[Clinical analysis of 4 children with hereditary hypercholesterolemia].

Objective: To investigate the clinical characteristics of hereditary hypercholesterolemia in childhood. Methods: The clinical data including general conditions, clinical manifestations, laboratory tests, and genetic testing results of 4 children with hereditary hypercholesterolemia who admitted to Henan Children's Hospital from January 2020 to December 2020 were retrospectively analyzed. Results: There were 4 female children aged 5.5,1.5,6.3,3.1 years, all presented with skin xanthoxoma as the chief complaint. Plasma total cholesterol (range 11.8 to 20.9 mmol/L) and low density lipoprotein-cholesterol (range 8.2 to 13.7 mmol/L) were significantly elevated. The serum ß-glutamate levels in case 1 (241.2 µmol/L) and case 2 (164.2 µmol/L) increased significantly. Genetic analysis revealed compound heterozygous variants of ABCG8 gene in case 1 and ABCG5 gene in case 2 who were diagnosed with sitosterolemia. Case 3 and 4 who all had family history of hypercholesterolemia and compound heterozygous variants of LDLR gene were diagnosed with familial hypercholesterolemia. After diet treatment, the blood lipids returned normal and the skin xanoma subsided in case 1 and 2. In case 3 and 4, the blood lipids gradually decreased after diet and rosuvastatin treatment. Conclusions: Xanthomatosis is the common clinical manifestation of sitosterolemia and familial hypercholesterolemia. Family history, blood plant sterol profile, genetic variation, and changes in blood lipids after early dietary treatment are helpful for disease identification.

The effect of SOX9 on islet ß cells in high glucose environment through regulation of ERK/P38 signaling pathway.

OBJECTIVE: Islet beta cells are involved in insulin secretion. SRY-related high mobility group 9 (SX-S9) is involved in the progression of various diseases, but the role of SOX9 in islet ß cells remains unclear. PATIENTS AND METHODS: The islet ß cell MIN6 cells were cultured in vitro and randomly divided into control group, high glucose group, and SOX9 siRNA group followed by analysis of SOX9 mRNA and protein expression by real-time PCR and Western blot, respectively, cell proliferation by MTT assay, cell apoptosis by flow cytometry, secretion of inflammatory factors TNF-α and IL-2 by ELISA, insulin secretion levels by spectrophotometer, myeloperoxidase (MPO), and superoxide dismutase (SOD) activities, as well as ERK/P38 signaling protein expression by Western blot. RESULTS: Under high glucose environment, SOX9 mRNA and protein expression were significantly increased, MIN6 cell proliferation was inhibited, apoptosis rate and secretion of TNF-α and IL-2 were increased, along with decreased insulin secretion, increased MPO content, decreased SOD activity and phosphorylation of ERK/P38, compared with control group (p < 0.05). However, transfection of SOX9 siRNA reduced SOX9 expression, promoted proliferation of MIN6 cells, decreased apoptotic rate and secretion of TNF-α and IL-2, increased insulin secretion, decreased MPO content, increased SOD and ERK/P38 protein phosphorylation. Compared with high glucose group, the differences were statistically significant (p < 0.05). CONCLUSIONS: The expression of SOX9 is increased under high glucose environment. Down-regulation of SOX9 expression can inhibit islet cell apoptosis, oxidative stress and inflammation, and promote islet cell proliferation and insulin secretion by regulating ERK/P38 signaling pathway.

[Effect of positive end-expiratory pressure on the cross-sectional area of the internal jugular vein and anatomic relationship between the internal jugular vein and the carotid artery in general anaesthesia of laryngeal mask airway].

Objective: To investigate the effect of positive end-expiratory pressure(PEEP) on the cross-sectional area (CSA) of the internal jugular vein (IJV) and anatomic relationship between the IJV and the common carotid artery (CCA) in general anaesthesia of laryngeal mask airway(LMA). Methods: Sixty American Society of Anesthesiologists (ASA) Ⅰ or Ⅱ grade patients undergoing elective operation received general anaesthesia using LMA from May to November 2017, aged 20-65, were included in this study and randomly divided into 4 groups: group P0 (PEEP: 0 cmH(2)O), group P5 (PEEP: 5 cmH(2)O), group P10 (PEEP: 10 cmH(2)O), group P15 (PEEP: 15 cmH(2)O). Following the induction of anesthesia, LMA was inserted, and mechanical ventilation was started while the right cervical vessels was imaged by ultrasonography after applying 4 different PEEPs in random order. Measurements were made after 1 min in each PEEP. The CSA, transverse diameter (TD), anteroposterior diameters (AD) of the IJV and the diameter, overlap distance and overlap index of CCA was measured. The arterial blood pressure, heart rate, and vasoactive drugs used were also recorded. Results: The CSA of group P0, P10 and P15 was (1.36±0.55), (1.80±0.54), (2.02±0.58) cm(2). The TD was (1.31±0.33), (1.61±0.49), (1.74±0.53) cm. The AD was (1.12±0.20), (1.33±0.30), (1.46±0.32) cm. Compared to group P0, the CSA, TD and AD of IJV in group P10 and P15 were significantly increased (P0/P10: t=7.81, 3.81, 4.30, all P<0.01; P0/P15: t=11.68, 5.40, 6.96, all P<0.01). There was no significant difference in the AD and TD of IJV between group P10 and P15 (all P>0.05), while the CSA of group P15 was bigger than that of group P10 (t=2.17, P<0.05). The overlap distance of group P0, P10 and P15 was (0.51±0.12), (0.62±0.16), (0.66±0.15) cm. The overlap index was (76.80±20.03)%, (91.10±26.13)%, (96.21±25.36)%. Compared to group P0, the overlap distance and overlap index in group P10 and P15 were significantly increased (P0/P10: t=4.49, 3.41, both P<0.01; P0/P15: t=5.91, 4.63, both P<0.01). There was no significant changes in the overlap distance and overlap index between group P10 and P15 (all P>0.05). The MAP of group P15 was lower than that of group P10 [(73.35±9.73 )vs (67.58±12.58) mmHg, t=2.745, P<0.05]. No patients were given atropine or norepinephrine. Conclusions: The application of PEEP effectively increases the CSA of IJV in general anaesthesia of LMA. At the same time, it also lead to higher overlap index between the IJV and CCA.Ten cmH(2)O PEEP provides the best balance between the increase of CSA and the stability of haemodynamics.

