[Evaluation of intravascular therapy for cerebral ischemic tandem stenosis].

Objective: To investigate the efficacy and the safety of intravascular therapy for cerebrovascular ischemic tandem stenosis. Methods: Clinical data of 35 patients with symptomatic anterior circulation and posterior circulation tandem stenosis who received intravascular therapy for two sites of stenosis at the same time at Department of Neurosurgery of Peking University First Hospital from January 2013 to December 2018 were analyzed retrospectively. There were 27 males and 8 females,aged (65.6±9.4)years (range:47 to 81 years).There were 14 cases of anterior circulation tandem stenosis and 21 of posterior circulation tandem stenosis.The medical records were collected with emphasis on postoperative symptoms,imaging manifestations and modified Rankin scale(mRS) scores. Results: Sixty-eight stents were implants in to 35 patients,including 49 extracranial implants and 19 intracranial implants.The surgical success rate was 100%.The perioperative death rate was 0,and 1 patient(1/35,2.9%) had cerebral hemorrhage.All patients were followed up for 18 months.During 3 to 12 months after the intervention,1 case(1/35,2.9%) had stent restenosis,and 4 cases(4/35,11.4%) had persisted symptoms such as dizziness and weakness in limbs.All patients'mRS scores were ≤2. No new stroke occurred. During 12 to 18 months after the intervention,3 cases had in-stent restenosis,increasing the rate to 11.4% (4/35). The mRS scores of 32 patients(32/35,91.4%) were ≤2. Conclusion: Intravascular therapy for patients with symptomatic tandem stenosis is a feasible and safe procedure with good short-term outcomes.

[Microsurgical treatment of paraclinoid aneurysms].

Objective: To explore the microsurgical treatment of paraclinoid aneurysms and evaluate its safety and efficacy. Methods: The data of 21 patients with 22 paraclinoid aneurysms receiving craniotomy between Jan. 2010 and Dec. 2017 in Peking University First Hospital were retrospectively analyzed. According to the Barami K classification, 2 aneurysms were type Ⅰa, 6 type Ⅰb,7 typeⅡ,6 type Ⅲa,1 type Ⅳ. Out of the 17 cases of saccular aneurysms, 16 aneurysms were clipped and one aneurysm was trapped following high-flow EC-IC bypass. Out of the 5 cases of blood blister like aneurysms, 2 aneurysms were wrap-clipped, 2 aneurysms were trapped following high-flow EC-IC bypass and 1 aneurysm was trapped following STA-MCA bypass. The patients were reexamined with CT angiography (CTA) or digital subtraction angiography (DSA) and followed up in outpatient or by phone call. Results: Seventeen patients with 18 paraclinoid aneurysms had received aneurysm clipping. Aneurysmal neck remnant was found in 2 cases, parent artery stenosis was found in 2 cases. In all of the four bypass cases, graft artery patency was confirmed and no recurrence of aneurysm was observed. The obliteration rate of the paraclinoid aneurysm was 91%(20/22). Eight cases with preoperative vision defect had recovered to some extent. New vision defect occurred in two cases. At discharge, 12 patients scored with Glasgow outcome scale 5, 6 patients scored 4, 2 patients scored 3, and one patient scored 1. Conclusion: Microsurgical treatment for paraclinoid aneurysm is a safe and effective method with high aneurysm obliteration rate and low aneurysm recurrence rate, and is thus a reasonable and effective complementary method for endovascular treatment.

[Discussion on optimal duration of pegylated interferon α combined with ribavirin for chronic hepatitis C in HIV-infected patients].

