人胰高血糖素样肽-1类似物基因治疗对STZ诱导糖尿病大鼠的拮抗作用

目的 观察人胰高血糖素样肽-1(hGLP-1)类似物基因对链脲佐菌素(STZ)诱导的糖尿病大鼠的拮抗作用.方法 将SD大鼠随机分为正常对照组、糖尿病模型组、空载体对照组及基因治疗拮抗组,每组10只.应用小剂量、多次给药的方式诱导糖尿病大鼠模型,观察hGLP-1类似物基因对大鼠血糖、血清胰岛素及胆固醇水平的影响.借助荧光显微镜观察绿色荧光蛋白在不同组织中的表达情况;采用免疫组化分析胰岛细胞中胰岛素的表达水平.结果 与糖尿病模型组和空载体对照组比较,基因治疗拮抗组糖尿病大鼠血糖水平下降、血清胰岛素水平升高、血清胆固醇水平下降.胰岛素免疫组化分析证实,胰岛细胞中胰岛素得以表达,基因治疗可使受损的胰岛细胞功能恢复.结论 hGLP-1类似物基因治疗对STZ诱导的糖尿病症状具有拮抗作用,改善糖尿病大鼠血糖、血清胰岛素和血脂水平.     

Gonadotrophin surge-inhibiting/attenuating factor governs luteinizing hormone secretion during the ovarian cycle: physiology and pathology

The stabilization of low luteinizing hormone (LH) concentrationsduring the period of the ovarian cycle preceding the mid-cycleLH surge seems to be of importance for embryo viability andsurvival. Several studies have stressed the importance of thetimely excess of threshold levels of LH for optimal oocyte quality:LH already initiates the resumption of meiosis when a relativelylow threshold level is reached, whereas a further outburst ofLH release is necessary to reach the threshold level to induceovulation. Hence, the mechanism of LH release should have theability, on the one hand, to limit the LH secretion rate, andon the other, to allow an increased secretion rate, as duringthe LH surge. The functional antagonistic coupling of gonadotrophin-releasinghormone (GnRH) and the ovarian protein gonadotrophin surge-inhibitingfactor or -attenuating factor (GnSIF/AF) provides such a modeof action for the control of LH concentration during the ovariancycle. One of the important regulatory steps in this processis the de-novo synthesis of the so far unidentified pituitaryproteins induced by GnRH. The induction of these proteins isa prerequisite for the increase in the rate of LH release. Becausetheir synthesis is a time-consuming process, the effect becomesvisible in the typical biphasic pituitary LH responses to thepulsatile or continuous administration of GnRH: initially low,with an increase after some time. This phenomenon is also knownas the GnRH self-priming action. It is assumed that the synthesisof these self-priming-associated proteins is necessary to eliminatethe inhibitory effect of GnSIF/AF. GnSIF/AF eliminates the effectof self-priming by neutralizing the biological activity of thepituitary proteins, which are responsible for the increasedrate of LH release. Thus, the pituitary gland is kept in a GnRH-hyporesponsivestate. The major advantage of such a slow protein synthesisdependentregulatory mechanism is that it prevents sudden increases inthe LH secretion rate in response to GnRH. Thus it stabilizeslow LH concentrations to prevent the premature reinitiationof meiosis. However, the enhanced secretion of GnRH and/or thesuppressed release or action of GnSIF/AF may finally lead tothe restoration of the intrinsic LH responsiveness of the gonadotrophsat the start of the mid-cycle LH surge. The antagonistic interactionbetween GnRH and GnSIF/AF, and its implication in the controlof LH release under physiological and pathological conditions,are discussed.     

Block of sodium currents by the calcium antagonist D600 in human heart cell segments

The effects of micromolar concentrations of racemic D600 on the transmembrane inward sodium current (I_Na) were investigated in voltage clamped, intracellularly perfused, human heart cell segments. Extracellular D600 blocked I_Na in a voltage- and ratedependent manner as shown by the enhanced I_Na depression with a reduction of the resting transmembrane polarization (Vm) and stimulation interval (SI). D600 action was manifested as a voltage-dependent slowing down of the Na⁺ channels' recovery kinetics after a pulsed excitation, with greater recovery times during longer depolarized states, excited or non-excited. This phenomenon seems linked to the improved control of the intracellular environment normally influencing channel activity, and to the increased ratio of non-sarcolemmal to sarcolemmal cell membranes for this preparation.     

Enprofylline - Effects of a New Bronchodilating Xanthine Derivative in Asthmatic Patients

The bronchodilating effect of two doses of peroral enprofylline was compared with placebo in 24 asthmatic patients. Enprofylline produced significantly greater bronchodilatation than placebo. A dose of 2 mg/kg b.wt. and 4 mg/kg b.wt. caused a mean maximal increase in FEV1 of 26% and 35%, respectively. The degree and the incidence of headache and nausea were estimated by means of a scoring system. Dose-related effects on both parameters were observed. Other side effects were negligible. In seven patients the mean plasma half-life of enprofylline was found to be 113 min. It is suggested that enprofylline should be studied further in patients suffering from obstructive lung disease.     

