MICrocephaly,disproportionate pontine and cerebellar hypoplasia syndrome: A clinico-radiologic phenotype linked to calcium/calmodulin-dependent serine protein kinase gene mutation

MICrocephaly, disproportionate pontine and cerebellar hypoplasia (MICPCH) syndrome, a rare X-linked disorder, generally seen in girls, is characterized by neurodevelopmental delay, microcephaly, and disproportionate pontine and cerebellar hypoplasia. It is caused by inactivating calcium/calmodulin-dependent serine protein kinase (CASK) gene mutations. We report a 2-year-old girl with severe neurodevelopmental delay, microcephaly, minimal pontine hypoplasia, cerebellar hypoplasia, and normal looking corpus callosum, with whom the conventional cytogenetic studies turned out to be normal, and an array-comparative genomic hybridization (a-CGH) analysis showed CASK gene duplication at Xp11.4. Our case highlights the importance of using clinico-radiologic phenotype to guide genetic investigation and it also confirms the role of a-CGH analysis in establishing the genetic diagnosis of MICPCH syndrome, when conventional cytogenetic studies are inconclusive.     

Protective effect of Phyllanthus fraternus against mitochondrial dysfunction induced by co-administration of cisplatin and cyclophosphamide

The evolving role of mitochondria, in mediating chemotherapy-induced apoptosis motivated us for the studies described here. The combination of cisplatin and cyclophosphamide is widely used in treating various types of cancers. The purpose of our study was to understand the mechanism of the toxicity induced by the co-administration of cisplatin and cyclophosphamide, on mitochondrial bioenergetics, and to study the protective effect of prior administration of the medicinal plant extract Phyllanthus fraternus. Our results reveal that co-administration of cisplatin (12 mg/kg, i.p) and cyclophosphamide (150 mg/kg, oral) to wistar rats (100 g) significantly alters mitochondrial structure and hence function. The rate of mitochondrial respiration was decreased significantly with both NAD + and FAD-linked substrates. The respiratory control ratio, an index of membrane integrity and the P/O ratio, a measure of phosphorylating efficiency also were decreased significantly accompanied by elevation in the lipid peroxide levels in liver, kidney homogenate and liver mitochondria respectively. Also, the phospholipid content of the mitochondrial membrane, showed a significant decrease, indicating mitochondrial membrane changes. Prior administration of an aqueous extract of P. fraternus (100 mg/kg) to rats, showed protection on all parameters investigated. Administration of P. fraternus alone did not show any significant changes on mitochondrial membrane bioenergetics. Thus, we propose, that the toxic side effects of cisplatin ₊ cyclophosphamide, are due to a chain of interconnected events, within the mitochondrial inner membrane, ultimately leading to hepatotoxicity and nephrotoxicity. Further, our work also suggests that administration of aqueous extract of P. fraternus can enhance the therapeutic potential of anticancer drugs by reducing drug related toxicity.     

Assessment of the in vitro cytotoxicity and in vivo anti-tumor activity of the alcoholic stem bark extract/fractions of Mimusops elengi Linn.

Various parts of Mimusops elengi Linn. (Sapotaceae) have been used widely in traditional Indian medicine for the treatment of pain, inflammation and wounds. The study was conducted to explore the use of stem bark of M. elengi on pharmacological grounds and to evaluate the scientific basis of cytotoxic and anti-tumor activity. Extract/fractions were prepared and in vitro cytotoxicity was assessed using SRB assay. Most effective fractions were subjected to fluorescence microscopy based acridine orange/ethidium bromide (AO/EB) and Hoechst 33342 staining to determine apoptosis induction and DNA fragmentation assay. Comet and micronuclei assay were performed to assess genotoxicity. Cell cycle analysis was also performed. In vivo anti-tumor potential was evaluated by Ehrlich ascites carcinoma (EAC) model in mice. The alcoholic stem bark extract of M. elengi along with four fractions showed potential in vitro cytotoxicity in SRB assay. Of these, dichloromethane and ethyl acetate fractions were selected for further studies. The fractions revealed apoptosis inducing potential in AO/EB and Hoechst 33342 staining, which was further confirmed by DNA fragmentation assay. Genotoxic potential was revealed by comet and micronuclei assay. Fractions also exhibited specific cell cycle inhibition in G₀/G₁ phase. In EAC model, ethyl acetate fraction along with the standard (cisplatin) effectively reduced the increase in body weight compared to control and improved mean survival time. Both fractions were able to restore the altered hematological and biochemical parameters. Hence, M. elengi stem bark may be a possible therapeutic candidate having cytotoxic and anti-tumor potential.     

