Formation of virus-like particles by bone cells in mice with a high incidence of spontaneous leukemia.

Bone samples from potentially leukemic and leukemic mice revealed numerous 90-to 110-nanometer particles morphologically identical to murine leukemia virus. Particles were observed budding from plasma membranes of osteocytes and osteoblasts but were most numerous in osteocyte lacunae. Particles were not observed in bone samples from mice which rarely develop leukemia.

Distribution of carbonic anhydrase activity in neurons of the rat.

Certain neurons of dorsal root ganglia (DRG) and some fibers of the sciatic nerve contain histochemically demonstrable carbonic anhydrase activity. Since the distribution of this enzyme throughout the nervous system has not yet been evaluated systematically, we conducted a comprehensive histochemical survey focusing particularly on structures derived from the neural crest and nonneural crest ectoderm. In the peripheral nervous system, we observed carbonic anhydrase activity in some, but not all, neurons of dorsal root, trigeminal, celiac, and myenteric ganglia as well as in glial cells throughout the CNS. Some neurons of the nodose ganglion also showed carbonic anhydrase activity. In all first order sensory ganglia that were studied, the enzyme was found only in large (50 micron or above) and medium (20-50 micron) size neurons; in the case of spinal ganglia, the reactive neurons constituted approximately 30% of the total neuronal population. Of these reactive neurons, 56% were heavily stained and 44% were moderately stained. Several possible roles for neuronal carbonic anhydrase are considered.

Histopathological reactions an axonal regeneration in the transected spinal cord of Hibernating squirrels.

The failure of axonal regeneration in the transected spinal cord of mammals has been attributed to many factors, including an intrinsic lack of regenerative capacity of mature CNS neurons, mechanical obstruction of axonal elongation by glial-connective tissue scars, necrosis of spinal tissue resulting in cavitation, lack of trophic influences sufficient to sustain outgrowth, and contact inhibition resulting from the formation of aberrant synapses. Assessment of te relative importance of each of these factors requires animal models in which one or more of these pathological processes can be eliminated. We therefore examined the effects of spinal transection in the hibernating animal because, during hibernation, collagen formation is depressed while nerve regeneration and slow axonal transport are maintained. Midthoracic spinal transections were performed in hibernating ground squirrels and the spinal cords were examined histologically 1-6 months later. The lesion site was composed primarily of a loose accumulation of macrophages and showed minimal glial and collagenous scarring, or cavitation. There was extensive regeneration of intrinsic spinal cord and dorsal root fibers. These axons grew to the margin of the lesion where they turned abruptly and continued growing along the interface between the lesion and the spinal cord. We conclude (1) that mammalian spinal-cord neurons have considerable regenerative potential; (2) that such mechanical impediments as collagenous and glial scarring, cyst formation, and cavitation cannot provide the sole explanation of why regeneration in the mammalian CNS is abortive; and (3) that specific physical and chemical properties of the cells in the environment of the growth cone regulate the extent and orientation of regenerative axonal outgrowth.

Determination of the optical properties of CuA(II) in bovine cytochrome c oxidase using magnetic circular dichroism as an optical detector of paramagnetic resonance.

This paper describes the use of a novel magnetic circular dichroism-microwave double resonance (MCD-ODMR) experiment to study the optical properties of the EPR detectable copper center, CuA2+, in bovine cytochrome c oxidase. By irradiating the sample with a monochromatic microwave beam of appropriate frequency it is possible to quench partially the MCD intensity of the features due to CuA2+. In this way the MCD bands from this center have been identified even in the presence of overlapping optical transitions from the haem centers of this enzyme. The resulting spectrum compares well with that reported some years ago from this laboratory and obtained by measuring MCD magnetisation characteristics. In addition the shapes of the MCD-ODMR lines obtained in a plot of MCD intensity against magnetic field strength have been analyzed to yield the relative polarizations of the optical transitions of CuA2+ which contribute to the MCD spectrum. All of the bands observed between 450 and 850 nm are predominantly polarized in the xy plane perpendicular to the direction of the g-tensor component of CuA2+ at g parallel = 2.18. This suggests that all of the CuA2+ ligands that contribute to the optical charge-transfer transitions in the visible region lie approximately in the basal plane. Possible structures for CuA2+ can now be suggested.

Performance trade-offs for conventional lenses for free-space digital optics.

We describe the limitations on the use of conventional lenses in optical computing that arise from manufacturing tolerances. The consequences on maximum array size, minimum device size, and propagation delay of systems are discussed. Two experimental optical computing systems are then compared with these results. We show that there are maximum and minimum bounds on the focal length and the ƒ-number of lenses imposed when manufacturing tolerances are considered. We also show that there are maximum bounds on image sizes and space-bandwidth products and trade-offs between spot size and system latency.

Mode of C-type virus production by bone cells of C3H/Fg mice.

