Evidence that Griscelli syndrome with neurological involvement is caused by mutations in RAB27A,not MYO5A

Griscelli syndrome (GS), a rare autosomal recessive disorder, is characterized by partial albinism, along with immunologic abnormalities or severe neurological impairment or both. Mutations in one of two different genes on chromosome 15q can cause the different subtypes of GS. Most patients with GS display the hemophagocytic syndrome and have mutations in RAB27A, which codes for a small GTPase. Two patients with neurological involvement have mutations in MYO5A, which codes for an actin-based molecular motor. The RAB27A and MYO5A gene products interact with each other and function in vesicle trafficking. We report the molecular basis of GS in a Muslim Arab kindred whose members have extremely variable neurological involvement, along with the hemophagocytic syndrome and immunologic abnormalities. The patients have normal MYO5A genes but exhibit a homozygous 67.5-kb deletion that eliminates RAB27A mRNA and immunocytofluorescence-detectable protein. We also describe the molecular organization of RAB27A and a multiplex polymerase chain reaction assay for the founder deletion in this kindred. Finally, we propose that all patients with GS have RAB27A mutations and immunologic abnormalities that sometimes result in secondary neurological involvement. The two patients described elsewhere who have MYO5A mutations and neurological complications but no immunologic defects may not have GS but instead may have Elejalde syndrome, a condition characterized by mild hypopigmentation and severe, primary neurological abnormalities.     

Arsenic trioxide and paclitaxel induce apoptosis by different mechanisms

Arsenic trioxide (ATO) and paclitaxel (TAXOL) are effective in the treatment of various types of cancers. Both drugs induce G2/M arrest. We have previously shown that ATO is a potent inducer of apoptosis in myeloma cells expressing mutant p53 engaging both the intrinsic and extrinsic apoptotic pathways. Here we compared the effect of ATO and TAXOL on myeloma cells expressing mutant p53 and varying levels of Bcl-2. ATO rapidly induced Apo2/TRAIL, activation of caspase 8, cleavage of BID, depolarization of mitochondrial membrane (MM) and release of AIF from mitochondria in a Bcl-2 independent fashion. Apoptosis was associated with early formation of ring-like perinuclear condensed chromatin colocalized with AIF. In contrast, paclitaxel-induced apoptosis MM depolarization, cytochrome C release and activation of caspase 9 were all blocked by Bcl-2. Apoptosis was associated with a random chromatin condensation and nuclear fragmentation with no early involvement of AIF.     

Triglycerides in the Human Kidney Cortex: Relationship with Body Size

Obesity is associated with increased risk for kidney disease and uric acid nephrolithiasis, but the pathophysiological mechanisms underpinning these associations are incompletely understood. Animal experiments have suggested that renal lipid accumulation and lipotoxicity may play a role, but whether lipid accumulation occurs in humans with increasing body mass index (BMI) is unknown. The association between obesity and abnormal triglyceride accumulation in non-adipose tissues (steatosis) has been described in the liver, heart, skeletal muscle and pancreas, but not in the human kidney. We used a quantitative biochemical assay to quantify triglyceride in normal kidney cortex samples from 54 patients undergoing nephrectomy for localized renal cell carcinoma. In subsets of the study population we evaluated the localization of lipid droplets by Oil Red O staining and measured 16 common ceramide species by mass spectrometry. There was a positive correlation between kidney cortex trigyceride content and BMI (Spearman R = 0.27, P = 0.04). Lipid droplets detectable by optical microscopy had a sporadic distribution but were generally more prevalent in individuals with higher BMI, with predominant localization in proximal tubule cells and to a lesser extent in glomeruli. Total ceramide content was inversely correlated with triglycerides. We postulate that obesity is associated with abnormal triglyceride accumulation (steatosis) in the human kidney. In turn, steatosis and lipotoxicity may contribute to the pathogenesis of obesity-associated kidney disease and nephrolithiasis.     

