Improved technique for electrophoresis of human galactose-1-P uridyl transferase (EC 2.7.7.12)

Summary A newly developed electrophoretic technique for human galactose-1-phosphate uridyl transferase confirms the multiple band patterns for the Duarte and Los Angeles variants. This represents the first confirmation for the Los Angeles variant. The observed frequencies of N, D, and LA types are similar to earlier reports for these variants.     

Halothane binding proteome in human brain cortex

Inhaled anesthetics bind specifically to a wide variety of proteins in the brain. This set of proteins must include those that contribute to the physiological and behavioral phenotypes of anesthesia and the related side effects. To identify the anesthetic-binding targets and functional pathways associated with these targets in human brain, halothane photolabeling and two-dimensional (2D) gel electrophoresis were used. Both membrane and soluble proteins from human temporal cortex were prepared. More than 300 membrane and 400 soluble protein spots were detected on the stained blots, of which 23 membrane and 34 soluble proteins were labeled by halothane and identified by mass spectroscopy. Their functional classification reveals five groups, including carbohydrate metabolism, protein folding, oxidative phosphorylation, nucleoside triphosphatase, and dimer/kinase activity with different correlative stringency. When network analysis of the interaction between these protein molecules is used, the weighted interaction accentuates the cellular protein components important in cell growth and proliferation, cell cycle and cell death, and cell-cell signaling and interactions, although no pathway was specific. This study provides evidence for multiple anesthetic binding targets and suggests potential pathways involved in their actions.     

Protein kinase C-dependent phosphorylation and mitochondrial translocation of aldose reductase

Although aldose reductase (AR) is a critical participant in osmoregulation, and the metabolism of glucose and aldehydes derived from lipid peroxidation, post-translational mechanisms regulating its activity have not been identified. In this paper, we report that stimulation of protein kinase C (PKC) in several cell types induces phosphorylation of AR and translocation of the phosphorylated protein to the mitochondria. In vitro, recombinant AR was directly phosphorylated by activated PKC, suggesting that AR may be an in vivo PKC substrate. Together, these observations reveal a novel link between PKC activation and the regulation of glucose and aldehyde metabolism.     

Effect of lecithin and MgCO3 as additives on the enzymatic activity of carbonic anhydrase encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres

A model enzyme, carbonic anhydrase, was encapsulated and released from poly(lactide-co-glycolide) (PLGA) microspheres (1-3 microm) made by a novel phase inversion technique. Lecithin was used as a surfactant in the encapsulation process and was incorporated in either the organic phase, aqueous phase, both phases, or not at all. Additional microspheres were also made with lecithin incorporated in the aqueous phase and a basic salt, MgCO3, in the polymeric phase. Released carbonic anhydrase, protein extracted from microspheres, or enzyme incubated with lecithin and PLGA were analyzed via HPLC and activity assay to determine the effect of these additives on protein integrity and activity. Lecithin in the aqueous phase appeared to increase the fraction of enzyme in monomeric form as well as its activity for both extracted protein and released protein as compared to the other formulations without MgCO3. Incubation of enzyme with PLGA degradation products indicated that the acidic environment within the microspheres aids in the irreversible inactivation of the encapsulated protein. Addition of MgCO3 further increased the amount of monomer in both the extracted and released protein by decreasing the amount of acid-induced cleavage and noncovalent aggregation, but still greatly decreased the activity of the enzyme.     