Electron microscopy of bone from the distal ends of femurs of six 3-month-old male C3H/Fg mice revealed varying modes and quantities of C-type virus production by cells in the preosteoblast to osteocyte developmental gradient. Production occurred by budding from the cell surface, either from very short pedicles or from longer microvillous-like processes. Although apparently absent in preosteoblasts, limited numbers of budding viruses (90-110 nM) were seen in 6 of 53 osteoblasts examined. Examination of 200 osteocytes revealed evidence of virus production in all but eight. In these cells, budding occurred from the cell body and its processes with release of viruses into the pericellular lacunar space. Budding from the longer microvillous-like processes sometimes resulted in binding together of viruses in bead-like fashion by thin strands of limiting membrane. In a few cases budding was also seen on the cell processes within the canaliculi. The quantities of viruses present within the lacunae usually increased with increasing age of the osteocytes, indicating continual virus production. Most of the viruses observed, however, were pre C-types along with lesser numbers of mature C particles. Thus, factors contributing to the production of mature C particles may be diminished in osteocytes of mice at this age.

A possible non-oncogenic effect of C type bone cell virus on serum calcium levels of potentially leukemic mice.

In bone of C3H/Fg mice, particles structurally identical to C-type leukemia virus arise from membranes of osteocytes and osteoblasts. Although these virus apparently do not induce morphologic or neoplastic change in bone they may have other, more subtle, effects. Thus, comparison of sera from male C3H/Fg mice, a high leukemia-prone strain, with C57BL and C3H/HeJ mice, low leukemia strains which do not contain C-type virus in bone, reveals that serum calcium levels are significantly lower in the former than in the latter. Further, when C3H/Fg mice develop frank leukemia there is a corresponding increase in virus particles while the serum calcium concentration levels fall to even lower values. The presence of leukemia itself appears not to be the cause as indicated by the failure of implanted lymphocytic leukemic cells in C3H/Fg mice to significantly affect serum calcium concentration. It is postulated that the effects of the virus could be due either to increased osteo blastic activity or to inhibition of osteocytic osteolytic activity or to both.

Elevated serum calcium concentrations in mammary tumor bearing C3H/Fg mice without bony metastases.

Sera from eight adult C3H/Fg mice bearing spontaneous mammary gland tumors were analyzed for calcium content in two successive weekly samples. The sera from six mice were fount to have significantly elevated serum calcium concentrations while sera from two were normocalcemic. Intramuscular implantation of mammary gland tumors into 12, 3-month-old female C3H/Fg mice resulted in significant and progressive increases in serum calcium concentrations over a 4-week period. Subcutaneous implants had a similar effect, but gave less consistent results. These data supported the hypothesis that mammary gland tumors are capable of inducing elevated serum calcium concentrations in C3H/Fg mice. It was concluded that low grade elevation of serum calcium occasionally seen in adult female C3H/Fg mice may be caused by incipient mammary gland tumors.

Differences between adult and neonatal rats in their astroglial response to spinal injury.

Transection of the thoracic spinal cord in adult rats produces an astroglial reaction at the lesion site which spreads gradually to lumbar segments. We compared the spread of gliosis in cordotomized adult and neonatal rats in order to evaluate whether or not maturity of long spinal tracts is a precondition for the genesis of this histopathological reaction. By this experiment, we sought to determine whether spread of gliosis is induced by degeneration of nerve fibers in ascending and descending pathways or results from some more general reaction to injury. The spinal cords of 40 neonatal and 30 young adult rats were transected at T5, and 4 to 60 days later the cervical, thoracic, and lumbar segments were examined immunocytochemically for glial fibrillary acidic protein. In the neonatal rats, there was a moderate gliosis at the lesion site by 7 days; this reaction intensified somewhat during the next 60 days but always remained confined to the site of injury. In contrast, the lesion site of adult rats showed a much more intense gliosis; in those animals the response was maximal by 14 days and was characterized by a gradient of decreasing glial reactivity both rostrally and caudally from the transection site. These results support the hypothesis that the spread of gliosis from spinal lesions results from degeneration of the long ascending and descending fiber tracts.

Variations in serum calcium between strains of inbred mice.

Serum calcium concentrations of C3H/Fg, C3H/HeJFg, AKR/Fg, A/Fg, and LCS/Fg mice were compared at 4 and 7 mo of age. In the C3H/Fg and A-strain mice, calcium levels did not differ significantly between the 2 strains nor between the 2 age groups. The C3H/HeJFg, LCS/Fg, and C57BL/Fg strains formed a distinct group with similar calcium levels, but at a significantly higher level than C3H/Fg and A/Fg group. Again there was no significant difference between the 4- and 7-mo groups. The AKR/Fg strain was distinct from both groups in that higher calcium levels characteristic of the second group (C3H/HeJFg, LCS/Fg, and C57BL/Fg, were seen at 4 mo of age, but lower calcium levels similar to those of the C3H/Fg and A/Fg strains were found at 7 mo of age.

Quantitative evaluation of axonal regeneration by immunochemical assay for neurofilament protein.