BLOC-1 is required for cargo-specific sorting from vacuolar early endosomes toward lysosome-related organelles

Hermansky-Pudlak syndrome (HPS) is a genetic disorder characterized by defects in the formation and function of lysosome-related organelles such as melanosomes. HPS in humans or mice is caused by mutations in any of 15 genes, five of which encode subunits of biogenesis of lysosome-related organelles complex (BLOC)-1, a protein complex with no known function. Here, we show that BLOC-1 functions in selective cargo exit from early endosomes toward melanosomes. BLOC-1-deficient melanocytes accumulate the melanosomal protein tyrosinase-related protein-1 (Tyrp1), but not other melanosomal proteins, in endosomal vacuoles and the cell surface due to failed biosynthetic transit from early endosomes to melanosomes and consequent increased endocytic flux. The defects are corrected by restoration of the missing BLOC-1 subunit. Melanocytes from HPS model mice lacking a different protein complex, BLOC-2, accumulate Tyrp1 in distinct downstream endosomal intermediates, suggesting that BLOC-1 and BLOC-2 act sequentially in the same pathway. By contrast, intracellular Tyrp1 is correctly targeted to melanosomes in melanocytes lacking another HPS-associated protein complex, adaptor protein (AP)-3. The results indicate that melanosome maturation requires at least two cargo transport pathways directly from early endosomes to melanosomes, one pathway mediated by AP-3 and one pathway mediated by BLOC-1 and BLOC-2, that are deficient in several forms of HPS.     

Practical aspects of planning, building, and interpreting tissue microarrays: The Cooperative Prostate Cancer Tissue Resource experience

This is a review of several new approaches developed at or adopted by the Cooperative Prostate Cancer Tissue Resource (CPCTR) to resolve issues involved in tissue microarray (TMA) construction and use. CPCTR developed the first needle biopsy TMA, allowing researchers to obtain 200 or more consecutive cancer sections from a single biopsy core. Using radiographs of original paraffin blocks to measure tissue thickness we developed a method to produce TMAs with a larger number of usable sections. The modular approach to plan TMA construction is also a novel concept wherein TMAs of different types, such as tumor grade TMAs, metastasis TMA and hormone refractory tumors TMA can be combined to form an ensemble of TMAs with expanded research utility, such as support for tumor progression studies. We also implemented an open access TMA Data Exchange Specification that allows TMA data to be organized in a self-describing XML document annotated with well-defined common data elements. It ensures inter-laboratory reproducibility because it offers information describing the preparation of TMA blocks and slides. There are many important aspects that may be missed by both beginners and experienced investigators in areas of TMA experimental design, human subjects protection, population sample size, selection of tumor areas to sample, strategies for saving tissues, choice of antibodies for immunohistochemistry, and TMA data management.     

Simultaneous measurements of proton motive force, delta pH, membrane potential, and H+/O ratios in intact Escherichia coli.

An instrument is described that enables the simultaneous monitoring of proton motive force (PMF), membrane potential (delta psi), the delta pH across a membrane, oxidase activity, proton movements, and H+/O ratios. We have studied the relationship existing among these parameters of energy transduction as a critical condition is changed during an experiment. The major findings are: (a) In the pH range of 4.5 to 7.5, increasing the external pH causes an increase in delta psi, internal pH, and oxidase activity, a decrease in H+/O ratio, and a peak-plateau in PMF from pH 5.5 to 6.6 where delta pH is converted to delta psi. (b) An increase in [K+] from 1 to 100 mM, in the presence of 0.5 microM valinomycin, causes the conversion of delta psi to delta pH, a gradual decline in PMF and an increase in H+/O ratio, internal pH, and oxidase activity. (c) Increasing valinomycin concentration from 0 to 2.5 microM, in the presence of 50 mM [K+], causes a decline in delta psi from 125 to 0 mV, and an increase in delta pH from 35 to 70 mV. From 2.5 to 10 microM, the delta pH and the PMF (which it solely represents), stay constant, H+/O ratio increases mainly from 0 to 0.5 microM and much more slowly from 2.5 to 10 microM. (d) Oxygen at only 10% of its concentration in air-saturated buffer can support the generation of 90% or more of the delta psi, delta pH, and PMF generated in an air-saturated solution. (e) The return of extruded protons to the cell (referred to here as "suck-back") represents a complicated process driven by delta psi and influenced by a variety of factors. (f) H+/O ratios measured by the kinetic technique used here are much higher than those measured by standard oxygen pulse techniques.