Biomedical subjects

Michael Brownstein

Publications and source records attributed to Michael Brownstein.

4 recordsLinked to original sources

PubMed2006

Increased response to high KCl-induced elevation in the intracellular-Ca(2+) concentration in differentiated NG108-15 cell and the inhibitory effect of the L-type Ca(2+) channel blocker, calciseptine.

Characteristics of the increasing effect for the concentration of intracellular calcium ions ([Ca(2+)](i)) by high-KCl application were investigated in the neuroblastomaxglioma hybrid NG108-15 cell line (NG108-15 cells). The present study confirmed that the increasing effect of [Ca(2+)](i) by high-KCl application in single NG108-15 cells, differentiated with dibutyryl cAMP (Bt(2)cAMP), was significantly enhanced, compared to undifferentiated cells. The following observations were made at first: (1) The response to high-KCl application, in both undifferentiated and differentiated cells, was significantly inhibited by calciseptine (CaS), an L-type Ca(2+) channel blocker, but not by N-, P- and R-type Ca(2+) channel blockers. The IC(50) values for CaS in both undifferentiated and differentiated cell was almost identical. (2) The inhibitory effect of CaS was irreversible. (3) The increasing effect for [Ca(2+)](i) by high-KCl application was completely dependent on the presence of extracellular calcium ions. (4) The increased [Ca(2+)](i) by high-KCl application under a plateau concentration was quickly decreased to basal levels when the high-KCl solution was exchanged for a high-KCl solution containing EGTA (without CaCl(2)). Together, these results suggest that the enhancement of the response effect of [Ca(2+)](i) by high-KCl application in differentiated single NG108-15 cells was mainly due to the quantitative increase of L-type voltage-sensitive calcium channels (VSCCs), which were irreversibly inhibited by CaS.

Animals↗

PubMed2006

Sample labeling: an overview.

It is not easy to write a critical review of the methods available for labeling RNA and DNA "extracts" for microarray studies. There are a number of reasons for this: Suppliers of the reagents and kits used for this purpose do research and development, quality control, and validation and then they provide a hard-wired, "optimized" product. They often give few details about the compositions of these products, are inclined to put the best face they can on what they sell and gloss over any deficiencies, and have no interest in paying for direct comparisons of their product to those of other companies. These comparisons can be expensive to perform, and there are few good examples in the literature. When comparative experiments have been done, it is not clear that each of the individual methods tested was executed with equal proficiency. Many experiments can be required to determine how best to hybridize any given labeled extract to a particular array and how to block, wash, and postprocess (e.g., stain) the array so that the signal-to-noise ratio is maximized. In addition, authors of comparative studies used different arrays, technical protocols (some of which are out of date), experimental designs, and analyses. Finally, some new techniques, which seem quite promising, have been employed so little that their strengths and shortcomings are difficult to assess.

Animals↗

PubMed2004

Evaluation of vector-primed cDNA library production from microgram quantities of total RNA.

cDNA sequences are important for defining the coding region of genes, and full-length cDNA clones have proven to be useful for investigation of the function of gene products. We produced cDNA libraries containing 3.5-5 x 10(5) primary transformants, starting with 5 mug of total RNA prepared from mouse pituitary, adrenal, thymus, and pineal tissue, using a vector-primed cDNA synthesis method. Of approximately 1000 clones sequenced, approximately 20% contained the full open reading frames (ORFs) of known transcripts, based on the presence of the initiating methionine residue codon. The libraries were complex, with 94, 91, 83 and 55% of the clones from the thymus, adrenal, pineal and pituitary libraries, respectively, represented only once. Twenty-five full-length clones, not yet represented in the Mammalian Gene Collection, were identified. Thus, we have produced useful cDNA libraries for the isolation of full-length cDNA clones that are not yet available in the public domain, and demonstrated the utility of a simple method for making high-quality libraries from small amounts of starting material.

Animals↗

PubMed2004

Identification of genes expressed in primate primordial oocytes.

BACKGROUND: The factors involved in oocyte survival and transition from quiescence to the growing phenotype remain unknown. Herein we report genes that are differentially expressed in the primordial oocyte revealed by DNA arrays. METHODS: Primordial oocytes were captured selectively in rhesus monkey ovary sections using laser capture microdissection. The RNA was extracted and amplified in two rounds by T7-based linear RNA amplification, fluorescence labelled and then hybridized to human cDNA arrays containing 7680 elements. RNA from human placenta served as a reference sample. RESULTS: Ninety-five genes were found to be consistently expressed at a higher level in primordial oocytes. Expression of several of these genes in the oocyte has been reported before, e.g. deleted in azoospermia (DAZ), prohibitin and transglutaminase 2. Oocyte expression of several novel transcripts revealed on array hybridization, such as gene 33, ubiquitin-conjugating enzyme E2A, G1 to S phase transition 1, growth arrest and DNA damage-inducible (GADD), and dendritic cell-derived ubiquitin-like protein (DC-UbP) was confirmed by in situ hybridization. Some array-identified gene products [integrin beta3, alpha-tubulin, regulatory telomere elongation protein (RAP1) and cellular repressor of EIA-stimulated genes (CREG protein)] were detected in human oocytes by immunofluorescence. Bioinformatic analysis of the oocyte-enriched transcripts reveals a functional profile summarized as follows: cell cycle (14%); transporter (13%); signal transduction (10%); cytoskeletal (7%); transcription factor (5%); immune response (5%); apoptosis-related (5%); RNA processing (5%); and the remainder of miscellaneous categories. CONCLUSIONS: These observations may contribute to the elucidation of molecular pathways involved in oocyte survival and maturation.

Animals↗