Regulation of Metabolism of 25-Hydroxycholecalciferol by Kidney Tissue <Emphasis Type="Italic">in vitro</Emphasis> by Dietary Calcium

IT is now recognized that hydroxylated metabolites of vitamin D (that is, cholecalciferol) function as effectors of the physiological actions originally attributed to the unaltered vitamin¹. The activation of vitamin D by specific hydroxylation reactions and sequestration of the resultant metabolites by target tissues represents a hormonal control loop which is feed-back sensitive. 25-Hydroxycholecalciferol (25-HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) have been shown to be participants in the control loop, vitamin D being first metabolized in the liver to 25-HCC² which in turn is hydroxylated in the C-1 position to 1,25-DHCC in the kidney^3,4. The metabolically active form in the intestine appears to be 1,25-DHCC^5,6.     

Plasmonic Au-MoO<Subscript>3</Subscript> Colloidal Nanoparticles by Reduction of HAuCl<Subscript>4</Subscript> by Blue MoO<Subscript>x</Subscript> Nanosheets and Observation of the Gasochromic Property

Defective colloids of blue MoO_x nanosheets were prepared by anodizing exfoliation method in water. This colloidal solution exhibits an optical plasmonic absorption band in the infrared region at about 760 nm. Merely mixing HAuCl₄ solution with the MoO_x leads to loss of the blue color, decaying of 760 nm plasmonic peak and simultaneous formation of the gold plasmon absorption peak at 550–570 nm. Some spectral variations in gold plasmonic peak and MoO_x optical band gap were observed for Mo:Au ratio of 10:1, 20:1, 30:1, and 40:1. The size of the gold nanoparticles was in the 5–6 nm range with fcc crystalline structure. X-ray photoelectron spectroscopy (XPS) revealed that the initial solution contains Mo⁵⁺ states and hydroxyl groups, which after reduction, hydroxyl groups are eliminated and the Mo⁵⁺ states converted to Mo⁶⁺. The obtained Au-MoO₃ colloids have a gasochromic property in which they are colored back to blue in the presence of hydrogen gas and the molybdenum oxide absorption peak recovered again. Furthermore, it was observed that both gold and Mo oxide plasmonic peaks redshift by insertion of hydrogen gas which is attributed to change in solution refractive index and formation of defect concentration.     

Suppression TGF-&beta; Signaling by Phospholipase D

MDA-MB-231 human breast cancer cells have a survival signal generated by phospholipase D (PLD) that involves the activation of mTOR and MAP kinase. TGF-&beta; signals that block cell cycle progression in G₁ are suppressed in MDA-MB-231 cells. We report here that the elevated PLD activity in MDA-MB-231 cells suppresses TGF-&beta; signaling. Suppression of PLD activity or PLD expression resulted in increased phosphorylation of Smad2 and Smad3 on Ser 465/467 – sites on Smads that get phosphorylated by the TGF-&beta; receptor and positively regulate TGF-&beta; signaling. The effect of PLD suppression on Smad2/3 phosphorylation was dependent on the presence of TGF-&beta;. Suppression of PLD also suppressed phosphorylation of Smad2 on Ser 245/250/255 – sites that are phosphorylated by MAP kinase and negatively regulate TGF-&beta; signaling. Suppression of PLD also led to increased expression of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p27Kip1, the expression of which is stimulated in response to TGF-&beta;. Consistent with the elevated expression of CDK inhibitors, suppression of PLD also suppressed phosphorylation of the CDK substrate pRb. Similar effects were also seen in PANC-1 human pancreatic cancer cells. The data presented here indicate that the suppressed TGF-&beta; signaling in MDA-MB-231 and perhaps many other human cancer cells is due to elevated PLD activity and mediated by mTOR and MAP kinase. These results indicate that the survival signals generated by PLD involve the suppression TGF-&beta; signals that promote G₁ arrest.     

