Changes in the Levels and Forms of Cholinesterases in the Blood Plasma of Normal and Dystrophic Chickens

Abstract: Acetylcholinesterase (AChE) and pseudocholinesterase (°ChE) were analysed in the blood plasma of developing chickens, both normal and those with inherited muscular dystrophy. The amounts and the molecular forms of each were examined. °ChE concentration rises in the plasma of normal and dystrophic chicks at the end of embryonic development and is maintained after hatching at a constant, relatively high level, accounting for 90-95% of total cholinesterase activity in normal plasma. This level is maintained in normal and dystrophic chickens. In embryonic plasma of both normal and dystrophic chicks, on the other hand, the levels of AChE are higher than those of °ChE. Immediately after hatching the AChE level decreases rapidly in normal plasma, reaching a very low level by 2-3 weeks ex ovo. The AChE level in plasma from dystrophic birds, although less than normal from day 19 in ovo to 2 weeks ex ovo, subsequently increases to peak around 4 months at levels 15-20-fold of those in normal birds. There is virtually no enzyme of either type in the erythrocytes of normal or dystrophic chickens. The changes of AChE in plasma were correlated with the alterations of AChE in dystrophic fast-twitch muscles, suggesting that the latter pool is a precursor of the plasma AChE. Both the AChE and the °ChE in plasma exist in multiple molecular forms, which are similar to certain of those found previously in the muscles of these birds. The major form (60-80%) of both enzymes in the plasma is the M form (sedimentation coefficient ≥11 S) in all cases, but it is accompanied by certain other forms. In no case is there any of the heaviest form (H₂, 19-20 S) of AChE or of °ChE found in normal and dystrophic muscle, which is attached at the synapses in normal muscle. The pattern of forms of plasma °ChE is constant at all ages, and in normal and dystrophic chickens. The pattern of forms of AChE in the plasma, in contrast, varies with age and with dystrophy in a characteristic manner. The sedimentation coefficients and the amounts of the enzymes in fast-twitch muscle of dystrophic animals are compared with those of the plasma forms, and an interpretation is given of the characteristic patterns of AChE and of χE in their blood.     

Sites of in vivo phosphorylation of vesicular stomatitis virus matrix protein.

We mapped the in vivo phosphorylation sites for the matrix (M) protein of the Orsay and San Juan strains of vesicular stomatitis virus, Indiana serotype, using limited proteolysis and phosphoamino acid analysis. M protein was solubilized from 32P-labeled virions by using detergent and high-salt conditions, then treated with either trypsin or Staphylococcus aureus V8 protease, and analyzed by polyacrylamide gel electrophoresis and autoradiography to determine which fragments contained phosphate residues. The M protein fragment extending from amino acid 20 to the carboxy terminus contained approximately 70% of the control 32P label, while the fragment extending from amino acid 35 to the carboxy terminus had only trace amounts of label. These data indicate that the major phosphorylation site was between amino acids 20 and 34 in the Orsay strain M protein. Phosphoamino acid analysis of M protein by thin-layer electrophoresis showed the presence of phosphothreonine and phosphoserine and that phosphothreonine continued to be released after prolonged vapor-phase acid hydrolysis. These data identify Thr-31 as the primary in vivo phosphate acceptor for M protein of the Orsay strain of vesicular stomatitis virus. The San Juan strain M protein has serine at position 32, which may also be an important phosphate acceptor. In addition, phosphorylation at Ser-2, -3, or -17 occurs to a greater extent in the San Juan strain M protein than in the Orsay strain M protein. The subcellular distribution of phosphorylated M protein was investigated to determine a probable intracellular site(s) of phosphorylation. Phosphorylated M protein was associated primarily with cellular membranes, suggesting phosphorylation by a membrane-associated kinase. Virion M protein was phosphorylated to a greater extent than membrane-bound M protein, indicating that M protein phosphorylation occurs at a late stage in virus assembly. Phosphorylation of wild-type and temperature-sensitive mutant M protein was studied in vivo at the nonpermissive temperature. The data show that phosphorylated M protein was detected only in wild-type virus-infected cells and virions, suggesting that association with nucleocapsids may be required for M protein phosphorylation or that misfolding of mutant M protein at the nonpermissive temperature prevents phosphorylation.     

