The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Breastfeeding in the Mohawk community of Kahnawake: revisited and redefined

Breastfeeding in Kahnawake, Québec, increased to 64% in 1985/1986 from 45% in 1978, and duration of breastfeeding at three and six months also improved. Breastfeeding was significantly associated with birthweight, previous nursing experience and support from baby's father. The majority of mothers chose the method of feeding prior to pregnancy and did not perceive professional advice as important. Mothers chose breastfeeding for their babies' health, and bottlefeeding for "other" reasons or so "others can feed the baby". Those who chose to breastfeed prepregnancy, breastfed significantly longer. "Lack of milk" was the predominant reason for terminating breastfeeding before three months. 29% of breastfed babies and 46% of bottlefed babies received cereal before three months. These results will be incorporated into an extended breastfeeding promotion in this community to increase the understanding of the national recommendations for universal breastfeeding, and to re-create a culturally supportive environment for breastfeeding mothers.     

Tumor necrosis factor-alpha is expressed by glomerular visceral epithelial cells in human membranous nephropathy.

The role of tumor necrosis factor alpha (TNF-alpha) was examined in biopsy-proven glomerulonephritis by immunohistochemistry, in situ hybridization, immunogold electron microscopy, immunoassay in serum and urine, and urinary immunoblot. Striking glomerular capillary wall and visceral glomerular epithelial cell TNF-alpha protein staining was observed in all cases of membranous nephropathy and membranous lupus nephropathy. Staining was less frequently observed in crescentic glomerulonephritis and in isolated cases of other histological subtypes of glomerulonephritis, usually in association with glomerular macrophages. By immunogold electron microscopy TNF-alpha was localized in membranous nephropathy within the visceral glomerular epithelial cells, and also in the glomerular basement membrane, especially in relation to immune deposits. In situ hybridization localized TNF-alpha mRNA exclusively to glomerular epithelial cells in all biopsies with membranous morphology but not in other histological subtypes. Concentrations of TNF-alpha were significantly increased compared with normal controls in the urine of patients with membranous nephropathy and with crescentic glomerulonephritis. The expression of TNF-alpha by glomerular epithelial cells exclusively and universally in biopsies showing a membranous morphology strongly suggests this cytokine has a role in the pathogenesis of membranous nephropathy.     

Sedative but not anxiolytic properties of benzodiazepines are mediated by the GABA(A) receptor alpha1 subtype

Inhibitory neurotransmission in the brain is largely mediated by GABA(A) receptors. Potentiation of GABA receptor activation through an allosteric benzodiazepine (BZ) site produces the sedative, anxiolytic, muscle relaxant, anticonvulsant and cognition-impairing effects of clinically used BZs such as diazepam. We created genetically modified mice (alpha1 H101R) with a diazepam-insensitive alpha1 subtype and a selective BZ site ligand, L-838,417, to explore GABA(A) receptor subtypes mediating specific physiological effects. These two complimentary approaches revealed that the alpha1 subtype mediated the sedative, but not the anxiolytic effects of benzodiazepines. This finding suggests ways to improve anxiolytics and to develop drugs for other neurological disorders based on their specificity for GABA(A) receptor subtypes in distinct neuronal circuits.     

Light scattering from cervical cells throughout neoplastic progression: influence of nuclear morphology,DNA content,and chromatin texture

A number of noninvasive fiber optic optical technologies are under development for real-time diagnosis of neoplasia. We investigate how the light scattering properties of cervical cells are affected by changes in nuclear morphology, DNA content, and chromatin texture, which occur during neoplastic progression. We used a Cyto-Savant computer-assisted image analysis system to acquire quantitative nuclear features measurements from 122 Feulgen-thionin-stained histopathologic sections of cervical tissue. A subset of the measured nuclear features was incorporated into a finite-difference time-domain (FDTD) model of cellular light scattering. The magnitude and angular distribution of scattered light was calculated for cervical cells as a function of pathologic grade. The nuclear atypia strongly affected light scattering properties. The increased size and elevated DNA content of nuclei in high-grade lesions caused the most significant changes in scattering intensity. The spatial dimensions of chromatin texture features and the amplitude of refractive index fluctuations within the nucleus impacted both the angular distribution of scattering angles and the total amount of scattered light. Cellular scattering is sensitive to changes in nuclear morphology that accompany neoplastic progression. Understanding the quantitative relationships between nuclear features and scattering properties will aid in the development of noninvasive optical technologies for detection of precancerous conditions.     

