Hemodynamic response pattern predicts susceptibility to stress-induced elevation in arterial pressure in the rat

Cocaine or air jet stress evokes pressor responses due to either a large increase in systemic vascular resistance (vascular responders) or small increases in both cardiac output and vascular resistance (mixed responders) in conscious rats. Repeated cocaine administration results in elevated arterial pressure in vascular responders but not in mixed responders. The present study examined the hypothesis that the pattern of cardiovascular responses to an unconditioned stimulus (UCS; air jet) is related to responses to a conditioned stimulus (CS; tone followed by brief foot shock) in individual rats. Our data demonstrate that presentation of the UCS produced variable cardiac output responses that correlated with responses to the CS (n = 60). We also determined whether individual cardiovascular response patterns to acute stress correlated with predisposition to a sustained stress-induced elevation in arterial pressure. Rats were exposed to three different stressors presented one per day successively for 4 wk and during a poststress period of 3 wk while arterial pressure was recorded periodically. Mean arterial pressure was elevated in all rats during chronic stress but, during the poststress period, remained at significantly higher levels in vascular responders but not mixed responders. Therefore, we conclude that acute behavioral stress to a conditioned stimulus elicits variable hemodynamic responses that predict the predisposition to a sustained stress-induced elevation in arterial pressure.     

Networking senescence-regulating pathways by using Arabidopsis enhancer trap lines

The last phase of leaf development, generally referred to as leaf senescence, is an integral part of plant development that involves massive programmed cell death. Due to a sharp decline of photosynthetic capacity in a leaf, senescence limits crop yield and forest plant biomass production. However, the biochemical components and regulatory mechanisms underlying leaf senescence are poorly characterized. Although several approaches such as differential cDNA screening, differential display, and cDNA subtraction have been employed to isolate senescence-associated genes (SAGs), only a limited number of SAGs have been identified, and information regarding the regulation of these genes is fragmentary. Here we report on the utilization of enhancer trap approach toward the identification and analysis of SAGs. We have developed a sensitive large-scale screening method and have screened 1,300 Arabidopsis enhancer trap lines and have identified 147 lines in which the reporter gene GUS (beta-glucuronidase) is expressed in senescing leaves but not in non-senescing ones. We have systematically analyzed the regulation of beta-glucuronidase expression in 125 lines (genetically, each contains single T-DNA insertion) by six senescence-promoting factors, namely abscisic acid, ethylene, jasmonic acid, brassinosteroid, darkness, and dehydration. This analysis not only reveals the complexity of the regulatory circuitry but also allows us to postulate the existence of a network of senescence-promoting pathways. We have also cloned three SAGs from randomly selected enhancer trap lines, demonstrating that reporter expression pattern reflects the expression pattern of the endogenous gene.     

CXC and CC chemokine receptors on coronary and brain endothelia

BACKGROUND: Chemokine receptors on leukocytes play a key role in inflammation and HIV-1 infection. Chemokine receptors on endothelia may serve an important role in HIV-1 tissue invasion and angiogenesis. MATERIALS AND METHODS: The expression of chemokine receptors in human brain microvascular endothelial cells (BMVEC) and coronary artery endothelial cells (CAEC) in vitro and cryostat sections of the heart tissue was determined by light and confocal microscopy and flow cytometry with monoclonal antibodies. Chemotaxis of endothelia by CC chemokines was evaluated in a transmigration assay. RESULTS: In BMVEC, the chemokine receptors CCR3 and CXCR4 showed the strongest expression. CXCR4 was localized by confocal microscopy to both the cytoplasm and the plasma membrane of BMVEC. In CAEC, CXCR4 demonstrated a strong expression with predominantly periplasmic localization. CCR5 expression was detected both in BMVEC and CAEC but at a lower level. Human umbilical cord endothelial cells (HUVEC) expressed strongly CXCR4 but only weakly CCR3 and CCR5. Two additional CC chemokines, CCR2A and CCR4, were detected in BMVEC and CAEC by immunostaining. Immunocytochemistry of the heart tissues with monoclonal antibodies revealed a high expression of CXCR4 and CCR2A and a low expression of CCR3 and CCR5 on coronary vessel endothelia. Coronary endothelia showed in vitro a strong chemotactic response to the CC chemokines RANTES, MIP-1alpha, and MIP-1beta. CONCLUSIONS: The endothelia isolated from the brain display strongly both the CCR3 and CXCR4 HIV-1 coreceptors, whereas the coronary endothelia express strongly only the CXCR4 coreceptor. CCR5 is expressed at a lower level in both endothelia. The differential display of CCR3 on the brain and coronary endothelia could be significant with respect to the differential susceptibility of the heart and the brain to HIV-1 invasion. In addition, CCR2A is strongly expressed in the heart endothelium. All of the above chemokine receptors could play a role in endothelial migration and repair.     

Protease and protease inhibitor assays using biotinylated casein coated on a solid phase

A new type of solid-phase assay for proteases and protease inhibitors has been developed using biotinylated casein. The assay involves coating of titer plate wells with biotinylated casein, hydrolysis of this substrate with a protease such as trypsin, reaction of the biotin from the unhydrolyzed substrate with an alkaline phosphatase-streptavidin complex, and finally quantification of the amount of casein remaining on the plate using alkaline phosphatase activity as the indicator. The activity of the bound indicator enzyme is oppositely related to the protease activity of the sample. In addition, the assay can be modified for quantitating the corresponding amount of protease inhibitor in the sample. The assay is simple, sensitive, accurate, inexpensive, and amenable to automation.     

