The 5-HT2A serotoninergic receptor is expressed in the MCF-7 human breast cancer cell line and reveals a mitogenic effect of serotonin

Serotonin (5-hydroxytryptamine, 5-HT) has been described as a mitogen in a variety of cell types and carcinomas. It exerts its mitogenic effect by interacting with a wide range of 5-HT receptor types. Certain studies suggest that some selective serotonin re-uptake inhibitors promote breast cancer in animals and humans. This study attempts to clarify the role of serotonin in promoting the growth of neoplastic mammary cells. Expression of the 5-HT(2A) serotoninergic receptor subtype in MCF-7 cells was determined by RT-PCR, Western blotting, and immunofluorescence analysis. The mitogenic effect of 5-HT on MCF-7 cells was determined by means of the MTT proliferation assay. We have demonstrated that the 5-HT(2A) receptor subtype is fully expressed in the MCF-7 human breast cancer cell line, in terms of encoding mRNA and receptor protein. Automated sequencing has confirmed that the 5-HT(2A) receptor present in this cell line is identical to the 5-HT(2A) receptor found in human platelets and in human cerebral cortex. Furthermore, this receptor was found by immunofluorescence to be on the plasma membrane. MTT proliferation assays revealed that 5-HT and DOI, a selective 5-HT(2A) receptor subtype agonist, stimulated MCF-7 cell. These results indicate that 5-HT plays a mitogenic role in neoplastic mammary cells. Our data also indicate that 5-HT exerts this positive growth effect on MCF-7 cells through, in part, the 5-HT(2A) receptor subtype, which is fully expressed in this cell line.     

A Quantitative Technique to Compare and Classify Humpback Whale (Megaptera novaeangliae) Sounds

In an attempt to minimize observer bias, numerical taxonomy methods were used to describe and classify humpback whale sounds. The spectrograms (N = 1255) were digitized into a 16 × 21 binary matrix. The rows were 16 frequencies selected on a logarithmic scale (0.12–8 kHz). The columns were 21 time samples taken every 0.1 s. Each point of the matrix was coded 1 if it lay over part of the sound. Other binary variables were added to code for relative intensity within a sound, frequency modulation and amplitude modulation. The sounds were then compared using the Jaccard similarity coefficient for binary data, and classified with average linkage cluster analysis. This technique produced 115 clusters, which were compared with my aural and visual impressions of the sounds. I agreed with most major categories identified by cluster analysis, but many small clusters had to be fused to other categories. This was partially due to the technique used, and to the complexity of the repertoire under study. Improvements are proposed to further reduce observer bias in classification of sounds, and thus make studies of animal communication performed by different researchers or on different species more easily comparable.     

Age-related differences in apoptosis with disuse atrophy in soleus muscle

Muscle atrophy is associated with a loss of muscle fiber nuclei, most likely through apoptosis. We investigated age-related differences in the extent of apoptosis in soleus muscle of young (6 mo) and old (32 mo) male Fischer 344 x Brown Norway rats subjected to acute disuse atrophy induced by 14 days of hindlimb suspension (HS). HS-induced atrophy (reduction in muscle weight and cross-sectional area) was associated with loss of myofiber nuclei in soleus muscle of young, but not old, rats. This resulted in a significant decrease in the myonuclear domain (cross-sectional area per nucleus) in young and old rats, with changes being more pronounced in old animals. Levels of apoptosis (TdT-mediated dUTP nick end labeling and DNA fragmentation) were higher in soleus muscles of old control rats than young animals. Levels were significantly increased with HS in young and old rats, with the greatest changes in old animals. Caspase-3 activity in soleus muscle tended to be increased with age, but changes were not statistically significant (P=0.052). However, with HS, caspase-3 activity significantly increased in young, but not old, rats. Immunohistochemistry showed that the proapoptotic endonuclease G (EndoG, a mitochondrion-specific nuclease) was localized in the subsarcolemmal mitochondria in control muscles, and translocation to the nucleus occurred in old, but not young, control animals. There was no difference between EndoG total protein content in young and old control rats, but EndoG increased almost fivefold in soleus muscle of old, but not young, rats after HS. These results show that deregulation of myonuclear number occurs in old skeletal muscle and that the pathways involved in apoptosis are distinct in young and old muscles. Apoptosis in skeletal muscle is partly mediated by the subsarcolemmal mitochondria through EndoG translocation to the nucleus in response to HS.     

An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing

Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource’s iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.     