Subunit interactions of vesicular stomatitis virus envelope glycoprotein stabilized by binding to viral matrix protein.

The mechanism by which viral glycoproteins are incorporated into virus envelopes during budding from host membranes is a major question of virus assembly. Evidence is presented here that the envelope glycoprotein (G protein) of vesicular stomatitis virus binds to the viral matrix protein (M protein) in vitro with the specificity, reversibility, and affinity necessary to account for virus assembly in vivo. The assay for the interaction is based on the ability of M protein to stabilize the interaction of G protein subunits, which exist as trimers of identical subunits in the virus envelope. The interaction with M protein was shown by using G proteins labeled with fluorescent probes capable of detecting subunit dissociation and reassociation in vitro. The results show that the M protein isolated from virions either as purified soluble protein or as nucleocapsid-M protein complexes interacts with the G protein in vitro and that the reaction is reversible. The interaction between the G and M proteins was not serotype specific, but no interaction between the vesicular stomatitis virus M protein and the influenza virus hemagglutinin could be detected. These results support the conclusion that the interactions described here are the ones that govern assembly of G protein into virus envelopes in vivo.     

Labeling of the cytoplasmic domain of the influenza virus hemagglutinin with fluorescein reveals sites of interaction with membrane lipid bilayers

The hemagglutinin (HA) glycoprotein of influenza virus was labeled in its cytoplasmic domain with fluorescein. Reactive amino groups in the external domain were blocked by modification of the intact virus with the membrane-impermeable reagent isethionyl acetimidate. The HA was then solubilized with the detergent octyl glucoside, and the single lysine in the cytoplasmic domain was reacted with fluorescein isothiocyanate. This protocol resulted in the incorporation of 1.3 mol of fluorescein/mol of HA. Using a virus strain lacking lysine in the cytoplasmic domain of HA, it was determined that 0.47 mol of fluorescein/mol of HA was located at an additional site(s). The fluorescein groups at both sites exist in an environment of reduced polarity as shown by a shift in excitation and emission maxima and a shift in the pKa of the fluorescein groups. The fluorescence polarization and the pKa of the fluorescein groups were greater when the HA was incorporated into liposomes than when in detergent solution. These data indicate that the fluorescein groups interact directly with the lipid bilayer, probably in the phospholipid head-group region. The fluorescence properties of the labeled HA were not responsive to the gel to liquid-crystal phase transition in the lipid bilayer. These results indicate that the boundary between the cytoplasmic domain and the hydrophobic sequence that anchors the protein to the lipid bilayer is located in the head-group region of the bilayer.     

Extending McNemar's test: estimation and inference when paired binary outcome data are misclassified

McNemar's test is popular for assessing the difference between proportions when two observations are taken on each experimental unit. It is useful under a variety of epidemiological study designs that produce correlated binary outcomes. In studies involving outcome ascertainment, cost or feasibility concerns often lead researchers to employ error-prone surrogate diagnostic tests. Assuming an available gold standard diagnostic method, we address point and confidence interval estimation of the true difference in proportions and the paired-data odds ratio by incorporating external or internal validation data. We distinguish two special cases, depending on whether it is reasonable to assume that the diagnostic test properties remain the same for both assessments (e.g., at baseline and at follow-up). Likelihood-based analysis yields closed-form estimates when validation data are external and requires numeric optimization when they are internal. The latter approach offers important advantages in terms of robustness and efficient odds ratio estimation. We consider internal validation study designs geared toward optimizing efficiency given a fixed cost allocated for measurements. Two motivating examples are presented, using gold standard and surrogate bivariate binary diagnoses of bacterial vaginosis (BV) on women participating in the HIV Epidemiology Research Study (HERS).     

