher than the unique HbGp M value reported in the literature. In the current work, sedimentation velocity analytical ultracentrifugation (AUC) experiments were performed to obtain M for HbGp in oxy and cyano-met forms. s⁰_20,w values of 58.1 ± 0.2 S and 59.6 ± 0.2 S, respectively, for the two oxidation forms were obtained. The ratio between sedimentation and diffusion coefficients supplied values for M of approximately 3600 ± 100 and 3700 ± 100 kDa for oxy and cyano-met HbGp forms, respectively. An independent determination of the partial specific volume, V_bar, for HbGp was performed based on density measurements, providing a value of 0.764 ± 0.008, in excellent agreement with the estimates from SEDFIT software. Our results show total consistency between M obtained by AUC and recent partial characterization by mass spectrometry. Therefore, HbGp possesses M very close to that of HbLt, suggesting an oligomeric assembly in agreement with the Vinogradov model.     

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~ 55 kDa), higher than predicted (~ 50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 °C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~ 35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~ 47 kDa protein; substitution of both sites gave a smaller (~ 35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH₂ or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.     

Phagocytic activity,respiratory burst,cytoplasmic free-Ca concentration and apoptotic cell ratio of haemocytes from the black tiger shrimp, Penaeus monodon under acute copper stress

The aim of this study was to investigate the cellular toxicity of copper-induced injury to the black tiger shrimp Penaeus monodon. The 24 h, 48 h, 72 h and 96 h LC₅₀ (median lethal concentration) of Cu²⁺ on P. monodon (11.63 ± 1.14 g) were found to be 3.49, 1.54, 0.73 and 0.40 mg L^− 1, respectively. Total haemocyte count (THC), phagocytic activity, respiratory burst (RB), cytoplasmic free-Ca²⁺ (cf-Ca²⁺) concentration and apoptotic cell ratio of shrimp were determined after exposure to different concentrations of Cu²⁺ (0, 0.05, 0.5, 1.5 and 3.5 mg L^− 1) for 0, 6, 12, 24 and 48 h. There was no significant effect on the analytic indicator of shrimp exposed to 0.05 mg L^− 1 Cu²⁺. THC decreased after Cu-exposure to 0.5 mg L^− 1 for 48 h, 1.5 mg L^− 1 for 24 h and 3.5 mg L^− 1 for 12 h. Phagocytic activity decreased in P. monodon following 48 h exposure to 3.5 mg L^− 1 Cu²⁺. RB was induced after 6 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. cf-Ca²⁺ concentration increased after 48 h exposure to 0.5 mg L^− 1 Cu²⁺, and 12 h exposure to 1.5 and 3.5 mg L^− 1 Cu²⁺. The percentage of apoptotic cells increased to 9.5%, 16.3% and 18.6% respectively following 48 h exposure to 0.5, 1.5 and 3.5 mg L^− 1 Cu²⁺. These results indicate that Cu can induce oxidative stress, elevation of cf-Ca²⁺ and cell apoptosis, and inhibit phagocytic activity in the shrimp P. monodon, and the lethal injury of Cu²⁺ to P. monodon may be mainly due to the sharp reduction of THC caused by ROS-induced apoptosis.     

Isolation and characterization of a large soluble form of fibronectin that stimulates adhesion, spreading, and alignment of mouse erythroleukemia cells

Fibronectin (FN) is a major component of the extracellular matrix which plays important roles in a variety of cellular processes including cell adhesion, and migration. The soluble cellular form of FN has a monomer molecular weight of approximately 250 kDa, and generally exists as a dimer of 500 kDa. We have isolated a different form of soluble FN from mouse breast cancer cell line SC115 conditioned medium (CM) and purified it to homogeneity as evidenced by both native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate PAGE. It still exhibits a monomeric form of about 250 kDa while its form in the CM is stable and soluble with an apparent tetrameric molecular weight in the range of 800-1000 kDa. This form of FN is a potent cell adhesion factor (AF) that induces adhesion to polystyrene, elongation, spreading, alignment or “track” formation, and migration of mouse erythroleukemia cells. Column fractions homogeneous for AF protein were able to stimulate 10% cell adhesion at concentrations of 23 ng/ml and 1.9 ng/cm². Purified AF induced 50% cell adhesion at 94 ng/ml and 7.5 ng/cm². AF also increased the migration of human aortic smooth muscle and vascular endothelial cells. However, this form of FN differs from other forms as it does not bind tightly to either gelatin or heparin. Studies of this AF should shed light on adhesion of cells to extracellular matrix molecules and on cell migration, both of which are critical in several biological processes such as wound healing, metastasis, matrix formation and structure, and organ development.     