A new in-vitro kinetic model to study the pharmacodynamics of antifungal agents: inhibition of the fungicidal activity of amphotericin B against Candida albicans by voriconazole

The aim of this study was to develop and validate a new in-vitro kinetic model for the combination of two drugs with different half-lives, and to use this model for the study of the pharmacodynamic effects of amphotericin B and voriconazole, alone or in combination, against a strain of Candida albicans. Bolus doses of voriconazole and amphotericin B were administered to a starting inoculum of C. albicans. Antifungal-containing medium was eliminated and replaced by fresh medium using a peristaltic pump, with the flow-rate adjusted to obtain the desired half-life of the drug with the shorter half-life. A computer-controlled dosing pump compensated for the agent with the longer half-life. Voriconazole and amphotericin B half-lives were set to 6 and 24 h, respectively. Pharmacokinetic parameters were close to target values when both single doses and sequential doses were simulated. Voriconazole and amphotericin B administered alone demonstrated fungistatic and fungicidal activity, respectively. Simultaneous administration resulted in fungicidal activity, whereas pre-exposure of C. albicans to voriconazole, followed by amphotericin at 8 and 32 h, resulted in fungistatic activity similar to that observed with voriconazole alone. Using this model, which allowed a combination of antifungal agents with different half-lives, it was possible to demonstrate an antagonistic effect of voriconazole on the fungicidal activity of amphotericin B. The characteristics and clinical relevance of this interaction require further investigation.     

Antagonism screen for inhibitors of bacterial cell wall biogenesis uncovers an inhibitor of undecaprenyl diphosphate synthase

Drug combinations are valuable tools for studying biological systems. Although much attention has been given to synergistic interactions in revealing connections between cellular processes, antagonistic interactions can also have tremendous value in elucidating genetic networks and mechanisms of drug action. Here, we exploit the power of antagonism in a high-throughput screen for molecules that suppress the activity of targocil, an inhibitor of the wall teichoic acid (WTA) flippase in Staphylococcus aureus. Well-characterized antagonism within the WTA biosynthetic pathway indicated that early steps would be sensitive to this screen; however, broader interactions with cell wall biogenesis components suggested that it might capture additional targets. A chemical screening effort using this approach identified clomiphene, a widely used fertility drug, as one such compound. Mechanistic characterization revealed the target was the undecaprenyl diphosphate synthase, an enzyme that catalyzes the synthesis of a polyisoprenoid essential for both peptidoglycan and WTA synthesis. The work sheds light on mechanisms contributing to the observed suppressive interactions of clomiphene and in turn reveals aspects of the biology that underlie cell wall synthesis in S. aureus. Further, this effort highlights the utility of antagonistic interactions both in high-throughput screening and in compound mode of action studies. Importantly, clomiphene represents a lead for antibacterial drug discovery.Small molecules have long proven their utility as an alternative to genetic mutation in perturbing and understanding biological systems (1). Interactions between small molecules are comparable to those observed among genetic mutations. In combination, one molecule may enhance (synergism) or suppress (antagonism) the effect of another molecule. In recent years, drug combinations have been extensively used to explore connections between cellular processes. Specifically, synergies have provided unique means to expose pathway interactions (2, 3) and, in turn, understand the mode of action of small molecules (4, 5). Underpinning these interactions are complex genetic networks that define the outcome of drug combinations (6, 7). Indeed, synergistic pairs often exhibit their effects by targeting related processes and impacting genetic interactions that bridge those pathways. Further, synergistic drug combinations are now essential therapeutic strategies in the clinic in areas such as cancer, HIV, and infectious diseases (8). Where antagonistic interactions are generally undesirable from a therapeutic perspective, the utility of this class of interactions to reveal biological function and underlying network connectivity has been largely overlooked. Like synergism, antagonism typically has a genetic basis that reflects relationships between cellular targets and mechanisms of drug action (7). Recent reports of antagonistic drug interactions have shed light on novel connections that govern important bacterial physiology. For example, the suppressive effects of antibiotics targeting protein and DNA synthesis in Escherichia coli revealed antagonistic connections between the regulation of ribosomal genes and the DNA stress response (9). A more recent study surveyed suppressive interactions among antifungals and described the mechanism of the suppressive activities of bromopyruvate and staurosporine (10). Interestingly, but perhaps counterintuitively, other studies have suggested that antagonistic drug pairs can even slow the evolution of drug resistance (11, 12). Nevertheless, the utility of antagonism among small molecules has yet to be fully explored as a tool to study biological function. Certainly, there have been no systematic searches for antagonistic interactions to exploit suppressive network connections and, in turn, uncover novel inhibitors of the targeted pathways.Bacterial cell wall synthesis is an antibacterial target that is celebrated for its druggability and, increasingly, for its genetic complexity. Indeed, the dispensability of wall teichoic acid (WTA) genes in gram-positive bacteria has emerged in recent years as a prototypical example of genetic antagonism. WTAs are phosphate-rich polymers that make up a large proportion of the cell wall of gram-positive bacteria and, in the pathogen Staphylococcus aureus, have a key role in cell division and virulence (13, 14). The synthesis of WTA in S. aureus is initiated by the action of two nonessential gene products: TarO and TarA. TarO (undecaprenyl-phosphate N-acetylglucosaminyl 1-phosphate transferase) transfers an N-acetyl-glucosamine-1-phosphate moiety to an undecaprenyl phosphate carrier lipid, followed by the transfer of N-acetylmannosamine by TarA (N-acetylglucosaminyldiphospho-undecaprenol N-acetyl-β-d-mannosaminyltransferase). The resulting glycolipid is a substrate for so-called late-step gene products that append a polymer with ribitol-phosphate repeats before export to the external surface and attachment to peptidoglycan (PG). Paradoxically, the genes encoding the late-steps in WTA synthesis have an essential phenotype, but become dispensable in strains with a deletion in either of the early-step genes, tarO or tarA (encoding for an N-acetylglucosamine-1-phosphate transferase and an N-acetylmannosamine transferase, respectively) (15). Interestingly, blocking the early steps in WTA biosynthesis leads to β-lactam sensitivity in methicillin-resistant S. aureus (MRSA) (2, 16). These observations highlight the complexity of cell wall synthesis in gram-positive bacteria and provide a rationale for combination therapy. Further, the idiosyncratic genetic antagonism of the WTA biosynthetic pathway and interactions with additional components of cell wall synthesis provide a unique opportunity to screen for new chemical matter with utility as probes to better understand this genetic complexity.To this end, we conducted a search for compounds that antagonize the lethal activity of targocil (17) (Scheme 1), a probe of TarG, the essential gene product that makes up the transmembrane transporter that exports WTAs to the cell surface. Screening a library of previously approved drugs we discovered that clomiphene (Scheme 1) a widely used fertility drug, was a potent antagonist of targocil. Mechanistic characterization revealed that its target was the undecaprenyl diphosphate synthase (UppS), responsible for the synthesis of the lipid carrier, undecaprenyl phosphate (Und-P), and we solved a cocrystal structure of clomiphene with UppS from E. coli. We report on the ability of clomiphene to potentiate the activity of β-lactam antibiotics against MRSA, revealing UppS as a key component of the network that supports β-lactam resistance in MRSA. As such, clomiphene is new cell-permeable probe of the synthesis of Und-P and represents a potential lead for antibiotic drug discovery.Open in a separate windowScheme 1.Chemical structures of clomiphene, targocil, and ticlopidine.     

