Production of ethanol from wood and agricultural residues by Neurospora crassa

The feasibility of utilizing the rapidly growing tropical woods for ethanol production by Neurospora crassa has been studied. Hydrolysis of cold alkali pretreated wood gave a saccharification of 68% based on the available carbohydrate. The direct fermentation of pretreated wood (20 g l^?1) by Neurospora crassa gave quantitative conversion of available hemicellulose/cellulose to ethanol in 5 days. Increasing the substrate concentration to 50 g l^?1lowered the conversion to 40–60% yielding 12 g l^?1of ethanol. Fermentation of wood (50 g l^?1) pretreated with hot 1 m NaOH followed by neutralization with HCl gave only 6 g l^?1of ethanol.     

Purification and properties of a low molecular weight 1,4-beta-d-glucan glucohydrolase having one active site for carboxymethyl cellulose and xylan from an alkalothermophilic Thermomonospora sp

A low molecular weight 1,4-beta-D-glucan glucohydrolase from an extracellular culture filtrate of Thermomonospora sp. was purified to homogeneity. The molecular weight of the purified enzyme was 14.2 kDa by MALDI-TOF analysis and is in agreement with SDS-PAGE and gel filtration chromatography. The purified enzyme exhibited both endocarboxymethyl cellulase and endoxylanase activities. A kinetic method was employed to study the active site of the enzyme that hydrolyzes both carboxymethyl cellulose and xylan. The experimental data coincide well with the theoretical values calculated for the case of a single active site. Conformation and microenvironment at the active site was probed with fluorescent chemo-affinity labeling using o-phthalaldehyde as the chemical initiator. Formation of isoindole derivative resulted in complete inactivation of the enzyme to hydrolyze both xylan and CMC as judged by fluorescence studies corroborating a single active site for the hydrolysis of xylan and CMC.     

Design, engineering, and characterization of zinc finger nucleases

Zinc finger nuclease (ZFN)-mediated gene targeting is rapidly becoming a powerful tool for "gene editing" and "directed mutagenesis" of plant and mammalian genomes including the human genome. ZFN-mediated gene targeting provides molecular biologists with the ability to site-specifically manipulate and permanently modify plant and mammalian genomes. Facile production of ZFNs and rapid characterization of their in vitro sequence-specific cleavage properties are a pre-requisite before ZFN-mediated gene targeting can become an efficient and effective practical tool for widespread use in biotechnology. Here, we report the design, engineering, and rapid in vitro characterization of ZFNs that target specific endogenous sequences within two mouse genes (mTYR and mCFTR), and two human genes (hCCR5 and hDMPK), respectively. These engineered ZFNs recognize their respective cognate DNA sites encoded in a plasmid substrate in a sequence-specific manner and, as expected, they induce a double-strand break at the chosen target site.     

Fsr1, a striatin homologue,forms an endomembrane‐associated complex that regulates virulence in the maize pathogen Fusarium verticillioides

Fsr1, a homologue of mammalian striatin, containing multiple protein‐binding domains and a coiled‐coil (CC) domain, is critical for Fusarium verticillioides virulence. In mammals, striatin interacts with multiple proteins to form a STRIPAK (striatin‐interacting phosphatase and kinase) complex that regulates a variety of developmental processes and cellular mechanisms. In this study, we identified the homologue of a key mammalian STRIPAK component STRIP1/2 (striatin‐interacting proteins 1 and 2) in F. verticillioides, FvStp1, which interacts with Fsr1 in vivo. Gene deletion analysis indicates that FvStp1 is critical for F. verticillioides stalk rot virulence. In addition, we identified three proteins, designated FvCyp1, FvScp1 and FvSel1, which interact with the Fsr1 CC domain via a yeast two‐hybrid screen. Importantly, FvCyp1, FvScp1 and FvSel1 co‐localize to endomembrane structures, each having a preferred localization in the cell, and they are all required for F. verticillioides stalk rot virulence. Moreover, these proteins are necessary for the correct localization of Fsr1 to the endoplasmic reticulum (ER) and nuclear envelope. Thus, we identified several novel components in the STRIPAK complex that regulates F. verticillioides virulence, and propose that the correct organization and localization of Fsr1 are critical for STRIPAK complex function.     

