Cloning and expression of the gene encoding a novel proteinase from Tritirachium album limber

We have isolated the genomic and cDNA clones encoding a novel proteinase from the fungus Tritirachium album Limber, named proteinase T, synthesis of which is induced in skim milk medium. The coding sequence for this enzyme is interrupted by two introns in the fungal genome. The amino acid sequence of proteinase T as deduced from the nucleotide sequence is about 53% identical to that of proteinase K. Four cysteines are present in the mature proteinase, probably in the form of two disulfide bonds, which might explain the thermal stability of the proteinase. We have expressed the proT cDNA in Escherichia coli. The authenticity of the product has been characterized by Western blotting and N-terminal analysis of the recombinant product.     

Metabolic engineering of sugarcane to accumulate energy‐dense triacylglycerols in vegetative biomass

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy‐rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon‐partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co‐expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1‐2 (DGAT1‐2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP‐glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95‐ or 43‐fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5‐ to 9.5‐fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.     

On Generalized Log-Logistic Model for Censored Survival Data

In the analysis of survival data with parametric models, it is well known that the Weibull model is not suitable for modeling cases where the hazard rate is non-monotonic. For such cases, log-logistic model is frequently used. However, due to the symmetric property of the log-logistic model, it may be poor for the cases where the hazard rate is skewed or heavily tailed. In this paper, we suggest a generalization of the log-logistic model by introducing a shape parameter. This generalized model is then applied to fit the lung cancer data of Prentice (1973). The results seem to improve over those obtained by using the log-logistic model.     

Improved Generalized Ratio-Cum-Product Estimation

In this paper, a generalized ratio-cum-product estimator for estimating the ratio (product) of two population means using auxiliary information on two other variables is given of which the estimators by SINGH (1969) and SHAH and SHAH (1978) are particular cases. The estimator is regeneralized when the covariance between two auxiliary variables is known.     

Primary care for AIDS and chemical dependence.

Primary care clinicians are acquiring an increasingly important role in preventing, diagnosing, and treating both chemical dependence and human immunodeficiency virus (HIV) illness. Towards this end they need to know the epidemiology of HIV infection in chemically dependent persons and methods of educating persons at high risk for these problems. It is critical that physicians screen for alcohol and drug addiction. Health care providers should understand the risks and benefits of HIV antibody testing and include in their practices the basic components of counseling before and after testing and informed consent. Both HIV illness and addiction are chronic diseases with long-term health implications. A knowledge of patient characteristics, intensity of treatments, and treatment modalities is important in making recommendations for individualized therapy. Combining service delivery is a future challenge necessitated by today''s joint epidemics of the acquired immunodeficiency syndrome and chemical dependence.     

The continuous positive and negative dielectrophoresis of microorganisms

The continuous dielectrophoresis of living cells is described. The technique uses stream-centered transport of suspended microorganisms through an especially shaped non-uniform electric field. The cells can be given a positive or negative displacement, i.e., can be pushed into or out of the region of higher field intensity, depending upon the frequency of the applied ac field, and upon the relative permittivities of the cells and the suspending medium. Yeast (Saccharomyces cerevisiae) and algal cells (Chlorella vulgaris) were found to provide spectra of dielectrophoretic responses varying with the applied frequency (10 to 600 kHz) and conductivity.     

A Product-Type Estimator in Double Sampling

The use of ratio and product estimators, using auxiliary information, for estimating the mean of a finite population is well known. The efficiency of ratio estimator or product estimator is high depending on whether the auxiliary character is highly positively or negatively coorelated with the main character of interest. This paper proposes a product-type estimator which is more efficient than the usual ratio and product estimators in practical situations. We consider the case of double sampling from which the single sampling results may easily be derived.     

Escape of intracellular Shigella from autophagy requires binding to cholesterol through the type III effector,IcsB

Type III secretion systems are present in many pathogenic bacteria and mediate the translocation of bacterial effectors into host cells. Identification of host targets of these effectors is crucial for understanding bacterial virulence. IcsB, a type III secretion effector, helps Shigella to evade the host autophagy defense system by binding to the autophagy protein, Atg5. Here, we show that IcsB is able to interact specifically with cholesterol. The cholesterol binding domain (CBD) of IcsB is located between residues 288 and 351. Specific mutations of single tyrosine residues Y297 or Y340 of IcsB by phenylalanine (F) slightly reduced cholesterol binding, whereas deletion of the entire CBD or double mutation Y297F-Y340F strongly abolished interactions with cholesterol. To determine whether Shigella expressing IcsB variants could evade autophagy as effectively as the wild-type Shigella, we infected MDAMC cells stably expressing the autophagy marker LC3 fused to GFP and bacterial autophagosome formation was quantified using fluorescence microscopy. Mutation Y297F or Y340F slightly impaired IcsB function, whereas complete removal of CBD or mutation Y297F-Y340F significantly impaired autophagy evasion. Furthermore, we report that BopA, the counterpart of IcsB in Burkholderia pseudomallei with similar autophagy-evading properties, contains the CBD domain and is also able to bind cholesterol.