[The risk factors of first recurrence in ischemic stroke patients in different periods after first attack].

Objective: To investigate the risk factors of first recurrence in ischemic stroke patients at different periods after first attack. Methods: The subjects were from the screening population of China National Stroke Screening Survey (CNSSS) from 2013 to 2015. The basic demographic information, stroke history, influencing factors and modified Rankin Scale (mRs) scores were collected by using standardized face-to-face questionnaires. A case-control study was conducted to investigate the risk factors of first recurrence in ischemic stroke patients who relapsed for the first time within 12 months, 24 months and 36 months as the case group and non-recurrent ischemic stroke patients as the control group. Further, the subjects were stratified into different subgroups by age, gender and urban-rural distribution to investigate the risk factors of first recurrence in different periods. Results: Diabetes (OR=1.71, 95%CI: 1.08-2.71) and coronary heart disease (OR=1.55, 95%CI: 1.09-2.19) were significantly associated with the risk of first recurrence within 12 months after the first onset of ischemic stroke. The diabetes was significantly associated with the risk of first recurrence within 24 months (OR=1.94, 95%CI: 1.33-2.83) and 36 months (OR=1.64, 95%CI: 1.15-2.34) after the first onset of ischemic stroke. With the increase of mRs score, the risk of first recurrence within 12 months, 24 months and 36 months in ischemic stroke patients increased significantly. In the same period, the risk factors of ischemic stroke recurrence differed in patients with different age, gender and urban-rural distribution. Conclusions: The risk factors of first recurrence in ischemic stroke patients are diverse at different periods after the first onset of ischemic stroke. In different subgroups, the risk factors of first recurrence and the strength of its corresponding association are also different.

[The epidemiological characteristics and adherence to hypoglycemic agents of ischemic stroke patients with diabetes].

Objective: To investigate the epidemiological characteristics and adherence to hypoglycemic agents of the ischemic stroke patients combined with diabetes. Methods: The study recruited 23 044 ischemic stroke cases from 2013-2015 screening period of China National Stroke Screening Survey. Standardized questionnaires were used to obtain information on demographic data, stroke history, the presence of influence factors, as well as the type of stroke, diagnosis date, frequency, chronic diseases history and hypoglycemic therapy. We used logistics model to investigate the possible risk factors of ischemic stroke combined with diabetes, and calculated the population attributable risk proportion (PARP). We also investigate the adherence to hypoglycemic agents. Results: The mean age of 23 044 ischemic stroke patients was (64.99±9.42) years old, 50.91% were males (11 731). In ischemic stroke patients, 21.52% had diabetes. According to the results of logistics model, ischemic stroke patients with hypertension, dyslipidemia, atrial fibrillation or family history of stroke had higher risk to combine with diabetes, their Odds Ratios (OR) were 2.18 (1.87-2.55), 1.99 (1.78-2.23), 1.64 (1.39-1.92) and 1.19 (1.06-1.33). Considering the prevalence of each influence factor in ischemic stroke patients, atrial fibrillation had the highest PARP (95%CI) of 62.65% (61.27%-63.76%). In ischemic stroke patients combined with diabetes, 70.73% (3 463/4 896) had taken hypoglycemic agents. Conclusion: There still were a large number of ischemic stroke patients combined with diabetes and a low rate of adherence to hypoglycemic agents.

[Gender-specific factors of ischemic stroke among atrial fibrillation patients].

Objective: To explore gender-specific factors and their contributions to ischemic stroke among atrial fibrillation (AF) patients. Methods: A case-control study was conducted. The relevant data were obtained from the database of China National Stroke Screening Survey. The cases were first-ever ischemic stroke cases diagnosed from September 2013 to September 2015. Frequency-matched for the age and distribution of city and country, controls were randomly selected by 1∶3 ratio from individuals with AF but without stroke in the program. Altogether, there were 85 male cases (320 controls) and 147 female cases (484 controls). Unconditional logistic regression model was applied for the analysis of relevant factors of the onset of ischemic stroke, and their population-attributable risk proportion [PARP, (95%CI)] was calculated. Results: The age of male subjects in the case group and control group were (65.26±11.20) and (64.83±11.08) years old, and that of females in two groups were (63.63±10.40) and (63.93±10.35) years old. According to the PARP (95%CI), relevant factors of the onset of ischemic stroke in a descending sequence were hypertension history [35.63 (18.64-47.73)], family history of stroke [28.70 (23.63-32.30)]and physical inactivity [15.73 [5.62-23.06)] among male AF patients, and family history of stroke (29.39 (24.21-33.08)), dyslipidemia (22.17 (2.26-36.45)) and smoking [2.09 (0.76-3.24)] among female AF patients. Conclusion: The relevant factors of ischemic stroke were different between male and female AF patients.

