1-Naphthol 2-hydroxylase from <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain C6: purification,characterization and chemical modification studies

1-Naphthol 2-hydroxylase (1-NH) which catalyzes the conversion of 1-naphthol to 1,2-dihydroxynaphthalene was purified to homogeneity from carbaryl-degrading Pseudomonas sp. strain C6. The enzyme was found to be a homodimer with subunit molecular weight of 66 kDa. UV, visible and fluorescence spectral properties, identification of flavin moiety by HPLC as FAD, and reconstitution of apoenzyme by FAD suggest that enzyme is FAD-dependent. 1-NH accepts electron from NADH as well as NADPH. Besides 1-naphthol (K _m, 9.1 μM), the enzyme also accepts 5-amino 1-naphthol (K _m, 6.4 μM) and 4-chloro 1-naphthol (K _m, 2.3 μM) as substrates. Enzyme showed substrate inhibition phenomenon at high concentration of 1-naphthol (K _i, 283 μM). Stoichiometric consumption of oxygen and NADH, and biochemical properties suggest that 1-NH belongs to FAD containing external flavomonooxygenase group of oxido-reductase class of enzymes. Based on biochemical and kinetic properties, 1-NH from Pseudomonas sp. strain C6 appears to be different than that reported earlier from Pseudomonas sp. strain C4. Chemical modification and protection by 1-naphthol and NADH suggest that His, Arg, Cys, Tyr and Trp are at or near the active site of 1-NH.     

Indian herb ‘Sanjeevani’ (Selaginella bryopteris) can promote growth and protect against heat shock and apoptotic activities of ultra violet and oxidative stress

Selaginella bryopteris is a lithophyte with remarkable ressurection capabilities. It is full of medicinal properties, hence also known as ‘Sanjeevani’ (one that infuses life). For lack of credible scientific evidence the plant is not in active use as a medicinal herb. We provide scientific evidence for whyS. bryopteris is known as ‘Sanjeevani’. The aqueous extract ofS. bryopteris possesses growth-promoting activity as well as protective action against stress-induced cell death in a number of experimental cell systems including mammalian cells. Treatment of the cells in culture with 10% aqueous extract enhanced cell growth by about 41% in Sf9 cells and 78% in mammalian cells. Pre-treatment of cells with the Selaginella extract (SE) (1-2x5%) protected against oxidative stress (H2O2)-induced cell death. The killing potential of ultra violet (UV) was also significantly reduced when the cells were pre-treated with SE for 1 h. Thermal radiation suppressed cell growth by about 50%. Pre-treatment of cells with SE for 1 h afforded complete protection against heat-induced growth suppression. SE may possess anti-stress and antioxidant activities that could be responsible for the observed effects. Chemical analysis shows that SE contains hexoses and proteins. Taken together,S. bryopteris extract may help in stress-induced complications including those due to heat shock.     

Properties of doublecortin expressing neurons in the adult mouse dentate gyrus

The dentate gyrus is a neurogenic zone where neurons continue to be born throughout life, mature and integrate into the local circuitry. In adults, this generation of new neurons is thought to contribute to learning and memory formation. As newborn neurons mature, they undergo a developmental sequence in which different stages of development are marked by expression of different proteins. Doublecortin (DCX) is an early marker that is expressed in immature granule cells that are beginning migration and dendritic growth but is turned off before neurons reach maturity. In the present study, we use a mouse strain in which enhanced green fluorescent protein (EGFP) is expressed under the control of the DCX promoter. We show that these neurons have high input resistances and some cells can discharge trains of action potentials. In mature granule cells, action potentials are followed by a slow afterhyperpolarization that is absent in EGFP-positive neurons. EGFP-positive neurons had a lower spine density than mature neurons and stimulation of either the medial or lateral perforant pathway activated dual component glutamatergic synapses that had both AMPA and NMDA receptors. NMDA receptors present at these synapses had slow kinetics and were blocked by ifenprodil, indicative of high GluN2B subunit content. These results show that EGFP-positive neurons in the DCX-EGFP mice are functionally immature both in their firing properties and excitatory synapses.     