A limited screen for protein interactions reveals new roles for protein phosphatase 1 in cell cycle control and apoptosis

Protein phosphatase 1 (PP1) catalytic subunits typically combine with other proteins that modulate their activity, direct them to distinct substrates, or serve as substrates for PP1. More than 50 PP1-interacting proteins (PIPs) have been identified so far. Given there are approximately 10 000 phosphoproteins in mammals, many PIPs remain to be discovered. We have used arrays containing 100 carefully selected antibodies to identify novel PIPs that are important in cell proliferation and cell survival in murine fetal lung epithelial cells and human A549 lung cancer cells. The antibody arrays identified 31 potential novel PIPs and 11 of 17 well-known PIPs included as controls, suggesting a sensitivity of at least 65%. A majority of the interactions between PP1 and putative PIPs were isoform- or cell type-specific. We confirmed by co-immunoprecipitation that 9 of these proteins associate with PP1: APAF-1, Bax, E-cadherin, HSP-70, Id2, p19Skp1, p53, PCNA, and PTEN. We examined two of these interactions in greater detail in A549 cells. Exposure to nicotine enhanced association of PP1 with Bax (and Bad), but also induced inhibitory phosphorylation of PP1. In addition to p19Skp1, PP1alpha antibodies also coprecipitated cullin 1, suggesting that PP1alpha is associated with the SCF1 complex. This interaction was only detectable during the G1/S transition and S phase. Forced loss of PP1 function decreased the levels of p27Kip1, a well-known SCF1 substrate, suggesting that PP1 may rescue proteins from ubiquitin/proteasome-mediated destruction. Both of these novel interactions are consistent with PP1 facilitating cell cycle arrest and/or apoptosis.     

Mutagenesis of CXCR4 Identifies Important Domains for Human Immunodeficiency Virus Type 1 X4 Isolate Envelope-Mediated Membrane Fusion and Virus Entry and Reveals Cryptic Coreceptor Activity for R5 Isolates

CXCR4 is a chemokine receptor and a coreceptor for T-cell-line-tropic (X4) and dual-tropic (R5X4) human immunodeficiency virus type 1 (HIV-1) isolates. Cells coexpressing CXCR4 and CD4 will fuse with appropriate HIV-1 envelope glycoprotein (Env)-expressing cells. The delineation of the critical regions involved in the interactions within the Env-CD4-coreceptor complex are presently under intensive investigation, and the use of chimeras of coreceptor molecules has provided valuable information. To define these regions in greater detail, we have employed a strategy involving alanine-scanning mutagenesis of the extracellular domains of CXCR4 coupled with a highly sensitive reporter gene assay for HIV-1 Env-mediated membrane fusion. Using a panel of 41 different CXCR4 mutants, we have identified several charged residues that appear important for coreceptor activity for X4 Envs; the mutations E15A (in which the glutamic acid residue at position 15 is replaced by alanine) and E32A in the N terminus, D97A in extracellular loop 1 (ecl-1), and R188A in ecl-2 impaired coreceptor activity for X4 and R5X4 Envs. In addition, substitution of alanine for any of the four extracellular cysteines alone resulted in conformational changes of various degrees, while mutants with paired cysteine deletions partially retained their structure. Our data support the notion that all four cysteines are involved in disulfide bond formation. We have also identified substitutions which greatly enhance or convert CXCR4's coreceptor activity to support R5 Env-mediated fusion (N11A, R30A, D187A, and D193A), and together our data suggest the presence of conserved extracellular elements, common to both CXCR4 and CCR5, involved in their coreceptor activities. These data will help us to better detail the CXCR4 structural requirements exhibited by different HIV-1 strains and will direct further mutagenesis efforts aimed at better defining the domains in CXCR4 involved in the HIV-1 Env-mediated fusion process.     