[The association of health-related behavior and the risk of stroke in people with hypertension].

Objective: To investigate the association between the combination of different health-related behaviors and the risk of stroke in people with hypertension. Methods: The data in this study were obtained from the China National Stroke Screening Survey (CNSSS). The case group was the people with hypertension who were also diagnosed as the first-ever stroke cases (total stroke and ischemic stroke) during 2013-2014 screening period. Their corresponding controls (1∶3 frequency-matched for age group and urban/rural ratio) were randomly selected from individuals with hypertension without stroke. The information on demographic data, stroke history, influence factors and health-related behaviors (non-smoking, normal body mass index maintenance and physical activity) was obtained using standardized face-to-face questionnaires. Univariate analysis included t-test and Chi-square test. Multivariate analysis included unconditional logistic regression. Results: There were 603 total stroke cases (1 909 controls) and 536 ischemic stroke cases (1 608 controls) in men with hypertension, and 600 total stroke cases (1 800 controls) and 534 ischemic stroke cases (1 602 controls) in women with hypertension. We found that women with three health-related behaviors had lower risk of total stroke (OR=0.29, 95%CI: 0.11-0.79) and ischemic stroke (OR=0.28, 95%CI: 0.10-0.77). Only the combination of non-smoking and physical activity was significantly associated with the decreased risk of total stroke (OR=0.30, 95%CI: 0.11-0.78) and ischemic stroke (OR=0.32, 95%CI: 0.12-0.87). We had not found significant association between the combination of different health-related behaviors and risk of total stroke and ischemic stroke (P>0.05) in men. Conclusion: This study indicated that health-related behavior intervention might be more effectively to prevent stroke in women with hypertension, especially the smoking control and physical activity.

Essential groups in synthetic agonist peptides for activation of the platelet thrombin receptor.

Thrombin appears to activate platelets by a novel mechanism that involves the cleavage of its receptor, and it has been proposed that the newly generated N-terminal region of the receptor then acts as a tethered ligand [Vu, T. H., Hung, D. T., Wheaton, V. I., & Coughlin, S. R. (1991) Cell 64, 1057-1068]. Peptides with sequences corresponding to those of the tethered ligand are capable of activating the receptor. In the present study, groups within this tethered ligand peptide that are important for activation of the receptor have been identified by synthesizing a series of peptides. A 14-residue peptide based on the tethered ligand stimulated the aggregation of gel-filtered platelets with an EC50 of 7 microM, and a concentration of 10 microM was the minimum concentration necessary to yield a full aggregation response in platelet-rich plasma. Truncation of the peptide from the C-terminus to nine residues did not markedly affect the response to the peptide. Shorter peptides of five, six, and eight amino acids retained their agonist activity, but the minimal concentration necessary to achieve a full aggregation response in platelet-rich plasma was 2-5-fold higher. Side chains within the tethered ligand peptide that are important for receptor activation were identified by synthesizing a series of peptides in which residues were sequentially replaced by alanine. The results indicated that the side chains of phenylalanine, leucine, and arginine in positions 2, 4, and 5, respectively, are essential for full activity. Most notably, substitution of phenylalanine in the second position resulted in complete loss of agonist activity at concentrations up to 800 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure-function relationships of hirulog peptide interactions with thrombin.

Using hirudin as a model, a novel class of bivalent thrombin inhibitors has been designed and characterized (Maraganore et al. (1990) Biochemistry 29, 7095-7101). These peptides, designated 'hirulogs', interact with both thrombin's catalytic center and its anion-binding exosite for fibrinogen recognition. In order to investigate structure-activity relationships in hirulog peptides, a number of peptide and peptidomimetic derivatives with alterations in catalytic-site binding and anion-binding exosite binding moieties were prepared. Replacements or modifications in the catalytic site and exosite binding moieties were achieved with the consequences of maintaining or improving antithrombin activity. In addition to showing improved affinity for thrombin, some derivatives with Ki's in the sub-nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.

Binding of the snake venom-derived proteins applaggin and echistatin to the arginine-glycine-aspartic acid recognition site(s) on platelet glycoprotein IIb.IIIa complex inhibits receptor function.

