Slc25a13-knockout mice harbor metabolic deficits but fail to display hallmarks of adult-onset type II citrullinemia

Adult-onset type II citrullinemia (CTLN2) is an autosomal recessive disease caused by mutations in SLC25A13, the gene encoding the mitochondrial aspartate/glutamate carrier citrin. The absence of citrin leads to a liver-specific, quantitative decrease of argininosuccinate synthetase (ASS), causing hyperammonemia and citrullinemia. To investigate the physiological role of citrin and the development of CTLN2, an Slc25a13-knockout (also known as Ctrn-deficient) mouse model was created. The resulting Ctrn-/- mice were devoid of Slc25a13 mRNA and citrin protein. Liver mitochondrial assays revealed markedly decreased activities in aspartate transport and the malate-aspartate shuttle. Liver perfusion also demonstrated deficits in ureogenesis from ammonia, gluconeogenesis from lactate, and an increase in the lactate-to-pyruvate ratio within hepatocytes. Surprisingly, Ctrn-/- mice up to 1 year of age failed to show CTLN2-like symptoms due to normal hepatic ASS activity. Serological measures of glucose, amino acid, and ammonia metabolism also showed no significant alterations. Nitrogen-loading treatments produced only minor changes in the hepatic ammonia and amino acid levels. These results suggest that citrin deficiency alone may not be sufficient to produce a CTLN2-like phenotype in mice. These observations are compatible, however, with the variable age of onset, incomplete penetrance, and strong ethnic bias seen in CTLN2 where additional environmental and/or genetic triggers are now suspected.     

Sustained diacylglycerol accumulation resulting from prolonged G protein-coupled receptor agonist-induced phosphoinositide breakdown in hepatocytes

Studies in various cells have led to the idea that agonist-stimulated diacylglycerol (DAG) generation results from an early, transient phospholipase C (PLC)-catalyzed phosphoinositide breakdown, while a more sustained elevation of DAG originates from phosphatidylcholine (PC). We have examined this issue further, using cultured rat hepatocytes, and report here that various G protein-coupled receptor (GPCR) agonists, including vasopressin (VP), angiotensin II (Ang.II), prostaglandin F2alpha, and norepinephrine (NE), may give rise to a prolonged phosphoinositide hydrolysis. Preincubation of hepatocytes with 1-butanol to prevent conversion of phosphatidic acid (PA) did not affect the agonist-induced DAG accumulation, suggesting that phospholipase D-mediated breakdown of PC was not involved. In contrast, the GPCR agonists induced phosphoinositide turnover, assessed by accumulation of inositol phosphates, that was sustained for up to 18 h, even under conditions where PLC was partially desensitized. Pretreatment of hepatocytes with wortmannin, to inhibit synthesis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate (PIP2), prevented agonist-induced inositol phosphate and DAG accumulation. Upon VP stimulation the level of PIP) declined, but only transiently, while increases in inositol 1,4,5-trisphosphate (InsP3) and DAG mass were sustained, suggesting that efficient resynthesis of PIP2 allowed sustained PLC activity. This was confirmed when cells were pretreated with wortmannin to prevent resynthesis of PIP2. Furthermore, metabolism of InsP3 was rapid, compared to that of DAG, with a more than 20-fold difference in half-life. Thus, rapid metabolism of InsP3 and efficient resynthesis of PIP2 may account for the larger amount of DAG generated and the more sustained time course, compared to InsP3. The results suggest that DAG accumulation that is sustained for many hours in response to VP, Ang.II, NE, and prostaglandin F2alpha in hepatocytes is mainly due to phosphoinositide breakdown.     

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.     

Structural and functional specificities of PDGF-C and PDGF-D, the novel members of the platelet-derived growth factors family

The platelet-derived growth factor (PDGF) family was for more than 25 years assumed to consist of only PDGF-A and -B. The discovery of the novel family members PDGF-C and PDGF-D triggered a search for novel activities and complementary fine tuning between the members of this family of growth factors. Since the expansion of the PDGF family, more than 60 publications on the novel PDGF-C and PDGF-D have been presented, highlighting similarities and differences to the classical PDGFs. In this paper we review the published data on the PDGF family covering structural (gene and protein) similarities and differences among all four family members, with special focus on PDGF-C and PDGF-D expression and functions. Little information on the protein structures of PDGF-C and -D is currently available, but the PDGF-C protein may be structurally more similar to VEGF-A than to PDGF-B. PDGF-C contributes to normal development of the heart, ear, central nervous system (CNS), and kidney, while PDGF-D is active in the development of the kidney, eye and brain. In adults, PDGF-C is active in the kidney and the central nervous system. PDGF-D also plays a role in the lung and in periodontal mineralization. PDGF-C is expressed in Ewing family sarcoma and PDGF-D is linked to lung, prostate and ovarian cancers. Both PDGF-C and -D play a role in progressive renal disease, glioblastoma/medulloblastoma and fibrosis in several organs.     

