Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Morphological characterization of a human glioma cell l ine

A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells presented their surfaces dominated by microvilli.     

How formalin affects the outcome of routine and special stains

The discovery of formaldehyde for preserving tissue structures produced a new dimension in microscopy. Preserving structure and morphology became important; therefore, identifying a proper fixing agent for particular structures, chemical entities, and tissues, also became important. The methods for demonstrating tissue structures evolved and were implemented with careful observation and documentation of the results and outcomes. Formalin was incorporated into many techniques, and provided helpful results in many cases and hindrances in others. The effects of formalin on the outcomes of routine and special staining techniques are reported here.     

Introgression despite substantial divergence in a broadcast spawning marine invertebrate

Understanding the relationship between reproductive isolation and time since divergence is critical to our understanding of speciation. One group for which we know little about the relationship between hybridization/introgression and time since divergence is the marine broadcast spawners. Here, we investigate the distribution of closely related cryptic species of marine broadcast spawners (Type A and B Ciona intestinalis) in areas of potential sympatry to determine whether these two types occur together and if so, whether they show evidence of hybridization and introgression. Then we combine our data with other studies to investigate general patterns of reproductive isolation versus divergence in marine broadcast spawners. We found that Type A and B C. intestinalis occurred sympatrically in 2007, and that 21 individuals show evidence of introgression in sympatry (out of approximately 500). Type A and B C. intestinalis are 12.4% divergent at mitochondrial COI (mtCOI), and in comparison with other marine broadcast spawning species at mtCOI, these two types may be near the upper limit of the range of divergence values in which introgression is still possible. However, introgression at divergence levels similar to those found in Ciona does exist, prompting questions about the strength of postmating prezygotic reproductive barriers in marine broadcast spawners.     

Is endothelial-nitric-oxide-synthase-derived nitric oxide involved in cardiac hypoxia/reoxygenation-related damage?

Nitric oxide (NO) has been reported to act both as a destructive and a protective agent in the pathogenesis of the injuries that occur during hypoxia/reoxygenation (H/R). It has been suggested that this dual role of NO depends directly on the isoform of NO synthase (NOS) involved. In this work, we investigate the role that NO derived from endothelial NOS (eNOS) plays in cardiac H/R-induced injury. Wistar rats were submitted to H/R (hypoxia for 30 min; reoxygenation of 0 h, 12 h and 5 days), with or without prior treatment using the selective eNOS inhibitor l-NIO (20 mg/kg). Lipid peroxidation, apoptosis and protein nitration, as well as NO production (NOx), were analysed. The results showed that l-NIO administration lowered NOx levels in all the experimental groups. However, no change was found in the lipid peroxidation level, the percentage of apoptotic cells or nitrated protein expression, implying that eNOS-derived NO may not be involved in the injuries occurring during H/R in the heart. We conclude that l-NIO would not be useful in alleviating the adverse effects of cardiac H/R.     

A comparison of fecal percent dry matter and number of Cryptosporidium parvum oocysts shed to observational fecal consistency scoring in dairy calves

Evaluation of dairy calf feces is often used in research and for clinical decision making to assess severity of diarrhea. However, this has not been validated for agreement between dry matter content and observed fecal consistency. Therefore, a comparison of observed fecal consistency score to fecal percent dry matter and Cryptosporidium parvum oocyst shedding was performed to assess the accuracy of observational scoring as a measure of diarrhea and its association with number of oocysts shed. Fecal samples from 20 dairy calves experimentally infected with C. parvum oocysts were collected daily post-infection and scored on a scale from 1 to 4, with 1 being normal feces to 4 being severe diarrhea. An aliquot of each sample was analyzed for percent dry matter and Cryptosporidium oocyst counts by using immunofluorescent microscopy. Fecal consistency scores of 1, 2, 3, and 4 had median percent dry matter of 20.9, 16.3, 9.6, and 5.8, respectively. Using percent dry matter assessed by fecal consistency scoring were significantly different from each other (P < 0.001). A higher fecal consistency score also was associated with a greater number of Cryptosporidium oocysts shed (P < 0 .0001). Scores of 1, 2, 3, and 4 had median oocyst counts of 0, 0, 1.3 × 10?, and 2.8 × 10?, respectively. These results suggest that observational scoring is a useful proxy to assess diarrhea in dairy calves.     

Use of reagent test strips for diagnosis of endometritis in dairy cows

The use of leukocyte esterase (LE), protein, and pH tests were evaluated on widely available urinary test strips (Multistix 10 SG; Bayer Corporation, Elkart, IN, USA) on uterine lavage samples as a potential cow-side test for the diagnosis of cytologic endometritis. Uterine lavage samples of 563 lactating Holstein cows between 40 and 60 days postpartum from 28 herds were evaluated. Endometrial cytology was used as the reference for endometritis, with a cutoff point of ≥10% neutrophils. All three (LE, protein, and pH) were increased in cows with cytologic endometritis and the associations were highly significant. Optimal cutoff points determined by receiver operating characteristic analysis for LE, protein, and pH were ≥++, ≥300 mg/dL, and ≥7.0, respectively. Combining the results for LE and pH improved the performance of the test strip, but this resulted in a group of cows (20.6% of cows) which were approximately equally likely (46% with endometritis and 54% without endometritis) to have cytologic endometritis or not, and therefore could not be accurately classified. The direct relationship between reagent strip test and reproductive performance was also evaluated. Reproductive impairment due to endometritis was restricted to multiparous cows; significantly decreased reproductive performance was observed for multiparous cows with lavage fluid LE ≥+++ (154 vs. 115 median days not-pregnant), as well as cows with pH ≥ 7.0 (150.5 vs. 111.5 median days not-pregnant), but not in cows with high protein, even at the highest cutoff point. In conclusion, reagent strip test results were strongly associated with cytologic endometritis and reproductive impairment; however, in comparison with conventional cytology, the performance of reagent strip as an alternative test was relatively poor and may require further refinement.