肝硬化门静脉高压患者TIPS术后门静脉分支远端血栓形成相关因素及其对预后的影响

摘要 目的：探究肝硬化门静脉高压患者经颈静脉肝内门体静脉分流术（TIPS）后门静脉分支远端血栓形成影响因素及其对预后的影响。方法：选取南方医科大学南方医院2021年1月~2023年1月行TIPS的300例肝硬化门静脉高压患者,根据术后6个月内门静脉分支远端血栓形成发生与否分为血栓组与非血栓组。对比两组临床资料,多因素Logistic回归分析TIPS术后门静脉分支远端血栓形成的相关因素。对比两组术后6个月预后情况。结果：术后门静脉分支远端血栓形成发生率10.67%（32/300）。血栓组年龄、术前3 d内血浆D-二聚体（D-D）、总胆固醇（TC）、低密度脂蛋白（LDL）水平、门静脉主干直径、门脉左支直径、Child-Pugh分级A级比例、门静脉流速与非血栓组对比差异均有统计学意义（P＜0.05）;多因素Logistic回归分析显示,高龄、LDL水平升高、门脉左支直径增大是TIPS术后门静脉分支远端血栓形成的危险因素,Child-Pugh分级为A级为保护因素（P＜0.05）。血栓组TIPS术后6个月TIPS支架通畅率、肝性脑病、肝功能损伤及死亡发生率与非血栓组对比无统计学意义（P＞0.05）。结论：肝硬化门静脉高压TIPS术后门静脉分支远端血栓形成与年龄、术前LDL、Child-Pugh分级、门脉左支直径相关,但对患者术后6个月TIPS支架通畅率、肝性脑病、肝功能损伤及死亡发生率没有影响。     

S100/RAGE在实验性自身免疫性重症肌无力中对T细胞的作用及其机制研究

目的:探讨RAGE及其配体S100B在实验性自身免疫性重症肌无力(EAMG)中对T细胞的作用及其相关机制。方法:选取体重160-180g的Lewis大鼠,并将其随机分为EAMG模型组和CFA对照组。EAMG模型组大鼠通过尾根部注入200μL含有R-ACh R97-116肽段的免疫乳剂,并于初始免疫后的第30天尾根部追加免疫一次;CFA对照组大鼠为免疫乳剂中不含有R-ACh R97-116肽段。采用流式细胞术检测和比较两组大鼠CD4+T细胞上RAGE的表达情况;ELISA方法检测淋巴细胞培养上清中IFN-γ、IL-4、IL-17、TGF-β、IL-6和S100B的表达水平;采用S100B体外干预淋巴细胞,进一步检测S100B对EAMG大鼠T细胞增殖、亚型分布、细胞因子分泌的影响。结果:在疾病发生的晚期时相(初次免疫后第45天),EAMG组淋巴细胞CD4+T细胞上RAGE的表达明显高于CFA组(P0.001),血清中RAGE的配体S100B的表达也高于CFA组(P0.001);体外加入S100B干预能促进T淋巴细胞的增殖,与未干预组相比较差异显著(P0.05);S100B刺激后,Th1细胞和Th17细胞的百分比进一步增高,Th2细胞和Treg细胞百分比下降(PTh10.05,PTh170.05,PTh20.05,PTreg0.05),IFN-γ和IL-17的表达上调(PIFN-γ0.05,PIL-170.05),而IL-4和TGF-β表达下降(PTGF-β0.01,PIL-40.05),Th17细胞的调控因子IL-6表达升高(PIL-60.05)。结论:RAGE及其配体S100B参与EAMG的发病过程,在晚期时相中表现出明显的致病性;RAGE与S100B相互作用可以上调T细胞的致病性作用,加重四种辅助性T细胞之间的网络失衡。     

