Rapid preparative scale purification of myristylated variant surface glycoproteins from African trypanosomes

A rapid method for the preparative-scale purification of variant surface glycoproteins (VSG) from African trypanosomes has been developed to investigate the recently described myristylated form of this molecule. This high-performance liquid chromatography (HPLC) procedure gives high yields and can be achieved in as little as 1 h from the preparation of a cell lysate in 0.1% trifluoroacetic acid (TFA). Solubilization of myristylated VSG in 0.1% TFA was shown to be much more effective than in neutral or zwitterionic detergents. Surface iodination of bloodstream trypanosomes and subsequent lysis in TFA showed that no proteolytic degradation of VSG occurred during solubilization and purification. Biosynthetic labelling of trypanosomes with [3H]myristic acid and purification of VSG by the described method, demonstrated that the VSG possessed covalently linked fatty acid and can therefore be defined as the membrane form of VSG. Evidence was provided that the myristic acid was covalently associated with the C-terminal portion of the VSG via a hydroxyester bond. Solubilization of the myristylated VSG by 0.1% TFA could be interpreted to indicate that the fatty acid was not embedded in the trypanosome membrane.     

Involvement of κ‐opioid receptors in visceral nociception in mice

Abstract It has been shown that the behavioural responses to chemically evoked visceral nociception are increased in transgenic mice lacking the κ‐opioid receptor (KOR). The aim of the present study was to evaluate the contribution of KOR in mechanically evoked visceral pain by performing colorectal distension (CRD) and monitoring the subsequent visceromotor response (VMR) in control mice (KOR^+/+) and in mice lacking KOR (KOR^?/?). Pseudo‐affective visceral pain responses were evoked in conscious mice using increasing (10–80 mmHg) and repeated (12 × 55 mmHg) phasic CRD paradigms. The resulting VMR was determined by monitoring the electromyographic activity of the abdominal muscle. The increasing and repeated CRD paradigms, respectively, evoked similar responses in both KOR^+/+ and KOR^?/? mice. The selective KOR‐agonists U‐69593 (5 and 25 mg kg^?1, s.c.) and asimadoline (25 mg kg^?1, s.c.) significantly decreased the VMR in KOR^+/+ mice, while having no effect in KOR^?/? mice. In contrast, the selective μ‐opioid receptor agonist fentanyl significantly reduced the VMR in both types of mice and appeared more efficacious in KOR^?/? mice. The opioid receptor antagonist naloxone (0.3–30 mg kg^?1 s.c.) did not affect the response to CRD in C57BL/6 mice at any dose tested. In conclusion, the data confirm that the KOR agonists used in this study inhibit the VMR to CRD in mice by acting via KOR receptors. In addition, the data suggest that the endogenous opioid system is not likely to modulate the VMR to mechanically evoked visceral pain in mice.     

The lectin pathway of complement: Advantage or disadvantage in HIV pathogenesis?

The pattern recognition molecules of the lectin complement pathway are important components of the innate immune system with known functions in host–virus interactions. This paper summarizes current knowledge of how these intriguing molecules, including mannose-binding lectin (MBL), Ficolin-1, -2 and -3, and collectin-11 (CL-11) may influence HIV-pathogenesis. It has been demonstrated that MBL is capable of binding and neutralizing HIV and may affect host susceptibility to HIV infection and disease progression. In addition, MBL may cause variations in the host immune response against HIV. Ficolin-1, -2 and -3 and CL-11 could have similar functions in HIV infection as the ficolins have been shown to play a role in other viral infections, and CL-11 resembles MBL and the ficolins in structure and binding capacity.     

Pregabalin modulation of spinal and brainstem visceral nociceptive processing

Brainstem and spinal mechanisms mediating visceral nociception are investigated here using electrophysiology and immunohistochemistry techniques in a model of acute visceral pain. Colorectal distension (CRD) produced graded visceromotor responses (VMR) in normal rats, and these were facilitated by intracolonic mustard oil (MO) that generated acute visceral hyperalgesia. The neuropathic pain drug pregabalin (PGB) is thought to have state-dependent effects in attenuating neuropathic, but not acute somatic pain, likely by impairing calcium-channel trafficking. We found that systemic PGB produced antinociceptive effects on CRD-evoked VMRs in naïve rats lacking pathophysiology and in MO-pretreated rats. Systemic PGB also significantly reduced Fos labelling in lumbosacral spinal cords of rats given noxious repetitive CRD; however, PGB did not alter this measure of neural activity in the brainstem. Differential brainstem processing of noxious somatic and visceral stimuli may underlie the unique lack of state-dependent actions of PGB in this visceral pain model. Single-unit recordings in the rostral ventromedial medulla (RVM) verify that brainstem processing of somatic and visceral stimuli differs. The effects of CRD on RVM cells classed as ON, OFF, or NEUTRAL were independent of their somatic responses, with surprising changes in RVM cell activity to innocuous visceral stimulation. PGB also markedly reduced the visceral responses of RVM ON-cells to noxious CRD. These results illustrate clear differences in the central processing of visceral and somatic stimuli, yet a common role for descending modulation by brainstem activity in mediating evoked pain measures.     

