A novel method for embedding neonatal murine calvaria in methyl methacrylate suitable for visualizing mineralization, cellular and structural detail.

The study of undecalcified bone by histological methods is essential in the field of bone research. Culturing skeletal tissues such as neonatal murine calvaria provides a reliable bridge between assessment of bone formation in vitro and anabolic activity in vivo and contains most of the essential elements of bone for studying bone formation. Neonatal calvarial assay, supported by histological methods, is used to study the anabolic effects of a wide variety of factors and compounds on bone tissue. To optimize visualization and histomorphometric measurements using neonatal calvaria, we developed a method that provides high quality tissue sections suitable for routine and histochemical staining. Undecalcified neonatal mouse calvaria were processed and embedded using a low temperature methyl methacrylate procedure. Various staining methods were performed on deplastisized and floated sections to examine mineralization and to identify cells. The Von Kossa stain counterstained with a modified H & E yielded precise images of unmineralized bone including mineralization sites, and distinct osteoblasts and osteoclasts. Toluidine blue, Ladewig's trichrome, tartrate-resistant acid phosphatase, Goldner, H & E and Villanueva stains also were tested on the undecalcified neonatal calvaria sections.

Endogenous generation of cyanide in neuronal tissue: involvement of a peroxidase system.

In a study of the mechanism by which cyanide is produced in neural tissue, it was hypothesized that nerve cells generate cyanide in a manner similar to that in leukocytes. As in white blood cells, glycine addition enhanced cyanide production in rat pheochromocytoma cells. Because myeloperoxidase catalyses cyanide production in leukocytes, a selective myeloperoxidase inhibitor (aminobenzoic acid hydrazide) was tested and found to inhibit opiate agonist-induced cyanide production in pheochromocytoma cells and also in rat brain. In addition, hydrogen peroxide enhanced cyanide release in pheochromocytoma cells, further suggesting that the process is oxidative in nature. Sonicated rat pheochromocytoma cells did not generate cyanide in response to an agonist acting on surface receptors even though disrupted cells responded to glycine. The mitochondrial fraction from rat brain produced more cyanide in response to glycine than any other fraction. Thus glycine seems to act at an intracellular site to enhance cyanide production and the process seems to involve a peroxidase mechanism similar to that reported for white blood cells.

Mice lacking Snrpn expression show normal regulation of neuronal alternative splicing events.

The SmN protein is closely related to the constitutively expressed RNA splicing protein SmB but is expressed only in brain and heart tissue. Mice which lack expression of SmN die shortly after birth suggesting a critical role for this protein possibly in the regulation of neuronal-specific alternative splicing events. We show here however that the neuronal-specific alternative splicing of the RNAs encoding several different classes of protein proceeds normally in mice lacking SmN expression. The potential role of SmN and the reasons for the lethal effect observed in non-expressing mice are discussed.

The tissue specific SmN protein does not influence the alternative splicing of endogenous N-Cam and C-SRC RNAs in transfected 3T3 cells.

The SmN protein is closely related to the constitutively expressed RNA splicing protein SmB but is expressed only in brain and heart tissue. The inclusion of the VASE exon in the N-Cam mRNA and of the N1 exon in the c-src mRNA correlates with the expression pattern of SmN, being observed in brain and heart but not in other tissues and increasing in amount as SmN levels increase during brain development. However, the artificial expression of SmN in cells in which it is normally absent does not affect the pattern of N-Cam and c-src splicing whilst a cell line lacking detectable SmN is able to include the VASE exon. Hence SmN does not appear to be necessary or sufficient for these tissue-specific and developmentally regulated alternative splicing events.

A distal region of the CALC-1 gene is necessary for regulated alternative splicing.

The CALC-1 gene exhibits tissue specific alternative splicing with exons 1-4 being spliced to produce the calcitonin mRNA in thyroid C cells and exons 1-3 and 5-6 being joined to produce the CGRP mRNA in neuronal cells. Previous studies have identified an element in intron 3 within the alternatively spliced region which is critical for this effect to occur. We show here that deletion of sequences downstream of the alternatively spliced region also disrupts the tissue specific pattern of alternative splicing. The manner in which these sequences act is discussed.

Alternative splicing of the mRNA encoding the alpha subunits of the G(o) GTP-binding protein during brain development and in neuronal cell lines.

The RNA encoding the alpha subunit of the G(o) GTP-binding protein is alternatively spliced in some tissues to produce mRNAs encoding two distinct forms of the protein which differ at the carboxyl terminus. This alternative splicing process is observed at all stages during development of the rat brain and heart and in some but not all neuronal cell lines. These effects are discussed both in terms of the regulation of the alternative splicing event and the functional role(s) of the two forms of the protein which it produces.

Expression of the SmN splicing protein is developmentally regulated in the rodent brain but not in the rodent heart.

The SmN protein is a tissue-specific splicing factor which is closely related to the ubiquitous SmB splicing protein but which is expressed only in the adult brain and heart. SmN is also detectable albeit at a low level in both the embryonic brain and heart. During heart development, SmN levels remain constant while during rodent brain development the levels of SmN rise such that SmN replaces SmB as the predominant protein in adult brain. This increase in SmN levels is dependent upon a corresponding increase in the SmN mRNA which is detectable by in situ hybridization within neurons in virtually all areas of the adult brain.

