Evaluation of greyhound susceptibility to malignant hyperthermia using halothane-succinylcholine anesthesia and caffeine-halothane muscle contractures.

We investigated Greyhounds because of prior reports of malignant hyperthermia (MH) episodes and because Greyhounds may express high genetic relatedness due to inbreeding for generations. Seven Greyhound and six mongrel dogs were given halothane and succinylcholine anesthesia as a challenge to trigger MH. They also underwent semitendinosus muscle biopsy for contracture study with halothane and caffeine. Measurements in vivo of mixed venous and arterial blood gases, cardiac output by thermodilution, temperature, blood pressure, and pulse rate provided sequential data regarding whole body O2 consumption (product of cardiac output and arterial-mixed venous O2 content difference), acid-base status, and arterial CO2 tension. Greyhounds and mongrels had uniformly similar in vivo and in vitro responses, without evidence for MH. Contracture thresholds were higher than those reported for normal swine and humans (8 mM vs. 4 mM). Information on MH susceptibility in this breed is important for laboratory investigation in Greyhounds as well as to veterinary medicine in general. Neither mongrels nor this group of Greyhounds were obviously susceptible to MH. If all Greyhounds are genetically homologous, then Greyhounds may not be specifically MH susceptible. These findings overall may provide a protocol and baseline normal comparative data for determining MH susceptibility in dogs and other species.     

Characterization of Human gamma-glutamyl hydrolase in solution demonstrates that the enzyme is a non-dissociating homodimer

Human gamma-glutamyl hydrolase (hGH) is a key enzyme in the metabolism of folic acid and in the pharmacology of many antifolate drugs. hGH catalyzes removal of the poly-gamma-glutamate chains of intracellular folic acid and antifolates. hGH crystallized as a homodimer with two putative active sites. However, the quaternary structure and the number of species of the enzyme in solution have not been determined. hGH has now been characterized using analytical ultracentrifugation and dynamic light scattering. HisTag fusion proteins of wild-type hGH, rat GH, and hGH expressed as a glycosylated protein were studied. Analyses of HisTag wild-type hGH were conducted over a range of protein concentrations (1.4-200 microM), ionic strengths (0-1 M NaCl), and pH (4.5-8.5). A single species with a molecular mass consistent with a homodimer was observed. Glycosylated hGH and HisTag rat gamma-glutamyl hydrolase also formed very stable homodimers. The lack of dissociation of the dimer, the large monomer-monomer interface, and the presence of catalytically essential Tyr-36 in the homodimer interface sequences suggest that homodimer formation is required for the hGH monomer to fold into an active conformation. The conservation of hGH monomer-monomer interface sequences in other mammalian and plant gamma-glutamyl hydrolase molecules suggests that they also exist as stable homodimers.     

Mutants of the deubiquitinating enzyme Ubp14 decipher pathway diversity of ubiquitin-proteasome linked protein degradation

Selective proteolysis is an important regulatory mechanism in all cells. In eukaryotes, this process gains specificity by tagging proteins with the small protein ubiquitin. K48 linked polyubiquitin chains of four and more ubiquitin moieties target proteins for hydrolysis by the proteasome. Prior to degradation the polyubiquitin chain is removed from the protein, cleaved into single units, and recycled. The deubiquitinating enzyme Ubp14 is an important catalyst of this process. Mutants of Ubp14 had been shown to accumulate non-cleaved oligo- and polyubiquitin chains, which resulted in inhibition of overall ubiquitin-proteasome linked proteolysis as well as in inhibition of degradation of some known substrates. Here we show that accumulation of ubiquitin chains due to defective Ubp14 does not uniformly lead to inhibition of ubiquitin-proteasome linked protein degradation. Instead, inhibition of degradation depends on the substrate tested. The results indicate the existence of different paths through which proteins enter the proteasome.     

