The direct thrombin inhibitors: their role and use for rational anticoagulation.

Major clinical advantages are achieved when direct thrombin inhibitors are used in venous thromboembolism. These agents provide more reliable anticoagulant response patterns because they are not significantly bound to plasma proteins and few, if any, drug-drug interactions are seen. The studies to date confirm that not all direct thrombin inhibitors are the same.

Circulating tumor cells in patients with breast cancer dormancy.

Purpose: The purpose of this study was to test the hypothesis that circulating tumor cells (CTCs) are present in patients many years after mastectomy without evidence of disease and that these CTCs are shed from persisting tumor in patients with breast cancer dormancy.

Experimental Design: We searched for CTCs in 36 dormancy candidate patients and 26 age-matched controls using stringent criteria for cytomorphology, immunophenotype, and aneusomy.

Results: Thirteen of 36 dormancy candidates, 7 to 22 years after mastectomy and without evidence of clinical disease, had CTCs, usually on more than one occasion.

A protein sensor for siRNA asymmetry.

To act as guides in the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be unwound into their component strands, then assembled with proteins to form the RNA-induced silencing complex (RISC), which catalyzes target messenger RNA cleavage. Thermodynamic differences in the base-pairing stabilities of the 5' ends of the two approximately 21-nucleotide siRNA strands determine which siRNA strand is assembled into the RISC. We show that in Drosophila, the orientation of the Dicer-2/R2D2 protein heterodimer on the siRNA duplex determines which siRNA strand associates with the core RISC protein Argonaute 2.

MIB1 labeling index as an indicator of chemoresponse in carcinoma of the breast.

In this study, we determined the extent of variation in proliferative markers and hormone receptor status in breast carcinoma between core biopsies and subsequent resections, and determined the impact of clinical and histologic parameters on such variation. We performed a paired comparison of biomarkers in 87 core biopsies and subsequent resections of breast carcinomas in patients with and without preoperative chemotherapy. The markers included estrogen receptor, progesterone receptor, Her2/neu, DNA ploidy (diploidy versus nondiploidy), DNA index (difference of > or = 0.

HER-2 gene amplification can be acquired as breast cancer progresses.

Amplification and overexpression of the HER-2 oncogene in breast cancer is felt to be stable over the course of disease and concordant between primary tumor and metastases. Therefore, patients with HER-2-negative primary tumors rarely will receive anti-Her-2 antibody (trastuzumab, Herceptin) therapy. A very sensitive blood test was used to capture circulating tumor cells (CTCs) and evaluate their HER-2 gene status by fluorescence in situ hybridization.

Kinetic analysis of the RNAi enzyme complex.

The siRNA-directed ribonucleoprotein complex, RISC, catalyzes target RNA cleavage in the RNA interference pathway. Here, we show that siRNA-programmed RISC is a classical Michaelis-Menten enzyme in the presence of ATP. In the absence of ATP, the rate of multiple rounds of catalysis is limited by release of the cleaved products from the enzyme.

RISC assembly defects in the Drosophila RNAi mutant armitage.

The putative RNA helicase, Armitage (Armi), is required to repress oskar translation in Drosophila oocytes; armi mutant females are sterile and armi mutations disrupt anteroposterior and dorsoventral patterning. Here, we show that armi is required for RNAi. armi mutant male germ cells fail to silence Stellate, a gene regulated endogenously by RNAi, and lysates from armi mutant ovaries are defective for RNAi in vitro.

Reduced levels of mercury in first baby haircuts of autistic children.

Reported rates of autism have increased sharply in the United States and the United Kingdom. One possible factor underlying these increases is increased exposure to mercury through thimerosal-containing vaccines, but vaccine exposures need to be evaluated in the context of cumulative exposures during gestation and early infancy. Differential rates of postnatal mercury elimination may explain why similar gestational and infant exposures produce variable neurological effects.

In vitro analysis of RNA interference in Drosophila melanogaster.

