beta-Adrenoceptor blockers protect against staurosporine-induced apoptosis in SH-SY5Y neuroblastoma cells

The beta-adrenoceptor blockers exhibit a well-characterized anti-apoptotic property in the heart and kidney while less is known about the effect of this class of drugs on neuronal apoptosis. We studied the effects of three beta-adrenoceptor blockers propranolol (1-(isoproplyamino)-3-(naphthalene-1-yloxy)propan-2-ol), atenolol (2-[4-[2-hydroxy-3-(1-methylethylamino)propoxyl]phenyl]ehanamide), and ICI 118551 (1-[2,3-(dihydro-7-methyl-1H-iden-4-yl)oxy]-3-[(1-methylethyl)amino]-2-butanol), against staurosporine-induced apoptosis in SH-SY5Y human neuroblastoma cells. Staurosporine increased caspase 3-like activity, DNA fragmentation, PARP cleavage, and the number of TUNEL positive cells consistent with the induction of apoptosis. Propranolol and ICI 118551, but not atenolol, demonstrated a concentration-dependent inhibition of caspase 3-like activity. Propranolol and ICI 118551 directly inhibited the enzymatic activity of recombinant caspase 9 while atenolol did not; however, none of the beta-adrenoceptor blockers that were examined directly blocked caspases 2 or 3 activity. In isolated mitochondria, propranolol and ICI 118551 inhibited staurosporine-induced cytochrome c release while atenolol did not. We conclude that propranolol and ICI 118551 protect SH-SY5Y cells against staurosporine-induced apoptosis through a dual action on the mitochondria and on caspase 9 in a cell type and an apoptotic paradigm where the conventional inhibitors of mitochondrial permeability transition such as cyclosporin A and bongkrekic acid demonstrate no protection.     

Oxazolone and diclofenac-induced popliteal lymph node assay reactions are attenuated in mice orally pretreated with the respective compound: potential role for the induction of regulatory mechanisms following enteric administration

The murine popliteal lymph node assay (PLNA) was examined as a preclinical assay with the potential to identify low-molecular-weight compounds (LMWCs) that are likely to be associated with immune-mediated drug hypersensitivity reactions (IDHRs) in humans. We hypothesized that the contact sensitizer oxazolone (OX) would cause a strong PLN reaction in naive mice and that the PLN reaction would be attenuated in mice orally pretreated with OX due to the induction of oral tolerance. In naive mice, OX induced a strong PLN reaction and caused dose-dependent increases in PLN size, weight, cellularity, percentage of CD4(+) PLN T cells, and percentage of PLN B cells, with a concomitant decrease in the percentage of CD8(+) PLN T cells. Next, the PLNA was conducted in mice gavaged three times with either OX or vehicle alone (olive oil). Mice pretreated with OX had suppressed PLN reactions following the footpad injection of OX (decrease in PLN size, weight, and cellularity), which was associated with an increase in the percentage of PLN CD8(+)T cells. In contrast, oral pretreatment with OX had no observable effect on the PLN reaction induced following footpad injection of the irrelevant hapten dinitrochlorobenzene (DNCB). Adoptive transfer studies were conducted to examine the mechanism of PLN hyporesponsiveness. It was found that either (1) unfractionated splenocytes or (2) purified CD8(+) splenocytes, but not (3) purified CD4(+) splenocytes isolated from mice gavaged with OX adoptively transferred PLN suppression to naive BALB/c mice. Because OX is not a pharmaceutical, we also examined the NSAID diclofenac (DF) (Voltaren). Like OX, DF caused dose-dependent increases in PLN size, weight, and cellularity in naive mice. Furthermore, like OX, the diclofenac-induced PLN reaction was attenuated in mice that had been orally pretreated three times with DF. However, splenocytes from mice orally treated with DF were not able to adoptively transfer PLN hyporesponsiveness. Collectively, these observations demonstrate that both OX and DF are potent immunostimulators in the PLNA. As importantly, these results demonstrate that the immunostimulating potential of OX and DF in the PLNA is significantly decreased in mice orally exposed to the respective drug, possibly due to the presence of a cellular mechanism of oral tolerance. For OX, the mechanism appears to involve, in part, CD8(+) T cells, whereas the mechanism(s) associated with PLN hyporesponsiveness using DF remain to be defined.     

