Tributyltin-binding protein type 1, a lipocalin, prevents inhibition of osteoblastic activity by tributyltin in fish scales

Tributyltin-binding protein type 1 (TBT-bp1) is a member of the lipocalin family of proteins which bind to small hydrophobic molecules. In this study, we expressed a recombinant TBT-bp1 (rTBT-bp1, ca. 35kDa) in a baculovirus expression system and purified the protein from the hemolymph of silkworm larvae injected with recombinant baculovirus. After incubation of a mixture of rTBT-bp1 and TBT and its fractionation by means of gel filtration chromatography, TBT was detected in the elution peak of rTBT-bp1, confirming the binding potential of rTBT-bp1 for TBT. An assay of the ability of rTBT-bp1 or native TBT-bp1 (nTBT-bp1) to restore osteoblastic activity inhibited by TBT showed that co-treatment of the scales with rTBT-bp1 or nTBT-bp1 in combination with TBT restored osteoblastic activity in goldfish scales, whereas treatment with TBT alone significantly inhibited osteoblastic activity. These results suggest that TBT-bp1 as a lipocalin member might function to decrease the toxicity of TBT by binding to TBT.     

Discovery and crystallographic structure of human apolipoprotein

We report the serendipitous discovery of a human plasma phosphate binding protein (HPBP). This 38 kDa protein is co-purified with paraoxonase (PON1). The association between HPON1 and HPBP is modulated by phosphate and calcium concentrations. The HPBP X-ray structure solved at 1.9 A resolution is similar to the prokaryotic phosphate solute-binding proteins (SBPs) associated with ATP binding cassette transmembrane transporters, though phosphate-SBPs have never been characterized or predicted from nucleic acid databases in eukaryotes. However, HPBP belongs to the family of ubiquitous eukaryotic proteins named DING, meaning that phosphate-SBPs are also widespread in eukaryotes. The absence of complete genes for eukaryotic phosphate-SBP from databases is intriguing, but the astonishing 90% sequence conservation of genes between evolutionary distant species suggests that the corresponding proteins play an important function. HPBP is the first identified transporter capable of binding phosphate ions in human plasma. Thus it is thought to become a new predictor and a potential therapeutic agent for phosphate-related diseases such as atherosclerosis.     

Lipocalin‐2—The myth of its expression and function

Lipocalin‐2 is a functional biomarker for acute and chronic kidney diseases, heart failure and obesity‐related medical complications. It is rapidly induced in epithelial cells under stress conditions, but constitutively produced from pre‐adipocytes and mature adipocytes. Measuring the lipocalin‐2 levels represents an effective approach for risk prediction, patient stratification and disease management. Nevertheless, due to ligand‐binding, post‐translational modification and protein‐protein interaction, lipocalin‐2 exists as multiple variants that elicit different pathophysiological functions. To characterize the specific structure‐functional relationships of lipocalin‐2 variants is critical for the development of biomarker assays with sufficient precision and reliability. Moreover, identifying the pathological forms of lipocalin‐2 will provide new therapeutic targets and treatment approaches for obesity‐related complications.     

大鼠lipocalin 11蛋白非保守半胱氨酸双点突变体样品制备及理化性质表征

Lipocalin 11蛋白是新发现的一个lipocalin分泌型蛋白家族成员,与男性生殖密切相关。大鼠lipocalin 11(rLcn11)蛋白中4个半胱氨酸往往不能正确配对,为重组蛋白样品的制备带来困难。该研究通过序列比对和同源模建的方法获得rLcn11蛋白非保守半胱氨酸的位点,构建了rLcn11蛋白野生型与C59A/C156A突变体的表达系统和纯化方法。通过分子筛和圆二色谱分析rLcn11蛋白的聚集形态和结构特征,并用荧光滴定的方法研究了rLcn11蛋白与荧光探针ANS的结合能力。结果表明:(1)rLcn11蛋白非保守半胱氨酸残基的突变有利于减少蛋白的错误折叠和聚集,提高重组蛋白的产量;(2)rLcn11 C59A/C156A突变体的空间结构良好并能结合疏水性配体;(3)rLcn11蛋白Cys59、Cys156双点突变不显著地改变rLcn11蛋白的空间结构及其与配体的结合能力。该工作为rLcn11蛋白的空间结构解析和生理功能的研究奠定了基础。     

Construction of a phage displayed library based on a lipocalin scaffold and preliminary screening of anticalins with specificity for the FcεRI-α receptor

