Thrombin-induced secretion of serotonin from platelets can occur in seconds

The platelet release reaction was studied by a new quenched-flow approach. Platelets labeled with 14C-serotonin were reacted for short times (up to 5 sec) with thrombin and then quenched with glutaraldehyde or paraformaldehyde. Serotonin secretion began within 1 sec and was nearly complete by 4 sec. Aggregation recorded by a resistive-particle counter was similarly fast. Therefore, the quenched-flow system reveals that serotonin secretion can occur more rapidly than estimated in earlier studies.     

Antibiotic cement-coated interlocking nail for the treatment of infected nonunions and segmental bone defects

Chronic infection of bone with nonunion and/or bone defects is traditionally treated by a 2-stage procedure involving initial debridement and antibiotic delivery and then definitive internal fixation. Alternatively, external fixators are used to provide stability. A technique with which antibiotic cement-coated interlocking intramedullary nails are prepared in the operating room with the use of nails and materials that generally are available is herein described. Although useful for all infected nonunions and/or segmental bone defects, this technique is particularly useful for patients who are not ideal candidates for external fixation and for those who do not want to have an external fixator applied. This technique was used in a series of 20 patients. In 17 patients, the goal of bony union was achieved (85%). In the remaining 3 patients (15%), the goal of control of infection was achieved with stable nonunion (1 patient) and stable nonunion with cement spacer (2 patients). In 95% of the patients (19 of 20 patients) control of infection was achieved except for in 1 patient, who had a bony union with intermittent wound discharge and subsequently underwent an above-the-knee amputation. Three patients (15%) needed exchange nailing to another antibiotic cement-coated nail (for continued infection) before complete control of infection could be achieved. Four patients (20%) experienced cement-nail debonding during removal of the antibiotic cement-coated nail (3 during exchange to an uncoated intramedullary nail, 1 during removal at the request of patient). One patient experienced partial debonding at insertion, coinciding with the site of segmental defect, which was treated with an antibiotic cement spacer. In summary, control of infection and stability to promote union has traditionally been provided by 2 separate procedures, which have proved to be efficacious in the past. However, both these goals can be achieved in half the patients with 1 surgical procedure in a variety of scenarios using the technique of an antibiotic cement-coated intramedullary nail.     

Hepatitis B virus subgenotypes and basal core promoter mutations in Indonesia

AIM： To identify the distribution of hepatitis B virus （HBV） subgenotype and basal core promoter （BCP） mutations among patients with HBV-associated liver disease in Indonesia.
METHODS： Patients with chronic hepatitis （CH, n =61）, liver cirrhosis （LC, n = 62）, and hepatocellular carcinoma （HCC, n = 48） were included in this study. HBV subgenotype was identified based on S or preS gene sequence, and mutations in the HBx gene including the overlapping BCP region were examined by direct sequencing.
RESULTS： HBV genotype B （subgenotypes B2, B3, B4, 85 and B7） the major genotype in the samples, accounted for 75.4%, 71.0% and 75.0% of CH, LC and HCC patients, respectively, while the genotype C （subgenotypes C1, C2 and C3） was detected in 24.6%, 29.0%, and 25.0% of CH, LC, and HCC patients, respectively. Subgenotypes B3 （84.9%） and C1 （82.2%） were the main subgenotype in HBV genotype B and C, respectively. Serotype adw2 （84.9%） and adrq＋ （89.4%） were the most prevalent in HBV genotype B and C, respectively. Double mutation （A1762T/G1764A） in the BCP was significantly higher in LC （59.7%） and HCC （54.2%） than in CH （19.7%）, suggesting that this mutation was associated with severity of liver disease. The T1753V was also higher in LC （46.8%）, but lower in HCC （22.9%） and CH （18.0%）, suggesting that this mutation may be an indicator of cirrhosis.
CONCLUSION： HBV genotype B/B3 and C/C1 are the major genotypes in Indonesia. Mutations in BCP, such as A1762T/G1764A and T1753V, might have an association with manifestations of liver disease.     

Identification of sites responsible for the potentiating effect of niflumic acid on ClC-Ka kidney chloride channels

Background and purpose:

ClC-K kidney Cl⁻ channels are important for renal and inner ear transepithelial Cl⁻ transport, and are potentially interesting pharmacological targets. They are modulated by niflumic acid (NFA), a non-steroidal anti-inflammatory drug, in a biphasic way: NFA activates ClC-Ka at low concentrations, but blocks the channel above ∼1 mM. We attempted to identify the amino acids involved in the activation of ClC-Ka by NFA.

Experimental approach:

We used site-directed mutagenesis and two-electrode voltage clamp analysis of wild-type and mutant channels expressed in Xenopus oocytes. Guided by the crystal structure of a bacterial CLC homolog, we screened 97 ClC-Ka mutations for alterations of NFA effects.

Key results:

Mutations of five residues significantly reduced the potentiating effect of NFA. Two of these (G167A and F213A) drastically altered general gating properties and are unlikely to be involved in NFA binding. The three remaining mutants (L155A, G345S and A349E) severely impaired or abolished NFA potentiation.

Conclusions and implications:

The three key residues identified (L155, G345, A349) are localized in two different protein regions that, based on the crystal structure of bacterial CLC homologs, are expected to be exposed to the extracellular side of the channel, relatively close to each other, and are thus good candidates for being part of the potentiating NFA binding site. Alternatively, the protein region identified mediates conformational changes following NFA binding. Our results are an important step towards the development of ClC-Ka activators for treating Bartter syndrome types III and IV with residual channel activity.     