脊柱骨折合并脊髓损伤患者血清Neuritin、NFL、S100B蛋白水平与术后预后不良的关系研究

摘要 目的：研究脊柱骨折合并脊髓损伤（SCI）患者血清神经突起因子（Neuritin）、神经丝轻链（NFL）、S100B蛋白水平与术后预后不良的关系。方法：将从2018年12月-2019年12月我院收治的60例脊柱骨折合并SCI患者纳入研究,记作损伤组,另取同期我院收治的单纯脊柱骨折未合并SCI患者60例作为无损伤组,再取同期体检的健康志愿者60例作为对照组。检测并比较三组血清Neuritin、NFL、S100B蛋白水平。此外,将损伤组患者按照术后预后的不同分作预后不良组25例和预后良好组35例,分析两组血清Neuritin、NFL、S100B蛋白水平以及临床资料的差异,并以多因素Logistic回归分析明确脊柱骨折合并SCI患者预后不良和各项影响因素的关系。通过受试者工作特征（ROC）曲线明确血清Neuritin、NFL、S100B蛋白水平联合检测预测脊柱骨折合并SCI患者预后不良的效能。结果：损伤组及无损伤组血清Neuritin、NFL、S100B蛋白水平均明显高于对照组,且损伤组上述三项血清学指标水平均高于无损伤组（均P＜0.05）。预后不良组椎管侵占率高于预后良好组,且血清Neuritin、NFL、S100B蛋白水平均高于预后良好组（均P＜0.05）。经多因素Logistic回归分析可得：椎管侵占率以及血清Neuritin、NFL、S100B蛋白水平较高均是脊柱骨折合并SCI患者预后不良的危险因素（P＜0.05）。血清Neuritin、NFL、S100B蛋白水平联合检测预测脊柱骨折合并SCI患者预后不良的曲线下面积、灵敏度、特异度、约登指数均高于上述三项指标单独检测。结论：脊柱骨折合并SCI患者血清Neuritin、NFL、S100B蛋白水平较高,且随着上述三项血清学指标水平的升高,患者预后不良风险更高。     

门冬氨酸鸟氨酸注射液联合乳果糖对肝性脑病患者肝功能和炎性因子水平的影响

目的:探讨门冬氨酸鸟氨酸注射液联合乳果糖对肝性脑病(HE)患者肝功能和炎性因子水平的影响。方法:选取2014年1月～2018年1月期间广西医科大学附属柳铁中心医院收治的HE患者120例为研究对象。根据数字表法将患者随机分为对照组(n=60)和研究组(n=60),对照组给予乳果糖治疗,研究组在对照组基础上联合门冬氨酸鸟氨酸注射液治疗,两组患者均持续治疗14 d。比较两组患者治疗后临床疗效,以及两组患者治疗前后肝功能、炎性因子水平、血尿淀粉酶(AMY)、血氨浓度。记录两组患者治疗期间不良反应发生情况。结果:研究组患者治疗后总有效率为91.67%(55/60),显著高于对照组患者的63.33%(38/60)(P0.05)。两组患者治疗后谷丙转氨酶(ALT)、总胆红素(TBIL)、天冬氨酸转氨酶(AST)水平均较治疗前降低,且研究组低于对照组(P0.05)。两组患者治疗后血清白介素-6(IL-6)、白介素-8(IL-8)、白介素-1(IL-1)、肿瘤坏死因子-α(TNF-α)、超敏C反应蛋白(hs-CRP)水平均较治疗前降低,且研究组低于对照组(P0.05)。两组患者治疗后血AMY、尿AMY、血氨浓度均较治疗前降低,且研究组血氨浓度低于对照组(P0.05),但研究组血AMY、尿AMY与对照组比较差异无统计学意义(P0.05)。两组患者治疗期间不良反应率比较差异无统计学意义(P0.05)。结论:门冬氨酸鸟氨酸注射液联合乳果糖治疗HE患者安全有效,可有效改善患者肝功能,减轻炎性应激,降低血尿AMY、血氨浓度,具有一定的临床应用价值。     