Shear deformation kinematics during cartilage articulation: effect of lubrication, degeneration, and stress relaxation

During joint articulation, the biomechanical behavior of cartilage not only facilitates load-bearing and low-friction, but also provides regulatory cues to chondrocytes. Elucidation of cartilage kinematics under combined compression and shearing conditions clarifies these cues in health and disease. The objectives of this study were to elucidate the effects of lubricant, tissue degeneration, and stress relaxation duration on cartilage shear kinematics during articulation. Human osteochondral cores with normal and mildly degenerate surface structures were isolated. Paired blocks from each core were apposed, compressed, allowed to stress relax for 5 or 60 min, and shear tested with a micro-scale video microscopy system using phosphate-buffered saline (PBS) or synovial fluid as lubricant. During applied lateral motion, local and overall shear strain (Exz) of articular cartilage were determined. The applied lateral displacement at which Exz reached 50% of the peak (Deltax(1/2)) was also determined. Quantitatively, surface Exz increased at the onset of lateral motion and peaked just as surfaces detached and slid. With continued lateral motion, surface Exz was maintained. After short stress relaxation, effects of lubrication on Exz and Deltax(1/2) were not apparent. With prolonged stress relaxation, Exz and Deltax(1/2) near the articular surface increased markedly when PBS was used as lubricant. Similar patterns were observed for overall Exz and Deltax(1/2). With degeneration, surface Exz was consistently higher for all cases after the onset of lateral motion. Thus, cartilage shear kinematics is markedly affected by lubricant, cartilage degeneration, and loading duration. Changes in these factors may be involved in the pathogenesis of osteoarthritis.     

A Genetic Analysis of the Functional Interactions within Mycobacterium tuberculosis Single-Stranded DNA Binding Protein

Single-stranded DNA binding proteins (SSBs) are vital in all organisms. SSBs of Escherichia coli (EcoSSB) and Mycobacterium tuberculosis (MtuSSB) are homotetrameric. The N-terminal domains (NTD) of these SSBs (responsible for their tetramerization and DNA binding) are structurally well defined. However, their C-terminal domains (CTD) possess undefined structures. EcoSSB NTD consists of β1-β1′-β2-β3-α-β4-β45₁-β45₂-β5 secondary structure elements. MtuSSB NTD includes an additional β-strand (β6) forming a novel hook-like structure. Recently, we observed that MtuSSB complemented an E. coli Δssb strain. However, a chimeric SSB (mβ4-β5), wherein only the terminal part of NTD (β4-β5 region possessing L₄₅ loop) of EcoSSB was substituted with that from MtuSSB, failed to function in E. coli in spite of its normal DNA binding and oligomerization properties. Here, we designed new chimeras by transplanting selected regions of MtuSSB into EcoSSB to understand the functional significance of the various secondary structure elements within SSB. All chimeric SSBs formed homotetramers and showed normal DNA binding. The mβ4-β6 construct obtained by substitution of the region downstream of β5 in mβ4-β5 SSB with the corresponding region (β6) of MtuSSB complemented the E. coli strain indicating a functional interaction between the L₄₅ loop and the β6 strand of MtuSSB.     

Isotope ratio determination in boron analysis

Traditionally, boron (B) isotope ratios have been determined using thermal ionization mass spectrometry (TIMS) and, to some extent, secondary ion mass spectrometry (SIMS). Both TIMS and SIMS use a high-resolution mass analyzer, but differ in analyte ionization methods. TIMS uses electrons from a hot filament, whereas SIMS employs an energetic primary ion beam of Ga+, Cs+, or O- for analyte ionization. TIMS can be used in negative or positive ion modes with high sensitivity and precision of B isotope ratio determination. However, isobaric interferences may be a problem, if the sample is not well purified and/or memory of the previous sample is not removed. Time-consuming sample preparation, analyte (B) purification, and sample determination processes limit the applications of TIMS for routine analyses. SIMS can determine B and its isotope ratio in intact solid samples without destroying them, but has poorer resolution and sensitivity than TIMS, and is difficult to standardize for biological samples. Development of plasma-source mass spectrometry (MS) enabled the determination of B concentration and isotope ratio without requiring sample purification. Commonly used plasma-source MS uses an Ar inductively coupled plasma (ICP) as an ionization device interfaced to a low-resolution quadrupole mass analyzer. The quadrupole ICP-MS is less precise than TIMS and SIMS, but is a popular method for B isotope ratio determination because of its speed and convenience. B determination by ICP-MS suffers no spectroscopic interferences. However, sample matrices, memory effects, and some instrument parameters may affect the accuracy and precision of B isotope ratio determination if adequate precautions are not taken. New generations of plasma-source MS instruments using high-resolution mass analyzers provide better sensitivity and precision than the currently used quadrupole ICP-MS. Because of the convenience and high sample throughput, the high-resolution ICP-MS is expected to be the method of choice for B isotope ratio determination. The current state of instrumental capabilities is adequate for B isotope determination. However, precision and accuracy are primarily limited by sample preparation, introduction, and analytical methodology, including 1. Analyte loss and isotope fractionation during sample preparation. 2. The precision of B isotope determination in small samples, especially those containing low concentrations. 3. Difficult matrices. 4. Memory effects. Sample preparation by alkali fusion allows rapid and complete decomposition of hard-to-digest samples, but high-salt environments of the fused materials require extensive sample purification for B ratio determination. The alternative wet-ashing sample decomposition with HF also results in B loss and isotopic fractionation owing to the high volatility of BF3. Open-vessel dry- or wet-ashing methods usually do not work well for animal samples, and are also prone to B loss and contamination. Closed-vessel microwave digestion overcomes these problems, but the digests of biological materials have high C contents, which cause spectral interference on 11B and affect 11B/10B ratios. Exchange separation/preconcentration of B using exchange (cation or anion exchange, B-specific resin, e.g., Amberlite IRA-743) tend to cause B isotope fractionation, and C eluting from these resin columns may interfere with B isotope ratio determination. Memory effects of B that occur during sample determination may cause serious errors in B isotope ratio determination, especially when samples varying in B concentrations and/or isotope composition are analyzed together. Although the utilization of high-resolution plasma-source MS will undoubtedly improve analytical precision, it is the sample preparation, sample introduction, and analytical methodology that represent the primary limitation to accurate and precise B isotope ratio determination.     