Modeling of biomass production by Spirulinaplatensis as function of phosphate concentrations and pH regimes

Batch cultivations were performed to evaluate the influence of phosphate concentrations (0.25, 0.5, 0.75, and 1.0 g L⁻¹) for pH regimes (9.5, 10.0, and 10.5) on the biomass production by Spirulina platensis. The best condition for cell growth (3.099 g L⁻¹) was found at 0.5 g L⁻¹ phosphate and pH value of 10.0. Cultivation time, phosphate, and pH caused to increase significantly (p < 0.01) in biomass production by S. platensis. Lag time was observed up to 4 h. After then, biomass production increased sharply (p < 0.01) from 0.020 g L⁻¹ to 2.063, 2.213, 1.532, and 0.797 g L⁻¹ at 0.25, 0.5, 0.75, and 1.0 g L⁻¹ phosphate values, respectively. Modified Gompertz model could be regarded as sufficient to describe the biomass production by S. platensis with high determination coefficients and low sum of square value indicated that. Biological parameters for biomass production were successfully predicted by modified Gompertz model.     

Carboxy-terminal extension effects on crystal formation and insecticidal properties of Cry15Aa

Cry15Aa protein, produced by Bacillus thuringiensis serovar thompsoni HD542, in a crystal together with a 40 kDa accompanying protein, is one of a small group of non-typical, less well-studied members of the Cry family of insecticidal proteins, and may provide an alternative for the more commonly used Cry proteins in insect pest management. In this study we examined the role of the C-terminal part of Cry15Aa and of the 40 kDa protein in crystal formation in recombinant B. thuringiensis. The contribution of the 40 kDa protein and of the Cry15Aa carboxy-terminal sequence for crystal formation, crystal solubilization, and insecticidal properties was assessed. No significant differences in toxicity against Cydia pomonella, before or after in vitro solubilization of crystal-spore preparations, were found. Although the 40 kDa protein significantly contributes to in vitro solubility and in vivo crystal formation of Cry15Aa, no direct evidence for involvement of the 40 kDa protein in toxicity of Cry15Aa was found.     

Characterization of vitellin protein in the twospotted spider mite, Tetranychus urticae (Acari: Tetranychidae)

In mites, vitellogenin synthesis, regulation and uptake by the oocytes as vitellin remain practically unknown. Although a partial sequence of the gene is now available, no previous studies have been conducted that describe the native vitellin protein in mites. The objective of this study was to characterize vitellin in the twospotted spider mite, Tetranychus urticae. The native twospotted spider mite vitellin migrated as a single major band with a molecular weight of 476 ± 14.5 kDa as compared to 590 ± 25.5 kDa for vitellin from the American dog tick, Dermacentor variabilis. However, isoelectric focusing analysis of native spider mite vitellin showed five bands with pI values slightly acidic to neutral (pH 5.8, 6.2, 6.7, 7.0 and 7.2), as is the case for insect and tick vitellins. Reducing conditions (SDS-PAGE) also revealed multiple subunits ranging from 290.9 to 3.6 kDa and was similar to that found in D. variabilis. Spider mite vitellin weakly bound lipids and carbohydrates compared to the tick. Unlike D. variabilis, the spider mite egg yolk protein does not bind heme. The significance of non-heme binding in mites is discussed.     