血清肌钙蛋白I、同型半胱氨酸、乳酸联合检测对动脉瘤性蛛网膜下腔出血患者术后转归的预测价值分析

摘要 目的：研究血清肌钙蛋白I（cTnI）、同型半胱氨酸（Hcy）以及乳酸（Lac）联合检测对动脉瘤性蛛网膜下腔出血（aSAH）患者术后转归的预测价值。方法：将本院从2018年1月～2020年1月期间收治的115例aSAH患者纳入研究,记作研究组。另取100例健康体检志愿者作为对照组。检测并比较所有受试者血清cTnI、Hcy以及Lac水平。此外,按照术后转归情况将研究组患者分作转归不良组以及转归良好组,比较两组各项基线资料以及血清cTnI、Hcy、Lac水平,并采用多因素Logistic回归分析aSAH患者术后转归不良的影响因素。另外,借助受试者工作特征（ROC）曲线分析明确cTnI、Hcy及Lac联合检测对aSAH术后转归不良的预测价值。结果：研究组血清cTnI、Hcy以及Lac水平均明显高于对照组（P＜0.05）。转归不良组血清Hcy以及Lac水平均高于转归良好组（P＜0.05）。经单因素分析可得：Hunt-Hess分级、动脉瘤大小、术前格拉斯哥昏迷指数（GCS）评分、术前脑疝、术前再出血均和aSAH患者术后转归密切相关（均P＜0.05）。经多因素Logistic回归分析发现：Hunt-Hess分级Ⅳ～Ⅴ级、动脉瘤大小≥5 mm、术前GCS评分、术前脑疝、术前再出血及血清cTnI、Hcy、Lac均是研究组患者术后转归不良的危险因素（均P＜0.05）。经ROC曲线分析可得：血清cTnI、Hcy及Lac联合预测aSAH患者术后转归不良的曲线下面积、灵敏度、特异度以及约登指数均高于上述三项指标单独预测。结论：血清cTnI、Hcy及Lac联合检测对aSAH患者术后转归不良的预测价值较高。     

血清降钙素原、C反应蛋白/白蛋白比值联合尿白细胞酯酶对复杂性肾结石患者经皮肾镜碎石术后发生尿路感染的预测价值分析

摘要 目的：探讨血清降钙素原（PCT）、C反应蛋白（CRP）/白蛋白（ALB）比值联合尿白细胞酯酶（LEU）对复杂性肾结石患者经皮肾镜碎石术（PCNL）后发生尿路感染的预测价值。方法：选择2015年3月-2021年6月我院收治的128例行PCNL的复杂性肾结石患者作为观察对象,术后定期随访,统计尿路感染情况,根据是否发生术后尿路感染分为尿路感染组和非尿路感染组。比较两组PCT水平、LEU,CRP/ALB比值及其他临床资料。采用多因素Logistic回归分析术后尿路感染的危险因素,绘制受试者工作特征（ROC）曲线,分析PCT、CRP/ALB比值、LEU单独以及联合检测预测术后尿路感染的价值。结果：尿路感染组的血清PCT、CRP/ALB高于非尿路感染组（P<0.05）。尿路感染组的LEU呈阳性,而非尿路感染组的LEU呈阴性。两组在既往泌尿道手术史、手术时间、导尿管留置时间、住院时间、术前尿路感染、结石负荷、使用>3种抗菌药物、合并肾功能障碍、术中通道类型、术前血糖水平方面对比有统计学差异（P<0.05）。既往泌尿道手术史、手术时间≧100 min、导尿管留置时间≧7 d、术前尿路感染、结石负荷≧1000 mm2、术前血糖水平高、合并肾功能障碍是复杂性肾结石患者PCNL后发生尿路感染的危险因素（P<0.05）。PCT、CRP/ALB、LEU单独预测PCNL后尿路感染的曲线下面积（AUC）分别为0.712（0.476～0.944）、0.686（0.436～0.931）、0.836（0.753～0.918）,三者联合预测PCNL后尿路感染的AUC为0.879（0.785～0.972）,均高于PCT、CRP/ALB、LEU单独预测。结论：复杂性肾结石患者PCNL后发生尿路感染的影响因素较多,包括导尿管留置时间、住院时间、结石负荷等,且PCNL后发生尿路感染患者PCT、CRP/ALB比值偏高,LEU呈阳性,三者可能作为PCNL后发生尿路感染的生物标记物。     