A potential role of modulating inositol 1,4,5-trisphosphate receptor desensitization and recovery rates in regulating ovulation

Regulation of the human menstrual cycle is a frequency dependent process controlled in part by the pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus. The binding of GnRH to gonadotroph cells in the pituitary stimulates inositol 1,4,5-trisphosphate (IP3) mediated release of calcium from the endoplasmic reticulum, resulting in calcium oscillations and the secretion of luteinizing hormone (LH). A sudden increase in serum LH concentrations known as the LH surge triggers ovulation. Here we model the intracellular calcium dynamics of gonadotroph cells by adapting the model of Li and Rinzel (J. Theor. Biol. 166 (1994) 461) to include the desensitization of IP3 receptors to IP3. Allowing the resensitization rate of these receptors to vary over the course of the cycle suffices to explain the LH surge in both the normal menstrual cycle, and in the treatment of Kallmann's syndrome (a condition where endogenous production of GnRH is absent).     

One new genus and three new species of Orsillinae (Hemiptera: Lygaeidae) from Australia

Abstract  Two new species of Nysius Dallas, N. orarius sp. n. and N. tasmaniensis sp. n. are described from New South Wales and Tasmania (Australia), respectively. A new monotypic genus, Reticulatonysius , with type-species R. queenslandensis sp. n. is described from Queensland, and its systematic relationship with other orsilline genera is discussed.     

Dissociation of newly synthesized Sendai viral proteins from the cytoplasmic surface of isolated plasma membranes of infected cells.

The interaction of Sendai viral proteins with the membranes of infected cells during budding of progeny virions was studied. BHK cells infected with Sendai virus were labeled with [35S]methionine, and the plasma membranes were purified on polycationic polyacrylamide beads. The isolated membranes were incubated with various agents which perturb protein structure to dissociate viral proteins from the membranes. Incubation of membranes with thiocyanate and guanidine removed both the M and nucleocapsid proteins. Urea (6 M) removed the nucleocapsid proteins but removed M protein only in the presence of 0.1 or 1.0 M KCl. In contrast, high salt concentrations alone eluted only the M protein, leaving the nucleocapsid proteins completely membrane bound. About 65% of the M protein was eluted in the presence of 4 M KCl. The remaining membrane-associated M protein was resistant to further extraction by 4 M KCl. Thus, M protein forms two types of interaction with the membrane, one of them being a more extensive association with the membrane than the other.     

Treatment with tumour infiltrating lymphocytes and interleukin-2 in patients with metastatic melanoma: A pilot study

Tumour infiltrating lymphocytes (TIL) were isolated and expanded from biopsy samples of 4 patients with metastatic melanoma. The patients were treated with autologous expanded TIL and continuous or bolus infusion of Interleukin 2 (IL-2) at a dose of 18 × 10⁶ International Units/m²/day for 5 days starting 36–48 hours after administration of cyclophosphamide at a dose of 1 g/m². The number of TIL infused ranged from 10¹⁰ to 5,56 × 10¹⁰ cells. Two patients had stable disease (SD) lasting for 2 1/2 and 4 months respectively and they died 24 and 13 months after therapy. One patient died during therapy due to a pseudomonas septicaemia and another patient developed progressive disease (PD). He died 3 months after the start of therapy. The side effects were substantial but most of them were reversible upon cessation of the treatment.The majority of the expanded TIL of all patients were of the CD8+ phenotype. Cutaneous metastases from two patients, removed after treatment with IL-2 and TIL, showed moderate lymphocytic infiltration also mainly of CD8+ T cells.The treatment with IL-2 and TIL is feasible, but further investigations should continue in an attempt to improve the efficacy of the therapy, to reduce toxicity and to diminish the costs and labour of the culture methods.