Isolation and characterization of a monoclonal antibody that inhibits HIV-1 infection.

To identify a cell surface molecule other than CD4 involved in infection of cultured cells with human immunodeficiency virus type 1 (HIV-1), mice were immunized with the CD4-negative Raji human B-cell line in order to isolate a monoclonal antibody (mAb). We isolated mAb 33A, which inhibited the infection of CD4-positive T cells, B cells, human peripheral blood lymphocytes (PBL), and brain-derived cells with HIV-1. Formation of viral DNA was also blocked when CD4-positive Raji cells were treated with 33A after adsorption of HIV-1, but not before its adsorption. mAb 33A had little effect on syncytium formation induced by cocultivation with HIV-1-producing cells. Flow cytometry revealed that 33A reacted with HTLV-I-positive T-cell lines, Burkitt's lymphoma cell lines, phytohemagglutinin (PHA) -stimulated PBL, brain-derived fibroblast-like cells, and some adherent cell lines, but hardly at all with immature T-cell lines. Immunoblotting experiments showed that 33A recognized an antigen with an apparent molecular mass of 32 kDa, but did not recognize chemokine receptors such as CXCR4, CCR5, or CCR3. The distribution characteristic of the antigen recognized by 33A on various cells and its molecular weight suggest that mAb 33A recognizes a new cellular antigen that is necessary for HIV-1 entry.

Identification of membrane antigens important for adsorption of human T-cell leukaemia virus type I.

We isolated three monoclonal antibodies (mAbs), H3e, H11b and H16h, which were capable of inhibiting syncytium formation induced in a human T-cell line MOLT-4 or a human glioma line U251 MG by coculture with human T-cell leukaemia virus type I (HTLV-I)-positive human T-cell lines. The mAbs partially inhibited the plating of pseudotypes of vesicular stomatitis virus (VSV) bearing envelope antigens of HTLV-I. Formation of proviral DNA was also inhibited when indicator cells were treated with the mAbs before adsorption of HTLV-I, but not after its adsorption. They did not inhibit syncytium formation induced by human immunodeficiency virus type 1. Flow cytometry revealed that H16h hardly reacted with various HTLV-I-positive T cells, while H3e and H11b reacted with HTLV-I-positive human cells as well as HTLV-I-negative human cells. H11b and H16h immunoprecipitated the membrane antigen with a molecular weight of 20 and 110-130 kDa, respectively. Western blot analysis showed that H3e, H11b and H16h bound to the protein of 20, 20 and 110-130 kDa, respectively. Thus, these findings suggest that the 20- and 110-130-kDa cell surface proteins may play a role at the early stage of HTLV-I infection.

Asymmetric contribution of the conserved disulfide loop to subunit oligomerization and assembly of the nicotinic acetylcholine receptor.

The acetylcholine receptor (AChR) at the motor synapse is a pentamer of homologous subunits with the composition alpha2betagammadelta. Owing to the circular arrangement of subunits that forms a central ion channel, each subunit interface contains contributions from opposite faces of each subunit, designated + and -. Common to all subunits of the AChR and members of its superfamily is a disulfide loop formed between cysteines 128 and 142 of the major extracellular domain. To gain insight into the structural contribution of the disulfide loop and its possible location, we mutated the invariant proline at position 136 to glycine (P136G) and examined subunit assembly. When introduced into any AChR subunit, P136G disrupted assembly by affecting the - face of the subunit, suggesting equivalent positioning of the loop in each subunit and localization to the - face. Also, the contribution of the loop in the overall assembly process differed for each subunit. In the beta and gamma subunits, P136G prevented assembly of higher order heteroligomers, whereas in the alpha and delta subunits, P136G prevented transport of assembled pentamers to the cell surface. The results demonstrate asymmetry in the contribution of the disulfide loop to formation of subunit interfaces, and that the loop in each subunit contributes at different stages of assembly.