The primers were designed according to the gene sequence of lipocalin protein family, and the gene sequence containing random mutation protein was obtained by overlapping extension of PCR. The random mutation lipocalin library was constructed using phagemid expression vector. Lipocalin library was screened by subtracted screening of NSF60 cells and affinity screening of mast cells, and the lipocalin secondary library binding to mast cells was obtained. Then the lipocalin secondary library was enriched and screened with FcεRI-α receptor protein as target molecule, and specific binding phages were eluted. After three rounds of screening, eight recombinant phage clones were randomly selected from elution clones of the third round. ELISA assay showed that three anticalin molecules could specifically bind to the FcεRI-α receptor of mast cells. These results may provide some candidate biological molecules for the development of blocking drugs of mast cell FcεRI-α receptor, and also lay the foundation for the development of biological small molecule drugs to treat IgE associated allergic diseases.     

Binding of minaprine to human serum proteins and erythrocytes

The binding of minaprine to human serum, isolated proteins and erythrocytes was studied in vitro. This drug shows both a saturable and a nonsaturable binding process in serum. The drug is bound to alpha 1-acid glycoprotein in a saturable fashion with a moderate affinity whereas albumin is involved in the nonsaturable and major binding. The combination of these two processes results in a slight decrease (7%) in the drug binding to serum within the range of therapeutic concentrations. Competitive binding with metabolites of minaprine or with other basic drugs seems unlikely when these compounds are present in serum at therapeutic levels. The binding of minaprine to erythrocytes remains constant within the range of therapeutic concentrations.     

Recent patents on Rho signaling pathway as therapeutic target for cardiovascular diseases

Rho protein represents a family of small GTP binding proteins that are involved in many important cellular functions including cell proliferation, migration and cytoskeletal reorganization. Rho protein is activated by GTP binding and is inactivated by hydrolyzing GTP to GDP. This process is influenced by variety of physiological and pathophysiological stimuli including growth factors, many vasoactive substances, smoking and mechanic stress or injury. Recent evidence suggest that targeting Rho protein per se or its downstream effector proteins such as Rho kinase or LIM kinase may have therapeutic potential in diseases such as hypertension, angina, myocardiac infarction (MI), atherosclerosis, tumor metastasis and spinal cord injury. Several recent patents have described modalities that regulate the activity of Rho, Rho kinase and LIM kinase as potential therapeutics. In this article, we will review the current knowledge on the cellular functions of Rho signaling pathway and strategies in targeting different components in Rho signaling pathway for human diseases with an emphasis on cardiovascular indications.     

Regenerating gene (REG) product and its potential clinical usage

Introduction: The regenerating gene (Reg) was identified in regenerating islets and its related genes were revealed to constitute the Reg gene family. Reg family proteins act as growth factors for several cells. Recently, autoimmunity against the Reg family proteins has been reported in several diseases. In addition, the Reg family genes were found to be expressed in a large number of cancers and to influence prognosis.

Areas covered: The historical background and current view of the structure, function, and expression of Reg family genes/proteins and their physiological/pathological significance in several diseases are described. Based on the findings, the diagnostic/therapeutic potential of Reg family genes/proteins is also discussed.

Expert opinion: Autoimmunity against Reg family proteins may be a new diagnostic marker and/or therapeutic target for immune-mediated diseases. Treatment aimed at the expansion of the β-cell mass by the Reg genes/proteins, combined with the abrogation of autoimmunity, constitutes a potential approach for the treatment of diabetes. Conversely, some cancer cells have gained the ability to overexpress the Reg genes/proteins, thereby enhancing their proliferative capacities, resulting in these cells having a considerable growth advantage. Thus, the Reg genes/proteins are expected to be a new prognostic marker in cancer and/or a future therapeutic target.     

Histamine H3 and H4 receptors as novel drug targets

The identification of histamine H3 (H3R) and H4 (H4R) receptors some years ago revived interest in histamine research and exposed attractive perspectives for the potential therapeutic exploitation of these new drug targets. While the H3R is mainly localised in the CNS, the H4R is primarily expressed in hematopoietic cells, indicating their function in neurotransmission and immunomodulation, respectively. Although structural similarities between H3R and H4R and species differences in their pharmacological profiles are causes of limitations in the evaluation of their biological profile, the development of selective ligands for these receptors facilitates the elucidation of their physiological and pathophysiological functions. The H3R is a recognised drug target for neuronal diseases, such as cognitive impairment, schizophrenia, sleep/wake disorders, epilepsy and neuropathic pain; a small number of selective H3R antagonists have already passed some clinical Phase II trials. The H4R is the newest identified member of the histamine receptor family. Preclinical data strongly suggest its potential therapeutic exploitation in allergy, inflammation, autoimmune disorders and possibly cancer. Considering the topicality of this area of research, this review focuses on the pharmacology of selected promising indications and the rationales for the application of H3R and H4R ligands.     