Role of vasopressin and its antagonism in stroke related edema

Although many approaches have been tried in the attempt to reduce the devastating impact of stroke, tissue plasminogen activator for thromboembolic stroke is the only proved, effective acute stroke treatment to date. Vasopressin, an acute‐phase reactant, is released after brain injury and is partially responsible for the subsequent inflammatory response via activation of divergent pathways. Recently there has been increasing interest in vasopressin because it is implicated in inflammation, cerebral edema, increased intracerebral pressure, and cerebral ion and neurotransmitter dysfunctions after cerebral ischemia. Additionally, copeptin, a byproduct of vasopressin production, may serve as a promising independent marker of tissue damage and prognosis after stroke, thereby corroborating the role of vasopressin in acute brain injury. Thus, vasopressin antagonists have a potential role in early stroke intervention, an effect thought to be mediated via interactions with aquaporin receptors, specifically aquaporin‐4. Despite some ambiguity, vasopressin V1a receptor antagonism has been consistently associated with attenuated secondary brain injury and edema in experimental stroke models. The role of the vasopressin V2 receptor remains unclear, but perhaps it is involved in a positive feedback loop for vasopressin expression. Despite the encouraging initial findings we report here, future research is required to characterize further the utility of vasopressin antagonists in treatment of stroke. © 2014 Wiley Periodicals, Inc.     

What systems can and can't do

This commentary discusses a paper in this issue by Dr Jillian Baker on the antagonism of histamine H(2) receptors. It is an excellent example of the use of pharmacological principles to determine what systems can and can't do from the point of view of agonist-dependent antagonism. The most common model of antagonism, namely orthosteric, cannot discern agonist type; i.e. all agonists are blocked equally by a given orthosteric antagonist. Therefore, if quantitative assessment of antagonism unveils agonist dependence, then this is something an orthosteric mechanism cannot do and another mechanism must be considered. A simple alternative is a permissive allosteric model whereby the agonist and antagonist interact through conformational changes in the receptor protein. Under these circumstances, an agonist-antagonist dialogue can ensue whereby the nature of the agonist determines the magnitude of antagonist effect. Jillian Baker contrasts antagonist systems with historical data obtained for beta-adrenoceptors and the present data for histamine H(2) receptors where the simpler model of orthosteric antagonism suffices and thus shows how quantitative receptor pharmacology can be used to determine the molecular mechanism of antagonism.