Interactions of antagonists with subtypes of inositol 1,4,5-trisphosphate (IP3) receptor

BACKGROUND AND PURPOSE

Inositol 1,4,5-trisphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels. Interactions of the commonly used antagonists of IP₃Rs with IP₃R subtypes are poorly understood.

EXPERIMENTAL APPROACH

IP₃-evoked Ca²⁺ release from permeabilized DT40 cells stably expressing single subtypes of mammalian IP₃R was measured using a luminal Ca²⁺ indicator. The effects of commonly used antagonists on IP₃-evoked Ca²⁺ release and ³H-IP₃ binding were characterized.

KEY RESULTS

Functional analyses showed that heparin was a competitive antagonist of all IP₃R subtypes with different affinities for each (IP₃R3 > IP₃R1 ≥ IP₃R2). This sequence did not match the affinities for heparin binding to the isolated N-terminal from each IP₃R subtype. 2-aminoethoxydiphenyl borate (2-APB) and high concentrations of caffeine selectively inhibited IP₃R1 without affecting IP₃ binding. Neither Xestospongin C nor Xestospongin D effectively inhibited IP₃-evoked Ca²⁺ release via any IP₃R subtype.

CONCLUSIONS AND IMPLICATIONS

Heparin competes with IP₃, but its access to the IP₃-binding core is substantially hindered by additional IP₃R residues. These interactions may contribute to its modest selectivity for IP₃R3. Practicable concentrations of caffeine and 2-APB inhibit only IP₃R1. Xestospongins do not appear to be effective antagonists of IP₃Rs.     

Recent advances in biological nanopores for nanopore sequencing,sensing and comparison of functional variations in MspA mutants

Biological nanopores are revolutionizing human health by the great myriad of detection and diagnostic skills. Their nano-confined area and ingenious shape are suitable to investigate a diverse range of molecules that were difficult to identify with the previous techniques. Additionally, high throughput and label-free detection of target analytes instigated the exploration of new bacterial channel proteins such as Fragaceatoxin C (FraC), Cytolysin A (ClyA), Ferric hydroxamate uptake component A (FhuA) and Curli specific gene G (CsgG) along with the former ones, like α-hemolysin (αHL), Mycobacterium smegmatis porin A (MspA), aerolysin, bacteriophage phi 29 and Outer membrane porin G (OmpG). Herein, we discuss some well-known biological nanopores but emphasize on MspA and compare the effects of site-directed mutagenesis on the detection ability of its mutants in view of the surface charge distribution, voltage threshold and pore–analyte interaction. We also discuss illustrious and latest advances in biological nanopores for past 2–3 years due to limited space. Last but not the least, we elucidate our perspective for selecting a biological nanopore and propose some future directions to design a customized nanopore that would be suitable for DNA sequencing and sensing of other nontrivial molecules in question.

Future of nanopore DNA sequencing: schematic illustration shows the future of nanopore DNA sequencing by using a customized biological nanopore with appropriate fabrication.     

The Ca2+-gated channel TMEM16A amplifies capillary pericyte contraction and reduces cerebral blood flow after ischemia

Pericyte-mediated capillary constriction decreases cerebral blood flow in stroke after an occluded artery is unblocked. The determinants of pericyte tone are poorly understood. We show that a small rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) in pericytes activated chloride efflux through the Ca²⁺-gated anion channel TMEM16A, thus depolarizing the cell and opening voltage-gated calcium channels. This mechanism strongly amplified the pericyte [Ca²⁺]_i rise and capillary constriction evoked by contractile agonists and ischemia. In a rodent stroke model, TMEM16A inhibition slowed the ischemia-evoked pericyte [Ca²⁺]_i rise, capillary constriction, and pericyte death; reduced neutrophil stalling; and improved cerebrovascular reperfusion. Genetic analysis implicated altered TMEM16A expression in poor patient recovery from ischemic stroke. Thus, pericyte TMEM16A is a crucial regulator of cerebral capillary function and a potential therapeutic target for stroke and possibly other disorders of impaired microvascular flow, such as Alzheimer’s disease and vascular dementia.     

Quercetin induces morphological and proliferative changes of rat’s uteri under estrogen and progesterone influences

This study investigated the effect of 10 or 100 mg/kg/day quercetin on the uterus of ovariectomized adult female rats receiving sex-steroid replacement regime mimicking changes in hormonal profiles during the reproductive cycle. Following seven days of treatment with estrogen and progesterone with or without quercetin, uteri were harvested for histological and proliferative cell nuclear antigen (PCNA) protein and mRNA expression and PCNA protein distribution analyses. Our findings indicated that co-administration of 10 mg/kg/day quercetin with estrogen and progesterone caused a significant decrease in the size of uterine lumen and epithelial heights with lower PCNA protein and mRNA expression as compared to estrogen plus progesterone-only treatment (P < 0.05). Concomitant treatment with estrogen and progesterone with 100 mg/kg/day quercetin resulted in a marked increase in the number of glands with increased PCNA protein and mRNA expression. Significantly higher PCNA distribution was observed in the stroma and glands as compared to estrogen plus progesterone-only treatment (P < 0.05). In conclusion, at 10 mg/kg/day, quercetin affects uterine morphology but not proliferation, however at 100 mg/kg/day, quercetin induced significant stromal and glandular proliferation which could predispose the uterus towards neoplastic development.     