An endo-(1→3)-β-d-glucanase from the scallop Chlamys albidus: catalytic properties, cDNA cloning and secondary-structure characterization

An endo-(1→3)-β-d-glucanase (L₀) with molecular mass of 37 kDa was purified to homogeneity from the crystalline style of the scallop Chlamys albidus. The endo-(1→3)-β-d-glucanase was extremely thermolabile with a half-life of 10 min at 37 °C. L₀ hydrolyzed laminaran with K_m ∼ 0.75 mg/mL, and catalyzed effectively transglycosylation reactions with laminaran as donor and p-nitrophenyl β d-glucoside as acceptor (K_m ∼ 2 mg/mL for laminaran) and laminaran as donor and as acceptor (K_m ∼ 5 mg/mL) yielding p-nitrophenyl β d-glucooligosaccharides (n = 2-6) and high-molecular branching (1→3),(1→6)-β-d-glucans, respectively. Efficiency of hydrolysis and transglycosylation processes depended on the substrate structure and decreased appreciably with the increase of the percentage of β-(1→6)-glycosidic bonds, and laminaran with 10% of β-(1→6)-glycosidic bonds was the optimal substrate for both reactions. The CD spectrum of L₀ was characteristic for a protein with prevailing β secondary-structural elements. Binding L₀ with d-glucose as the best acceptor for transglycosylation was investigated by the methods of intrinsic tryptophan fluorescence and CD. Glucose in concentration sufficient to saturate the enzyme binding sites resulted in a red shift in the maximum of fluorescence emission of 1-1.5 nm and quenching the Trp fluorescence up to 50%. An apparent association constant of L₀ with glucose (K_a = 7.4 × 10⁵ ± 1.1 × 10⁵ M⁻¹) and stoichiometry (n = 13.3 ± 0.7) was calculated. The cDNA encoding L₀ was sequenced, and the enzyme was classified in glycoside hydrolases family 16 on the basis of the amino acid sequence similarity.     

Fasciola gigantica: Immunodiagnosis of fasciolosis by detection of circulating 28.5 kDa tegumental antigen

A monoclonal antibody (MoAb)-based sandwich ELISA was developed for the detection of circulating 28.5 kDa tegumental antigen (28.5 kDa TA) in the sera from mice experimentally infected with Fasciola gigantica. The MoAb was immobilized on a microtiter plate, and the antigen in the serum was captured and detected with biotinylated polyclonal rabbit anti TA antibody. The test could detect 28.5 kDa in the extracts of tegument (TA), whole body (WB) and excretory-secretory (ES) fractions at the concentrations of these crude antigens as low as 600 pg/ml, 16 and 60 ng/ml, respectively. This sandwich ELISA assay could detect the infection from day 1 to 35 post infection and showed that circulating level of 28.5 kDa TA peaked at day 1 post infection. In contrast, the antibody detection by indirect ELISA could only demonstrate the antibody level from 35 days post infection. The reliability of the assay method was evaluated using sera from mice infected with F. gigantica or Schistosoma mansoni, and hamsters infected with Opisthorchis viverrini, as well as healthy mice and hamsters. The sandwich ELISA exhibited a sensitivity and specificity at 94.55% and 100%, respectively, and with a positive predictive value of 100%, a negative predictive value of 97.39%, false positive rate of 0%, false negative rate of 5.50% and an accuracy of 98.2%. Thus, this detection method exhibited high specificity and sensitivity as well as could be used for early diagnosis of fasciolosis by F. gigantica.     

Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii

The thermotolerant yeast strain isolated from sugarcane juice through enrichment technique was identified as a strain of Pichiakudriavzevii (Issatchenkiaorientalis) through molecular characterization. The P. kudriavzevii cells adapted to galactose medium produced about 30% more ethanol from sugarcane juice than the non-adapted cells. The recycled cells could be used for four successive cycles without a significant drop in ethanol production. Fermentation in a laboratory fermenter with galactose adapted P. kudriavzevii cells at 40 °C resulted in an ethanol concentration and productivity of 71.9 g L⁻¹ and 4.0 g L⁻¹ h⁻¹, respectively from sugarcane juice composed of about 14% (w/v) sucrose, 2% (w/v) glucose and 1% (w/v) fructose. In addition to ethanol, 3.30 g L⁻¹ arabitol and 4.19 g L⁻¹ glycerol were also produced, whereas sorbitol and xylitol were not formed during fermentation. Use of galactose adapted P. kudriavzevii cells for ethanol production from sugarcane juice holds potential for scale-up studies.     

