Cortical Neurovascular Coupling Driven by Stimulation of Channelrhodopsin-2

While functional imaging is widely used in studies of the brain, how well the hemodynamic signal represents the underlying neural activity is still unclear. And there is a debate on whether hemodynamic signal is more tightly related to synaptic activity or action potentials. This study intends to address these questions by examining neurovascular coupling driven by pyramidal cells in the motor cortex of rats. Pyramidal cells in the motor cortex of rats were selectively transduced with the light sensitive cation channel channelrhodopsin-2 (ChR2). Electrophysiological recordings and optical intrinsic signal imaging were performed simultaneously and synchronously to capture the neural activity and hemodynamics induced by optical stimulation of ChR2-expressing pyramidal cells. Our results indicate that both synaptic activity (local field potential, LFP) and action potentials (multi-unit activity, MUA) are tightly related to hemodynamic signals. While LFPs in γ band are better in predicting hemodynamic signals elicited by short stimuli, MUA has better predictions to hemodynamic signals elicited by long stimuli. Our results also indicate that strong nonlinearity exists in neurovascular coupling.     

Abnormal fatty alcohol metabolism in cultured keratinocytes from patients with Sjögren-Larsson syndrome

Sj?gren-Larsson syndrome (SLS) is an inherited neurocutaneous disorder characterized by ichthyosis, mental retardation, spasticity, and deficient activity of fatty aldehyde dehydrogenase (FALDH). FALDH is an enzyme component of fatty alcohol:NAD oxidoreductase (FAO), which is necessary for fatty alcohol metabolism. To better understand the biochemical basis for the cutaneous symptoms in this disease, we investigated lipid metabolism in cultured keratinocytes from SLS patients. Enzyme activities of FALDH and FAO in SLS cells were <10% of normal. SLS keratinocytes accumulated 45-fold more fatty alcohol (hexadecanol, octadecanol, and octadecenol) than normal, whereas wax esters and 1-O-alkyl-2,3-diacylglycerols were increased by 5.6-fold and 7.5-fold, respectively. SLS keratinocytes showed a reduced incorporation of radioactive octadecanol into fatty acid (24% of normal) and triglyceride (13% of normal), but incorporation into wax esters and 1-O-alkyl-2,3-diacylglycerol was increased by 2.5-fold and 2.8-fold, respectively. Our results indicate that FALDH deficiency in SLS keratinocytes causes the accumulation and diversion of fatty alcohol into alternative biosynthetic pathways. The striking lipid abnormalities in cultured SLS keratinocytes are distinct from those seen in fibroblasts and may be related to the stratum corneum dysfunction and ichthyosis in SLS.     

Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-break repair

Mre11 forms the core of the multifunctional Mre11-Rad50-Nbs1 (MRN) complex that detects DNA double-strand breaks (DSBs), activates the ATM checkpoint kinase, and initiates homologous recombination (HR) repair of DSBs. To define the roles of Mre11 in both DNA bridging and nucleolytic processing during initiation of DSB repair, we combined small-angle X-ray scattering (SAXS) and crystal structures of Pyrococcus furiosus Mre11 dimers bound to DNA with mutational analyses of fission yeast Mre11. The Mre11 dimer adopts a four-lobed U-shaped structure that is critical for proper MRN complex assembly and for binding and aligning DNA ends. Further, mutations blocking Mre11 endonuclease activity impair cell survival after DSB induction without compromising MRN complex assembly or Mre11-dependant recruitment of Ctp1, an HR factor, to DSBs. These results show how Mre11 dimerization and nuclease activities initiate repair of DSBs and collapsed replication forks, as well as provide a molecular foundation for understanding cancer-causing Mre11 mutations in ataxia telangiectasia-like disorder (ATLD).     

Metabolically engineered Escherichia coli for efficient production of glycosylated natural products

Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP‐4‐keto‐6‐deoxyglucose resulting in increased production of the heterologous sugars TDP‐L‐mycarose and TDP‐d ‐desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6‐deoxyerythronolide B (6‐dEB) or 3‐α‐mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60‐ and 25‐fold more erythromycin D (EryD) than the original strain K207‐3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.     

Mining marine shellfish wastes for bioactive molecules: chitin and chitosan--Part A: extraction methods

Legal restrictions, high costs and environmental problems regarding the disposal of marine processing wastes have led to amplified interest in biotechnology research concerning the identification and extraction of additional high grade, low-volume by-products produced from shellfish waste treatments. Shellfish waste consisting of crustacean exoskeletons is currently the main source of biomass for chitin production. Chitin is a polysaccharide composed of N-acetyl-D-glucosamine units and the multidimensional utilization of chitin derivatives including chitosan, a deacetylated derivative of chitin, is due to a number of characteristics including: their polyelectrolyte and cationic nature, the presence of reactive groups, high adsorption capacities, bacteriostatic and fungistatic influences, making them very versatile biomolecules. Part A of this review aims to consolidate useful information concerning the methods used to extract and characterize chitin, chitosan and glucosamine obtained through industrial, microbial and enzymatic hydrolysis of shellfish waste.     