Objective: To investigate the optimal duration of pegylated-alpha interferon (Peg-INFα) combined with ribavirin (RBV) in treating chronic hepatitis C infection in human immunodeficiency virus (HIV)-infected patients. Methods: A multicenter prospective study was conducted. The study subjects were divided into two groups; HIV/HCV co-infections (Group A, n = 158) and control with HCV-monoinfections (Group B, n = 60). All recruited patients received standard Peg-INFα plus RBV therapy. Group A was divided into 3 subgroups according to CD4(+) cell counts: A1 subgroup, 79 cases, CD4(+) counts > 350 cells /µl, who received anti-HCV before combination antiretroviral therapy(cART); A2 subgroup, 45 cases, CD4(+) counts between 200 and 350 cells/µl, who did not start anti-HCV until they could tolerate cART well; A3 subgroup, 34 cases, CD4(+) counts < 200 cells /µl, cART was administered first, and anti-HCV therapy was started when CD4(+) counts > 200 cells/µl. The anti-HCV efficacy of two groups and 3 subgroups were compared. Statistical analysis for normal distribution and homogeneity of variance data was calculated by t-test and the counting data was analyzed by χ (2) test. The Mann-Whitney U test was used for non-normal data. A one-way analysis of variance (ANOVA) was used for the comparison of multiple groups, followed by SNK method. Multiple independent samples were used for non-parametric tests. Results: There was no significant difference in age and baseline HCV RNA levels between groups and subgroups (P > 0.05). By an intent-to-treat (ITT) analysis, in Group A, the ratio of complete early virological response (cEVR) rate was 75.3% (119/158), the ratio of end of treatment virological response (eTVR) rate was 68.4% (108/158), and the ratio of sustained virological response (SVR) rate was 48.7% (77/158); in Group B, the ratio of cEVR rate was 93.3% (56/60), the ratio of eTVR rate was 90.0% (54/60), and the ratio of SVR rate was 71.7% (43/60); The therapeutic index of Group A were lower than those of Group B (P≤0.05). By per-protocol (PP) analysis, the ratio of cEVR rate in Group A [75.2% (88/112)] was still lower than that in Group B [93.3% (56/60)], but no significant differences were found in the ratio of eTVR rate and SVR rate between 2 groups (P > 0.05). Comparing the efficacy of subgroups (A1, A2 and A3) by ITT analysis, the ratios of cEVR rate were respectively 78.5% (62/79), 75.6% (34/45) and 67.6% (23/34); the ratios of eTVR rate were respectively 68.4%(54/79), 80.0%(36/45)and 52.9%(18/34); and the ratios of SVR rate were respectively 41.8%(33/79), 64.4%(29/45)and 44.1%(15/34). The ratio of eTVR in subgroup A2 was obviously higher than that in subgroup A3 and the ratio of SVR in subgroup A2 was statistically higher than that of subgroup A1(P≤0.05). However, by PP analysis, no significant differences of the therapeutic indexes were found among the respective subgroups (P > 0.05). Conclusion: HIV-HCV co-infected patients would have better anti-HCV efficacy with Peg-INFα-2a plus RBV than HCV- monoinfected patients. The best time for initiating anti-HCV therapy in HIV-HCV co-infected patients is when CD4(+) counts 200 cells/ µl.

The primary structure of tubulin. Sequences of the carboxyl terminus and seven other cyanogen bromide peptides from the alpha-chain.

The alpha-chain of calf brain tubulin was fragmented by treatment with cyanogen bromide and eight peptides together accounting for 108 residues were purified and sequenced. The COOH-terminal peptide contains a fractional amount (about 0.3 residues) of tyrosine at its COOH-terminus; this presumably represents tyrosine that is added post-translationally to alpha-tubulin. The beta-chain can be phosphorylated, and the probable site of this modification is identified also in the COOH-terminal peptide. Comparison of the sequences described here with the sequence of actin reveals no homology between actin and tubulin.

Reactivities of thiols in myosin rod: effect of magnesium and ionic strength.

There are six cysteines in each chain of myosin rod of rabbit skeletal muscle: three are in the S-2 portion, at residues 66, 108 and 410 (Lu, R.C. and Lehrer, S. (1984) Biochemistry 23, 5975-5981). The other three are in the light meromyosin portion, assigned at residues 572, 600 and 770 on the basis of homology between the amino acid sequence in the vicinity of these thiols and that of the rod of nematode myosin (McLachlan, A.D. and Karn, J. (1982) Nature 299, 226-231). Since the thiols are distributed in different regions of the rod, measuring their reactivities under various conditions may provide information on the conformations of these regions. Myosin rod was carboxymethylated with radioactive iodoacetic acid under various conditions. The cysteine-containing peptides were isolated using HPLC from the tryptic digests, and the radioactivity incorporated into each thiol was measured. In the denatured state all six thiols were equally reactive. In the native state, all thiols have low reactivity, the reactivity of Cys-108 or -410 is only 0.1% of that in the denatured state, Cys-600 exhibited the highest reactivity, about 20-times that of Cys-410; Cys-66, -572 and -770 had 2-4-times that of Cys-410. When the rods formed filaments, the reactivities of all cysteines further decreased: Cys-66, -108 and -770 were reduced to 50%, while Cys-410, -572 and -600, located in the middle of the rod, were reduced to 20-30% of their reactivities in the monomeric form. In the presence of Mg2+ the reactivity of Cys-108 increased by 20%, whereas Cys-572 decreased by 50%. The results are consistent with the view that metal ions affect the conformation of the rod. This may play a role in the mechanism of filament formation and the movement of crossbridges.