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-¹³C_Fru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO₃]⁻/or [H₂PO₄]⁻ ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure ¹³C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments.Plant cell walls are the most abundant renewable resources (Pauly and Keegstra, 2008a). Much of the current biotechnological research on plant cell wall synthesis involves manipulating these biosynthetic processes to obtain higher concentrations of starches or oil, which show much promise in biofuel production, or to alter cell wall composition for easier breakdown. A detailed knowledge of these processes is essential to understanding and utilizing plant cell wall materials as well as for progress in understanding plant growth and structural development (Pauly and Keegstra, 2008b). However, research into cell wall biosynthesis has been hindered by our limited understanding of the metabolic processes that produce cell walls and particularly their regulation. Progress in this area is limited by the difficulty of differentiating among the compounds involved and of analyzing the fluxes through the biochemical network of wall biosynthesis. Many of the metabolic steps involve isomeric sugars, including hexose-Ps and nucleotide-sugars (NDP-sugars) that serve as direct precursors to plant cell wall biosynthesis. Separate quantification of these sugars has been difficult to achieve.Much of the current research on identifying and differentiating among different metabolic pathways involves the use of chromatography and mass spectrometry (MS; Wolfender et al., 2009). It has been found that liquid chromatography (LC), when linked to a triple quadrupole mass spectrometer (tandem MS [MS/MS]), can be a powerful tool to detect and specifically quantify several classes of metabolic compounds (Allwood and Goodacre, 2010). After initial compound separation by LC, analytes are directed to a triple quadrupole mass spectrometer (MS/MS), where the initial two quadrupoles separate the compounds for detection in the third quadrupole, first by selection of particular mass-to-charge (m/z) ratios of the ionized parent compounds in the first quadrupole, then by fragmentation of the compounds in the second quadrupole (Arrivault et al., 2009). This coupling method of LC-MS/MS to identify compounds has been used with several metabolites involved in plant primary metabolism recently (Cruz et al., 2008; Arrivault et al., 2009).Several studies have reported the separation of phosphorylated metabolites using LC-MS/MS. We are particularly interested in analyzing such compounds because plant cell wall biosynthesis involves a range of phosphorylated precursors, mainly NDP-sugars and hexose-Ps (Feingold and Barber, 1990; Fry 2000, 2004; Seifert, 2004; Somerville et al., 2004; Sharples and Fry, 2007). For example, Huck et al. (2003) and Luo et al. (2007) were able to separate, respectively, six and 28 intracellular metabolites involved in glycolysis, the oxidative pentose-P pathway, and the tricarboxylic acid cycle, some of which are phosphorylated. Bajad et al. (2006) were able to separate a large number of water-soluble cellular metabolites by hydrophilic interaction chromatography, but this method does not appear to separate isomers. Anion exchange chromatography was shown to be effective at separating phosphorylated intermediates involved in glycolysis (van Dam et al., 2002) and in the Calvin cycle (Cruz et al., 2008; Arrivault et al., 2009), especially when coupled with the high specificity and sensitivity of triple quadrupole MS. Moreover, anion-exchange chromatography coupled with MS/MS can be used to determine the mass isotopomer distribution of labeled compounds and Kiefer et al. (2007) were able to quantify isotope abundances in six phosphorylated metabolites in Escherichia coli.However, some of the methods that achieved good separation of four NDP-sugars did not allow quantification by MS/MS because of the eluents used (Räbinä et al., 2001), and none of the methods using coupled LC-MS/MS developed to date separates all or nearly all of the hexose-Ps and NDP-sugars known to be involved in plant cell wall biosynthesis (Turnock and Ferguson, 2007). This presents a special challenge given the fact that many of these sugar compounds are diastereoisomers and ionize similarly in traditional LC-MS/MS methods. Current methods of separating hexose-Ps and NDP-sugars also involve multiple steps of chromatographic and enzymatic separation. In a notable recent study, Sharples and Fry (2007) separated many of the compounds involved in plant cell wall biosynthesis, including hexose-Ps and NDP-sugars, and used radioactive [U-¹⁴C]Fru and [1-³H]Gal as substrates to determine their relative contributions to different cell wall components. The method used in that study involved high-voltage paper electrophoresis separation followed by mild acid hydrolysis and/or phosphatase digestion of different fractions to release neutral hexoses that were then separated by a second paper chromatography procedure. At the cost of considerable effort, this approach allowed eight compounds to be separated. However, neither this nor many of the other approaches used to date appear to have yielded absolute metabolite levels or specific activities in labeling. Metabolic flux analysis requires quantifying these compounds, their fractional and preferably also positional labeling, and the ability to analyze many time point samples. These requirements necessitated the development of a method that can be performed in medium to high throughput and achieves compound separation and quantitation, such as LC-MS/MS, and that also yields detailed labeling information.In this study we have developed and validated a robust and sensitive LC-MS/MS method that successfully allows us to separate and quantify the levels and isotopic labeling of plant cell wall precursors. Using plant tissues from fenugreek (Trigonella foenum-graecum) endosperms and Arabidopsis (Arabidopsis thaliana) cell cultures, 12 hexose-Ps and NDP-sugars known to be involved in plant cell wall biosynthesis were separated and quantified. The direct analysis of intracellular cell wall precursors and their isotopic labeling significantly expands the set of tools for assessing the dynamics and regulation of cell wall biosynthesis, including the potential for dynamic metabolic flux analysis.     

Herpes simplex virus type 1 preferentially targets human colon carcinoma: role of extracellular matrix

Viral therapy of cancer (viral oncolysis) is dependent on selective destruction of the tumor tissue compared with healthy tissues. Several factors, including receptor expression, extracellular components, and intracellular mechanisms, may influence viral oncolysis. In the present work, we studied the potential oncolytic activity of herpes simplex virus type 1 (HSV-1), using an organ culture system derived from colon carcinoma and healthy colon tissues of mouse and human origin. HSV-1 infected normal colons ex vivo at a very low efficiency, in contrast to high-efficiency infection of colon carcinoma tissue. In contrast, adenoviral and lentiviral vectors infected both tissues equally well. To investigate the mechanisms underlying the preferential affinity of HSV-1 for the carcinoma tissue, intracellular and extracellular factors were investigated. Two extracellular components, collagen and mucin molecules, were found to restrict HSV-1 infectivity in the healthy colon. The mucin layer of the healthy colon binds to HSV-1 and thereby blocks viral interaction with the epithelial cells of the tissue. In contrast, colon carcinomas express small amounts of collagen and mucin molecules and are thus permissive to HSV-1 infection. In agreement with the ex vivo system, HSV-1 injected into a mouse colon carcinoma in vivo significantly reduced the volume of the tumor. In conclusion, we describe a novel mechanism of viral selectivity for malignant tissues that is based on variance of the extracellular matrix between tumor and healthy tissues. These insights may facilitate new approaches to the application of HSV-1 as an oncolytic virus.     