Increased endothelin-1 and endothelin receptor expression in myocytes of ischemic and reperfused rat hearts and ventricular myocytes exposed to ischemic conditions and its inhibition by nitric oxide generation

Endothelin-1 (ET-1) and nitric oxide (NO) exert opposite effects in the cardiovascular system, and there is evidence that the NO counters the potential deleterious effects of ET-1. We investigated whether NO affects the increased mRNA expression of ET-1 and endothelin receptors induced by (i) 30 min of ischemia with or without 30 min reperfusion in myocytes from isolated rat hearts or (ii) ischemic conditions (acidosis or hypoxia) in cultured rat neonatal ventricular myocytes. Ischemia with or without reperfusion produced more than a twofold increase in mRNA expression of ET-1 as well as the ET(A) and ET(B) receptor (P < 0.05), although these effects were completely blocked by the NO donor 3-morpholinosydnonimine (SIN-1; 1 microM). To assess the possible factors regulating ET expression, myocytes were exposed to acidosis (pH 6.8-6.2) or to hypoxic conditions in an anaerobic chamber for 24 h in the presence or absence of SIN-1. At all acidic pHs, ET-1 and ET(A) receptor mRNA expression was significantly (P < 0.05) elevated approximately threefold, although the magnitude of elevation was independent of the degree of acidosis. These effects were completely prevented by SIN-1. ET(B) receptor expression was unaffected by acidosis. Hypoxia increased ET-1 as well as ET(A) and ET(B) receptor expression threefold (P < 0.05), although this was unaffected by SIN-1. Our results demonstrate that myocardial ischemia and reperfusion upregulate the ET system, which is inhibited by NO. Although increased expression of the ET system can be mimicked by both acidosis and hypoxia, only the effects of the former are NO sensitive. NO may serve an endogenous inhibitory factor which regulates the expression of the ET system under pathological conditions.     

Construction of Secretory Expression System Suitable to Express Glucagon Under the Control of PL Promoter

It was reported that P_L promoter and alkaline phosphatase (phoA) signal peptide were used to construct secretory expression plasmid suitable to express glucagon and [Des-His¹] glucagon in E. coli BL21 herein. Expression studies showed these two peptides could be expressed and secreted into the culture medium. The expression yield of recombinant glucagon reached 3.46 mg/L/OD₆₀₀ unit of cells in shake flask. The yield of [Des-His¹] glucagon was found to be higher than that of glucagon. In addition, some factors involved in secretion were studied too.Received: 26 September 2002 / Accepted: 24 October 2002     

小鼠胚胎干细胞分化为血管内皮细胞的永生化研究

本文探讨了小鼠胚胎干细胞（ES细胞）、诱导分化的血管内皮细胞永生化。在体外培养系统中,以维甲酸（RA）和转化生长因子－β1（TGF－β1）诱导小鼠胚胎干细胞（ES细胞）的拟胚体（EB）分化为“圆形细胞”和由这些“圆形细胞”组成的血管样结构。经光学和扫描电镜及免疫荧光等法分析检测,证明组成血管样结构的细胞具有专一性vWF荧光染色,表明是血管内皮样细胞。利用脂质体将人端粒酶催化亚基逆转录酶（hTERT)基因转染诱导分化中的“圆形细胞”。应用Dot-blot,RT－PCR,Western blot及免疫组织化学等方法分析、观察和证明了诱导分化的组成血管样结构的园形细胞和被hTERT基因转染的“圆形”细胞的形态和生物学特性。结果表明,携带hTERT基因的从ES细胞分化来的圆形细胞在体外可大量增殖,持续传代,95％具有血管内皮细胞的一些特有标志和管道化生长特征。因此,通过人端粒酶基因的转染途径可解决由ES细胞诱导分化而来的内皮细胞扩增和永生化问题,为构建组织工程化血管及其它人工血管的内皮化提供种子细胞来源打下基础。     

Critical role of mitochondrial damage in determining outcome of macrophage infection with Mycobacterium tuberculosis

Human macrophages (Mphi) respond to Mycobacterium tuberculosis (Mtb) infection by undergoing apoptosis, a cornerstone of effective antimycobacterial host defense. Virulent mycobacteria override this reaction by inducing necrosis leading to uncontrolled Mtb replication. Accordingly, Mphi death induced by inoculation with Mtb had the characteristics of apoptosis and necrosis and correlated with moderate increase of mitochondrial permeability transition (MPT), mitochondrial cytochrome c release, and caspase-9 and -3 activation. We hypothesized that changes in intramitochondrial Ca(2+) concentration ([Ca(2+)](m)) determine whether Mphi undergo either apoptosis or necrosis. Therefore, we induced mechanism(s) leading to predominant apoptosis or necrosis by modulating [Ca(2+)](m) and examined their physiological consequences. Adding calcium ionophore A23187 to Mphi inoculated with Mtb further increased calcium flux into the cells which is thought to lead to increased [Ca(2+)](m), blocked necrosis, stabilized MPT, decreased mitochondrial cytochrome c release, lowered caspase activation, and accompanied effective antimycobacterial activity. In contrast, Mphi infected with Mtb in presence of the mitochondrial calcium uniporter inhibitor ruthenium red showed increased mitochondrial swelling and cytochrome c release and decreased MPT and antimycobacterial activity. Thus, in Mtb-infected Mphi, high levels of mitochondrial membrane integrity, low levels of caspase activation, and diminished mitochondrial cytochrome c release are hallmarks of apoptosis and effective antimycobacterial activity. In contrast, breakdown of mitochondrial membrane integrity and increased caspase activation are characteristic of necrosis and uncontrolled Mtb replication.