Double-stranded RNA (dsRNA) triggers the destruction of mRNA sharing sequence with the dsRNA, a phenomenon termed RNA interference (RNAi). The dsRNA is converted by endonucleolytic cleavage into 21- to 23-nt small interfering RNAs (siRNAs), which direct a multiprotein complex, the RNA-induced silencing complex to cleave RNA complementary to the siRNA. RNAi can be recapitulated in vitro in lysates of syncytial blastoderm Drosophila embryos.

Evidence that siRNAs function as guides, not primers, in the Drosophila and human RNAi pathways.

In Drosophila, two features of small interfering RNA (siRNA) structure--5' phosphates and 3' hydroxyls--are reported to be essential for RNA interference (RNAi). Here, we show that as in Drosophila, a 5' phosphate is required for siRNA function in human HeLa cells. In contrast, we find no evidence in flies or humans for a role in RNAi for the siRNA 3' hydroxyl group.

Aneusomy 17 in breast cancer: its role in HER-2/neu protein expression and implication for clinical assessment of HER-2/neu status.

HER-2/neu protein overexpression in breast cancer is mostly caused by HER-2/neu gene amplification. However, it is unclear whether aneusomy 17 may also play a role. Using immunohistochemistry assay (IHC) with DAKO antibody and manual quantitation, 189 specimens were selected from archival invasive breast cancer specimens, including most IHC-positive and some IHC-negative cases (n = 158 and 31, respectively).

ATP requirements and small interfering RNA structure in the RNA interference pathway.

We examined the role of ATP in the RNA interference (RNAi) pathway. Our data reveal two ATP-dependent steps and suggest that the RNAi reaction comprises at least four sequential steps: ATP-dependent processing of double-stranded RNA into small interfering RNAs (siRNAs), incorporation of siRNAs into an inactive approximately 360 kDa protein/RNA complex, ATP-dependent unwinding of the siRNA duplex to generate an active complex, and ATP-independent recognition and cleavage of the RNA target. Furthermore, ATP is used to maintain 5' phosphates on siRNAs.

Assessment of HER-2/neu status in breast cancer. Automated Cellular Imaging System (ACIS)-assisted quantitation of immunohistochemical assay achieves high accuracy in comparison with fluorescence in situ hybridization assay as the standard.

This retrospective study of formalin-fixed infiltrating breast cancer specimens compared manual immunohistochemical assay with a new image analyzer-assisted immunohistochemical quantitation method, using fluorescence in situ hybridization assay (FISH) as the standard. Following the manual immunohistochemical assay, 189 cases, including most manual immunohistochemically positive and some random negative cases, were analyzed by FISH assay for Her-2/neu gene amplification and by the Automated Cellular Imaging System (ACIS) for immunohistochemical staining. Using the FISH standard, the ACIS immunohistochemical assay attained a higher concordance rate and sensitivity than the manual immunohistochemical assay (91.

Creatine kinase in human retinal pigment epithelium.

Retinal pigment epithelial ion transport activity, and consequent ATP consumption vary significantly as a function of photoreceptor activity. In a variety of cell types, ATP levels are maintained during high-energy usage by phosphocreatine hydrolysis, catalysed by the enzyme creatine kinase. The present work was designed to assess the importance of creatine kinase in retinal pigment epithelial cell metabolism.

Orientation of GTP and ADP within their respective binding sites in glutamate dehydrogenase.

Previous studies have identified the guanine and adenine binding domains of the GTP and ADP binding sites of GDH. In this study the peptide sequences within or near to the terminal phosphate-binding domains of the GTP and ADP binding sites of bovine liver glutamate dehydrogenase (GDH) were identified using photoaffinity labeling with the benzophenone nucleotide derivatives, [gamma-32P]GTPgammaBP and [gamma-32P]ATPgammaBP. Without activating light, GTPgammaBP exhibited inhibiting effects on the GDH reaction similar to GTP; ATPgammaBP, as expected, produced activating effects similar to those of ADP.

ATP nucleotidylation of creatine kinase.

