Depletion of O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine in three-dimensional collagen cultures of normal human breast epithelial cells.

O6-Alkylguanine--DNA alkyltransferase (AGT) is a protein which removes the promutagenic O6-alkylguanine lesion induced in DNA by alkylating agents. Our results demonstrate that freshly isolated organoids from reduction mammoplasty specimens contain significant levels of AGT activity. AGT activity in breast epithelial cells shows interindividual variation. Constitutive levels of AGT activity remain unchanged during short-term serum-free culture of breast epithelial cells inside three-dimensional rat-tail collagen gel matrix. In the present study, we optimized conditions for depleting AGT activity in human breast epithelial cells cultured in three-dimensional collagen gel matrix using O6-methylguanine and O6-benzylguanine which are substrates for AGT. AGT activity was efficiently inactivated by exposure of cells to O6-methylguanine or O6-benzylguanine. Inactivation with O6-benzylguanine was more rapid, of greater magnitude and consistency and occurred at lower concentrations than with O6-methylguanine. Near-complete inactivation (> 99.5%) of AGT activity was reproducibly achieved with 50 microM O6-benzylguanine. In contrast, 500 microM O6-methylguanine was needed to obtain a maximal effect and this reduced AGT activity by only 53-93% of control. Within 30 min of adding the free base, 50 microM O6-benzylguanine depleted 95% of the levels of AGT compared to 30% inhibition with 500 microM O6-methylguanine. The profile for restoration of AGT activity was different following a 24 h incubation and subsequent removal of each of the guanine derivatives. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. AGT activity levels remained undetectable for at least 2 days after removal of 50 microM O6-benzylguanine from the medium and recovered to only 53% of control values after an additional 3 days. In contrast, following removal of 500 microM O6-methylguanine, the activity was restored from its nadir of 16% of control values reaching pretreatment levels after 5 days. These results suggest that treatment with O6-benzylguanine may be used to modulate the incidence of transforming mutations in cultured human breast epithelial cells treated with chemical carcinogens which give rise to O6-alkylguanine adducts.     

Model for assessment of proficiency of human immunodeficiency virus type 1 sequencing-based genotypic antiretroviral assays

Use of sequencing-based genotyping as a diagnostic assay for human immunodeficiency virus (HIV) antiretroviral resistance is increasing. Periodic evaluation of the proficiency of laboratories performing this assay should be established. It is important to identify components of the assay that influence the generation of reliable sequencing data and that should and can be monitored. A model was developed to determine what parameters were reasonable and feasible for assessing the performance of genotyping assays. Ten laboratories using the genotyping platform, HIV-1 Genotyping System (HGS) v. 1 and software versions 1.1 or 2.0, participated in two rounds of testing. For each round, each group was sent a panel consisting of three clinical samples to sequence in real time. Six months later, seven laboratories using the TRUGENE HIV-1 Genotyping Kit participated in a separate round, working with both panels at the same time. Analysis of the data showed that one main indicator of genotyping proficiency was achievement of > or =98% sequence homology of a sample tested to a group consensus sequence for that sample. A second was concordant identification of codons at sites identified with resistance mutations in the sample, although scoring of these criteria is still undetermined from this study. These criteria are applicable to all sequence-based genotyping platforms and have been used as a baseline for assessing the performance of genotyping for the determination of antiretroviral resistance in our ongoing proficiency program.     

CGS 22745: A selective orally active inhibitor of 5-lipoxygenase

CGS 22745, and aralkyl hydroxamic acid, inhibited 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄) synthesis in guinea pig leukocytes (IC₅₀=0.6M). The compound did not appreciably affect cyclooxygenase (ram seminal vesicles), 12-lipoxygenase and thromboxane synthase (human platelets) or 15-lipoxygenase (human neutrophils). CGS 22745 inhibited A23187-induced formation of LTB₄ in blood (IC₅₀'s of 4.3, 0.56 and 3.2 M for human, dog and rat, respectively). At 1 mg/kg i.v. in dogs, it caused 96% inhibition of A23187-stimulated LTB₄ formationex vivo after 5 min. Its effective biological half-life was >160 min. In dogs at 3 and 10 mg/kg p.o., CGS 22745 inhibitedex vivo A23187-stimulated LTB₄ formation at 3 hr by 48% and 97%, respectively. The inhibition persisted up to 6 hr (26% at 3 mg/kg; 49% at 10 mg/kg). CGS 22745 (3, 10 and 30 mg/kg p.o.) inhibited exudate formation, mononuclear cells and PMN accumulation in a dose-dependent manner during the late phase (48 and 72 hr) of carrageenan-induced pleurisy in the rat.     