Binding of IgA by <Emphasis Type="Italic">Mycoplasma penetrans</Emphasis>

The current study shows that Mycoplasma penetrans strain GTU binds human serum immunoglobulin A (IgA) and secretory IgA but not IgG. Binding of IgA was associated almost exclusively with the lipoprotein fraction obtained by Triton X-114 fractionation of isolated M. penetrans membranes. Western immunoblot analysis of isolated membranes of M. penetrans strain GTU and of the Triton X-114 fraction showed that the major IgA-binding component was a lipoprotein with a molecular mass of 38 kDa, one of the major lipoproteins of this organism. The authors suggest that the high IgA-binding capacity of M. penetrans strain GTU may serve as a defense mechanism, conferring on this microorganism the ability to evade clearance mediated by specific IgA antibodies.     

Microhabitat use by wild-ranging Cabrera voles <Emphasis Type="Italic">Microtus cabrerae</Emphasis> as revealed by live trapping

The conservation problems affecting the endangered rodent Microtus cabrerae are mainly related to habitat loss and fragmentation. In the present study, we examined the presence/absence of the species in a group of potentially suitable habitat patches, and then evaluated the microhabitat attributes of locations used by wild-ranging individuals within occupied patches. Our results revealed that shallow flooding and abundant grass cover appear to be important factors in determining the presence among localities, but high rush cover strongly determined the presence and habitat use by voles both among and within localities. The preservation of these microhabitat characteristics may be required to effectively ensure the occurrence and stability of M. cabrerae colonies.     

‘Osmotic pump‘ feeding by coreids

Species of Coreidae (Heteroptera) cause ‘water soaked’ lesions in their food plants. Such insects typically feed from parenchyma in and surrounding vascular tissues and also cause acropetal wilting and necrosis of small diameter shoots. Feeding byMictis profana (Fabr.) in South Australia on the shoots ofAcacia iteaphylla F. Muell. ex Benth. was found to cause a local, concurrent increase in both water content and free amino acid concentration, consistent with phloem unloading. Coreids, unlike other groups of phytophagous Heteroptera, secrete a salivary sucrase (α-D-glucohydrolase, EC 3.2.1.48) as probably the sole salivary carbohydrase, and tissues attacked byM. profana showed more sucrose hydrolysing activity than unattacked. The salivary enzyme is postulated to cause unloading of solutes into the apoplast due to the osmotic effects of conversion of endogenous sucrose to glucose and fructose, allowing the insect to suck the leaked contents of many cells from a single locus. The term ‘osmotic pump feeding’ is proposed for such a process. In demonstrations of its feasibility, infiltration of shoots with mixtures of glucose and fructose stoichiometrically equivalent to isosmotic sucrose increased the amounts of tissue sap and amino acid that could be sucked from the tissues; similarly, invertase and 1 M sugars forced into the extracellular space of stem sections increased the amino acids offloaded into the bathing solutions.     

Enhancement of antibody production by the addition of Coenzyme-Q<Subscript>10</Subscript>

Recently, there has been a growing demand for therapeutic monoclonal antibodies (MAbs) on the global market. Because therapeutic MAbs are more expensive than low-molecular-weight drugs, there have been strong demands to lower their production costs. Therefore, efficient methods to minimize the cost of goods are currently active areas of research. We have screened several enhancers of specific MAb production rate (SPR) using a YB2/0 cell line and found that coenzyme-Q₁₀ (CoQ₁₀) is a promising enhancer candidate. CoQ₁₀ is well known as a strong antioxidant in the respiratory chain and is used for healthcare and other applications. Because CoQ₁₀ is negligibly water soluble, most studies are limited by low concentrations. We added CoQ₁₀ to a culture medium as dispersed nanoparticles at several concentrations (Q-Media) and conducted a fed-batch culture. Although the Q-Media had no effect on cumulative viable cell density, it enhanced SPR by 29%. In addition, the Q-Media had no effect on the binding or cytotoxic activity of MAbs. Q-Media also enhanced SPR with CHO and NS0 cell lines by 30%. These observations suggest that CoQ₁₀ serves as a powerful aid in the production of MAbs by enhancing SPR without changing the characteristics of cell growth, or adversely affecting the quality or biological activity of MAbs.     