Early-life stress induces visceral hypersensitivity in mice

Early-life stress is a risk factor for irritable bowel syndrome (IBS), a common and debilitating functional gastrointestinal disorder that is often co-morbid with stress-related psychiatric disorders. In the rat, maternal separation (MS) stress has been shown to induce visceral hypersensitivity in adulthood and thus has become a useful model of IBS. However, development of mouse models of maternal separation has been difficult. Given the advent of transgenic mouse technology, such models would be useful to further our understanding of the pathophysiology of IBS and to develop new pharmacological treatments. Thus, the present study aimed to develop a mouse model of MS stress-induced visceral hyperalgesia as measured using manometric recordings of colorectal distension (CRD). Moreover, since the GABA(B) receptor has been reported to play a role in pain processes, we also assessed its role in visceral nociception using novel GABA(B(1b)) receptor subunit knockout mice. CRD was performed in adult male wildtype and GABA(B(1b)) receptor knockout mice that had undergone unpredictable MS combined with unpredictable maternal stress (MSUS) from postnatal day 1 through 14 (PND 1-14). MSUS induced visceral hypersensitivity in both wildtype and GABA(B(1b)) receptor knockout mice when compared with non-stressed mice. Wildtype and GABA(B(1b)) receptor knockout mice did not differ in baseline or stress-induced visceral sensitivity. To the best of our knowledge, this is the first study to show that early-life stress induces visceral hypersensitivity in a mouse model. These findings may provide a novel mouse model of visceral hypersensitivity which may aid our understanding of its underlying mechanisms in future studies.     

人干细胞生长因子cDNA克隆、表达和纯化及其造血种属特异性

人干细胞生长因子（human stem cell growth factor，hSCGF）是一种早期造血调控因子。已知人干细胞生长因子具有两种形式，包括全长分子hSCGF以及在Ca^2＋依赖糖识别结构域（calcium-dependent carbohydrate recognition domain，CRD）内缺失78氨基酸的截短型分子hSCGFβ。hSCGFβ的造血刺激活性具有严格的种属特异性。本研究目的在于探讨hSCGF能否协同刺激小鼠彬单系祖细胞增殖。为克服hSCGF的cDNAGC含量较高的困难，本研究以两步PCR的方法从人胎肝cDNA文库（Clontech）中成功克隆hSCGFcDNA，进而将hSCGF成熟肽编码序列亚克隆于原核表达载体pGEX4T-2中，融合表达。研究结果表明，通过低温诱导（28℃）。重组表达产物主要以可溶蛋白的形式存在于裂解上清中，利用亲和层析纯化融合表达蛋白。对重组蛋白的造血刺激活性分析表明，不同于截短型分子hSCGFβ。全长分子hSCGF能够刺激小鼠骨髓彬单系造血祖细胞增殖。结论：hSCGFCRD不直接结合受体。可能具有其他生物学功能。     

Advances in patient-derived tumor xenografts: From target identification to predicting clinical response rates in oncology

Most oncology compounds entering clinical development have passed stringent preclinical pharmacology evaluation criteria. However, only a small fraction of experimental agents induce meaningful antitumor activities in the clinic. Low predictability of conventional preclinical pharmacology models is frequently cited as a main reason for the unusually high clinical attrition rates of therapeutic compounds in oncology. Therefore, improvement in the predictive values of preclinical efficacy models for clinical outcome holds great promise to reduce the clinical attrition rates of experimental compounds.     

A novel compound heterozygous TACI mutation in an autosomal recessive common variable immunodeficiency (CVID) family

Common variable immunodeficiency (CVID) is a primary immune disorder characterized by low immunoglobulin serum levels and increased susceptibility to infections. Underlying genetic causes are only known in less than 15% of patients and encompass mutations in the genes encoding for ICOS, TACI, BAFF-R, CD19, CD20, CD81 and MSH5. TACI is the most frequently mutated gene among CVID patients. We report on two pediatric Italian male siblings with hypogammaglobulinemia and recurrent respiratory and gastrointestinal infections in association with a novel compound heterozygous TACI mutation. Both patients carry the I87N/C104R mutation that has not been reported yet. This results in aberrant TACI expression and abrogates APRIL binding on EBV B cells. This study identifies a novel combined mutation in TNFRSF13B increasing the spectrum of TACI mutations associated with CVID.