Changes of lysine reactivities of actin in complex with myosin subfragment-1, tropomyosin and troponin.

The interactions of actin with myosin subfragment-1 and tropomyosin were explored by comparing the reactivities of lysine residues in F-actin alone with those in F-actin complexed to the other proteins. Limited reductive methylation was carried out on F-actin and the F-actin complexes with [14C]HCHO and [3H]HCHO, respectively. After dissociation from the other components, [3H]actin was combined with [14C]actin and 3H/14C of each lysine residue was measured. Myosin subfragment-1 reduced the reactivities of Lys-335 and Lys-372, while tropomyosin reduced those of Lys-237, -325, 327 and -335. When troponin was present in the absence of Ca2+, the effect of tropomyosin on Lys-335 remained the same, but its reactivity was completely restored upon the addition of Ca2+. Thus, the results suggest that different parts of actin are affected by the interaction with myosin subfragment-1 and tropomyosin, but the region containing Lys-335 is commonly affected by the presence of either of them. The change in reactivity is attributable either to a direct steric effect or to an induced conformational effect.

Cleavage of a specific bond in troponin C by thrombin.

Limited proteolysis of rabbit skeletal troponin C with bovine thrombin yielded two fragments, TH1 (Mr = 11000) containing Ca2+ binding regions I--III and TH2 (Mr = 6000) containing region IV. Determination of the partial sequences of the fragments established the site of cleavage at Arg120-Ala121. Secondary cleavage by thrombin at other arginyl or lysyl residues in troponin C was ruled out by the sequence data and by the amino acid compositions of the two fragments.

Changes of lysine reactivities of actin in complex with DNAase I.

The reactivities of lysines of actin in the actin-DNAase I complex were measured by the method of reductive methylation. The reactivities of lysines in the amino-terminal part, lysines 18, 50, 61 and 68, decreased 50%, while those of lysines 237, 283 and 290 increased about 30%, in comparison with those in G-actin, when actin was bound to DNAase I. These results are consistent with the view that the amino-terminal region of actin is the binding site for DNAase I. In conjunction with our earlier work on the reactivities of lysines in F-actin (Lu, R.C. and Szilagyi, L. (1981) Biochemistry 20, 5914-5919), these results are also consistent with the view that DNAase I binds to actin at one of the regions that is involved in polymerization.

Digestion of troponin C with trypsin in the presence and absence of Ca2+. Identification of cleavage points.

The rate of tryptic digestion of troponin C has been shown to be dependent on Ca2+ (Drabikowski et al., Biochim. Biophys. Acta 490, 216-224). We have characterized the tryptic peptides produced both in the presence and absence of Ca2+ using amino acid composition and end-group analyses. In the presence of Ca2+ trypsin cleaves TnC at Arg-8, Lys-84 and Lys-88, leading to the formation of two large peptides, one containing the two low-affinity sites (TR1C), the other, the two high-affinity Ca2+-binding sites (TR2C). In the absence of Ca2+ (1 mM EDTA), digestion proceeds much more rapidly and takes place first at Arg-100, followed by Arg-104, Arg-120, Lys-153, Arg-8 and others. The data suggest that the points of cleavage are determined by the Ca2+-dependent conformational states of TnC, particularly in the C-terminal half of the protein where the cation is known to induce secondary structure.

Thermal stability of myosin subfragment-1 decreases upon tryptic digestion in the presence of nucleotides.

Myosin subfragment-1 (S-1), digested with trypsin in the presence of ATP, rapidly loses its ATPase activity upon mild heat treatment even if ATP or ADP is present. The heat-treated molecule is very sensitive to further tryptic digestion. Undigested S-1 and S-1 digested in the absence of ATP are protected by nucleotides. The loss of the protective effect of nucleotides correlates with the tryptic splitting of the 25 kDa amino-terminal fragment between Arg 23 and Ile 24.

[Electroanalytical chemistry study of pilocarpine].