Metabolism of dimethylsulphoniopropionate by Ruegeria pomeroyi DSS‐3

Ruegeria pomeroyi DSS‐3 possesses two general pathways for metabolism of dimethylsulphoniopropionate (DMSP), an osmolyte of algae and abundant carbon source for marine bacteria. In the DMSP cleavage pathway, acrylate is transformed into acryloyl‐CoA by propionate‐CoA ligase (SPO2934) and other unidentified acyl‐CoA ligases. Acryloyl‐CoA is then reduced to propionyl‐CoA by AcuI or SPO1914. Acryloyl‐CoA is also rapidly hydrated to 3‐hydroxypropionyl‐CoA by acryloyl‐CoA hydratase (SPO0147). A SPO1914 mutant was unable to grow on acrylate as the sole carbon source, supporting its role in this pathway. Similarly, growth on methylmercaptopropionate, the first intermediate of the DMSP demethylation pathway, was severely inhibited by a mutation in the gene encoding crotonyl‐CoA carboxylase/reductase, demonstrating that acetate produced by this pathway was metabolized by the ethylmalonyl‐CoA pathway. Amino acids and nucleosides from cells grown on ¹³C‐enriched DMSP possessed labelling patterns that were consistent with carbon from DMSP being metabolized by both the ethylmalonyl‐CoA and acrylate pathways as well as a role for pyruvate dehydrogenase. This latter conclusion was supported by the phenotype of a pdh mutant, which grew poorly on electron‐rich substrates. Additionally, label from [¹³C‐methyl] DMSP only appeared in carbons derived from methyl‐tetrahydrofolate, and there was no evidence for a serine cycle of C‐1 assimilation.     

Transformation of <Emphasis Type="Italic">Lyophyllum decastes</Emphasis> by particle bombardment

The basidiomycete Lyophyllum decastes was transformed by means of particle bombardment. We isolated five transformants under twelve conditions differing in the two parameters of target distance and helium pressure. The transformation frequency was one transformant/μg DNA. In the transformants, plasmid DNAs were integrated into the genomic DNA and stably maintained. This is the first report on transformation of L. decastes by particle bombardment.     

Degradation of sulfide by dehaloperoxidase-hemoglobin from <Emphasis Type="Italic">Amphitrite ornata</Emphasis>

Dehaloperoxidase-hemoglobin (DHP) is a unique multifunctional enzyme with a globin fold. The enzyme serves as the respiratory hemoglobin for the marine worm Amphitrite ornata and has been shown to catalyze the conversion of highly toxic trihalophenols to dihaloquinones as a detoxification function for the organism. Given the simplicity of the structure of A. ornata, it is entirely possible that DHP may play an even more general role in detoxification of the organism from sulfide commonly found in the coastal estuaries where A. ornata thrives. Comparison of DHP with other sulfide-binding hemoglobins shows that DHP possesses several distal cavity structural properties, such as an aromatic cage and a hydrogen-bond-donor amino acid (His55), that facilitate sulfide binding. Furthermore, a complete reduction of the ferric heme occurs after sulfide exposure under aerobic or anaerobic conditions to yield either the oxy or the deoxy ferrous states of DHP, respectively. Oxidation of sulfide by the heme leads to sulfur products that are less toxic to A. ornata. This proposed new function for DHP relies on the highly flexible distal His55 for deprotonation of the bound hydrogen sulfide, similar to H₂O₂ activation of the peroxidase function, and provides further support for the importance of the flexibility of the distal His55 in this novel globin.     

Enhancement of tyrosine hydroxylase expression by <Emphasis Type="Italic">Cordyceps militaris</Emphasis>

Cordyceps militaris is a popular medicinal mushroom, and has received extensive attention for medical application because of its various physiological activities. However, there is limited information about the function of Cordyceps militaris on dopaminergic system. This study has attempted to evaluate the effect of cultured fruiting bodies of Cordyceps militaris extract (CME) on the expression of the tyrosine hydroxylase (TH) gene in PC12 cells and rat brain and stomach. Related mRNA levels were determined by the RT-PCR. Protein levels were measured by Western blot and immunohistochemistry. Our results demonstrated CME induced TH gene expression both in vitro and in vivo. Treatment of 10 μg/ml and 20 mg/kg CME to PC12 cells and rat cells yielded significant increases of TH protein levels. Significantly, TH immunoreactive neurons were detected not only in the brain but also in the stomach. TH-immunohistochemical staining was markedly enhanced in animals treated with CME compared to those in the untreated control. These results suggest that CME can upregulate the dopaminergic (DArgic) system, and may contribute to neuroprotection in neurodegenerative diseases.     