In vitro and in vivo acute antihyperglycemic effects of five selected indigenous plants from Jordan used in traditional medicine

Ethnopharmacological relevance

Achillea santolina L., Pistacia atlantica Desf, Rheum ribes L., Sarcopoterium spinosum (L.) Spach and Teucrium polium L. have traditionally been used as herbal antidiabetic medicines. However their alleged benefits and mechanisms remain elusive.

Aim of the study

This study aimed to evaluate the effect of water extracts of these plants in in vitro and in vivo experiments.

Materials and methods

In vitro enzymatic starch digestion with aqueous extracts from plants at concentrations of 1, 5, 10, 12.5, 25, 50 and 100 mg/ml was assayed using α-amylase and α-amyloglucosidase. Acarbose was used as control and glucose liberation was determined by glucose oxidase method. Oral starch tolerance test (OSTT) and oral glucose tolerance test (OGTT) were determined for the plant extracts at concentrations 125, 250 and 500 mg/kg b.wt. on Sprague-Dawley rats. Blood glucose levels in rats treated with plant extracts and drugs (acarbose or metformin and glipizide) were measured at −30, 0, 45, 90 and 135 min.

Results

Compared to acarbose (IC₅₀ = 1.2 μg/ml), water extracts of Pistacia atlantica, Rheum ribes and Sarcopoterium spinosum exerted significant dose dependent dual inhibition of α-amylase and α-glucosidase in in vitro enzymatic starch digestion bioassay, with IC₅₀s; 46.98, 58.9 and 49.9 mg/ml, respectively. Comparable in vivo results were obtained for starch-fed rats, exhibiting significant acute postprandial antihyperglycemic efficacies. While Achillea santolina and Teucrium polium extracts lacked any favourable in vitro anti-α-amylase and anti-α-glucosidase effect, other modes of action can possibly explain their substantial acute antihyperglycemic activities in starch-treated rats. Except for Pistacia atlantica extracts, none of the investigated extracts qualified for improving the glucose intolerance in fasted rats on glucose loading.

Conclusions

Pistacia atlantica, Rheum ribes and Sarcopoterium spinosum can be considered as potential candidates for amelioration/management of type 2 diabetes.     

Hypophosphatemic rickets/osteomalacia in linear sebaceous nevus syndrome: a variant of tumor-induced osteomalacia

A severe form of vitamin D-resistant rickets is associated with the linear sebaceous nevus syndrome. We investigated the pathophysiology underlying defective bone mineralization in two individuals and examined the effects of 1,25-dihydroxyvitamin D (1,25(OH)2D, calcitriol) therapy on the clinical and biochemical abnormalities. Both patients had fasting hypophosphatemia, markedly diminished TmP/GFR, and elevated alkaline phosphase activity in the presence of normocalcemia. Before treatment with calcitriol, serum 1,25(OH)2D concentrations were reduced but serum 25-hydroxyvitamin D (25(OH)D) concentrations were normal. Administration of calcitriol increased serum 1,25(OH)2D concentrations and led to an increase in TmP/GFR and serum phosphorus levels and to a decrease in alkaline phosphatase activity. However, the renal tubular maximum for reabsorption of inorganic phosphate, normalized according to glomerular filtration rate, and serum phosphorus levels remained abnormally low even in the patient who also received phosphate supplementation. Bone histomorphologic studies in the adult patient showed extreme osteomalacia, which partially improved with calcitriol. These data demonstrate that the putative skin lesion-derived factor results in both a renal tubular defect in phosphate reabsorption and in 1,25-(OH)2 D deficiency. The vitamin D-resistant rickets of linear sebaceous nevus syndrome is a variant of tumor-induced osteomalacia.     

Embolic myocardial infarction diagnosed with Fourier domain optical coherence tomography

We report the case of a young female with embolic myocardial infarction. The embolic etiology was confirmed by Fourier Domain Optical Coherence Tomography as well as histo-pathology.     

Oscillatory vagal maneuvers produce ventricular entrainment in patients with atrial fibrillation

Background  

Atrial fibrillation (AF) is the most common sustained dysrhythmia and appears to be an independent predictor of sudden cardiac death. The irregular ventricular rhythm contains both linear and non-linear patterns; however, it remains unclear whether vagally mediated effects are present within these patterns.