Optimisation and validation of a sensitive high-performance liquid chromatography assay for routine measurement of pyridoxal 5-phosphate in human plasma and red cells using pre-column semicarbazide derivatisation

There are few studies in which direct measurement of vitamin B6 status in both plasma and red cells has been assessed. The aims of the present study were to evaluate the use of a simple, robust HPLC method of direct pyridoxal 5'-phosphate (PLP) measurement in plasma and red cells and to assess its use in establishing reference ranges in a healthy population. A reverse phase HPLC method with pre-column derivatisation using semicarbazide for the simultaneous measurement of PLP, its degradation product, 4-pyridoxic acid (PA) and pyridoxal (PL) in plasma and red cells was developed. Pre-column derivatisation, reverse phase chromatography and detection procedures were optimised. The recovery, precision, linearity and sensitivity of the assay for plasma and red cell PLP, PA and PL was established. The recovery of PLP was greater than 95% for both plasma and red cell samples. The Intra and Inter batch imprecision for PLP was less than 6% and 7%, respectively. The method for PLP was linear up to at least 1000 nmol/l and the detection limit was 2.1 nmol/l (limit of quantification; 5.8 nmol/l). Accuracy of PLP measurements in plasma were acceptable, showing a mean bias of 4.5% from the mean value of laboratories (N=34) participating in an external quality assurance scheme. Geometric mean (95% reference intervals) for plasma and red cell PLP in the healthy subjects (N=126) were 56 (21-138) nmol/l and 410 (250-680) pmol/g Hb, respectively. There was a strong positive correlation (r(2)=0.81) between plasma and red cell PLP levels in the reference population. The HPLC method described was found to be suitable for the routine measurement of PLP in both plasma and red cells.     

Isodicentric Y chromosome: cytogenetic,molecular and clinical studies and review of the literature

Dicentrics are among the most common structural abnormalities of the human Y chromosome. Predicting the phenotypic consequences of different duplications and deletions of dicentric Y chromosomes is usually complicated by varying degrees of mosaicism (45,X cell lines), which may, in some cases, remain undetected. Molecular studies in patients with dicentric Y chromosomes have been few, and only two studies have attempted to determine the presence of SRY (the putative testis-determining factor gene). We report an 18-year-old female with short stature, amenorrhea, hirsutism, hypoplastic labia minora, and clitoromegaly who has a 45,X/46,X,idic(Y)(p11.32)/47,X,idic(Y)(p11.32),idic(Y) (p11.32) karyotype. Southern analysis using Y-specific probes (Y97, 2D6, 1F5, pY3.4) and polymerase chain reaction (PCR) analysis using primers for ZFY and SRY were positive for all loci tested, indicating that almost all of the Y chromosome was present. Our findings and an extensive review of the literature emphasize the importance of molecular analyses of abnormal Y chromosomes before any general conclusions can be reached concerning the relative effects of the Y-chromosome abnormality and mosaicism on sexual differentiation.     

Protective immunoglobulin A and G antibodies bind to overlapping intersubunit epitopes in the head domain of type 1 reovirus adhesin sigma1

Nonfusogenic mammalian orthoreovirus (reovirus) is an enteric pathogen of mice and a useful model for studies of how an enteric virus crosses the mucosal barrier of its host and is subject to control by the mucosal immune system. We recently generated and characterized a new murine immunoglobulin A (IgA)-class monoclonal antibody (MAb), 1E1, that binds to the adhesin fiber, sigma1, of reovirus type 1 Lang (T1L) and thereby neutralizes the infectivity of that strain in cell culture. 1E1 is produced in hybridoma cultures as a mixture of monomers, dimers, and higher polymers and is protective against peroral challenges with T1L either when the MAb is passively administered or when it is secreted into the intestines of mice bearing subcutaneous hybridoma tumors. In the present study, selection and analysis of mutants resistant to neutralization by 1E1 identified the region of T1L sigma1 to which the MAb binds. The region bound by a previously characterized type 1 sigma1-specific neutralizing IgG MAb, 5C6, was identified in the same way. Each of the 15 mutants isolated and analyzed was found to be much less sensitive to neutralization by either 1E1 or 5C6, suggesting the two MAbs bind to largely overlapping regions of sigma1. The tested mutants retained the capacity to recognize specific glycoconjugate receptors on rabbit M cells and cultured epithelial cells, even though viral binding to epithelial cells was inhibited by both MAbs. S1 sequence determinations for 12 of the mutants identified sigma1 mutations at four positions between residues 415 and 447, which contribute to forming the receptor-binding head domain. When aligned with the sigma1 sequence of reovirus type 3 Dearing (T3D) and mapped onto the previously reported crystal structure of the T3D sigma1 trimer, the four positions cluster on the side of the sigma1 head, across the interface between two subunits. Three such interface-spanning epitopes are thus present per sigma1 trimer and require the intact quaternary structure of the head domain for MAb binding. Identification of these intersubunit epitopes on sigma1 opens the way for further studies of the mechanisms of antibody-based neutralization and protection with type 1 reoviruses.