Reversible ring opening and closure reactions of the triazine ligands derived from 1-phenylazo-2-naphthylamine and pyridine-2-aldehyde or quinoline-2-aldehyde - structure and reactivity of the palladium (II) complexes

New ligands containing a heterocyclic ring, L¹ (1-anilino-2-(2-pyridyl)-naphth[1,2-d]imidazol-1-io-3-ide), L² (2-phenyl-3-(2-pyridyl)-3,4-dihydro-naphtho[2,1-e][1,2,4]triazin-1-io-4-ide), and L³ (1-anilino-2-(2-quinolyl)-naphth[1,2-d]imidazol-1-io-3-ide), and their palladium (II) complexes have been prepared. Structures of the ligands and the complexes were determined by X-ray crystallography. The mononuclear square-planar complexes of [PdCl₂(Lⁿ)] (n = 1 (1), n = 2 (2) and n = 3 (3)) had didentate Lⁿ (n = 1-3) ligands. The Lⁿ (n = 1-3) ligands were stable and their absorption spectra did not change in dichloromethane and methanol. On the other hand, the absorption spectrum of [PdCl₂(L²)] (2) in dichloromethane changed rapidly when methanol was added to the solution, and [PdCl(L^4b)] (5) (L^4b = N-[methoxy(2-pyridyl)methyl]-1-(phenylazo)-2-naphthylamide) was obtained from the concentrated reaction mixture. In this reaction, the dihydrotriazine ring of the didentate L² ligand in complex 2 opened and the resulting tridentate L^4b ligand coordinated to the Pd atom in complex 5. When an excess amount of (ⁿBu)₄NCl was added to complex 5 in dichloromethane, the absorption spectrum reverted to that of complex 2. Thus, the reversible ring opening and closure reactions of the coordinating dihydrotriazine ligand were observed. We also prepared [PdCl₂(L⁵)] (9) (L⁵ = 1-(phenylazo)-N-[1-(2-pyridyl)ethylidene]-2-naphthylamine) and determined the structure. It is noted that neither the ring closure reaction nor the coordination of the azo nitrogen atom of the L⁵ ligand occurred in complex 9.     

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein.All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.     

Toxoplasma gondii: serological characterization and immunogenicity of recombinant surface antigen 2 (SAG2) expressed in the yeast Pichia pastoris

The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients’ serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the P. pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge.     

Cultivation of shear stress sensitive microorganisms in disposable bag reactor systems

Technical scale (≥5 l) cultivations of shear stress sensitive microorganisms are often difficult to perform, as common bioreactors are usually designed to maximize the oxygen input into the culture medium. This is achieved by mechanical stirrers, causing high shear stress. Examples for shear stress sensitive microorganisms, for which no specific cultivation systems exist, are many anaerobic bacteria and fungi, such as basidiomycetes. In this work a disposable bag bioreactor developed for cultivation of mammalian cells was investigated to evaluate its potential to cultivate shear stress sensitive anaerobic Eubacterium ramulus and shear stress sensitive basidiomycetes Flammulina velutipes and Pleurotus sapidus. All cultivations were compared with conventional stainless steel stirred tank reactors (STR) cultivations. Good growth of all investigated microorganisms cultivated in the bag reactor was found. E. ramulus showed growth rates of μ = 0.56 h⁻¹ (bag) and μ = 0.53 h⁻¹ (STR). Differences concerning morphology, enzymatic activities and growth in fungal cultivations were observed. In the bag reactor growth in form of small, independent pellets was observed while STR cultivations showed intense aggregation. F. velutipes reached higher biomass concentrations (21.2 g l⁻¹ DCW vs. 16.8 g l⁻¹ DCW) and up to 2-fold higher peptidolytic activities in comparison to cell cultivation in stirred tank reactors.     