In the present report we describe the platelet-binding characteristics of applaggin and echistatin, potent inhibitors of fibrinogen-dependent platelet aggregation derived from Agkistrodon piscivorus piscivorus and Echis carinatus snake venoms, respectively. Both molecules bound to unstimulated platelets in a specific and saturable manner. At saturation there were 37,100 +/- 3,150 (mean, +/- S.D.) molecules of applaggin and 27,200 +/- 2,816 molecules of echistatin bound/platelet, with dissociation constants (Kd) of 1.4 +/- 0.6 x 10(-7) M and 4.9 +/- 1.2 x 10(-7) M, respectively. Stimulation of platelets with ADP (10 microM) + epinephrine (2 microM) resulted in an increase in the number of molecules bound at saturation to 42,300 +/- 2,105 for applaggin and 32,185 +/- 3,180 for echistatin, with a Kd of 5.6 +/- 0.3 x 10(-8) M and 1.8 +/- 0.6 x 10(-7) M, respectively. The synthetic peptide (Arg)8-Gly-Asp-Val was a competitive antagonist of applaggin and echistatin binding to unstimulated platelets (Ki = 25 and 36 microM, respectively). Applaggin and echistatin inhibited the binding of fibrinogen to stimulated platelets in a dose-dependent manner, with an IC50 of 9 and 25 nM, respectively. In concert with inhibition of platelet aggregation, applaggin and echistatin inhibited platelet secretion and synthesis of thromboxane A2 induced by ADP, collagen, and human gamma-thrombin. The monclonal antibody, LJ-CP3, which inhibits the binding of Arg-Gly-Asp containing ligands to platelet GPIIb.IIIa, also inhibited applaggin binding to unstimulated platelets in a competitive manner (Ki = 4.5 microM). Thus, applaggin and echistatin bind to the platelet GPIIb.IIIa complex, and the Arg-Gly-Asp sequence plays a central role in mediating this interaction.

Agkistrodon piscivorus piscivorus platelet aggregation inhibitor: a potent inhibitor of platelet activation.

Applaggin (Agkistrodon piscivorus piscivorus platelet aggregation inhibitor) is a potent inhibitor of platelet activation. The protein is isolated from the venom of the North American water moccasin snake in three steps, including gel filtration, cation exchange, and reverse-phase HPLC procedures. The purified protein migrates as a 17,700-Da polypeptide by SDS/PAGE under nonreducing conditions and as a 9800-Da peptide in the presence of thiol. The behavior of applaggin on SDS/PAGE would indicate that the protein is a disulfide-linked dimer. Applaggin has been completely sequenced by Edman degradation and consists of 71 amino acids. The sequence is rich in cysteine and contains Arg-Gly-Asp at residues 50-52. Applaggin blocks platelet aggregation induced by ADP, collagen, thrombin, or arachidonic acid with IC50 values ranging from 12 to 128 nM (0.2-2.3 micrograms/ml) depending on the agonist and its concentration. This inhibition is found to correlate with inhibition of thromboxane A2 generation and of dense granule release of serotonin. Inhibition by applaggin of serotonin release induced by ADP, gamma-thrombin, and collagen was monitored in plasma under stirred conditions with [3H]serotonin-loaded platelets, and IC50 values for inhibition are found to range from less than 10 to 145 nM. At saturating concentrations, 125I-labeled applaggin (125I-applaggin) binds to 28,500 sites per unstimulated, washed platelet with a Kd of 1.22 x 10(-7) M. Binding of 125I-applaggin to platelets is inhibited by the synthetic undecapeptide Arg8-Gly-Asp-Val at 200 microM.

Structure-function studies on bacteriorhodopsin. VIII. Substitutions of the membrane-embedded prolines 50, 91, and 186: the effects are determined by the substituting amino acids.

To study their role in the structure and function of bacteriorhodopsin, three prolines, presumed to be in the membrane-embedded alpha-helices, have been individually replaced as follows: Pro-50 and Pro-91 each by Gly and Ala and Pro-186 by Ala, Gly, and Val. The mutants of Pro-50 and Pro-91 all showed normal chromophore and proton pumping. However, the rates of regeneration of the chromophore in Pro-50----Ala, Pro-91----Ala and ----Gly with all-trans-retinal were about 30-fold slower than that in the wild-type, whereas the chromophore regeneration rate in Pro-50----Gly was 10-fold faster than in the wild-type. While, Pro-186----Ala regenerated the wild-type chromophore, the mutants Pro-186----Val and Pro-186----Gly showed large blue shifts (about 80 nm) in the chromophore regenerated with all-trans-retinal and showed no apparent dark-light adaptation. Pro-186----Gly first regenerated the wild-type chromophore with 13-cis-retinal which was thermally unstable and rapidly converted to the blue-shifted chromophore obtained with all-trans-retinal. High salt concentration restored the wild-type purple chromophore in the Pro-186----Gly mutant. Thus, in this mutant, the protein interconverts between two conformational states. Pro-186----Ala and Pro-186----Gly showed about 65%, whereas Pro-186----Val showed 10-20% of the normal proton pumping.