In experiments on nerve regeneration requiring assessment of the rate and extent of axonal outgrowth, the availability of a simple and accurate method of quantification would be extremely useful. We approached this issue by modifying the conventional ELISA procedure so as to provide a sensitive, specific, and quantitative biochemical assay of the phosphorylated neurofilament content of homogenates or sections of nerve tissue. The technique involves four sequential steps: (i) adhesion of fixed or fresh homogenates or tissue sections to wells of microtiter plates, (ii) binding of a monoclonal antibody against phosphorylated neurofilament to the tissue, (iii) secondary binding to the anti-phosphorylated neurofilament of a phosphatase-labeled second antibody (antimouse IgG), and (iv) enzymatic assay of alkaline phosphatase activity using a fluorescent substrate (4-methylumbelliferyl phosphate). The technique is sufficiently sensitive to measure the phosphorylated neurofilament content of a 1:100,000 (w/v) homogenate of brain, spinal cord, or peripheral nerve and of single 10-microns paraffin sections of Bouin-fixed rat spinal cord. To validate the applicability of the procedure to the study of nerve regeneration, the sciatic nerve of adult rats was either crushed (to permit regeneration) or transected and ligated (to preclude regeneration). The animals were autopsied 1 to 16 weeks later, when four segments 3-mm in length taken from regions proximal and distal to the lesion site were assayed for phosphorylated filament content. The temporal course of its disappearance during degeneration and its reappearance during regeneration coincided with the known histologic changes in crushed and transected nerves. These findings demonstrate the validity of using the immunochemical assay for PNF in studies of nerve regeneration in the peripheral nervous system and the potential applicability of this procedure to studies on regeneration in the central nervous system.

Essentiality of a specific cellular terrain for growth of axons into a spinal cord lesion.

To date, there are no reports of growth of significant numbers of axons into or across a lesion of the mammalian spinal cord. However, recent studies showing that CNS axons will grow into PNS environments indicate that comparable growth into spinal cord lesions could be achieved if ischemic necrosis could be prevented and the lesion site repopulated by astrocytes and ependymal cells rather than by the macrophages, lymphocytes, and fibroblasts that generally accumulate at sites of CNS injury. To examine this possibility, we made a laminectomy at T5 in rats and crushed the spinal cord for 2 s with a smooth forceps (leaving the dura mater intact to prevent ingrowth of connective tissue). At 1 week, the lesion was filled with mononuclear cells, degenerating nerve fibers, and capillaries that were oriented parallel to the long axis of the spinal cord. By 2 weeks, longitudinally oriented cords of ependymal cells and astrocytes had migrated into the lesion from the adjacent spinal cord, and similarly oriented nerve fibers had begun to grow into the lesion along these capillaries and cellular cordons. The mononuclear cells had now assumed phagocytic activity and were engorged with myelin and other cellular debris. After 3 weeks, the astrocytes had elaborated thick cell processes. The nerve fibers in the lesion were still oriented longitudinally but had increased in number and were often arranged in small fascicles. These observations provide the first histological evidence of growth of nerve fibers into a lesion of the rat spinal cord. We conclude that the intrinsic regenerative capacity of the spinal cord can be expressed if ischemic necrosis and collagenous scarring are prevented and the spinal cord parenchyma is first reconstructed by its nonneuronal constituents.

Enhancement of axonal growth into a spinal lesion by topical application of triethanolamine and cytosine arabinoside.

We have demonstrated that a brief compression lesion of the rat spinal cord produces axotomy with minimal necrosis or scarring and that axons grow into such a lesion along longitudinally oriented capillaries and similarly oriented cordons of ependymal cells and astrocytes. Inasmuch as extensive, oriented growth of axons into a spinal lesion is never seen after transection, concussion, or other models of spinal cord injury, this new surgical procedure appeared to be applicable to the in vivo testing of pharmacological agents designed to promote neuritic outgrowth. The spinal cord of anesthetized rats was crushed extradurally for 1 s with a smooth jeweler's forceps. After 2 days when edema had subsided, the animals were reoperated. The dura mater was opened, and a polyethylene tube was implanted so that one end was fastened over the injury site and the other end was exteriorized at the back of the neck. The lesion site was superfused with 0.1 ml of control or test solutions four times daily for 2 weeks and then the animals were anesthetized and killed by vascular perfusion with fixative. After decalcification, the vertebral column and spinal cord were embedded in paraffin and stained by several histologic procedures including the protargol silver impregnation method for nerve fibers. Treatment with triethanolamine and cytosine arabinoside, substances which promote neuritogenesis in cultured spinal ganglia of chick embryos, markedly stimulated the growth of axons into the lesion of the rat spinal cord. We conclude (i) that it is possible to pharmacologically enhance the intrinsic growth capacity of CNS neurons and (ii) that brief compression provides a type of injury that is well suited to the evaluation of treatments aimed at promoting axonal regeneration.

Components for the implementation of free-space optical crossbars.

We describe an optical system developed to form the basis of a 64 × 64 free-space optical matrix-matrix crossbar switch. The design and performance of each of the main optical components is discussed: lenses, diffractive optical elements, and polarizing beamsplitters, together with the optomechanical hardware design. For these components, throughput levels of -6.9 dB have been achieved, which is compatible with full system operation at 10(-12) bit error rates at ≥270 Mbits s(-1).