Biological activity and phytochemical analysis of three Indonesian medicinal plants, Murraya koenigii, Syzygium polyanthum and Zingiber purpurea

Extracts of Indonesian medicinal plants, Murraya koenigii, Syzygium polyanthum, and Zingiber purpurea were investigated for their biological activity. The presence of phytochemicals, cytotoxicity, and antimicrobial and antioxidant activities were investigated. Parts of M. koenigii, S. polyanthum, and Z. purpurea were extracted with ethanol. The extracts were evaluated for antimicrobial activity using the disc diffusion method, while antioxidant activity was determined with a 1,1-diphenyl-2-picrylhydrazyl radical scavenging assay. Cytotoxicity was investigated using the brine shrimp lethality test, and phytochemical screening was performed using a standard method. M. koenigii leaf extract exhibited the most activity in the test microorganism activity index (AI), 0.38-1.25, when compared with standard drugs. S. polyanthum ripened fruit displayed significant antioxidant activity (90%) in comparison to ascorbic acid (95%). Z. purpurea rhizome extract possessed the highest cytotoxic effect with a LC(50) of 52 μg/mL. Phytochemical analysis revealed that carbohydrate, tannin, alkaloid, steroid, triterpenoid, and flavonoid were present in the extracts of M. koenigii leaves and twigs, S. polyanthum leaves and ripened and unripe fruits, and Z. purpurea rhizome, while saponin was only present in the S. polyanthum ripened fruit extract. Our work revealed that the M. koenigii leaves, S. polyanthum ripened fruit, and Z. purpurea rhizome extracts have potential as sources of new antimicrobial, antioxidant, and cytotoxic compounds, respectively.     

Contribution des facteurs génétiques et nutritionnels dans les hyperhomocystéinémies dans la population algérienne saine

The particular genetic polymorphisms associated to homocysteine metabolism enzymes, or more usually, a relative lack in different vitamins B group, are the main cause of the increase in this sulfur amino acid in the blood. As a fact, hyperhomocysteinemia are associated to many pathologies. The aim of this study is to determine the frequencies of the different genotypes caused by these polymorphisms, folate and vitamin B12 status and their eventual connections to hyperhomocysteinemia among a healthy adult population. The investigation has been applied to 165 apparently healthy volunteers. The homocysteine concentration was determined by IMx fluorescence polarization immunoassay. The genotypes determination was done by real-time PCR (RT-PCR) (Light Cycler 480). Folates and vitamin B12 were analyzed by SimulTRAC-SNB immunoassay. The homocysteine level was 14,69 ± 7,30 μmol/L. 22.5% of the subjects exhibited a moderate hyperhomocysteinemia (> 15 μmol/L). The major nutritional determinants of the plasmatic homocysteine rates among these subjects were blood folate and vitamin B12 levels. Blood homocysteine was the highest for the homozygote (TT) individuals for the MTHFR gene than for the heterozygote (CT) and homozygote (CC) subjects in particular for the lowest blood folate quartile. These two assessments have to be taken in consideration when evaluating the predisposition of the Algerian population for morbid and/or mortal pathologies and allow emphasizing on the necessity of screening out and eventually treating vitamin deficiencies on folates vitamin B12.     

Human bone marrow-derived cells: An attractive source to populate dermal substitutes

We have previously shown the importance of dermal fibroblasts within skin substitutes for promoting the emergence of a functional neodermis after grafting in humans. However, the use of fibroblasts from sources other than the dermis needs to be evaluated for patients with extensive skin loss. Here we examined the capacity of human bone marrow-derived cells (BMDCs), selected for their ability to adhere to plastic culture dishes, to behave like human dermal fibroblasts when incorporated within a 3D in vitro reconstructed tissue that promotes dermal fibroblast differentiation. Like dermal fibroblasts, BMDCs contracted a collagen matrix and were growth regulated by the matrix environment. They had the same shape and their nuclei had the same form factor as dermal fibroblasts. In addition, both cell types expressed desmin and vimentin but not α-smooth muscle actin. BMDCs deposited collagen types I and III, and fibrillin-1 with similar efficiency to dermal fibroblasts. In addition, BMDCs have the potential to regulate this deposition, as they produced metalloproteinases (MMP1, MMP2, and MMP9) and metalloproteinase inhibitors (TIMP1) very similarly to dermal fibroblasts. BMDCs can thus be induced to express functions resembling those of dermal fibroblasts, including those involved in the wound healing process.     