内毒素、PCT联合NLR对经皮肾镜碎石术后患者尿源性脓毒血症的预测价值

摘要 目的：探讨内毒素、降钙素原（PCT）联合中性粒细胞与淋巴细胞比值（NLR）对经皮肾镜碎石术（PCNL）术后患者发生尿源性脓毒血症的预测价值。方法：选取2020年5月-2023年5月于西安医学院第二附属医院和空军军医大学第一附属医院泌尿外科行PCNL的患者750例作为研究对象。根据尿源性脓毒症发生情况分为尿源性脓毒血症组（n=45）和非脓毒血症组（n=705）。检测PCNL术前血清内毒素、PCT、中性粒细胞与淋巴细胞水平,并计算NLR。对比两组血清内毒素、PCT水平及NLR。采用多因素Logistic回归模型分析PCNL术后患者发生尿源性脓毒血症的影响因素。绘制受试者工作特征（ROC）曲线分析血清内毒素、PCT联合NLR预测PCNL术后患者发生尿源性脓毒血症的临床效能。结果：与非脓毒血症组相比,尿源性脓毒血症组血清内毒素、PCT及NLR更高（P<0.05）。多因素Logistic回归模型分析结果显示,血清内毒素升高、PCT升高、NLR升高、尿白细胞阳性、术前发热及鹿角型结石是PCNL术后患者发生尿源性脓毒血症的独立危险因素（P<0.05）;ROC曲线分析结果显示,血清内毒素、PCT联合NLR检测预测PCNL术后患者发生尿源性脓毒血症的曲线下面积（AUC）为0.913,高于上述各指标单独检测。结论：PCNL术前血清内毒素、PCT和NLR升高可能与术后患者发生尿源性脓毒血症有关。血清内毒素、PCT水平升高、NLR升高、术前发热、尿白细胞阳性、鹿角型结石是PCNL术后患者发生尿源性脓毒血症的危险因素。血清内毒素、PCT联合NLR检测对PCNL术后患者发生尿源性脓毒血症具有较高预测价值。     

互动式头针联合认知训练对脑卒中后认知功能障碍患者认知功能、事件相关电位P300和血清NSE、S100β蛋白的影响

摘要 目的：观察认知训练联合互动式头针对脑卒中后认知功能障碍患者事件相关电位P300、认知功能和血清神经元特异性烯醇化酶（NSE）、S100β蛋白的影响。方法：选取自2019年3月~2022年1月期间广州中医药大学第二附属医院收治的120例脑卒中后认知功能障碍患者。按抛掷硬币法将患者分为对照组（n=60,常规治疗）和研究组（n=60,常规治疗基础上接受互动式头针训练）。对比两组疗效、认知功能、事件相关电位P300和血清NSE、S100β蛋白的差异。结果：研究组的临床总有效率91.67%（55/60）高于对照组75%（45/60）,差异有统计学意义（P<0.05）。研究组治疗后简易智力状态检查量表（MMSE）、蒙特利尔认知评估量表（MoCA）评分高于对照组（P<0.05）。研究组治疗后血清NSE、S100β蛋白水平低于对照组（P<0.05）。研究组治疗后潜伏期短于对照组,波幅长于对照组（P<0.05）。结论：认知训练联合互动式头针可有效改善脑卒中后认知功能障碍患者的认知功能,且与调节事件相关电位P300和血清NSE、S100β蛋白水平有关。     

高频重复经颅磁刺激联合言语听觉反馈训练对脑卒中后认知功能障碍患者事件相关电位P300和血清NSE、S100β蛋白的影响

摘要 目的：探讨高频重复经颅磁刺激（rTMS）联合言语听觉反馈训练对脑卒中后认知功能障碍（PSCI）患者认知功能的改善作用及对事件相关电位P300和血清神经元特异性烯醇化酶（NSE）、S100β蛋白的影响。方法：研究对象来源于徐州市中心医院2020年3月~2022年2月期间收治的130例PSCI患者。根据随机数字表法将患者分为对照组（n=65）和实验组（n=65）,对照组患者接受常规治疗方案和言语听觉反馈训练,实验组在对照组的基础上联合高频rTMS。对比两组干预8周后认知功能、生活质量、事件相关电位P300和血清NSE、S100β蛋白的变化情况。结果：干预8周后,两组蒙特利尔认知评估量表（MoCA）、Rivermead行为记忆测验（RBMT）升高,且实验组高于对照组（P<0.05）;干预8周后,两组维多利亚版Stroop测试（VST）下降,且实验组低于对照组（P<0.05）。干预8周后,两组改良的Barthel指数（MBI）、日常生活能力量表（ADL）评分升高,且实验组高于对照组（P<0.05）。干预8周后,两组波幅升高,且实验组高于对照组（P<0.05）;干预8周后,两组潜伏期下降,且实验组低于对照组（P<0.05）。干预8周后,两组血清NSE、S100β蛋白水平下降,且实验组低于对照组（P<0.05）。结论：高频rTMS联合言语听觉反馈训练可有效改善PSCI患者的认知功能,同时还可调节事件相关电位P300和血清NSE、S100β蛋白水平,提高患者的生活质量。     