Competitive antagonists bridge the alpha-gamma subunit interface of the acetylcholine receptor through quaternary ammonium-aromatic interactions.

We recently demonstrated that conserved tyrosines Tyr198 of the alpha subunit and Tyr117 of the gamma subunit of the acetylcholine receptor stabilize binding of the curariform antagonist dimethyl-d-tubocurarine (DMT). To test the hypothesis that DMT interacts directly with these tyrosines, and therefore bridges the alpha-gamma subunit interface, we introduced point mutations into these key positions and expressed one or both mutant subunits in alpha 2 beta gamma 2 acetylcholine receptors in 293 HEK cells. Binding of DMT, measured by competition against the initial rate of 125I-alpha-bungarotoxin binding, shows high affinity for aromatic mutations, reduced affinity for polar mutations, and lowest affinity for arginine mutations. Similar side chain dependences were observed for both Tyr alpha 198 and Tyr gamma 117, indicating interaction of these residues with two symmetrical chemical groups in DMT. Two more bisquaternary antagonists, pancuronium and gallamine, show side chain dependences similar to that of DMT, indicating that the primary stabilizing interactions are aromatic-quaternary in both subunits. For the rigid ligands DMT and pancuronium, co-expressing mutant alpha and gamma subunits revealed independent contributions by each determinant, but strict independence was not observed for the flexible ligand gallamine. The free energy contributed by each aromatic-quaternary interaction was estimated to be 2-4 kcal/mol, as determined from the free energy difference between aromatic and alkyl hydroxyl mutations. Our results suggest that bis-quaternary competitive antagonists bridge the alpha-gamma subunit interface by fitting into a pocket bounded by tyrosines at positions 198 of the alpha subunit and 117 of the gamma subunit.

Depression of calcium transients after exposure to high K+ solution in Li(+)-loaded frog twitch muscle fibres and its reversal by exogenous myo-inositol.

Using Arsenazo III as a myoplasmic calcium indicator, we have studied the calcium transients evoked by voltage-clamp depolarizing pulses in frog twitch muscle fibres which had been temporarily depolarized by 80 mmol/L K+ in the absence or presence of myoplasmic Li+. After the high K+ exposure, for either a short (15 min) or long (1 h) time, the post-K+ calcium transients could gradually be restored to the level of the pre-K+ ones, if the fibres were not loaded with Li+. In contrast, the post-K+ calcium transients of Li(+)-loaded fibres could not fully recover, and were depressed in a Li+ concentration-dependent manner. The mean amplitude of the post-K+ responses recorded more than 3.5 h after 15 min high K+ exposure was reduced to 56% of pre-K+ control in the fibres which had been loaded with Li+ in 20 mmol/L Li+ Ringer's solution. This depression could be prevented or partially reversed by exogenous myo-inositol. More depression could be induced by 1 h high K+ exposure, but the presence of exogenous myo-inositol could not clearly prevent the post-K+ calcium transients from reduction. Assuming that high K+ exposure caused a depletion of myo-inositol and probably other changes in the metabolism of inositol phospholipids in Li(+)-loaded fibres, we conclude that some metabolites of phosphoinositides may play modulation roles in excitation-contraction coupling in frog twitch muscle fibres.

Effect of barium ions on excitation-contraction coupling of frog twitch muscle fibres.

The mechanism for the Ba2(+)-induced potentiation of the twitch tension in frog skeletal muscle was investigated. No significant difference in the peak amplitude of the calcium transients evoked by voltage-clamp depolarizing pulse was found between the fibres bathed in Ca2+ (control) and Ba2+ Ringer's solution, whereas the calcium transients evoked by the action potentials from the fibres in Ba2+ Ringer's solution were increased by about 64%, compared with control. In comparison with control, the action potentials of the fibres in Ba2+ Ringer's solution had a similar overshoot but a significantly longer time course. Our results suggest that excitation-contraction coupling in frog twitch muscle fibres is not altered by replacing Ca2+ with Ba2+. The Ba2(+)-induced potentiation of contraction may be accounted for by broadening of the action potentials.

Effect of prolonged in vitro lithium treatment on calcium transients in frog twitch muscle fibres and its reversal by exogenous myo-inositol.

Using arsenazo III as an intracellular indicator to monitor the calcium transients elicited by voltage-clamp depolarizing pulse, the effect of prolonged in vitro lithium treatment on excitation--contraction coupling in frog twitch muscle fibres was investigated. Incubation in 10 mM Li+ Ringer's solution for 2 days caused a 46% increase in the amplitude of the calcium transients, while treatment with 30 mM Li+ for 2 days produced a depression of 44%. Shortening the bathing time to 1 day, the decrease of calcium transients caused by 30 mM Li+ was reversed to a small increase. For the 2-day incubation, both the increase in the amplitude with 10 mM and the decrease with 30 mM Li+ were abolished by the presence of 1 mM myo-inositol in the bathing medium. These results imply that the turnover of inositol phospholipids is involved in regulating excitation-contraction coupling in skeletal muscle fibres.