Creatine kinase (CK) will autoincorporate radiolabel from [gamma32P]ATP and has thus been reported to be autophosphorylated. Also, in contrast to normal brain enzyme, CK in Alzheimer-diseased brain homogenate shows greatly decreased activity, abolished photolabeling with [32P]8N3ATP, and no detectable autoincorporation of radiolabel by [gamma32P]ATP. Surprisingly, our studies with both human brain and purified CK showed that [alpha32P]ATP, [gamma32P]ATP, [alpha32P]ADP, [2,8H3]ATP, [gamma32P]2',3'-O-(2,4, 6-trinitrophenyl)-ATP, and [gamma32P]benzophenone-gammaATP all autoincorporate radiolabel into CK with good efficiency.

Bovine oocyte cytoplasm supports development of embryos produced by nuclear transfer of somatic cell nuclei from various mammalian species.

The transfer of nuclei from one cell to another provides a powerful tool for studying the interactions between the cytoplasm of one cell and the nucleus of another. This study was designed to examine the ability of the bovine metaphase oocyte cytoplasm to support mitotic cell cycles under the direction of differentiated somatic cell nuclei of various mammalian species. Skin fibroblast cells from cows, sheep, pigs, monkeys, and rats were used as sources of donor nuclei.

Abnormal properties of creatine kinase in Alzheimer's disease brain: correlation of reduced enzyme activity and active site photolabeling with aberrant cytosol-membrane partitioning.

The report shows that Alzheimer's disease (AD) brain creatine kinase (CK) is modified such that the nucleotide binding site of CK is blocked and that abnormal partitioning of CK between the soluble and pellet fractions occurs. First, CK activity was 86% decreased in AD brain homogenates in comparison to age-matched controls. Secondly, over a 23.

Identification of an adenine-nucleotide-binding site on interferon alpha2.

Using 32P-labeled 2-azidoadenosine 5'-triphosphate (2N3ATP) and 8-azidoadenosine 5'-triphosphate (8N3ATP), we have identified a site on human interferon alpha2 (IFN-alpha2) that binds adenine nucleotides. The results from saturation and competition experiments demonstrated the specificity of the nucleotide interaction. Half-maximal saturation of IFN-alpha2 was observed at 10 microM 2N3ATP or 35 microM 8N3ATP.

A comparison of changes in nucleotide-protein interactions in the striatal, hippocampus and paramedian cortex after cerebral ischemia and reperfusion: correlations to regional vulnerability.

[32P]Azido-purine analogs of ATP and GTP were used to detect changes in phosphorylation and nucleotide binding induced by ischemia and subsequent reperfusion in rat brain striatum, hippocampus and paramedian cortex (PM cortex) tissues. Major changes in phosphorylation were observed for a 130-kDa protein, tentatively identified as the Ca2+ transport ATPase, and calcium/calmodulin-dependent protein kinase II (CaM Kinase II) in all tissues. However, recovery of the phosphorylation of the 130-kDa protein occurred only in the PM cortex on reperfusion.

Oxidatively induced structural alteration of glutamine synthetase assessed by analysis of spin label incorporation kinetics: relevance to Alzheimer's disease.

The activity of the astrocytic enzyme glutamine synthetase (GS) is decreased in the Alzheimer's disease brain, which may have relevance to mechanisms of chronic excitotoxicity. The molecular perturbation(s) that results in GS inactivation is not known, although oxidative lesioning of the enzyme is one likely cause. To assess structural perturbation induced in GS by metal-catalyzed oxidation, a series of spin-labeling studies were undertaken.

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers.

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc.

Mercury vapor inhalation inhibits binding of GTP to tubulin in rat brain: similarity to a molecular lesion in Alzheimer diseased brain.

Hg2+ interacts with brain tubulin and disassembles microtubules that maintain neurite structure. Since it is well known that Hg vapor (Hg0) is continuously released from "silver" amalgam tooth fillings and is absorbed into brain, rats were exposed to Hg0 4h/day for 0, 2, 7, 14 and 28 d at 250 or 300 micrograms Hg/m3 air, concentrations present in mouth air of some humans with many amalgam fillings. Average rat brain Hg concentrations increased significantly (11-47 fold) with duration of Hg0 exposure.