Effects of prior decomposition of <Emphasis Type="Italic">Camellia japonica</Emphasis> leaf litter by an endophytic fungus on the subsequent decomposition by fungal colonizers

Effects of prior decomposition of Camellia japonica leaf litter by an endophytic phyllosphere fungus Coccomyces sp. on the subsequent decomposition of the litter by Coccomyces sp. and two succeeding fungi Dermateaceae sp. and Xylaria sp. (anamorph) were examined in a pureculture decomposition test. The prior decomposition of litter by Coccomyces sp. stimulated the subsequent decomposition by the three fungi. Dermateaceae sp. caused negligible weight loss on litter previously partly decomposed by Coccomyces sp. and then by Dermateaceae sp. and on litter decomposed singly by Dermateaceae sp. Xylaria sp. (anamorph) caused greater weight loss in these litters than control, uninoculated litter.     

Suppression of disease in tomato infected by <Emphasis Type="Italic">Pythium ultimum</Emphasis> with a biosurfactant produced by <Emphasis Type="Italic">Pseudomonas koreensis</Emphasis>

The use of biosurfactants is a promising alternative in biological control of zoospore-producing oomycetes, which are a major plant pathogen world-wide in a wide variety of crops. Oomycetes are of particular concern in closed hydroponic cultivation systems. The present study investigated the efficacy of a biosurfactant produced by Pseudomonas koreensis and added as a crude extract against the oomycete Pythium ultimum in hydroponic tomato cultivation. A significant reduction in disease was observed. Biosurfactant addition did not affect the indigenous root microflora when evaluated as sole carbon source utilisation. Chemical analysis, using electrospray hybrid mass spectrometry (ESI-MSMS), of the biosurfactant indicated it to be lokisin, a cyclic lipopeptide. These results confirm that biosurfactants are important in developing sustainable biological control strategies for oomycetes.     

Biosorption of heavy metals by the exopolysaccharide produced by <Emphasis Type="Italic">Paenibacillus jamilae</Emphasis>

The biosorption of several toxic heavy metals (Pb, Cd, Co, Ni, Zn and Cu) by the exopolysaccharide (EPS) produced by Paenibacillus jamilae, a potential biosorbent for metal remediation and recovery was studied. Firstly, the biochemical composition of this bacterial polymer was determined. Glucose was the most abundant neutral sugar, followed by galactose, rhamnose, fucose and mannose. The polymer presented a high content of uronic acids (28.29%), which may serve as binding sites for divalent cations. The presence of carboxylic groups was also detected by infrared spectroscopy. The EPS presented an interesting affinity for Pb in comparison with the other five metals. Lead biosorption (303.03 mg g⁻¹) was tenfold higher (in terms of mg of metal adsorbed per gram of EPS) than the biosorption of the rest of metals. Biosorption kinetics, the effect of pH and the effect of competitive biosorption were determined. Finally, we found that the EPS was able to precipitate Fe(III), but the EPS-metal precipitate did not form with Fe(II), Pb(II), Cd(II), Co(II), Ni(II), Cu(II) and Zn(II).     

Chromosome phylogeny of <Emphasis Type="Italic">Zamia</Emphasis> and <Emphasis Type="Italic">Ceratozamia</Emphasis> by means of Robertsonian changes detected by fluorescence <Emphasis Type="Italic">in situ</Emphasis> hybridization (FISH) technique of rDNA

