Selection of a dye for use in semen transport studies in the cow

A series of experiments was carried out to select a dye and diluent that would facilitate identification of bovine inseminate in the uterus of the live cow when using hysteroscopic techniques. Of the dyes tested, Aniline Blue and the permitted food dye Green S as 1% solutions were the most suitable when added to semen with milk buffer diluent. Neither stained the uterine mucosa nor adversely affected the equipment, and both were visually distinctive during hysteroscopy. Using 1% concentrations of these dyes, sperm motility scores were acceptable before and after slow and fast freezing processes.     

Spectral sensitivity of vision and bioluminescence in the midwater shrimp Sergestes similis

In the oceanic midwater environment, many fish, squid, and shrimp use luminescent countershading to remain cryptic to silhouette-scanning predators. The mid-water penaeid shrimp, Sergestes similis Hansen, responds to downward-directed light with a dim bioluminescence that dynamically matches the spectral radiance of oceanic down-welling light at depth. Although the sensory basis of luminescent countershading behavior is visual, the relationship between visual and behavioral sensitivity is poorly understood. In this study, visual spectral sensitivity, based on microspectrophotometry and electrophysiological measurements of photoreceptor response, is directly compared to the behavioral spectral efficiency of luminescent countershading. Microspectrophotometric measurements on single photoreceptors revealed only a single visual pigment with peak absorbance at 495 nm in the blue-green region of the spectrum. The peak electrophysiological spectral sensitivity of dark-adapted eyes was centered at about 500 nm. The spectral efficiency of luminescent countershading showed a broad peak from 480 to 520 nm. Both electrophysiological and behavioral data closely matched the normalized spectral absorptance curve of a rhodopsin with lambda(max) = 495 nm, when rhabdom length and photopigment specific absorbance were considered. The close coupling between visual spectral sensitivity and the spectral efficiency of luminescent countershading attests to the importance of bioluminescence as a camouflage strategy in this species.     

NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression.

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF. Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity--high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have paralleled as well as reciprocal roles in vivo.     

Microarray-formatted clinical biomarker assay development using peptide aptamers to anterior gradient-2

Anterior gradient-2 protein was identified using proteomic technologies as a p53 inhibitor which is overexpressed in human cancers, and this protein presents a novel pro-oncogenic target with which to develop diagnostic assays for biomarker detection in clinical tissue. Combinatorial phage-peptide libraries were used to select 12 amino acid polypeptide aptamers toward anterior gradient-2 to determine whether methods can be developed to affinity purify the protein from clinical biopsies. Selecting phage aptamers through four rounds of screening on recombinant human anterior gradient-2 protein identified two classes of peptide ligand that bind to distinct epitopes on anterior gradient-2 protein in an immunoblot. Synthetic biotinylated peptide aptamers bound in an ELISA format to anterior gradient-2, and substitution mutagenesis further minimized one polypeptide aptamer to a hexapeptide core. Aptamers containing this latter consensus sequence could be used to affinity purify to homogeneity human anterior gradient-2 protein from a single clinical biopsy. The spotting of a panel of peptide aptamers onto a protein microarray matrix could be used to quantify anterior gradient-2 protein from crude clinical biopsy lysates, providing a format for quantitative screening. These data highlight the utility of peptide combinatorial libraries to acquire rapidly a high-affinity ligand that can selectively bind a target protein from a clinical biopsy and provide a technological approach for clinical biomarker assay development in an aptamer microarray format.     

A study of cell electrophoresis as a means of purifying growth hormone secreting cells

Growth hormone secreting cells of the rat anterior pituitary are heavily laden with granules of growth hormone and can be partially purified on the basis of their resulting high density. Two methods of preparative cell electrophoresis were investigated as methods of enhancing the purification of growth hormone producing cells: density gradient electrophoresis and continuous flows electrophoresis. Both methods provided a two- to four-fold enrichment in growth hormone production per cell relative to that achieved by previous methods. Measurements of electrophoretic mobilities by two analytical methods, microscopic electrophoresis and laser-tracking electrophoresis, revealed very little distinction between unpurified anterior pituitary cell suspensions and somatotroph-enriched cell suspensions. Predictions calculated on the basis of analytical electrophoretic data are consistent with the hypothesis that sedimentation plays a significant role in both types of preparative electrophoresis and the electrophoretic mobility of the growth hormone secreting subpopulation of cells remains unknown.