A miniature lighted stylet for fast oral endotracheal intubation in rabbits.

Efficient oral endotracheal intubation of laboratory animals is a challenging technique in veterinary research. This study introduces a miniaturized lighted stylet for rabbit intubation. An experiment with repeated measures on two factors was used to assess the feasibility and efficacy of this method. The first factor compared stylet intubation vs. laryngoscopic intubation. The second compared three practitioners, one with prior experience and two without. Success rates on the initial attempt were not statistically different (χ(2)=2.46, P=0.12). The time difference between methods was significant (F=41.007, P<0.001), although the effect of practitioners was not (F=1.038, P=0.365). The mean±SD of the intubation time, combining results from the three practitioners, was 20.34±17.15s for the stylet method and 57.58±64.21s for the laryngoscopic method. The results of this study demonstrate that lighted stylet intubation is efficient, robust, and independent of practitioner experience.

Expression of recombinant envelope protein of Japanese encephalitis virus YL strain in Escherichia coli possesses hemagglutination activity.

The nucleotide sequence of glycoprotein E of YL vaccine strain was cloned, sequenced and expressed in E. coli. Phylogenetic analysis of envelope (E) amino acid sequences of 18 JEVs in GenBank showed that the vaccine strain YL closer to the virulent strain HVI which is a Taiwanese isolate. We found only two amino acid mutations (K-138 and G-389) of E protein might lead viral attenuation in YL. In this study, we used pRSET vector system to construct three recombinant plasmids (pRSET/F1R1, pRSET/F2R2 and pRSET/F1R2), which encoded and expressed different or overlapping amino acid region of E protein. The antigenicity and hemagglutination activity of these recombinant proteins were examined by western blotting and hemagglutination test, respectively. Our results demonstrated that the recombinant protein of pRSET/F1R2 possesses predominant antigenicity and hemagglutination activity.

Essential role for the C-terminal noncatalytic region of SHIP in FcgammaRIIB1-mediated inhibitory signaling.

The inositol phosphatase SHIP binds to the FcgammaRIIB1 receptor and plays a critical role in FcgammaRIIB1-mediated inhibition of B-cell proliferation and immunoglobulin synthesis. The molecular details of SHIP function are not fully understood. While point mutations of the signature motifs in the inositol phosphatase domain abolish SHIP's ability to inhibit calcium flux in B cells, little is known about the function of the evolutionarily conserved, putative noncatalytic regions of SHIP in vivo. In this study, through a systematic mutagenesis approach, we identified the inositol phosphatase domain of SHIP between amino acids 400 and 866. Through reconstitution of a SHIP-deficient B-cell line with wild-type and mutant forms of SHIP, we demonstrate that the catalytic domain alone is not sufficient to mediate FcgammaRIIB1/SHIP-dependent inhibition of B-cell receptor signaling. Expression of a truncation mutant of SHIP that has intact phosphatase activity but lacks the last 190 amino acids showed that the noncatalytic region in the C terminus is essential for inhibitory signaling. Mutation of two tyrosines within this C-terminal region, previously identified as important in binding to Shc, showed a reduced inhibition of calcium flux. However, studies with an Shc-deficient B-cell line indicated that Shc-SHIP complex formation is not required and that other proteins that bind these tyrosines may be important in FcgammaRIIB1/SHIP-mediated calcium inhibition. Interestingly, membrane targeting of SHIP lacking the C terminus is able to restore this inhibition, suggesting a role for the C terminus in localization or stabilization of SHIP interaction at the membrane. Taken together, these data suggest that the noncatalytic carboxyl-terminal 190 amino acids of SHIP play a critical role in SHIP function in B cells and may play a similar role in several other receptor systems where SHIP functions as a negative regulator.

Identification and characterization of a dimerization domain in CED-6, an adapter protein involved in engulfment of apoptotic cells.

Phagocytosis of apoptotic cells is a key step in the completion of programmed cell death that occurs throughout life in multicellular organisms. The molecular events involved in clearance of apoptotic cells are just beginning to be elucidated. Recently, CED-6, an adapter protein involved in engulfment has been cloned in Caenorhabditis elegans and in humans. CED-6 is composed of a phosphotyrosine-binding (PTB) domain and a proline-rich C-terminal domain with no apparent catalytic domain. Since PTB domains, originally identified in Shc, mediate intracellular signaling downstream of cell surface receptors, CED-6 has also been proposed to mediate intracellular signals leading to engulfment. In this report, we demonstrate that CED-6 dimerizes through a leucine zipper domain that is immediately adjacent to the PTB domain. Several lines of evidence based on co-immunoprecipitation studies, yeast two-hybrid assays, and gel filtration studies suggest that CED-6 exists as a dimer in vivo. Through mutational analyses, we show that the leucine zipper is necessary and sufficient for CED-6 dimerization and that this dimerization is conserved among C. elegans, rodent, and human CED-6 proteins. We propose that dimerization may have unique implications for ligand binding via CED-6 and its function during the phagocytosis of apoptotic cells.