 Appearance and location of 45S rDNA and 5S rDNA signals were compared in chromosomes of nine species of the aneuploid Zamia and their taxonomically and phylogenetically closely related Ceratozamia mexicana. The 45S rDNA signal was detected in the proximal region of six chromosomes in Zamia angustifolia, Z. integrifolia, Z. pumila and Z. pygmaea (all 2n=16); in the proximal region of 6–14 chromosomes in Z. furfuracea, Z. loddigesii, Z. skinneri and Z. vazquezii (all 2n=18); and on the proximal region of 20 chromosomes in Z. muricata (2n=23). The 5S rDNA signals were commonly seen near the terminal region of the short arm of two metacentric chromosomes in the four species with 2n=16 and Z. furfuracea, Z. loddigesii and Z. vazquezii with 2n=18. Other 5S rDNA signals were seen near the terminal region of two terminal-centromeric chromosomes in Z. skinneri and near the terminal region of a metacentric and a telocentric chromosomes in Z. muricata. In contrast, those with 45S and 5S rDNA signals were exhibited in chromosomes of Ceratozamia mexicana in a different manner from those in the nine species of Zamia; the 45S rDNA signal in the terminal region of four metacentric and two submetacentric chromosomes and the 5S rDNA signal near the proximal region of two metacentric chromosomes. Received November 1, 1999 Accepted January 10, 2001     

Adsorption of turbid materials by the cyanobacterium <Emphasis Type="Italic">Phormidium parchydematicum</Emphasis>

The present study investigated the adsorption of turbid materials such as clays, by microalgae. Among six tested microalgae, including Chlorophyceae and Cyanophyceae, a cyanobacterium, Phormidium parchydematicum strain KCTC 10851BP, and unicellular alga, Chlorella vulgaris strain UTEX 265, showed a higher turbidity-removal efficiency (TRE) of 99% and 93%, respectively, for clay-containing water after 24 h, which was much higher than the 36% for the control. The TREs of all the treatments were >95% after 24 h, except for the treatment with a lower algal density and optical density (OD) = 0.1. Phormidium parchydematicum demonstrated a slightly higher TRE than a polyaluminum chloride coagulant (Al₁₃(OH)₂₈Cl₉SO₄) for a turbid field water. Scanning electron microscope (SEM) observations revealed a dense adsorption of clay particles to the surface of P. parchydematicum. Thus, it would appear that P. parchydematicum and C. vulgaris can be used for clay removal in turbid water by sedimentation through microalgae–clay flocculation.     

Comparison of CO<Subscript>2</Subscript> and H<Subscript>2</Subscript>O fluxes over grassland vegetations measured by the eddy-covariance technique and by open system chamber

Measurements of CO₂ and H₂O fluxes were carried out using two different techniques—eddy-covariance (EC) and open system gas exchange chamber (OC)—during two-years’ period (2003–2004) at three different grassland sites. OC measurements were made during fourteen measurement campaigns. We found good agreement between the OC and EC CO₂ flux values (n = 63, r ² = 0.5323, OC F_CO2 = −0.6408+0.9508 EC F_CO2). The OC F_H2O values were consistently lower than those measured by the EC technique, probably caused by the air stream difference inside and outside the chamber. Adjusting flow rate within the chamber to the natural conditions would be necessary in future OC measurements. In comparison with EC, the OC proved to be a good tool for gas exchange measurements in grassland ecosystems.     

Measuring “Unmeasurable” Folding Kinetics of Proteins by Single-Molecule Force Spectroscopy

Folding and unfolding are fundamental biological processes in cell and are important for the biological functions of proteins. Characterizing the folding and unfolding kinetics of proteins is important for understanding the energetic landscape leading to the active native conformations of these molecules. However, the thermal or chemical-induced unfolding of many proteins is irreversible in vitro, precluding characterization of the folding kinetics of such proteins, just as it is impossible to “un-boil” an egg. Irreversible unfolding often manifests as irreversible aggregation of unfolded polypeptide chains, which typically occurs between denatured protein molecules in response to the exposure of hydrophobic residues to solvent. An example of such a protein where thermal denaturation results in irreversible aggregation is the β-1,4 endoxylanase from Bacillus circulans (BCX). Here, we report the use of single-molecule atomic force microscopy to directly measure the folding kinetics of BCX in vitro. By mechanically unfolding BCX, we essentially allowed only one unfolded molecule to exist in solution at a given time, effectively eliminating the possibility for aggregation. We found that BCX can readily refold back to the native state, allowing us to measure its folding kinetics for the first time. Our results demonstrate that single-molecule force-spectroscopy-based methods can adequately tackle the challenge of “un-boiling eggs”, providing a general methodology to characterize the folding kinetics of many proteins that suffer from irreversible denaturation and thus cannot be characterized using traditional equilibrium methodologies.