Needle biopsy findings in prostatic adenocarcinoma: experience at a tertiary care center in a developing country

The objectives of this study are to report the findings in prostatic needle biopsies positive for cancer seen in our practice with regard to the frequency of cancer detected at various sites, the cancer volume, Gleason grade, presence of perineural invasion, and others; to correlate cancer volume with Gleason grade, perineural invasion, and serum prostate-specific antigen (PSA) levels; and to correlate Gleason grade with serum PSA levels. The study was conducted at The Section of Histopathology, Department of Pathology and Microbiology, Aga Khan University Hospital, Karachi, Pakistan. All consecutive needle biopsies received from January 1, 2011 to June 30, 2012, which were positive for prostatic adenocarcinoma, were included in the study. Statistical analysis was carried out using SPSS 19.0 software package (SPSS Hong Kong Headquarters, Quarry Bay, Hong Kong). A total of 97 needle biopsies positive for carcinoma in this period were included. Prostate-specific antigen levels were available in 60.8% cases and ranged from 5.0 to 1747 ng/mL. Tumor was bilaterally present in 54.6% cases. Tumor positivity in right apex, mid, and base was 52.6%, 54.6%, and 51.5%, respectively. Tumor positivity in left apex, mid, and base was 55.7%, 63.9%, and 59.8%, respectively. Average tumor volume in right apex, mid, and base was 51.2%, 50.6%, and 49.9%, respectively. Average tumor volume in left apex, mid, and base was 49.8%, 49.1%, and 51.6%, respectively. Gleason score was 6 in 52.6% cases and 7 in 28.9% cases. Perineural invasion was positive in 46.4% cases. High-grade prostatic intraepithelial neoplasia was seen in 4 (4.1%) of 97 cases. On statistical analysis, no significant correlation was found between tumor volume and serum PSA levels. However, significant correlation was found between tumor volume and Gleason grade and between tumor volume and presence of perineural invasion. No significant correlation was found between Gleason grade and serum PSA level. To our knowledge, these are the first reported findings in prostatic needle biopsies from Pakistan. Most prostatic carcinomas in our country are still diagnosed on transurethral resection specimens, and needle biopsies are quite uncommon. Findings in needle biopsies will help in predicting adverse prognostic factors on radical prostatectomies and in planning surgery accordingly.     

Comparative uptakes and biodistributions of internalizing vs. noninternalizing copper-64 radioimmunoconjugates in cell and animal models of colon cancer

Copper-64-labeled monoclonal antibodies (mAbs) have previously demonstrated unexpectedly effective tumor control in rodent models of cancer at relatively low tumor-absorbed radiation doses. This property has been associated with delivery platforms resulting in cellular internalization. The purpose of the present studies was to evaluate the in vitro internalization and in vivo distribution of a two-antibody model of ⁶⁴Cu radioimmunotherapy (RIT) in the same cell and animal models of cancer. Biodistributions of an internalizing antibody, cBR96, and a noninternalizing antibody, cT84.66, labeled with ⁶⁴Cu, were obtained in nude mice bearing LS174T colon carcinoma xenografts from 15 min to 48 h. The ⁶⁴Cu-DOTA-cBR96 conjugate demonstrated rapid tumor uptake, reaching 20.2% ID/g at 3 h and peaking at 35.4% ID/g by 24 h. Tumor accumulation of ⁶⁴Cu-DOTA-cT84.66 was more gradual, 8.19% ID/g at 3 h and 43.8% ID/g by 24 h, but maximum uptake was not statistically different from ⁶⁴Cu-DOTA-cBR96. Mouse xenograft dosimetry was estimated to be 1128 rad/mCi (304.9 mGy/MBq) for ⁶⁴Cu-DOTA-cBR96 and 1409 rad/mCi (380.5 mGy/MBq) for ⁶⁴Cu-DOTA-cT84.66. In LS174T cells, internalized radioactivity increased by a factor of 3.8 over 4 h for ⁶⁴Cu-DOTA-cBR96, but remained unchanged ⁶⁴Cu-DOTA-cT84.66. When normalized to uptake at 1 h, cellular efflux of ⁶⁴Cu was essentially identical for both mAbs. The biodistributions and tumor dosimetry of these internalizing and noninternalizing radiolabeled mAbs were sufficiently similar for direct comparison of the therapeutic efficacies of low doses of ⁶⁴Cu RIT agents in the same animal model of cancer.     

An Investigation of Factors Predictive of Continued Self‐Injurious Behaviour in an Intellectual Disability Service

Background Self‐injurious behaviour (SIB) is among the most serious problems faced by intellectual disability services. It is very difficult to treat and can become a chronic problem. Method Information on a number of variables was collected through a survey of service‐users identified as displaying SIBs. Clinical opinion and a literature review guided the selection of potential predictors of continued SIB. Univariate statistical analyses were used to investigate associations between continued SIB and each of the variables identified. Variables shown to have a significant association with continued SIB were subjected to a multivariate analysis to isolate those variables that still predicted continued SIB once the influence of the others had been accounted for. Results Two factors, self‐biting and verbal ability, were found to independently predict continued SIB. Conclusion The results have implications for intellectual disability services, in terms of the importance of multidisciplinary team working, training and guidelines for problem management.