Human immunodeficiency virus type 1 pathogenesis in SCID-hu mice correlates with syncytium-inducing phenotype and viral replication.

Human immunodeficiency virus type 1 (HIV-1) patient isolates and molecular clones were used to analyze the determinants responsible for human CD4(+) thymocyte depletion in SCID-hu mice. Non-syncytium-inducing, R5 or R3R5 HIV-1 isolates from asymptomatic infected people showed little or no human CD4(+) thymocyte depletion in SCID-hu mice, while syncytium-inducing (SI), R5X4 or R3R5X4 HIV-1 isolates from the same individuals, isolated just prior to the onset of AIDS, rapidly and efficiently eliminated CD4-bearing human thymocytes. We have mapped the ability of one SI HIV-1 isolate to eliminate CD4(+) human cells in SCID-hu mice to a region of the env gene including the three most amino-terminal variable regions (V1 to V3). We find that for all of the HIV-1 isolates that we studied, a nonlinear relationship exists between viral replication and the depletion of CD4(+) cells. This relationship can best be described mathematically with a Hill-type plot indicating that a threshold level of viral replication, at which cytopathic effects begin to be seen, exists for HIV-1 infection of thymus/liver grafts in SCID-hu mice. This threshold level is 1 copy of viral DNA for every 11 cells (95% confidence interval = 1 copy of HIV-1 per 67 cells to 1 copy per 4 cells). Furthermore, while SI viruses more frequently achieve this level of replication, replication above this threshold level correlates best with cytopathic effects in this model system. We used GHOST cells to map the coreceptor specificity and relative entry efficiency of these early- and late-stage patient isolates of HIV-1. Our studies show that coreceptor specificity and entry efficiency are critical determinants of HIV-1 pathogenesis in vivo.

Prolonged fasting in pediatric outpatients does not cause hypoglycemia.

Two hundred and thirty healthy children scheduled for receiving elective minor surgery were assigned into 4 different groups. Group I (small infant group) included 27 infants of age from 1 to 3 months (2.0 +/- 0.6 months), Group II (infant group) included 42 infants age from 3 to 12 months (7.4 +/- 2.8 months), Group III (pre-school children group) included 122 patients of age from 1 to 6 years (3.1 +/- 1.4 years). The remained 39 cases of age older than 6-years-old (8.0 +/- 1.5 years) were collected in group IV (old children group). All studied children were starved for at least 4, 6, or 8 hours in infants, pre-school children, and old children group, respectively, pre-operatively. The fasting time and fasting blood glucose levels of the 4 groups were 6.7 +/- 1.4 hours and 109.0 +/- 22.9 mg% in group I, 7.7 +/- 2.3 hours and 98.6 +/- 18.0 mg% in group II, 10.4 +/- 2.9 hours and 96.9 +/- 24.7 mg% in group III, and 12.6 +/- 2.6 hours and 95.7 +/- 20.5 mg% in group IV, respectively. No one in the 230 children had blood glucose less than 40 mg% even in 5 infants who were starved for 12 hours or more. Therefore, we concluded that preoperative starvation is well tolerated than the originally expected in the infants and children. The fasting time before anesthesia can be executed safely even though the operation schedule may not be right on time.

Study of plasma neuropeptide Y (NPY) and catecholamines levels during isoflurane anesthesia.

Twelve ASA physical status I-II patients were studied after obtaining institutional approval and informed contents. All patients were free from endocrine and metabolic disease undergoing elective low risk operation. Pethidine 1 mg/kg i.m. and benzodiazepine 0.01 mg/kg p.o. were given as premedication one hour before anesthesia. Anesthesia was induced with thiopental 4 mg/kg and succinylcholine 1.5 mg/kg for tracheal intubation. Anesthesia was maintained with 2% isoflurane and 50% N2O in oxygen. Ventilation was controlled and adjusted to maintain an end-tidal CO2 concentration of 25-35 torr. Atracurium 0.4 mg/kg was given as muscle relaxant. Blood samples were obtained from radial arterial catheter, 15 minutes before induction of anesthesia and 5 min after anesthesia, 15 min, 30 min, 60 min during operation and 30 min after operation in postanesthesia recovery room. The results showed that there were no statistically significant changes in plasma levels of NPY and catecholamines during operation underwent isoflurane anesthetic technique. This result indicates that isoflurane anesthesia can block the plasma NPY as well as catecholamines during surgical stress.