Protein S100B release from rat brain slices during and after ischemia: comparison with lactate dehydrogenase leakage

One hour of ischemia significantly increased protein S100B release from rat brain slices without altering lactate dehydrogenase leakage. Reoxygenation of the ischemic slices, however, increased the levels of these biochemical markers in the medium. Although removal of extracellular Ca⁺² ions from the medium did not alter the basal lactate dehydrogenase leakage from cortical slices, an excessive increase in basal protein S100B release was seen under this condition. Ischemia and/or reoxygenation induced enhancements in these markers were attenuated by removal of Ca⁺² ions from the medium. Ischemia significantly increased glutamate release, but neither ischemia nor reoxygenation induced rises in protein S100B and lactate dehydrogenase levels were altered by glutamate receptor antagonists. Rising the glutamate levels in the medium by each ouabain or exogenous glutamate, moreover, failed in exerting an ischemia like effect on protein S100B and LDH outputs. In contrast, exogenous glutamate added into the medium protected the slices against reoxygenation induced increments in protein S100B and lactate dehydrogenase levels.

These results indicate that protein S100B has a greater sensitivity against ischemia than lactate dehydrogenase in in vitro brain slice preparations. Since neither exogenous glutamate nor enhancements of the extracellular glutamate levels by ouabain had an ischemia like effect, and since glutamate receptor antagonists were also unsuccessful, it seems unlikely that ischemia-induced increase in glutamate release is directly involved in protein S100B release or lactate dehydrogenase leakage determined in the present study.     

Identification of the binding site on S100B protein for the actin capping protein CapZ.

The calcium-binding protein S100B binds to several potential target proteins, but there is no detailed information showing the location of the binding site for any target protein on S100B. We have made backbone assignments of the calcium-bound form of S100B and used chemical-shift changes in spectra of 15N-labeled protein to locate the site that binds a peptide corresponding to residues 265-276 from CapZ alpha, the actin capping protein. The largest chemical-shift changes are observed for resonances arising from residues around the C terminus of the C-terminal helix of S100B and residues Val-8 to Asp-12 of the N-terminal helix. These residues are close to but not identical to residues that have been identified by mutational analysis to be important in other S100 protein-protein interactions. They make up a patch across the S100B dimer interface and include some residues that are quite buried in the structure of calcium-free S100B. We believe we may have identified a binding site that could be common to many S100 protein-protein interactions.     

S100B protein is released from rat neonatal neurons, astrocytes, and microglia by in vitro trauma and anti-S100 increases trauma-induced delayed neuronal injury and negates the protective effect of exogenous S100B on neurons

S100B protein is found in brain, has been used as a marker for brain injury and is neurotrophic. Using a well-characterized in vitro model of brain cell trauma, we have previously shown that strain injury causes S100B release from neonatal rat neuronal plus glial cultures and that exogenous S100B reduces delayed post-traumatic neuronal damage even when given at 6 or 24 h post-trauma. The purpose of the current studies was to measure post-traumatic S100B release by specific brain cell types and to examine the effect of an antibody to S100 on post-traumatic delayed (48 h) neuronal injury and the protective effect of exogenous S100B. Neonatal rat cortical cells grown on a deformable elastic membrane were subjected to a strain (stretch) injury produced by a 50 ms displacement of the membrane. S100B was measured with an ELISA kit. Trauma released S100B from pure cultures of astrocytes, microglia, and neurons. Anti-S100 reduced released S100B to below detectable levels, increased delayed neuronal injury in traumatized cells and negated the protective effect of exogenous S100B on injured cells. Heat denatured anti-S100 did not exacerbate injury. These studies provide further evidence for a protective role for S100B following neuronal trauma.     