Histamine H4 receptor ligands and their potential therapeutic applications

Background: The histamine H4 receptor (H4R) was first cloned in 2000. As a new member of the histamine receptor family it was of great interest and a promising drug target for the treatment of inflammatory diseases. Recent studies suggest that H4 antagonists have therapeutic potential, even in several pain conditions. Initially, pharmacological processes mediated by H4R were analyzed primarily by non-selective H4 ligands. Since then, some highly potent and selective H4 antagonists have been reported by different groups. Several companies are working on the development of potent H4 antagonists, although the number of published patent applications is still low. Objective: This review aims to give an overview of the ligands acting on H4R, this is drawn from published in papers and patent applications. Methods: Analysis of scaffolds possessing significant H4 activity and review of their therapeutic potential. Results/conclusion: The pharmacology of currently available selective H4 ligands suggests therapeutic utility for H4 antagonists in inflammatory conditions as well as in chronic pain.     

Histamine is an antagonist of the acetylcholine receptor at the frog endplate.

The effects of histamine on the acetylcholine (ACh) receptor-channel complex were examined by means of voltage-clamp at the frog endplate. ACh was ionophoretically applied to the endplate. Histamine was added to the perfusate. Histamine (100 nM - 1 mM) reversibly depressed the peak amplitude of the ACh-induced inward current in a dose-dependent manner. The double reciprocal plot of the dose-response relationship between the peak ACh current and the amount of ACh applied suggested that histamine (100 microM) depressed the ACh-induced current in a competitive manner. Histamine prevented the specific ACh binding site within the receptor-channel complex from binding erabutoxin, a sea-snake venom, which binds irreversibly to the specific ACh binding site. Histamine had no detectable effects on the equilibrium potential of the endplate current but shortened the half-decay time of the endplate current in a voltage-dependent manner. It was therefore concluded that histamine blocks not only the specific ACh binding site but also interacts with the ACh-channel site. The present experiments strongly suggest that histamine can act as an antagonist to modulate nicotinic cholinergic transmission.     

Partial characterization of red gram (Cajanus cajan L. Millsp) polypeptides recognized by patients exhibiting rhinitis and bronchial asthma

We sought to assess the allergenic potential of red gram by identifying and characterizing the responsible proteins. Immunoblotting was performed to detect IgE binding proteins. Identities of these proteins were confirmed by mass spectrometry. To evaluate allergenic potential, BALB/c mice were sensitized with red gram proteins and levels of specific immunoglobulins, histamine, Th2 cytokines were measured. Allergenic response was evident by significant increase in specific IgE, IgG1, histamine and Th2 cytokine levels. Prominent anaphylactic symptoms, discernible histopathological responses and down regulation of IFN-γ levels give strong support towards allergenicity of red gram proteins. IgE immunoblot detected five proteins; one of 66 kDa, three of 45 kDa (pI of ∼5.3, 5.9 and 6.6) and one of 30 kDa. All these proteins showed homology to known allergens of soybean (different subunits of β-conglycinin), lentil (Len c1 and Len c2), peanut (Ara h1) and pea (vicilin). In conclusion, five novel IgE binding proteins (namely Caj c1, Caj c2, Caj c3, Caj c4 and Caj c5) were identified as putative clinically relevant allergens.     

Detailed pharmacological characterization of GT-2331 for the rat histamine H3 receptor

Histamine H(3) receptor antagonists are potential therapeutic agents for cognitive dysfunction, epilepsy, hypersomnia and obesity. GT-2331 (4-[(R,R)-2-(5,5-dimethyl-1-hexynyl)cyclopropyl]-1H-imidazole) was originally identified as a potent histamine H(3) receptor antagonist. However, recent reports demonstrated a complex pharmacology for GT-2331. To further understand the pharmacological profile of GT-2331, we characterized GT-2331 using various in vitro and in vivo assays. In vitro, GT-2331 behaved as a full agonist on adenylyl cyclase inhibition and as a partial agonist on [(35)S]GTPgammaS binding at the recombinant rat histamine H(3) receptor. In contrast, in vivo, GT-2331 had no effect on brain histamine turnover while the histamine H(3) receptor agonist R-alpha-methylhistamine significantly decreased histamine turnover. Furthermore, GT-2331 completely blocked R-alpha-methylhistamine-induced water intake, suggesting that GT-2331 behaves as a full antagonist. Thus, GT-2331 displayed the spectrum of pharmacological activities from full agonism to full antagonism, these observations suggest that histamine H(3) receptor ligands need to be carefully evaluated in various paradigms.     