Platelet-derived growth factor (PDGF)-C,a PDGF family member with a vascular endothelial growth factor-like structure

Platelet-derived growth factor (PDGF)-C is a novel member of the PDGF family that binds to PDGF alphaalpha and alphabeta receptors. The growth factor domain of PDGF-C (GFD-PDGF-C) was expressed in high yields in Escherichia coli and was purified and refolded from inclusion bodies obtaining a biologically active growth factor with dimeric structure. The GFD-PDGF-C contains 12 cysteine residues, and Ellman assay analysis indicates that it contains three intramonomeric disulfide bonds, which is in accordance with GFD-PDGF-C being a member of the cystine knot superfamily of growth factors. The recombinant GFD-PDGF-C was characterized by CD, fluorescence, NMR, and infrared spectroscopy. Together, our data indicate that GFD-PDGF-C is a highly thermostable protein that contains mostly beta-sheet secondary structure and some (6%) alpha-helix structure. The structural model of PDGF-C, obtained by homology-based molecular modeling using the structural representatives of this family of growth factors, shows that GFD-PDGF-C has a higher structural homology to the vascular endothelial growth factor than to PDGF-B. The modeled structure can give further insights into the function and specificity of this molecule.     

Paracrine Interactions within the Pancreatic Islet Determine the Glycemic Set Point

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Control Strategy for Small Molecule Impurities in Antibody-Drug Conjugates

Antibody-drug conjugates (ADCs) are an emerging class of biopharmaceuticals. As such, there are no specific guidelines addressing impurity limits and qualification requirements. The current ICH guidelines on impurities, Q3A (Impurities in New Drug Substances), Q3B (Impurities in New Drug Products), and Q6B (Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products) do not adequately address how to assess small molecule impurities in ADCs. The International Consortium for Innovation and Quality in Pharmaceutical Development (IQ) formed an impurities working group (IWG) to discuss this issue. This white paper presents a strategy for evaluating the impact of small molecule impurities in ADCs. This strategy suggests a science-based approach that can be applied to the design of control systems for ADC therapeutics. The key principles that form the basis for this strategy include the significant difference in molecular weights between small molecule impurities and the ADC, the conjugation potential of the small molecule impurities, and the typical dosing concentrations and dosing schedule. The result is that exposure to small impurities in ADCs is so low as to often pose little or no significant safety risk.     

Genome sizes and phylogenetic relationships suggest recent divergence of closely related species of the <Emphasis Type="Italic">Limonium vulgare</Emphasis> complex (Plumbaginaceae)

Limonium vulgare and related species form a complex group, but until now cytological and genetic studies have been based on single species and specific geographical areas. We investigated genome size, karyological and genetic diversity in samples from Western Mediterranean and evaluated the phylogenetic relationships among the species of this complex. Genome size was assessed using flow cytometry on samples from natural populations of L. vulgare, L. maritimum and L. narbonense. Chromosome counts were conducted in plants obtained from seeds collected in the field. The internal transcribed spacer ITS1 of the nuclear rDNAs was used to assess ITS polymorphisms as well as the phylogenetic relationships within the L. vulgare complex. Our analyses showed that all species were tetraploid, with the chromosome number of L. maritimum being presented here for the first time. Significant differences were observed in genome size, with L. narbonense having lower genome sizes than the other two species, and possible aneuploids being detected. Ten new ITS sequences from L. vulgare, L. narbonense and L. maritimum were provided. Most species’ populations showed unique ribotypes, and L. narbonense has the highest ribotype diversity. One of the L. maritimum populations presented a closer genetic relationship with L. vulgare, whereas the other two seemed to be more related with L. narbonense. Phylogenetic analyses confirmed that L. vulgare and L. narbonense form a monophyletic group, sister to the remaining Limonium species. Our results put into evidence that the studied species may represent a relatively early stage of divergence.     

Cell cycle studies in chorionic villi

Summary We have studied the cell cycle of cells obtained from chorionic villi in direct and culture preparations by incorporation of the thymidine analogue BrdU to produce latelabelling or sister chromatid differentiation patterns. We have, therefore, been able to estimate the duration of the cell cycle and, more specifically, the length of some of its phases. While results for chorionic villus sample cells in culture resembled those obtained for fibroblasts, data for the spontaneously dividing trophoblastic cells in direct preparations were different. Villi exposed to BrdU immediately after sampling showed a slight delay in the incorporation of the analogue and a lower percentage of labelled cells compared to villi treated after an overnight incubation, probably due to a temporary effect of the sampling technique. Results from semi-direct protocols suggest that cells have a G2 of no more than 4h, and a mid-S phase of 10–16h. The G1 period is very variable. After 48 h incubation with BrdU, only 4% of cells reach their second generation, whereas this percentage increases up to 70% after 72h, indicating that under these experimental conditions most cells have a cell cycle of approximately 36 h. The average number of sister chromatid exchanges was similar in both direct preparations and cultures: 5.2±2.1 SCE per cell.     

Elevated expression of ISY1, APOA-1, SYNE1, MTG1, and MMP10 at HCC initiation: HCC specific protein network involving interactions of key regulators of lipid metabolism,EGFR signaling,MAPK, and splicing pathways

Protoplasma - Identification of molecular regulators of hepatocellular carcinoma (HCC) initiation and progression is not well understood. We chemically induced HCC in male Wistar rats by...