The electrochemical behaviour of pilocarpine was investigated by differential pulse polarography and cyclic voltammetry. The optimum conditions for the determination of pilocarpine were as follows: The electrolyte was 0.4 mol/L HAc-NaAc(pH 4.0) buffer solution, Zn (II) 1.0 x 10(-3) mol/L; initial potential -0.80 V (vs Ag-AgCl), final potential -1.30 V (vs Ag-AgCl), and voltage sweep rate 5 mV/s. A fair reproducibility in the above procedure and a good relationship between peak current and pilocarpine concentration (4.0 x 10(-6)-4.0 x 10(-5) mol/L) were achieved. The detection limit was 8.0 x 10(-7) mol/L. This paper also reports that the application of cyclic voltammetry to study electrode process behavior has been established. The PVC membrane ion-selective electrode for pilocarpine also was studied. It is based on the use of tetraphenylborate pilocarpine ion pair complex as the active material. The PVC membrane electrode showed Nernstian response over the pilocarpine concentration range from 1.0 x 10(-2) to 3.0 x 10(-5) mol/L, with a slope of 36 mV/decade, the detection limit being 3.0 x 10(-6) mol/L. The electrode gave fast response and good reproducibility. This method provides a rapid and simple way to the determination of pilocarpine in pharmaceutical preparations.

Bioactive tantalum metal prepared by micro-arc oxidation and NaOH treatment.

The study aim was to evaluate the effect of micro-arc oxidation (MAO) and alkali treatment on porous tantalum specimens by foam immersion technology. Tantalum specimens modified by MAO formed an apatite layer on their surfaces in simulated body fluid (SBF). However, the leach liquor of these specimens shows toxicity to 3T3-E1 cells. When they were previously soaked in a 0.5 M NaOH aqueous solution at 60 °C for 24 h, there was no statistical difference between the leach liquor of specimens with NaOH pretreatment and the control in cell proliferation experiments. NaOH-treated tantalum metal can form more apatite on its surface in SBF than the control and specimens produced by MAO. Furthermore, a sodium tantalate hydrogel layer was observed on the specimen's surface by X-ray diffraction. Well defined actin stress fibers and bone-like tissue are distributed throughout the body of 3T3-E1 cells cultured on specimens after 5 days. Animal tests showed new bone formation in the gap between the specimens and the host bone after 4 weeks. Neovascularization and new bone ingrowth occurred within the pores of the specimens after 4 and 12 weeks, respectively. Our results indicated that highly bioactive tantalum metal can be obtained by simple chemical treatment.

Identification of a region susceptible to proteolysis in myosin subfragment-2.

Comparison of the NH2-terminal sequence of myosin short subfragment-2 (Mr of subunit = 37,000) and long subfragment-2 (Mr of subunit = 59,000) demonstrates that the former represents the NH2-terminal portion of the latter and suggests that the hinge region in myosin rod is in the COOH-terminal portion of the long subfragment-2.

The distribution of the charged residues in myosin hinge region and its relationship to the distribution of charged residues in the rest of myosin rod.

Fourier transform analysis of the amino acid sequence of the hinge region from both rabbit skeletal myosin and nematode myosin indicates that the basic residues show strong periodicity of 25.7 residues whereas the periodicity of acidic residues is very weak. Other 100 residue segments of the rod sequence of nematode myosin show an alternation between regions in which the 28 residue repeat is predominantly basic and regions in which it is predominantly acidic. The strong basic and weak acidic near 28 residue repeat of the hinge region appears to form part of this pattern. This alternation suggests that the packing of myosin rods is more favourable when the neighbouring molecules are staggered by about 100 residues or odd multiples of 100 residues, which is consistent with the observed repeats (14.6 and 44.0 nm) of cross-bridges.

Glutamic acid-88 is close to SH-1 in the tertiary structure of myosin subfragment 1.

The thiol-specific photoactivatable reagent benzophenone iodoacetamide (BPIA) can be selectively incorporated into the most reactive thiol, SH-1, of myosin S1, and upon photolysis, an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa region or with the central 50-kDa region [Lu et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392]. Comparison of the peptide maps of cross-linked and un-cross-linked S1 heavy chains indicates that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide [Sutoh & Lu (1987) Biochemistry 26, 4511]. In this report, S1 was labeled with radioactive BPIA, photolyzed in the absence of nucleotide, and then degraded with proteolytic enzymes. Peptides containing cross-links were isolated by liquid chromatography and subjected to amino acid sequence analyses. The results show that Glu-88 is the major site and Asp-89 and Met-92 are the minor sites involved in cross-linking with SH-1 (Cys-707) via BPIA. These residues are very near the reactive lysine residue (Lys-83) but relatively remote in the primary structure from the putative nucleotide binding region.