Neutrophil gelatinase-associated lipocalin (NGAL) as a new biomarker for non--acute kidney injury (AKI) diseases

Neutrophil gelatinase-associated lipocalin, or NGAL, an acute phase protein, is part of the lipocalin family. NGAL is highly induced in inflammatory conditions and ischemia, and is a critical component of innate immunity to bacterial infection. Recently, NGAL has been proven as an emerging biomarker for predicting acute kidney injury (AKI). Meanwhile, numerous studies have also demonstrated that NGAL may be a potential biomarker for the diagnosis, prediction, prevention, and prognosis of non--AKI diseases such as chronic kidney diseases, vascular disorders, cancer, preeclampsia, and allergies. This article systematically reviews the clinical utilities of NGAL as a new biomarker for non--AKI diseases.     

Histamine-gated ion channels in mammals?

There is ample pharmacological and physiological evidence for yet unidentified histamine receptors in mammalian brain that are linked to a Cl(-) conductance. In invertebrates, two histamine-gated chloride channels (HisCl α1 and α2) are already well known. HisCl channels are members of the Cys-loop receptor superfamily of ligand-gated ion channels and are closely related to the mammalian GABA(A) and glycine receptors (GlyR). Indeed, they share particularly strong homology within the ligand binding and ion channel domains. Here we discuss the possibility that mammalian HisCl channels might exist among the known GABA(A) or GlyR subunits. Studies published to date support this hypothesis, including evidence for direct histamine gating of GABA(A) β homomers, histamine potentiation of GABA(A) αβ and αβγ heteromeric receptors, and GABA(A) receptor blockade by some antihistamines. We explore what is known about the binding-site structure, function and pharmacology of invertebrate HisCl channels and other histamine binding sites to support and inform a broader search for HisCl channels among the mammalian GABA(A) and GlyR subunits. The discovery and identification of HisCl-like channels in mammals would not only enhance understanding of inhibitory signaling and histamine function in the mammalian brain, but also provide new avenues for development of therapeutic compounds targeting this novel histamine site. This commentary is therefore intended to foster consideration of a novel and potentially important target of histamine and histaminergic drugs in the CNS.     

In vitro and in vivo pharmacological analysis of imidazole-free histamine H3 receptor antagonists: promising results for a brain-penetrating H3 blocker with weak anticholinesterase activity

The pharmacological profiling of potent histamine H(3)-ligands initiated in a previous study is completed here. In vitro functional and binding studies revealed that several derivatives were selective H(3)-antagonists with nanomolar potency at human and guinea-pig histamine receptors, able to inhibit rat brain cholinesterase at micromolar concentrations and devoid of any cytotoxicity on cultured cells. Ex vivo binding experiments in rats showed that the most potent H(3)-antagonist, compound 5, had a prompt and long-lasting presence in the central nervous system (CNS), inhibiting [(3)H](R)-alpha-methylhistamine cortical binding [ED(50) (dose that elicits a 50% response) = 0.63 mg/kg intraperitoneally (i.p.)]. In the passive-avoidance test, compound 5, at 1.25 mg/kg i.p., was as effective as the anti-Alzheimer drug donepezil in attenuating scopolamine-induced amnesia in rats. These results suggest that a good CNS penetration and multitarget activity could account for the antiamnesic effect of this weak cholinesterase inhibitor and potent H(3)-antagonist (compound 5). This result represents a potential benchmark for the development of non-imidazole H(3)-antagonists/cholinesterase inhibitors with therapeutic potential in cognitive disorders.     

人脂质运载蛋白2研究进展

人脂质运载蛋白2(lipocalin 2,Lcn2),属于Lcn家族的新成员.它存在于人体许多正常组织细胞中,可作为运铁蛋白介导转铁途径,并与免疫炎症反应、肿瘤发生发展以及肾脏疾病有关.