Applying and comparing two chemometric methods in absorption spectral analysis of photopigments from Arctic microalgae

Pigment absorption property of two arctic microalgae species (Skeletonema marinoi and Chlorella sp.) cultured at three temperatures (0, 4 and 8 °C) was analyzed. Carotenoids and chlorophyll (Chl) c were positive factors to the high cell activities and primary productivities of S. marinoi at 4 °C and 0 °C, respectively; whereas Chl a had a positive effect on Chlorella sp. at all three temperatures, and carotenoids had a relatively high effect at 0 °C. The absorption locations of photopigments were analyzed in detail using both fourth derivative and Symlet-6 wavelet analysis. Both methods precisely detected pigments with a relative large content; the fourth derivative analysis specifically detected the existence of a Chl a peak at about 410 nm and showed better differentiation of diatoxanthin, whereas the wavelet analysis distinctively indicated the existence of chlorophyllide a, β-carotene, and Chl c. The separation limit to pigment peaks of the fourth derivative spectra (4 nm) was 1 nm higher than that of the wavelet high-frequency spectra (3 nm). The wavelet high-frequency spectra were more stable in detecting pigment locations and were more effective in discriminating microalgae. Small algebraic difference of 10⁻¹⁶ between the reconstructed absorption spectra obtained by the inverse wavelet transform and their corresponding original spectra also showed the validity of Symlet-6 wavelet in the detection of pigments. Another specific discovery of this research is the existence of a Chl a allomer in Chlorella sp., which was detected by both methods.     

Vertical tubular photobioreactor for semicontinuous culture of Cyanobium sp

We evaluated the kinetic culture characteristics of the microalgae Cyanobium sp. grown in vertical tubular photobioreactor in semicontinuous mode. Cultivation was carried out in vertical tubular photobioreactor for 2 L, in 57 d, at 30 °C, 3200 Lux, and 12 h light/dark photoperiod. The maximum specific growth rate was found as 0.127 d⁻¹, when the culture had blend concentration of 1.0 g L⁻¹, renewal rate of 50%, and sodium bicarbonate concentration of 1.0 g L⁻¹. The maximum values of productivity (0.071 g L⁻¹ d⁻¹) and number of cycles (10) were observed in blend concentration of 1.0 g L⁻¹, renewal rate of 30%, and bicarbonate concentration of 1.0 g L⁻¹. The results showed the potential of semicontinuous cultivation of Cyanobium sp. in closed tubular bioreactor, combining factors such as blend concentration, renewal rate, and sodium bicarbonate concentration.     

Determination of hyaluronan molecular mass distribution in human breast milk

Hyaluronan (HA) in human milk mediates host responses to microbial infection via TLR4- and CD44-dependent signaling. Signaling by HA is generally size specific. Because pure HA with average molecular mass (M) of 35 kDa can elicit a protective response in intestinal epithelial cells, it has been proposed that human milk HA may have a bioactive low-M component. Here we report the size distribution of HA in human milk samples from 20 unique donors. A new method for HA analysis, employing ion exchange (IEX) chromatography to fractionate HA by size and specific quantification of each size fraction by competitive enzyme-linked sorbent assay (ELSA), was developed. When separated into four fractions, milk HA with M ? 20 kDa, M ∼ 20 to 60 kDa, and M ∼ 60 to 110 kDa comprised averages of 1.5, 1.4, and 2.0% of the total HA, respectively. The remaining 95% was HA with M ? 110 kDa. Electrophoretic analysis of the higher M HA from 13 samples showed nearly identical M distributions, with an average M of approximately 440 kDa. This higher M HA component in human milk is proposed to bind to CD44 and to enhance human beta defensin 2 (HBD2) induction by the low-M HA components.