The tempo and mode of barnacle evolution

Previous phylogenetic attempts at resolving barnacle evolutionary relationships are few and have relied on limited taxon sampling. Here we combine DNA sequences from three nuclear genes (18S, 28S and H3) and 44 morphological characters collected from 76 thoracican (ingroup) and 15 rhizocephalan (outgroup) species representing almost all the Thoracica families to assess the tempo and mode of barnacle evolution. Using phylogenetic methods of maximum parsimony, maximum likelihood, and Bayesian inference and 14 fossil calibrations, we found that: (1) Iblomorpha form a monophyletic group; (2) pedunculated barnacles without shell plates (Heteralepadomorpha) are not ancestral, but have evolved, at least twice, from plated forms; (3) the ontogenetic pattern with 5-->6-->8-->12+ plates does not reflect Thoracica shell evolution; (4) the traditional asymmetric barnacles (Verrucidae) and the Balanomorpha are each monophyletic and together they form a monophyletic group; (5) asymmetry and loss of a peduncle have evolved twice in the Thoracica, resulting in neither the Verrucomorpha nor the Sessilia forming monophyletic groups in their present definitions; (6) the Scalpellomorpha are not monophyletic; (7) the Thoracica suborders evolved since the Early Carboniferous (340mya) with the final radiation of the Sessilia in the Upper Jurassic (147mya). These results, therefore, reject many of the underlying hypotheses about character evolution in the Cirripedia Thoracica, stimulate a variety of new thoughts on thoracican radiation, and suggest the need for a major rearrangement in thoracican classification based on estimated phylogenetic relationships.     

Common Adaptive Strategies Underlie Within-Host Evolution of Bacterial Pathogens

Within-host adaptation is a hallmark of chronic bacterial infections, involving substantial genomic changes. Recent large-scale genomic data from prolonged infections allow the examination of adaptive strategies employed by different pathogens and open the door to investigate whether they converge toward similar strategies. Here, we compiled extensive data of whole-genome sequences of bacterial isolates belonging to miscellaneous species sampled at sequential time points during clinical infections. Analysis of these data revealed that different species share some common adaptive strategies, achieved by mutating various genes. Although the same genes were often mutated in several strains within a species, different genes related to the same pathway, structure, or function were changed in other species utilizing the same adaptive strategy (e.g., mutating flagellar genes). Strategies exploited by various bacterial species were often predicted to be driven by the host immune system, a powerful selective pressure that is not species specific. Remarkably, we find adaptive strategies identified previously within single species to be ubiquitous. Two striking examples are shifts from siderophore-based to heme-based iron scavenging (previously shown for Pseudomonas aeruginosa) and changes in glycerol-phosphate metabolism (previously shown to decrease sensitivity to antibiotics in Mycobacterium tuberculosis). Virulence factors were often adaptively affected in different species, indicating shifts from acute to chronic virulence and virulence attenuation during infection. Our study presents a global view on common within-host adaptive strategies employed by different bacterial species and provides a rich resource for further studying these processes.     

Mitochondrial metabolism in developing embryos of Brassica napus

The metabolism of developing plant seeds is directed toward transforming primary assimilatory products (sugars and amino acids) into seed storage compounds. To understand the role of mitochondria in this metabolism, metabolic fluxes were determined in developing embryos of Brassica napus. After labeling with [1,2-(13)C2]glucose + [U-(13)C6]glucose, [U-(13)C3]alanine, [U-(13)C5]glutamine, [(15)N]alanine, (amino)-[(15)N]glutamine, or (amide)-[(15)N]glutamine, the resulting labeling patterns in protein amino acids and in fatty acids were analyzed by gas chromatography-mass spectrometry. Fluxes through mitochondrial metabolism were quantified using a steady state flux model. Labeling information from experiments using different labeled substrates was essential for model validation and reliable flux estimation. The resulting flux map shows that mitochondrial metabolism in these developing seeds is very different from that in either heterotrophic or autotrophic plant tissues or in most other organisms: (i) flux around the tricarboxylic acid cycle is absent and the small fluxes through oxidative reactions in the mitochondrion can generate (via oxidative phosphorylation) at most 22% of the ATP needed for biosynthesis; (ii) isocitrate dehydrogenase is reversible in vivo; (iii) about 40% of mitochondrial pyruvate is produced by malic enzyme rather than being imported from the cytosol; (iv) mitochondrial flux is largely devoted to providing precursors for cytosolic fatty acid elongation; and (v) the uptake of amino acids rather than anaplerosis via PEP carboxylase determines carbon flow into storage proteins.