Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A-E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.     

Transgenic mice secreting coronavirus neutralizing antibodies into the milk

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.     

Endocrine factors associated with recurrent spontaneous abortion

The role of endocrine factors as a cause of recurrent spontaneous abortion is controversial. Diabetes mellitus and thyroid disease do not represent a significant risk factor for recurrent pregnancy loss. Luteal-phase defect has been questioned because there are no accurate methods for diagnosis and no convincing evidence of correction with treatment exists. The corpus luteum is an unusual endocrine gland, highly diverse in function and important for successful reproduction in all mammalian species. Much controversy exists about the luteal function in humans and how defects in luteal function affects reproduction. Disagreement has been due to lack of accurate diagnosis and controlled studies to determine whether correction of the luteal-phase defect is worthwhile when treating female reproductive problems. The donor egg recipient model from assisted reproductive technology programs has shown that corpus luteum function can be replaced by estrogen and progesterone administration. The mechanism by which these steroids stimulate a uterus to be receptive to implantation of the embryo is not known. Several proteins produced by the endometrium are candidate markers for uterine receptivity. Further work needs to be done to correlate these markers with subsequent pregnancy outcome. A noninvasive marker for uterine receptivity is ultrasonographic evaluation of the endometrium. Although the sensitivity of this test is high (100%), its specificity is low (only 20% to 60%).     

Signal transduction pathways that regulate cell survival and cell death

Apoptosis or programmed cell death (PCD) is a physiological process critical for organ development, tissue homeostasis and elimination of defective or potentially dangerous cells in complex organisms. Apoptosis permits cell death without a concomitant inflammatory response in the surrounding tissues. The process of apoptosis depends on the reception of multiple extracellular and intracellular signals, integration and amplification of these signals by second messengers and finally, activation of the death effector proteases. Defects in control of apoptotic pathways may contribute to a variety of diseases including cancer, autoimmune and neurodegenerative conditions and AIDS. While many components of the regulatory network controlling apoptosis have been defined, the mechanisms of action and patterns of interaction of these factors remain controversial. This article summarizes some of the known aspects of signaling pathways involved in apoptosis.     

Differential acetylcholine and GABA release from cultured chick retina cells

In the present work we investigated the mechanisms controlling the release of acetylcholine (ACh) and of gamma-aminobutyric acid (GABA) from cultures of amacrine-like neurons, containing a subpopulation of cells which are simultaneously GABAergic and cholinergic. We found that 81.2 +/- 2.8% of the cells present in the culture were stained immunocytochemically with an antibody against choline acetyltransferase, and 38.5 +/- 4.8% of the cells were stained with an antibody against GABA. Most of the cells containing GABA (87.0 +/- 2.9%) were cholinergic. The release of acetylcholine and GABA was mostly Ca2+-dependent, although a significant release of [3H]GABA occurred by reversal of its transporter. Potassium evoked the Ca2+-dependent release of [3H]GABA and [3H]acetylcholine, with EC50 of 31.0 +/- 1.0 mm and 21.6 +/- 1.1 mm, respectively. The Ca2+-dependent release of [3H]acetylcholine was significantly inhibited by 1 micrometer tetrodotoxin and by low (30 nm) omega-conotoxin GVIA (omega-CgTx GVIA) concentrations, or by high (300 nm) nitrendipine (Nit) concentrations. On the contrary, the release of [14C]GABA was reduced by 30 nm nitrendipine, or by 500 nm omega-CgTx GVIA, but not by this toxin at 30 nm. The release of either transmitters was unaffected by 200 nm omega-Agatoxin IVA (omega-Aga IVA), a toxin that blocks P/Q-type voltage-sensitive Ca2+ channels (VSCC). The results show that Ca2+-influx through omega-CgTx GVIA-sensitive N-type VSCC and through Nit-sensitive L-type VSCC induce the release of ACh and GABA. However, the significant differences observed regarding the Ca2+ channels involved in the release of each neurotransmitter suggest that in amacrine-like neurons containing simultaneously GABA and acetylcholine the two neurotransmitters may be released in distinct regions of the cells, endowed with different populations of VSCC.     

Using neural networks to select wavelet features for breast cancerdiagnosis

This study focuses on improving microcalcification classification by establishing an efficient computer-aided diagnosis system that extracts Daubechies-4 and biorthogonal wavelet features. These wavelets were chosen because they have been used in military target recognition and fingerprint recognition research with images characterized by low contrast, similar to mammography. Feature selection techniques are employed to further increase classification performance. The artificial neural network feature selection techniques are complemented by a conventional decision boundary-based feature selection method. The results using the wavelet features are compared to more conventional measures of image texture, angular second moment, and Karhunen Loeve coefficients. The use of alternative signal processing to compare wavelet and neural techniques allows for a measure of the problem difficulty. It is concluded that advances and contributions have been made with the introduction of two novel feature extraction methods for breast cancer diagnosis, wavelets and eigenmasses. Additionally, feature selection techniques are demonstrated, compared, and validated, transforming adequate discrimination power into promising classification results