Two DHH Subfamily 1 Proteins in Streptococcus pneumoniae Possess Cyclic Di-AMP Phosphodiesterase Activity and Affect Bacterial Growth and Virulence

Cyclic di-AMP (c-di-AMP) and cyclic di-GMP (c-di-GMP) are signaling molecules that play important roles in bacterial biology and pathogenesis. However, these nucleotides have not been explored in Streptococcus pneumoniae, an important bacterial pathogen. In this study, we characterized the c-di-AMP-associated genes of S. pneumoniae. The results showed that SPD_1392 (DacA) is a diadenylate cyclase that converts ATP to c-di-AMP. Both SPD_2032 (Pde1) and SPD_1153 (Pde2), which belong to the DHH subfamily 1 proteins, displayed c-di-AMP phosphodiesterase activity. Pde1 cleaved c-di-AMP into phosphoadenylyl adenosine (pApA), whereas Pde2 directly hydrolyzed c-di-AMP into AMP. Additionally, Pde2, but not Pde1, degraded pApA into AMP. Our results also demonstrated that both Pde1 and Pde2 played roles in bacterial growth, resistance to UV treatment, and virulence in a mouse pneumonia model. These results indicate that c-di-AMP homeostasis is essential for pneumococcal biology and disease.     

The first characterized asparaginase from a basidiomycete, Flammulina velutipes

Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of L-asparagine and L-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its L-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases.     

Fatty Acid Binding Protein 7 Is a Molecular Marker in Adenoid Cystic Carcinoma of the Salivary Glands: Implications for Clinical Significance

Adenoid cystic carcinoma (ACC) is an aggressive malignant neoplasm of the salivary glands. Its diagnosis is difficult due to overlapping features with other salivary tumors. Gene expression analysis may complement traditional diagnostic methods. We searched gene expression patterns in the Gene Expression Omnibus (GEO) database and in our tumor and normal samples. The biologic and prognostic potential of the identified genes was analyzed. The GEO data set of primary xenografted ACCs revealed that expression of five genes, engrailed homeobox 1 (EN1), fatty acid binding protein 7 (FABP7), hemoglobin epsilon 1, MYB, and versican (VCAN), was dramatically increased. mRNA expression of EN1, FABP7, MYB, and VCAN distinguished our sporadic ACCs from normal tissues and benign tumors. FABP7 expression appeared to be regulated differently from EN1 and MYB and was crossly correlated with poor prognosis in our ACC cohort. Immunohistochemistry showed that FABP7 protein was predominantly expressed in the nucleus of myoepithelial cells of both tubular and cribriform subtypes. In contrast, in the solid subtype, which is often associated with a lower survival rate, FABP7 protein was uniformly expressed in cancerous cells. One case with cribriform architecture and the highest level of FABP7 mRNA showed strong FABP7 staining in both duct-type epithelial and myoepithelial cells, suggesting that diffuse expression of FABP7 protein might be related to aggressive tumor behavior and poor prognosis. We propose FABP7 as a novel biomarker in ACC. The molecule may be useful in diagnosis and for identifying more effective therapies targeting this protein or upstream molecules that regulate it.     

Small scale spatial heterogeneity of Normalized Difference Vegetation Indices (NDVIs) and hot spots of photosynthesis in biological soil crusts

Normalized Difference Vegetation Indices (NDVIs) are typically determined using satellite or airborne remote sensing, or field portable spectrometers, which give an averaged signal on centimetre to metre scale plots. Biological soil crust (BSC) patches may have smaller sizes, and ecophysiological, hydrological as well as pedological processes may be heterogeneously distributed within this level of resolution. A ground-based NDVI imaging procedure using low-cost equipment (Olympus Camedia 5000z digital camera equipped with a Hoya R72 infrared filter) was developed in this study to fill this gap at the level of field research, where carrying costly and bulky equipment to remote locations is often the limiting factor for data collection. Method principle and field data are presented, and the field experiment was deepened comparing NDVI measurements and CO₂ turnover of soil crust samples in the laboratory, backing the reliability of the approach.     

In vitro transformation of androgen receptor from murine skeletal muscle by cAMP

Sedimentation constants and DNA-cellulose-binding of cytosolic androgen receptor from murine skeletal muscle were determined in presence of cyclic nucleotides. Without cAMP, two testosterone-binding fractions of similar amount at 4-5S and 8-9S were obtained. With 3 microM cAMP the receptor sedimented predominantly at 4-5S. Binding of testosterone-receptor-complexes to DNA-cellulose was enhanced by increasing cAMP-concentrations and reached a maximum at 20-90 nM cAMP depending on the DNA-concentrations in the test. A similar DNA-binding characteristic was obtained after partial purification of the receptor by affinity chromatography. cGMP had no effect. We conclude that the androgen receptor is transformed in vitro by cAMP.