Analysis of erythrocytic polyamines by automated reverse-phase liquid chromatography

A sensitive and reproducible method for quantitation of polyamines in erythrocytes by high-performance liquid chromatography (HPLC) is described. Spermidine and spermine are first converted to fluorescent dansyl derivatives and are then separated in less than 15 minutes on a C18 reverse-phase cartridge using methanol in water mobile phase. Sensitivity of the method is 20 pmol with recoveries that average 96% and 95% for spermidine and spermine, respectively. Precision study revealed an average mean coefficient of variation of 5.42 ± 2.95 (mean ± S.D.) from four levels of the polyamines (8.2 to 354.9 nmol/g hemoglobin [Hgb]). The normal ranges for 30 apparently healthy men and women, aged 20–60 years, were 2.9–33.7 nmol/g Hgb (± 2 S.D. from mean) for spermidine and 0-20.9 nmol/g Hgb for spermine. The quantity of putrescine was negligible. Feasibility of this method was evaluated in serial specimens from an advanced-stage patient with prostate cancer who was receiving multiple-modality therapy. Results revealed that this HPLC method can be used in quantifying circulating polyamines in clinical specimens.     

Commercial transplants in local Pakistanis from vended kidneys: a socio-economic and outcome study

Donor shortage and absence of transplant law lead to unrelated commercial transplants in Pakistan. We report the socio‐economic and outcome parameters of 126 local recipients of unrelated kidney vendor transplants presenting to our institute between 1997 and 2007. Their outcome was compared with 180 recipients of living‐related donor transplants matched for age, gender and transplant duration as controls. Age of commercial recipients was 35.63 ± 11.57 years with an M:F ratio of 2.4:1. Majority (92%) were transplanted in northern Pakistan paying US$7271 ± 2198. All were educated with 50% being graduates or above and rich earning a monthly salary of US$517 ± 518 with 44% earning >US$500. Comparison of commercial recipients with controls showed high comorbidities 35 (28%) vs. 14 (8%) (P = 0.0001) with diabetes, hepatitis‐C and cardiovascular diseases. Donor age was 29.97 ± 6.16 vs. 32.63 ± 9.3 years (P = 0.035). Biologic agents induction in 101 (80%) vs. 14 (8%) (P = 0.0001), acute rejections in 42 (33%) vs. 31 (17%) (P = 0.005), 1‐year creatinine 1.84 ± 1.28 vs. 1.27 ± 0.4 mg/dl (P = 0.0001), surgical complications 28 (22%) vs. 14 (8%) (P = 0.001), tuberculosis 14 (11%) vs. 6 (6%) (P = 0.007), acute hepatitis 20 (16%) vs. 3 (2%) (P = 0.0001), cytomegalovirus 33 (26%) vs. 21 (11%) (P = 0.001) and recurrent urinary tract infection 35 (28%) vs. 30 (16%) (P = 0.034). Overall 1‐ and 5‐year graft survival was 86% and 45% vs. 94% and 80%, respectively (P = 0.00001). Total deaths were 34 (27%) vs. 12 (6.0%) (P = 0.001). In conclusion, recipients of the vended kidneys are poor candidates, educated, rich and often self‐selecting. Their outcome is poor, which will leave them poorer still and back to dialysis if not death.     

Dysfunction of mouse cerebral arteries during early aging

Aging leads to a gradual decline in the fidelity of cerebral blood flow (CBF) responses to neuronal activation, resulting in an increased risk for stroke and dementia. However, it is currently unknown when age-related cerebrovascular dysfunction starts or which vascular components and functions are first affected. The aim of this study was to examine the function of microcirculation throughout aging in mice. Microcirculation was challenged by inhalation of 5% and 10% CO₂ or by forepaw stimulation in 6-week, 8-month, and 12-month-old FVB/N mice. The resulting dilation of pial vessels and increase in CBF was measured by intravital fluorescence microscopy and laser Doppler fluxmetry, respectively. Neurovascular coupling and astrocytic endfoot Ca²⁺ were measured in acute brain slices from 18-month-old mice. We did not reveal any changes in CBF after CO₂ reactivity up to an age of 12 months. However, direct visualization of pial vessels by in vivo microscopy showed a significant, age-dependent loss of CO₂ reactivity starting at 8 months of age. At the same age neurovascular coupling was also significantly affected. These results suggest that aging does not affect cerebral vessel function simultaneously, but starts in pial microvessels months before global changes in CBF are detectable.