A 113-amino acid fragment of CD4 produced in Escherichia coli blocks human immunodeficiency virus-induced cell fusion.

A gene encoding a 113-amino acid, NH2-terminal fragment of CD4, rsT4.113, was constructed and expressed in Escherichia coli under the control of the tryptophan operon promoter. Following induction, rsT4.113 is produced at 5-10% of total E. coli protein, and it is found in inclusion bodies. The protein is purified in two steps under denaturing and reducing conditions. Solubilized rsT4.113 is first purified on a column of Q-Sepharose to remove low molecular weight contaminants and then purified to greater than 95% homogeneity by gel filtration. Renaturation of rsT4.113 is achieved at approximately 20% yield by dilution and dialysis. High performance liquid chromatography analysis of renatured rsT4.113 reveals a less than 15% contaminant of reduced protein. Purified and renatured rsT4.113 contains epitopes for both OKT4a and Leu3a, anti-CD4 monoclonal antibodies which block CD4-gp 120 association, but lacks measurable affinity toward a nonblocking anti-CD4 monoclonal antibody, OKT4. By comparison to a longer form (375 amino acids) of recombinant soluble T4 produced in mammalian cells that contains the entire extracellular domain, rsT4.113 has a comparable affinity for binding to OKT4a and Leu3a in a radioimmunoassay. Analysis of antiviral activity of rsT4.113 demonstrates that the E. coli-derived protein inhibits human immunodeficiency virus-induced syncytium formation with an IC50 of 5-10 micrograms/ml. These data demonstrate that the human immunodeficiency virus-binding domain of CD4 is localized within the NH2-terminal 113 amino acids of CD4 and is contained within a structure homologous to the kappa variable-like domain of immunoglobulins.

Aspartic acid substitutions affect proton translocation by bacteriorhodopsin.

We have substituted each of the aspartic acid residues in bacteriorhodopsin to determine their possible role in proton translocation by this protein. The aspartic acid residues were replaced by asparagines; in addition, Asp-85, -96, -115, and -112 were changed to glutamic acid and Asp-212 was also replaced by alanine. The mutant bacteriorhodopsin genes were expressed in Escherichia coli and the proteins were purified. The mutant proteins all regenerated bacteriorhodopsin-like chromophores when treated with a detergent-phospholipid mixture and retinal. However, the rates of regeneration of the chromophores and their lambda max varied widely. No support was obtained for the external point charge model for the opsin shift. The Asp-85----Asn mutant showed not detectable proton pumping, the Asp-96----Asn and Asp-212----Glu mutants showed less than 10% and the Asp-115----Glu mutant showed approximately equal to 30% of the normal proton pumping. The implications of these findings for possible mechanisms of proton translocation by bacteriorhodopsin are discussed.

Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle.

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.

Structure-function studies on bacteriorhodopsin. V. Effects of amino acid substitutions in the putative helix F.

To test structural and mechanistic proposals about bacteriorhodopsin, a series of analogues with single amino acid substitutions has been studied. Mutants in the proposed helix F of bacteriorhodopsin were chosen for investigation because of the probable interaction of this part of the protein with the retinal chromophore. Seven mutants of the bacteriorhodopsin gene were constructed by site-directed mutagenesis, and the gene products were expressed in Escherichia coli. The resulting mutant proteins were purified and assayed for their ability to interact with retinal in phospholipid/detergent micelles to form a bacteriorhodopsin-like chromophore. Four mutants, Ser-183----Ala, Tyr-185----Phe, Ser-193----Ala, and Glu-194----Gln, bound retinal to give pigments with absorption maxima approximately the same as the wild type. Three mutant opsins bound retinal to give chromophores that were blue-shifted relative to the wild type. Two Trp----Phe substitutions at positions 182 and 189 gave absorption maxima of 480 and 524 nm, respectively, and the mutant Pro-186----Leu gave a pigment with an absorption maximum of 470 nm. However, none of the amino acid substitutions eliminated the ability of the mutant bacteriorhodopsin to pump protons in response to illumination.