Incomplete Administration of Intravenous Vancomycin Prophylaxis is Common and Associated With Increased Infectious Complications After Primary Total Hip and Knee Arthroplasty

BackgroundVancomycin is often used as antimicrobial prophylaxis in patients undergoing total hip or knee arthroplasty. Vancomycin requires longer infusion times to avoid associated side effects. We hypothesized that vancomycin infusion is often started too late and that delayed infusion may predispose patients to increased rates of surgical site infections and prosthetic joint infections.MethodsWe reviewed clinical data for all primary total hip arthroplasty (THA) and total knee arthroplasty (TKA) patients at our institution between 2013 and 2020 who received intravenous vancomycin as primary perioperative gram-positive antibiotic prophylaxis. We calculated duration of infusion before incision or tourniquet inflation, with a cutoff of 30 minutes defining adequate administration. Patients were divided into two groups: 1) appropriate administration and 2) incomplete administration. Surgical factors and quality outcomes were compared between groups.ResultsWe reviewed 1047 primary THA and TKA patients (524 THAs and 523 TKAs). The indication for intravenous vancomycin usage was allergy (61%), methicillin-resistant staphylococcus aureus colonization (17%), both allergy and colonization (14%), and other (8%). 50.4% of patients began infusion >30 minutes preoperatively (group A), and 49.6% began infusion <30 minutes preoperatively (group B). Group B had significantly higher rates of readmissions for infectious causes (3.6 vs 1.3%, P = .017). This included a statistically significant increase in confirmed prosthetic joint infections (2.2% vs 0.6%, P = .023). Regression analysis confirmed <30 minutes of vancomycin infusion as an independent risk factor for PJI when controlling for comorbidities (OR 5.22, P = .012).ConclusionLate infusion of vancomycin is common and associated with increased rates of infectious causes for readmission and PJI. Preoperative protocols should be created to ensure appropriate vancomycin administration when indicated.     

Mevalonate prevents lovastatin-induced apoptosis in medulloblastoma cell lines

BACKGROUND: 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) is a key rate-limiting enzyme in the mevalonate pathway, which generates precursors for cholesterol biosynthesis and the production of non-steroidal mevalonate derivatives that are involved in a number of growth-regulatory processes. We have reported that lovastatin, a competitive inhibitor of HMG-CoA reductase, not only inhibits medulloblastoma proliferation in vitro, but also induces near-complete cell death via apoptosis. The present study explores some of the pathways which may be involved in lovastatin-induced apoptosis. METHODS: Medulloblastoma cell lines were exposed in vitro to lovastatin with or without mevalonate, and document the effects using morphology, flow cytometry. DNA electrophoresis and Northern analysis. RESULTS: 1) Mevalonate prevents apoptosis when co-incubated with lovastatin, or when administered to lovastatin-pretreated cells. 2) Mevalonate restores the lovastatin-arrested cell cycle, allowing S phase entry. 3) Mevalonate does not prevent lovastatin-induced apoptosis after a critical duration of lovastatin pretreatment. For cell lines Daoy and UW228 this was 24 hours, and for D283 Med and D341 Med it was 48 hours. 4) Increases in HMG-CoA reductase mRNA levels induced by lovastatin are abrogated by co-incubation with lovastatin and mevalonate. CONCLUSIONS: These results confirm that lovastatin inhibition of this enzyme results in blockage of the mevalonate pathway, and that such a block is a critical step in the mechanism of lovastatin-induced apoptosis.     

Correlation of surface receptors with histological appearance in 29 cases of non-Hodgkin lymphoma.

The receptor patterns of cell suspensions from 29 cases of non-Hodgkin lymphoma were correlated with the histology of the nodes from which the cells were taken. Twenty-two were judged to be predominantly or largely B-cell, and because of this preponderance these were divided by a method based on the distribution of surface immunoglobulin and the expression of Fc and C3 receptors. "Mature" B-cell and B-mixed tumours showing capping surface Ig with Fc and/or C2 receptors correlated well with a nodular growth pattern, and consisted of what Rappaport (1966) calls "poorly differentiated" lymphocytes equivalent to the "small cleaved" cells as defined by Lukes and Collins (1975). Ten of the 14 patients in this receptor category are alive between 12 and 30 months after diagnosis. Receptor-silent and "immature" B-cell tumours with non-capping surface Ig correlated predominantly with the Rappaport histiocytic lymphoma and Lukes and Collins' large cleaved and large non-cleaved lymphomas, though these histological categories also included a wide variety of other receptor types such as T-cell, Receptor-overlap and the single true Macrophage tumour.Five of the 11 patients with receptor-silent or immature B-cell tumours are alive between 7 and 15 months after the diagnosis. Diffuse mixed and diffuse poorly differentiated lymphocytic lymphomas in Rappaport's classification correlated poorly with receptors, mature and immature B-cell tumours being equally represented.