Manipulation of liver regeneration with macrophages to influence the hepatic progenitor cell niche

Insufficient regeneration of the adult liver is believed to cause failure to recover from severe liver disease. An undifferentiated cell population with stem-cell-like qualities known as hepatic progenitor cells (HPCs) is hypothesised to have a central role in regeneration of the adult liver during massive or chronic liver disease. Stem cells in other organ systems are believed to reside in a specialised microenvironment or niche that supports their maintenance and function. The existence of a hepatic stem cell niche might provide a means of therapeutically manipulating endogenous HPCs in vivo as a regenerative therapy.To investigate the physiological potential of HPCs to regenerate the mammalian liver, we have established a novel model of hepatocellular injury and HPC activation using genetic manipulation of hepatocytes. After hepatocyte senescence and death in this model (AhCre Mdm2^flox), HPCs expand and bring about the complete regeneration of the liver parenchyma.We demonstrate that a stereotypical niche, consisting partly of macrophages, exists in both animal models and correlating human disease. Using cell tracking, we show active recruitment of extrahepatic macrophages into this niche during injury. In health, intravenous injection of macrophages results in macrophage engraftment to the liver niche, with subsequent HPC activation and changes to liver structure and function.Within the niche, macrophages use paracrine signalling to control both HPC proliferation and cell fate via TWEAK (tumour-necrosis-factor-like weak inducer of apoptosis) and the Wnt signalling pathway, respectively. After hepatocellular injury, macrophages ingest hepatocyte debris, and release Wnt which promotes HPC differentiation into hepatocytes. TWEAK is vital for HPC proliferation in the AhCre Mdm2^flox model of regeneration. Here, the absence of TWEAK signalling results in liver failure and mortality.This work has demonstrated for the first time the ability of a solid organ to fully regenerate in the adult mammal from progenitor cells, and additionally highlights mechanisms by which this process can be modulated by either small molecule or cell therapy.FundingUniversity of Edinburgh.     

4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4

Background and Purpose

The Ca²⁺-permeable cation channel TRPV4 is activated by mechanical disturbance of the cell membrane and is implicated in mechanical hyperalgesia. Nerve growth factor (NGF) is increased during inflammation and causes mechanical hyperalgesia. 4α-phorbol 12,13-didecanoate (4αPDD) has been described as a selective TRPV4 agonist. We investigated NGF-induced hyperalgesia in TRPV4 wild-type (+/+) and knockout (–/–) mice, and the increases in [Ca²⁺]_i produced by 4αPDD in cultured mouse dorsal root ganglia neurons following exposure to NGF.

Experimental Approach

Withdrawal thresholds to heat, von Frey hairs and pressure were measured in mice before and after systemic administration of NGF. Changes in intracellular Ca²⁺ concentration were measured by ratiometric imaging with Fura-2 in cultured DRG and trigeminal ganglia (TG) neurons during perfusion of TRPV4 agonists.

Key Results

Administration of NGF caused a significant sensitization to heat and von Frey stimuli in TRPV4 +/+ and –/– mice, but only TRPV4 +/+ mice showed sensitization to noxious pressure. 4αPDD stimulated a dose-dependent increase in [Ca²⁺]_i in neurons from +/+ and –/– mice, with the proportion of responding neurons and magnitude of increase unaffected by the genotype. In contrast, the selective TRPV4 agonist GSK1016790A failed to stimulate an increase in intracellular Ca²⁺ in cultured neurons. Responses to 4αPDD were unaffected by pretreatment with NGF.

Conclusions and Implications

TRPV4 contributes to mechanosensation in vivo, but there is little evidence for functional TRPV4 in cultured DRG and TG neurons. We conclude that 4αPDD activates these neurons independently of TRPV4, so it is not appropriate to refer to 4αPDD as a selective TRPV4 agonist.     

Design and synthesis of positional isomers of 1-alkyl-2-trifluoromethyl-5 or 6-substituted benzimidazoles and their antimicrobial activity

A series of novel positional isomers of 1-alkyl-2-trifluoromethyl benzimidazole derivatives 3a–j and 4a–j have been synthesized and screened for antibacterial activity against gram positive bacteria viz, Bacillus subtilis, Staphylococcus aureus and gram negative bacteria viz., Escherichia coli, Pseudomonas aeruginosa and antifungal activity against Candida albicans, Aspergillus niger, Rhizopus oryzae and Saccharomyces cerevisiae. Results indicate that compounds 3b, 4b were having promising antibacterial and antifungal activity.     

QSAR and docking studies of novel β-carboline derivatives as anticancer

Quantitative structure–activity relationship (QSAR) studies were performed on β-carboline derivatives for prediction of anticancer activity. The statistically significant 2D-QSAR model having r ² = 0.726 and q ² = 0.654 with pred_r ² = 0.763 was developed by stepwise multiple linear regression method. In order to understand the structural requirement of these β-carboline derivatives, a ligand-based pharmacophore 3D-QSAR model was developed. The five-point pharmacophore hypothesis yielded a 3D-QSAR model with good partial least-square statistics results (r ² = 0.73, Q _ext ²  = 0.755, F = 67.5, SD = 0.245, RMSE = 0.241, Pearson-R = 0.883). A docking study revealed the binding orientations of these derivatives at the active site amino residues of DNA intercalate (PDB ID: 1D12). The results of 2D-QSAR, atom-based 3D-QSAR, and docking studies gave detailed structural insights as well as highlighted important binding features of β-carboline derivatives as anticancer agent which provided guidance for the rational design of novel potent anticancer agents.