A common mutation is associated with a mild, potentially asymptomatic phenotype in patients with isovaleric acidemia diagnosed by newborn screening

Isovaleric acidemia (IVA) is an inborn error of leucine metabolism that can cause significant morbidity and mortality. Since the implementation, in many states and countries, of newborn screening (NBS) by tandem mass spectrometry, IVA can now be diagnosed presymptomatically. Molecular genetic analysis of the IVD gene for 19 subjects whose condition was detected through NBS led to the identification of one recurring mutation, 932C-->T (A282V), in 47% of mutant alleles. Surprisingly, family studies identified six healthy older siblings with identical genotype and biochemical evidence of IVA. Our findings indicate the frequent occurrence of a novel mild and potentially asymptomatic phenotype of IVA. This has significant consequences for patient management and counseling.     

Two distinct modes of ESCRT-III recognition are required for VPS4 functions in lysosomal protein targeting and HIV-1 budding

The ESCRT pathway mediates membrane remodeling during enveloped virus budding, cytokinesis, and intralumenal endosomal vesicle formation. Late in the pathway, a subset of membrane-associated ESCRT-III proteins display terminal amphipathic "MIM1" helices that bind and recruit VPS4 ATPases via their MIT domains. We now report that VPS4 MIT domains also bind a second, "MIM2" motif found in a different subset of ESCRT-III subunits. The solution structure of the VPS4 MIT-CHMP6 MIM2 complex revealed that MIM2 elements bind in extended conformations along the groove between the first and third helices of the MIT domain. Mutations that block VPS4 MIT-MIM2 interactions inhibit VPS4 recruitment, lysosomal protein targeting, and HIV-1 budding. MIT-MIM2 interactions appear to be common throughout the ESCRT pathway and possibly elsewhere, and we suggest how these interactions could contribute to a mechanism in which VPS4 and ESCRT-III proteins function together to constrict the necks of budding vesicles.     

Direct Detection of the Acetate-forming Activity of the Enzyme Acetate Kinase

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily^1-5, is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya⁶. The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila^7-14. An acetate kinase which can only utilize PP_i but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus^15,16.In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann^17-20, a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD⁺ by the enzymes pyruvate kinase and lactate dehydrogenase^21,22, or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine²³. Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP⁺ to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase²⁴.Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PP_i.     

大麦初生叶中质外体的蛋白、酯酶和苹果酸脱氢酶同工酶(英文)

把6天株龄的大麦(Hordeum vylgare L.)初生叶的下表皮剥去后,以含pH6.5的200mmol/L NaCl缓冲液真空渗洗叶片36min,获得的细胞间隙洗液含有水溶性或弱离子结合态的质外体蛋白质和酶。渗洗过的叶片用缓冲液磨成匀浆和离心后,上清液含原质体蛋白质和酶。用1mmol/L NaCl可溶解离子结合态的质外体蛋白质和酶,两条(25和31kD)和7条(22,28,30,51,55,60和71kD)蛋白质带只分别在含有200mmol/L和1mmol/L NaGl的质外体提取液中测得。机械创伤诱导两条(32和33kD)可溶于200mmol/L NaCl的质外体蛋白带,在质外体还测到3条可溶于200mmol/L NaCl和4条可溶于1mmol/L NaCl的苹果酸脱氢酶同工酶。在质外体和共质体两部分均发现有酯酶Ⅰ同工酶,但移向阴极的酯酶同工酶Ⅰ只见于溶在200mmol/L NaCl的质外体中。移向阳极的酯酶Ⅱ同工酶仅见于共质体中。     

Evaluation of Methods for Reestablishment of L-Cell Suspension Cultures Directly from Liquid Nitrogen Stored Stocks

Methods were developed and evaluated for the preservation of tissue cells grown in suspension culture and the reestablishment of suspension cultures directly from inoculum stored at -175 C. The factors investigated were processing pH, temperature of processing, freezing medium, and method of inoculation of the starter suspension cultures from the frozen stock (-175 C). Three parameters, cell viability, cell size, and growth potential in suspension culture after freezing, were used to evaluate the various factors. The results indicate that cells processed at 4 C, frozen at 1 C per min to -50 C in a medium containing 5% dimethyl sulfoxide plus 10% bovine serum at concentrations of 2 x 10(7) to 4 x 10(7) cells/ml, and stored at -175 C will reestablish suspension cultures directly from frozen seed. A 1-ml amount of frozen stock inoculated into 99 ml of medium routinely produced 2 x 10(6) to 3 x 10(6) viable cells/ml (2 x 10(8) to 3 x 10(8) total cells) in suspension culture in 4 to 5 days. Inoculum preserved by this procedure grew equally well in either serum-free or serum-containing growth medium.     

Control of eIF4E cellular localization by eIF4E-binding proteins, 4E-BPs

Eukaryotic initiation factor (eIF) 4E, the mRNA 5'-cap-binding protein, mediates the association of eIF4F with the mRNA 5'-cap structure to stimulate cap-dependent translation initiation in the cytoplasm. The assembly of eIF4E into the eIF4F complex is negatively regulated through a family of repressor proteins, called the eIF4E-binding proteins (4E-BPs). eIF4E is also present in the nucleus, where it is thought to stimulate nuclear-cytoplasmic transport of certain mRNAs. eIF4E is transported to the nucleus via its interaction with 4E-T (4E-transporter), but it is unclear how it is retained in the nucleus. Here we show that a sizable fraction (approximately 30%) of 4E-BP1 is localized to the nucleus, where it binds eIF4E. In mouse embryo fibroblasts (MEFs) subjected to serum starvation and/or rapamycin treatment, nuclear 4E-BPs sequester eIF4E in the nucleus. A dramatic loss of nuclear 4E-BP1 occurs in c-Ha-Ras-expressing MEFs, which fail to show starvation-induced nuclear accumulation of eIF4E. Therefore, 4E-BP1 is a regulator of eIF4E cellular localization.     

The production of pig preimplantation embryos in vitro: Current progress and future prospects

Human assisted reproductive technology procedures are routinely performed in clinics globally, and some of these approaches are now common in other mammals such as cattle. This is currently not the case in pigs. Given that the global population is expected to increase by over two billion people between now and 2050, the demand for meat will also undoubtedly increase. With this in mind, a more sustainable way to produce livestock; increasing productivity and implementing methods that will lead to faster genetic selection, is imperative. The establishment of routine and production scale pig embryo in vitro production could be a solution to this problem. Producers would be able to increase the overall number of offspring born, animal transportation would be more straightforward and in vitro produced embryos could be produced from the gametes of selected elite. Here we review the most recent developments in pig embryology, outline the current barriers and key challenges that exist, and outline research priorities to surmount these difficulties.