Structure-function studies on bacteriorhodopsin. IV. Purification and renaturation of bacterio-opsin polypeptide expressed in Escherichia coli.

Expression of the bacterio-opsin gene in Escherichia coli has been described in the accompanying papers. We now describe rapid and efficient methods for the purification of the E. coli-expressed bacterio-opsin. Bacterio-opsin can be extracted from E. coli membranes in a denatured form by using an organic solvent containing chloroform, methanol, water, and triethylamine. The bacterio-opsin, enriched to 30-50% in the extract, can be further purified to 90% by ion-exchange chromatography on DEAE-Trisacryl or hydroxylapatite chromatography in organic solvents or by preparative sodium dodecyl sulfate gel electrophoresis. In appropriate aqueous phospholipid/detergent mixtures, up to 80% of purified protein refolds and binds retinal covalently to regenerate the bacteriorhodopsin chromophore. When reconstituted into phospholipid vesicles, bacteriorhodopsin from E. coli shows the expected proton pumping activity in response to illumination.

Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli.

To express the bacterio-opsin (bop) gene in Escherichia coli, we have employed the inducible expression vectors pIN-II-A, -B, and -C (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The vectors contain three cloning sites early in the E. coli lipoprotein gene (lpp) which is transcribed from tandem lpp and lac promoters. The bop gene was modified so as to delete the N-terminal leader sequence and then cloned into each of the three cloning sites to encode three different lipoprotein/bacterio-opsin fusions. Expression of the fusions was demonstrated both in vitro and in vivo. The fusion protein was estimated to be about 0.05% of the total cell protein. The cause for the low level of expression apparently was neither an inadequate level of mRNA nor degradation of the protein. However, expression of the fusions caused inhibition of the growth of the host to varying extents. One fusion protein was purified from E. coli membranes to homogeneity by immunoaffinity chromatography followed by preparative gel electrophoresis. The purified fusion protein generated a bacteriorhodopsin-like chromophore on treatment with defined lipid/detergent mixtures and retinal. When reconstituted into vesicles, the protein pumped protons on illumination comparably to the reconstituted native bacterio-opsin.

Amino acid sequence of bovine white matter proteolipid.

The sequence of the bovine white matter proteolipid has been studied by a combination of proteolytic digestion and chemical cleavage at tryptophan residues. Alignment of peptides obtained by digestion with trypsin, chymotrypsin, clostripain, and Staphylococcus aureus protease gave the sequence of 52 residues at the amino terminus, 96 residues at the carboxyl terminus, and several additional segments. Peptides obtained by treatment of the protein with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-bromoindolenine confirmed the alignment and extended the sequence. This information, combined with that of other investigators, permits us to propose the primary structure for the entire protein. On the basis of the sequence determination, the molecular weight of the proteolipid protein is 29,869.

A hydrophobic tryptic peptide from bovine white matter proteolipid.

A hydrophobic, chloroform-soluble tryptic peptide with a molecular weight of approximately 4000 has been purified from the bovine white matter proteolipid protein. Its primary structure was obtained by a combination of solid-phase Edman degradation and mass spectrometry. A major part of the tryptic peptide appears to be inaccessible to the action of proteolytic enzymes. The peptide spans the three cyanogen bromide peptides located by Jollès et al. (Biochem. Biophys. Res. Commun. (1979) 87, 619--626) at the COOH-terminal region of the intact protein. Secondary structure calculations for this region indicate a segregation into discrete domains, with most of the tryptic peptide corresponding to a highly ordered, hydrophobic domain; an equal probability for alpha-helical or beta-structure is predicted for this region.

A study of elastase peptides from bovine white matter proteolipid.

Bovine white matter proteolipid has been digested with elastase in the presence of deoxycholate. After acidification, the digest was separated into an acid-soluble and an acid-insoluble fraction. The acid-insoluble fraction was enriched in nonpolar amino acids and, by a combination of solvent fractionation and chromatography, a fraction was obtained which consisted of a mixture of two peptides with a molecular weight of approximately 4000 daltons. The acid-soluble peptides were separated by molecular sieve, ion exchange and high performance liquid chromatography (HPLC) in the reverse phase mode. The purified peptides were smaller than expected on the basis of their elution position from a molecular sieve column, suggesting they were in an aggregated state during the initial chromatography. Reverse phase HPLC was shown to be useful for fingerprinting these peptide mixtures. The data demonstrate the difficulties associated with the study of this proteolipid and emphasize the tendency of both the protein and the peptides derived from it to aggregate.