Properties and applications of Penicillium funiculosum cellulase immobilized on a soluble polymer

Summary The cellobiase and xylanase activities of Penicillium funiculosum were immobilized on a soluble polymer poly(vinyl alcohol) (PVA). The kinetic parameters and the adsorption characteristics of the bound and free enzymes were compared. The Km value of the immobilized preparation was the same as the free enzyme. The hydrolysis of different cellulosic substrates by the bound enzyme is investigated.     

Interleukin 2 production in patients with thyroid diseases

Con A-induced production of interleukin 2 was studied in peripheral blood lymphocytes obtained from 15 normal subjects and 42 patients with different thyroid diseases. It was found that thyroid diseases are accompanied by changes in IL 2 production. The level of IL 2 production by T lymphocytes is determined by the functional status of thyroid glands, their anatomy, stages of the disease and mode of treatment.     

Parental Somatic Mosaicism Is Underrecognized and Influences Recurrence Risk of Genomic Disorders

New human mutations are thought to originate in germ cells, thus making a recurrence of the same mutation in a sibling exceedingly rare. However, increasing sensitivity of genomic technologies has anecdotally revealed mosaicism for mutations in somatic tissues of apparently healthy parents. Such somatically mosaic parents might also have germline mosaicism that can potentially cause unexpected intergenerational recurrences. Here, we show that somatic mosaicism for transmitted mutations among parents of children with simplex genetic disease is more common than currently appreciated. Using the sensitivity of individual-specific breakpoint PCR, we prospectively screened 100 families with children affected by genomic disorders due to rare deletion copy-number variants (CNVs) determined to be de novo by clinical analysis of parental DNA. Surprisingly, we identified four cases of low-level somatic mosaicism for the transmitted CNV in DNA isolated from parental blood. Integrated probabilistic modeling of gametogenesis developed in response to our observations predicts that mutations in parental blood increase recurrence risk substantially more than parental mutations confined to the germline. Moreover, despite the fact that maternally transmitted mutations are the minority of alleles, our model suggests that sexual dimorphisms in gametogenesis result in a greater proportion of somatically mosaic transmitting mothers who are thus at increased risk of recurrence. Therefore, somatic mosaicism together with sexual differences in gametogenesis might explain a considerable fraction of unexpected recurrences of X-linked recessive disease. Overall, our results underscore an important role for somatic mosaicism and mitotic replicative mutational mechanisms in transmission genetics.     

Cellular level models as tools for cytokine design

Cytokines and growth factors are critical regulators that connect intracellular and extracellular environments through binding to specific cell‐surface receptors. They regulate a wide variety of immunological, growth, and inflammatory response processes. The overall signal initiated by a population of cytokine molecules over long time periods is controlled by the subtle interplay of binding, signaling, and trafficking kinetics. Building on the work of others, we abstract a simple kinetic model that captures relevant features from cytokine systems as well as related growth factor systems. We explore a large range of potential biochemical behaviors, through systematic examination of the model's parameter space. Different rates for the same reaction topology lead to a dramatic range of biochemical network properties and outcomes. Evolution might productively explore varied and different portions of parameter space to create beneficial behaviors, and effective human therapeutic intervention might be achieved through altering network kinetic properties. Quantitative analysis of the results reveals the basis for tensions among a number of different network characteristics. For example, strong binding of cytokine to receptor can increase short‐term receptor activation and signal initiation but decrease long‐term signaling due to internalization and degradation. Further analysis reveals the role of specific biochemical processes in modulating such tensions. For instance, the kinetics of cytokine binding and receptor activation modulate whether ligand–receptor dissociation can generally occur before signal initiation or receptor internalization. Beyond analysis, the same models and model behaviors provide an important basis for the design of more potent cytokine therapeutics by providing insight into how binding kinetics affect ligand potency. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010