Redistribution of secretory granule components precedes that of synaptic vesicle proteins during differentiation of a neuronal cell line in serum-free medium.

Incubation of the rat sensory neuron-derived cell line ND7 in serum-free medium results in the arrest of mitosis and the appearance of numerous neuronal processes. During this differentiation event, secretory granule components such as chromogranins, neuropeptide Y and the C-flanking peptide of pro-neuropeptide Y move to the tips of the majority of the neuronal processes regardless of process length. In contrast, the synaptic vesicle component, synaptophysin, is found only at the tips of the very long processes which appear following prolonged periods of culture in serum-free medium. A similar restriction of synaptophysin to long processes is also observed following differentiation and process formation induced by other treatments such as incubation in reduced serum or treatment with cyclic AMP or phorbol myristate acetate. Hence the regulated secretory pathway associated with the chromogranins and neuropeptides appears to be segregated into the processes at an earlier stage of ND7 differentiation than the synaptophysin-associated synaptic vesicle pathway. ND7 cells therefore provide a model system for studying the processes regulating these pathways and the redistribution of their components during neuronal differentiation.

Expression of the tissue specific splicing protein SmN in neuronal cell lines and in regions of the brain with different splicing capacities.

The SmN protein is closely related to the ubiquitously expressed SmB and B' RNA splicing proteins but is expressed in only a limited range of tissues and cell types. The expression of SmN in a range of neuronal and non-neuronal cell lines correlates with their ability to splice the calcitonin/CGRP transcript to produce the mRNA encoding CGRP rather than that encoding calcitonin. Moreover, the SmN mRNA shows a widespread distribution within the brain and spinal ganglia being present in neuronal cells in all regions which naturally produce CGRP as well as in those areas which do not naturally express the calcitonin/CGRP gene but which can correctly splice the CGRP mRNA in transgenic mice expressing the calcitonin/CGRP gene in all cell types. Interestingly however the mRNA encoding SmN is also found in a few areas of the brain which can only carry out calcitonin-specific splicing in transgenic mice, such as the Purkinje layer of the cerebellum and the inferior colliculus. The possible role of SmN in the regulation of splicing in neuronal cells is discussed in the light of these results.

Intracellular redistribution of neuropeptides and secretory proteins during differentiation of neuronal cell lines.

We have demonstrated that the mouse neuroblastoma N18Tg2 cell line and several clones of hybrid ND cells (ND7, ND9 and ND21), derived from the fusion of neonatal rat sensory neurons with that neuroblastoma, show immunostaining to protein gene product 9.5, neuropeptide Y, C-flanking peptide of neuropeptide Y, tyrosine hydroxylase and chromogranins. Synaptophysin could only be detected in ND cells. Immunoreactivities to substance P, calcitonin gene-related peptide, galanin and somatostatin could not be detected in any of these cell lines. After three days of incubation in a differentiation medium, cell processes of various lengths were observed both in neuroblastoma and ND cell cultures. In ND7 cells there was also a redistribution of neuropeptide Y and its C-flanking peptide to the tips of cell processes. The differentiation of cell processes was also accompanied by the appearance of immunostaining to rat chromogranins in their tips. In contrast, synaptophysin expression was found mainly in cell bodies. Neuropeptide Y, its C-flanking peptide and chromogranins have been associated with secretory granules, whereas synaptophysin is a marker for small synaptic-like vesicles. Therefore, our morphological findings further support and expand the view that these markers are primarily associated with different subcellular structures. Moreover, they indicate that the regulated secretory pathway associated with chromogranins is segregated into nerve processes at an early stage of differentiation, when the synaptophysin-associated pathway is not yet mature. ND7 cells thus provide a useful model system for studying changes in the distribution of neuropeptides, cytoskeletal elements and proteins associated with cell secretion during neuronal differentiation.

Comparison of the Effects of the Anderson Knee Stabler and McDavid Knee Guard on the Kinematics of the Lower Extremity during Gait*.

* This study was completed in partial fulfillment of Ms. Van Horn's master's degree, University of North Carolina at Chapel Hill. The purpose of this study was to compare gait patterns among subjects wearing Anderson Knee Stabler braces, McDavid Knee Guards, and no braces. Fifteen male subjects were filmed while running on a treadmill at 4 mph and 8 mph without a brace, with an Anderson Knee Stabler, and with a McDavid Knee Guard. Fourteen gait variables were measured for each brace and speed condition. Analysis of the variables with multivariate ANOVA indicated that there was an increase in hip and knee flexion and knee angular velocity with and without braces at 8 mph as compared to 4 mph, a decrease in knee extension when either brace was worn, and minimal gait pattern differences with the Anderson Knee Stabler as compared with the McDavid Knee Guard (all results p < 0.05). The results of this study demonstrate that no clear superiority exists between the braces' effect on the gait characteristics measured. Therefore, other parameters should be considered when making brace selections.J Orthop Sports Phys Ther 1988;9(7):254-260.

University-industry programs.

In the report by R. G. Wyatt et al. "Human rotavirus type 2: Cultivation in vitro" (11 Jan., p. 189), in the sentence describing the porcine rotavirus plaque reduction test (p. 190, column 3, line 16), the concentration of pancreatin in the agar overlay should have been given as "0.15 percent of 2.5 percen pantreatin 4 x N. F.; Gibco."