Regulation of immature cartilage growth by IGF-I,TGF-<Emphasis Type="Italic">β</Emphasis>1, BMP-7, and PDGF-AB: role of metabolic balance between fixed charge and collagen network

Cartilage growth may involve alterations in the balance between the swelling tendency of proteoglycans and the restraining function of the collagen network. Growth factors, including IGF-I, TGF-beta1, BMP-7, and PDGF-AB, regulate chondrocyte metabolism and, consequently, may regulate cartilage growth. Immature bovine articular cartilage explants from the superficial and middle zones were incubated for 13 days in basal medium or medium supplemented with serum, IGF-I, TGF-beta1, BMP-7, or PDGF-AB. Variations in tissue size, accumulation of proteoglycan and collagen, and tensile properties were assessed. The inclusion of serum, IGF-I, or BMP-7 resulted in expansive tissue growth, stimulation of proteoglycan deposition but not of collagen, and a diminution of tensile integrity. The regulation of cartilage metabolism by TGF-beta1 resulted in tissue homeostasis, with maintenance of size, composition, and function. Incubation in basal medium or with PDGF-AB resulted in small volumetric and compositional changes, but a marked decrease in tensile integrity. These results demonstrate that the phenotype of cartilage growth, and the associated balance between proteoglycan content and integrity of the collagen network, is regulated differentially by certain growth factors.     

One-Carbon Metabolic Pathway Rewiring in Escherichia coli Reveals an Evolutionary Advantage of 10-Formyltetrahydrofolate Synthetase (Fhs) in Survival under Hypoxia

In cells, N¹⁰-formyltetrahydrofolate (N¹⁰-fTHF) is required for formylation of eubacterial/organellar initiator tRNA and purine nucleotide biosynthesis. Biosynthesis of N¹⁰-fTHF is catalyzed by 5,10-methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) and/or 10-formyltetrahydrofolate synthetase (Fhs). All eubacteria possess FolD, but some possess both FolD and Fhs. However, the reasons for possessing Fhs in addition to FolD have remained unclear. We used Escherichia coli, which naturally lacks fhs, as our model. We show that in E. coli, the essential function of folD could be replaced by Clostridium perfringensfhs when it was provided on a medium-copy-number plasmid or integrated as a single-copy gene in the chromosome. The fhs-supported folD deletion (ΔfolD) strains grow well in a complex medium. However, these strains require purines and glycine as supplements for growth in M9 minimal medium. The in vivo levels of N¹⁰-fTHF in the ΔfolD strain (supported by plasmid-borne fhs) were limiting despite the high capacity of the available Fhs to synthesize N¹⁰-fTHF in vitro. Auxotrophy for purines could be alleviated by supplementing formate to the medium, and that for glycine was alleviated by engineering THF import into the cells. The ΔfolD strain (harboring fhs on the chromosome) showed a high NADP⁺-to-NADPH ratio and hypersensitivity to trimethoprim. The presence of fhs in E. coli was disadvantageous for its aerobic growth. However, under hypoxia, E. coli strains harboring fhs outcompeted those lacking it. The computational analysis revealed a predominant natural occurrence of fhs in anaerobic and facultative anaerobic bacteria.