Effects of interchain disulfide cross-links on the trypsin cleavage pattern and conformation of myosin subfragment 2.

The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)

Change of reactivity of lysine residues upon actin polymerization.

The reactivity of lysine residues of actin was measured by a surface labeling method--limited reductive methylation. After labeling, actin was subjected to CNBr and enzymatic cleavage, and all lysines were obtained either singly in a peptide or as a free residue. The specific activity of each lysine was taken as the measure of its reactivity. In actin denatured in 8 M urea, the reactivity of each lysine residue is approximately equal whereas those in G-actin fall into three categories: Lys-61 and Lys-113 are the most reactive ones; Lys-18, -213, -215, -314, and -358 are hardly reactive; the remainder, including Lys-50, -68, -84, -118, -191, -237, -283, -290, -325, -327, -335, and -372, are moderately reactive. The least reactive ones are probably buried in the native G-actin and all the others are most likely on the surface. Upon actin polymerization the reactivities of Lys-61, -68, -113, and -283 are significantly reduced while that of Lys-335 is strikingly enhanced. The decrease in reactivity could be readily explained if these residues were located in the monomer-monomer contact area although a polymerization-induced conformational change cannot be excluded. Such a conformational change may be invoked to explain the increase in the reactivity of Lys-335. Alternatively, the latter may be interacting with the bound ATP of G-actin, and the increased reactivity might be directly attributable to the loss of gamma-P for ATP accompanying polymerization.

The amino acid sequence and stability predictions of the hinge region in myosin subfragment 2.

From an NH2-terminal sequence analysis of the long and short form of myosin subfragment 2 we have suggested that the putative hinge region in the myosin rod is located in the COOH-terminal portion of the long subfragment 2 (Lu, R. C. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2010-2013). The amino acid sequence of this hinge region has now been determined: ASRA KAEKQRSDLSRELEEISERLEEAGGATSAQIEMNK KREAEFEKMRRDLEEATLQHEATAAALRKKHAD SVAELGEQIDNLQRVKQKLEKEKSELKMEIDDLA GNMETVSKAKGNLEKMCRTLEDQ(L/V)SE(V/L)KT KEEEHQRLIN(D/E)L(S/G)AQ(K/R)AR. Comparison of the sequence with that of other portions of the rod, viz. short subfragment 2 and light meromyosin, and of tropomyosin shows that the hinge region shares some feature of a coiled-coil helical structure, but it has somewhat fewer hydrophobic coil-coil interactions and there is a significant number of charged residues in the hydrophobic core region. This suggests that the stability of the putative hinge region would be reduced in comparison with other coiled-coil structures.

Identification of two segments, separated by approximately 45 kilodaltons, of the myosin subfragment 1 heavy chain that can be cross-linked to the SH-1 thiol.

The thiol-specific photoactivatable reagent 4-(2-iodoacetamido)benzophenone (BPIA) can be selectively incorporated into the SH-1 of myosin subfragment 1 (S1), and upon photolysis an intramolecular cross-link is formed between SH-1 and the N-terminal 25-kDa region of S1. If a Mg2+-nucleotide is present during photolysis, cross-links can be formed either with the 25-kDa or with the central 50-kDa region [Lu, R. C., Moo, L., & Wong, A. G. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6392-6396]. Heavy chains with these two types of intramolecular cross-links and un-cross-linked heavy chain have different mobility on sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels and therefore can be purified electrophoretically. Each type of heavy chain was cleaved with Staphylococcus aureus protease, chymotrypsin, or lysyl endopeptidase. The cleavage points were determined on the basis of the molecular weights of weights of peptides containing the N-terminus, which was identified with the use of an antibody. Locations of the cross-links were deduced by comparing the peptide maps of cross-linked and un-cross-linked heavy chains. The results indicate that the segment located about 12-16 kDa from the N-terminus of the heavy chain can be cross-linked to SH-1 via BPIA independently of the presence of a nucleotide, whereas the segment located 57-60 kDa from the N-terminus can be cross-linked to SH-1 only in the presence of a Mg2+-nucleotide. With use of the avidin-biotin system, it has been shown that SH-1 is located 13 nm from the head/rod junction [Sutoh, K., Yamamoto, K., & Wakabayashi, T. (1984) J. Mol. Biol. 178, 323-339]. Since BPIA spans less than 1 nm, our results show that two regions, separated by approximately 400 amino acid residues and located in the 25- and 50-kDa domains of S1, respectively, are also part of the head structure about 12-14 nm from the head/rod junction.