Differentiating ischemic from hemorrhagic stroke using plasma biomarkers: the S100B/RAGE pathway

Although neuroimaging is useful in differentiating ischemic (IS) from hemorrhagic (ICH) stroke in the Emergency Department, a wide-available rapid biochemical test would add advantages in the pre-hospital triage and management of stroke patients. Our aim was to examine the predictive value of a panel of blood-borne biomarkers to differentiate IS from ICH. Admission blood samples obtained within 24h from stroke symptoms onset were tested by ELISA for CRP, D-dimer, sRAGE, MMP9, S100B, BNP, NT-3, caspase-3, chimerin-II, secretagogin, cerebellin and NPY. The complete protocol was achieved in 915 patients (776 IS, 139 ICH). Among blood samples obtained <6 h from symptoms onset (n=337), S100B levels were increased in ICH (107.58 vs 58.70 pg/mL; p<0.001) whereas sRAGE levels were decreased (0.77 vs 1.02 ng/mL; p=0.009) as compared to IS. In this subset of patients S100B (OR 3.97 95% CI 1.82-8.68; p=0.001) and sRAGE (OR 0.22 95% CI 0.10-0.52; p<0.001) were independently associated with ICH. A regression tree was created by CART method showing good classification ability (AUC=0.762). Similar results were found for samples obtained within 3 h. In conclusion, a combination of biomarkers including those of the S100B/RAGE pathway seems promising to achieve a rapid biochemical diagnosis of IS versus ICH in the first hours from symptoms onset. This article is part of a Special Issue entitled: Translational Proteomics.     

The S100B protein in biological fluids: more than a lifelong biomarker of brain distress

S100B is a calcium-binding protein concentrated in glial cells, although it has also been detected in definite extra-neural cell types. Its biological role is still debated. When secreted, S100B is believed to have paracrine/autocrine trophic effects at physiological concentrations, but toxic effects at higher concentrations. Elevated S100B levels in biological fluids (CSF, blood, urine, saliva, amniotic fluid) are thus regarded as a biomarker of pathological conditions, including perinatal brain distress, acute brain injury, brain tumors, neuroinflammatory/neurodegenerative disorders, psychiatric disorders. In the majority of these conditions, high S100B levels offer an indicator of cell damage when standard diagnostic procedures are still silent. The key question remains as to whether S100B is merely leaked from injured cells or is released in concomitance with both physiological and pathological conditions, participating at high concentrations in the events leading to cell injury. In this respect, S100B levels in biological fluids have been shown to increase in physiological conditions characterized by stressful physical and mental activity, suggesting that it may be physiologically regulated and raised during conditions of stress, with a putatively active role. This possibility makes this protein a candidate not only for a biomarker but also for a potential therapeutic target.     

The Zn2+ and Ca2+‐binding S100B and S100A1 proteins: beyond the myths

The S100 genes encode a conserved group of 21 vertebrate‐specific EF‐hand calcium‐binding proteins. Since their discovery in 1965, S100 proteins have remained enigmatic in terms of their cellular functions. In this review, we summarize the calcium‐ and zinc‐binding properties of the dimeric S100B and S100A1 proteins and highlight data that shed new light on the extracellular and intracellular regulation and functions of S100B. We point out that S100B and S100A1 homodimers are not functionally interchangeable and that in a S100A1/S100B heterodimer, S100A1 acts as a negative regulator for the ability of S100B to bind Zn²⁺. The Ca²⁺ and Zn²⁺‐dependent interactions of S100B with a wide array of proteins form the basis of its activities and have led to the derivation of some initial rules for S100B recognition of protein targets. However, recent findings have strongly suggested that these rules need to be revisited. Here, we describe a new consensus S100B binding motif present in intracellular and extracellular vertebrate‐specific proteins and propose a new model for stable interactions of S100B dimers with full‐length target proteins. A chaperone‐associated function for intracellular S100B in adaptive cellular stress responses is also discussed. This review may help guide future studies on the functions of S100 proteins in general.     