Nitric oxide synthase inhibition in human sperm affects sperm-oocyte fusion but not zona pellucida binding

There is recent evidence that mouse and human spermatozoa contain constitutive nitric oxide synthase (cNOS) and can synthesize nitric oxide. The aim of this study was to investigate whether the inhibition of human sperm cNOS could affect sperm-oocyte fusion and sperm binding to the zona pellucida (ZP). N(G)-nitro-L-arginine methyl ester (L-NAME) was used as cNOS inhibitor. Sperm-oocyte fusion was evaluated using the hamster egg penetration test (HEPT). The ZP binding was evaluated using the hemizona assay. L-NAME added from the onset of capacitation strongly inhibited sperm-oocyte fusion. This inhibitory effect was dose dependent, stereospecific, and suppressed by L-arginine in a dose-dependent manner. L-NAME also inhibited sperm-oocyte fusion in the HEPT enhanced with progesterone (P), where P (5 microM) was added for 15 min to capacitated sperm. A lesser but significant inhibition was also observed when sperm suspensions were exposed to L-NAME following capacitation in both versions of HEPT. On the contrary, L-NAME did not affect ZP binding. In conclusion, the present study provides the evidence that cNOS plays a role in the human sperm's capacity to fuse with oocyte but not in the ZP binding.     

Mitochondrial Profiling of Acute Myeloid Leukemia in the Assessment of Response to Apoptosis Modulating Drugs

BH3 profiling measures the propensity of transformed cells to undergo intrinsic apoptosis and is determined by exposing cells to BH3-mimicking peptides. We hypothesized that basal levels of prosurvival BCL-2 family proteins may modulate the predictive power of BH3 profiling and termed it mitochondrial profiling. We investigated the correlation between cell sensitivity to apoptogenic agents and mitochondrial profiling, using a panel of acute myeloid leukemias induced to undergo apoptosis by exposure to cytarabine, the BH3 mimetic ABT-199, the MDM2 inhibitor Nutlin-3a, or the CRM1 inhibitor KPT-330. We found that the apoptogenic efficacies of ABT-199 and cytarabine correlated well with BH3 profiling reflecting BCL2, but not BCL-XL or MCL-1 dependence. Baseline BCL-2 protein expression analysis increased the ability of BH3 profiling to predict resistance mediated by MCL-1. By utilizing engineered cells with overexpression or knockdown of BCL-2 family proteins, Ara-C was found to be independent, while ABT-199 was dependent on BCL-XL. BCL-2 and BCL-XL overexpression mediated resistance to KPT-330 which was not reflected in the BH3 profiling assay, or in baseline BCL-2 protein levels. In conclusion, mitochondrial profiling, the combination of BH3 profiling and prosurvival BCL-2 family protein analysis, represents an improved approach to predict efficacy of diverse agents in AML and may have utility in the design of more effective drug combinations.     

Unresolved molecular phylogenies of gibbons and siamangs (Family: Hylobatidae) based on mitochondrial, Y-linked, and X-linked loci indicate a rapid Miocene radiation or sudden vicariance event

According to recent taxonomic reclassification, the primate family Hylobatidae contains four genera (Hoolock, Nomascus, Symphalangus, and Hylobates) and between 14 and 18 species, making it by far the most species-rich group of extant hominoids. Known as the "small apes", these small arboreal primates are distributed throughout Southeast, South and East Asia. Considerable uncertainty surrounds the phylogeny of extant hylobatids, particularly the relationships among the genera and the species within the Hylobates genus. In this paper we use parsimony, likelihood, and Bayesian methods to analyze a dataset containing nearly 14 kilobase pairs, which includes newly collected sequences from X-linked, Y-linked, and mitochondrial loci together with data from previous mitochondrial studies. Parsimony, likelihood, and Bayesian analyses largely failed to find a significant difference among phylogenies with any of the four genera as the most basal taxon. All analyses, however, support a tree with Hylobates and Symphalangus as most closely related genera. One strongly supported phylogenetic result within the Hylobates genus is that Hylobates pileatus is the most basal taxon. Multiple analyses failed to find significant support for any singular genus-level phylogeny. While it is natural to suspect that there might not be sufficient data for phylogenetic resolution (whenever that situation occurs), an alternative hypothesis relating to the nature of gibbon speciation exists. This lack of resolution may be the result of a rapid radiation or a sudden vicariance event of the hylobatid genera, and it is likely that a similarly rapid radiation occurred within the Hylobates genus. Additional molecular and paleontological evidence are necessary to better test among these, and other, hypotheses of hylobatid evolution.