Thrombin peptide TP508 prevents nitric oxide mediated apoptosis in chondrocytes in the endochondral developmental pathway

TP508 is a 23-amino acid peptide derived from human prothrombin that increases cartilage matrix production and reduces alkaline phosphatase activity without changing chondrocyte proliferation. This study tested the hypothesis that TP508 acts by blocking the onset of apoptosis associated with hypertrophy. Rat costochondral resting zone chondrocytes and human auricular chondrocytes were cultured in DMEM containing 50microM ascorbic acid and 10% FBS. Apoptosis was induced by treatment of confluent cultures with chelerythrine, tamoxifen, or inorganic phosphate (Pi) for 24h. One half of the cultures received TP508 (0, 0.7, or 7microg/ml). Apoptosis was assessed as a function of DNA fragmentation ([3H]-thymidine labeled DNA fragments), TUNEL staining, and cell viability using the MTT assay, as well as by assessing the Bcl-2/Bax mRNA and protein ratios and caspase-3 activity. The universal NO synthase inhibitor l-NMMA was used to assess the effect of NO production on chondrocyte apoptosis and specific NO synthase subspecies were identified using iNOS inhibitor 1400W and nNOS inhibitor vinyl-l-NIO, as well as l-NAME, which inhibits both iNOS and eNOS. Finally, we assessed if TP508 would block NO production induced by the apoptogens. Chelerythrine, tamoxifen and Pi-induced apoptosis and this was reversed by TP508. All apoptogens increased NO production and this was reduced by TP508. TP508 reduced NO levels to the same extent as 1400W but not to the same extent as l-NAME, suggesting that its effects are mediated primarily by iNOS. In addition, TP508 reduced the effect of chelerythrine to the same extent as 1400W and l-NAME, again indicating that it acts via inhibition of an iNOS pathway. TP508 also regulated Bcl-2/Bax mRNA in a time and dose-dependent manner. The Bcl-2/Bax mRNA ratio was 0.11 in the absence of TP508 at 1h and 4.95 at 7microg/ml TP508; by 3h the ratio was approximately 1 in both groups. The Bcl-2/Bax protein ratio also increased by 63% at 1h. TP508 did not affect caspase-3 activity. TP508 also caused a dose-dependent increase in protein kinase C (PKC) activity within 9min that was maximal at 270min. These results show that TP508 prevents apoptosis in growth plate chondrocytes via inhibition of iNOS-dependent NO and suggest a possible role for PKC in the mechanism.     

Invasion in space and time: non-native species richness and relative abundance respond to interannual variation in productivity and diversity

Ecologists have long sought to understand the relationships among species diversity, community productivity and invasion by non‐native species. Here, four long‐term observational datasets were analyzed using repeated measures statistics to determine how plant species richness and community resource capture (i.e. productivity) influenced invasion. Multiple factors influenced the results, including the metric used to quantify invasion, interannual variation and spatial scale. Native richness was positively correlated with non‐native richness, but was usually negatively correlated with non‐native abundance, and these patterns were stronger at the larger spatial scale. Logistic regressions indicated that the probability of invasion was reduced both within and following years with high productivity, except at the desert grassland site where high productivity was associated with increased invasion. Our analysis suggests that while non‐natives were most likely to establish in species rich communities, their success was diminished by high resource capture by the resident community.     

Accommodation of GDP-linked sugars in the active site of GDP-perosamine synthase

Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate.     

Photoinduced electron transfer and energy transfer reactions of hydroxo-(2,2′:6′,2″-terpyridine) platinum(II)

The luminescent complex [Pt(terpy)OH]BF₄ undergoes photoinduced electron transfer reactions with phenyl amine electron donors and nitrophenyl electron acceptors. Stern-Volmer analysis of the quenching of metal-to-ligand charge transfer phosphorescence (³MLCT) was used to calculate bimolecular rate constants for electron transfer. Rate constants vary from 10⁸ to >10¹⁰ M⁻¹ s⁻¹, depending on the thermodynamic driving force of the electron transfer reaction, with rate constants indicating that [Pt(terpy)OH]BF₄* is a powerful photo-oxidant. Aromatic triplet energy acceptors can also quench the ³MLCT emission.