Identification of a dimeric intermediate in the unfolding pathway for the calcium-binding protein S100B

The S100 proteins comprise 25 calcium-signalling members of the EF-hand protein family. Unlike typical EF-hand signalling proteins such as calmodulin and troponin-C, the S100 proteins are dimeric, forming both homo- and heterodimers in vivo. One member of this family, S100B, is a homodimeric protein shown to control the assembly of several cytoskeletal proteins and regulate phosphorylation events in a calcium-sensitive manner. Calcium binding to S100B causes a conformational change involving movement of helix III in the second calcium-binding site (EF2) that exposes a hydrophobic surface enabling interactions with other proteins such as tubulin and Ndr kinase. In several S100 proteins, calcium binding also stabilizes dimerization compared to the calcium-free states. In this work, we have examined the guanidine hydrochloride (GuHCl)-induced unfolding of dimeric calcium-free S100B. A series of tryptophan substitutions near the dimer interface and the EF2 calcium-binding site were studied by fluorescence spectroscopy and showed biphasic unfolding curves. The presence of a plateau near 1.5 M GuHCl showed the presence of an intermediate that had a greater exposed hydrophobic surface area compared to the native dimer based on increased 4,4-dianilino-1,1′-binaphthyl-5,5′-disulfonic acid fluorescence. Furthermore, ¹H-¹⁵N heteronuclear single quantum coherence analyses as a function of GuHCl showed significant chemical shift changes in regions near the EF1 calcium-binding loop and between the linker and C-terminus of helix IV. Together these observations show that calcium-free S100B unfolds via a dimeric intermediate.     

S100B(betabeta) inhibits the protein kinase C-dependent phosphorylation of a peptide derived from p53 in a Ca2+-dependent manner.

S100B(betabeta) is a dimeric Ca2+-binding protein that is known to inhibit the protein kinase C (PKC)-dependent phosphorylation of several proteins. To further characterize this inhibition, we synthesized peptides based on the PKC phosphorylation domains of p53 (residues 367-388), neuromodulin (residues 37-53), and the regulatory domain of PKC (residues 19-31), and tested them as substrates for PKC. All three peptides were shown to be good substrates for the catalytic domain of PKC. As for full-length p53 (Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. 1992. Proc Natl Acad Sci USA 89:11627-11631), S100B(betabeta) binds the p53 peptide and inhibits its PKC-dependent phosphorylation (IC50 = 10 +/- 7 microM) in a Ca2+-dependent manner. Similarly, phosphorylation of the neuromodulin peptide and the PKC regulatory domain peptide were inhibited by S100B(betabeta) in the presence of Ca2+ (IC50 = 17 +/- 5 microM; IC50 = 1 +/- 0.5 microM, respectively). At a minimum, the C-terminal EF-hand Ca2+-binding domain (residues 61-72) of each S100beta subunit must be saturated to inhibit phosphorylation of the p53 peptide as determined by comparing the Ca2+ dependence of inhibition ([Ca]IC50 = 29.3 +/- 17.6 microM) to the dissociation of Ca2+ from the C-terminal EF-hand Ca2+-binding domain of S100B(betabeta).     

Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: potential role for oxidative regulation

S100A8 and S100A9 and their heterocomplex calprotectin (S100A8/A9) are abundant cytosolic constituents in human neutrophils previously shown to possess antifungal activity. This study was designed to investigate mechanisms involved in the modulation of the antifungal properties of S100A8/A9. S100A8, S100A9 and site-directed mutants of both proteins were tested for their antifungal effect against Candida albicans in microplate dilution assays. Whereas S100A8 alone did not inhibit fungal growth, S100A9 by itself had a moderate antifungal effect. Combining both proteins had the strongest effect. Supporting a potential role for oxidation in S100A8/A9, substitution of methionine 63 or 83 of S100A9 resulted in the loss of antifungal activity. Additionally, the substitution to alanine of cysteine 42 of S100A8 also caused a loss of S100A8's ability to enhance S100A9's antifungal effect. Overall, our data indicate that both S100A8 and S100A9 are required for their fully active antifungal effect and that oxidation regulates S100A8/A9 antifungal activity through mechanisms that remain to be elucidated and evaluated. Finally, together with our previous work describing the oxidation-sensitive anti-inflammatory effects of S100A8/A9, we propose that S100A8/A9 exerts an anti-inflammatory activity in healthy state and that conditions associated with oxidative stress activate the antifungal activity of S100A8/A9.