Effects of fixation and substrate protection on the isoenzymes of aspartate aminotransferase studied in a quantitative cytochemical model system.

The cytochemical technique of Lee and Torack for the demonstration of aspartate aminotransferase activity was tested on a model system consisting of either total liver homogenate or the mitochondrial or soluble cytoplasmic fraction, incorporated in polyacrylamide film. After incubation of portions of film in a medium of alpha-ketoglutarate, L-aspartate, and lead nitrate, the lead oxaloacetate formed was converted to lead sulfide. The absorbance determined at 520 nm in a film spectrophotometer and expressed in terms of unit weight of film provided a measure of the contained enzymatic activity, and was directly proportional to the concentration of chemically determined oxaloacetate in the film. Both mitochondrial and "soluble" isozymes of aspartate aminotransferase reacted with the cytochemical media to a quantitatively similar degree, but were considerably inactivated after 15 min of treatment with 1% glutaraldehyde or 3.7% formaldehyde in imidazole buffer, the rate of inactivation being greater for the soluble isozyme. Application of the principle of substrate protection delayed inactivation. Thus, for both isozymes the rate of inactivation decreased if ketoglutarate was added to the fixative. Similarly, it was shown that the optimal incubation medium for the demonstration of the soluble isozyme must contain 4 mM of alpha-ketoglutarate and 20 mM of L-aspartate. Under these conditions the turnover-number for the cytochemical system is 70% of the value obtained from biochemical estimations. Cytochemical K(m) values differed for each isozyme and were in accord with values determined by biochemical techniques, indicating that the model system can be used as a link between biochemical and cytochemical data in enzymatic studies.

The ultrastructural localization of the isozymes of aspartate aminotransferase in murine tissues.

Two isozymes of aspartate aminotransferase have been demonstrated biochemically. One isozyme is found in the mitochondrial fraction of the cytoplasm, the other ("soluble") in the supernatant. Both isozymes can be demonstrated by the cytochemical technique of Lee and Torack, as reported in the preceding report. Aldehyde fixation rapidly inactivates both isozymes, especially the soluble one. Inactivation can be delayed by addition of ketoglutarate to the fixative. The ketoglutarate probably competes with the fixative for the active site of the enzyme, thus protecting that region of the molecule. This enables adequate tissue preservation with enough remaining enzymatic activity to be demonstrated by the precipitation of oxaloacetate as the lead salt from a medium containing alpha-ketoglutaric acid aspartic acid, and lead nitrate. Electron-opaque material was found not only in mitochondria but, as the result of substrate protection, on the plasma membranes of many cells including erythrocytes and bacteria, the limiting membrane of peroxisomes, and the transverse tubular system of striated muscle. Occasional centrioles, neurotubules, tubules in the tails of spermatozoa, the A-I band junction in myofibrils of striated muscle, and the ground substance between cisternae of endoplasmic reticulum in intestinal goblet cells also showed precipitate. In all cases, replacement of L-aspartic acid by D-aspartic acid in the medium resulted in unstained sections. The sensitivity of extramitochondrial sites to fixation, the need of ketoglutarate as an agent for protecting the enzymatic activity during the fixation process, and the known presence of only soluble isozyme in erythrocytes indicate that enzymatic activity at these sites can be attributed to the soluble isozyme. Localization of the soluble isozyme on the plasma membrane may be related to possible involvement in depolarization phenomena, amino acid transport, or synthesis of plasma membrane-bound mucopolysaccharides.

Erratum: Author Correction: Exosomes-the enigmatic regulators of bone homeostasis.

[This corrects the article DOI: 10.1038/s41413-018-0039-2.].

Endoplasmic reticulum mediates mitochondrial transfer within the osteocyte dendritic network.

Mitochondrial transfer plays a crucial role in the regulation of tissue homeostasis and resistance to cancer chemotherapy. Osteocytes have interconnecting dendritic networks and are a model to investigate its mechanism. We have demonstrated, in primary murine osteocytes with photoactivatable mitochondria (PhAM)floxed and in MLO-Y4 cells, mitochondrial transfer in the dendritic networks visualized by high-resolution confocal imaging. Normal osteocytes transferred mitochondria to adjacent metabolically stressed osteocytes and restored their metabolic function. The coordinated movement and transfer of mitochondria within the dendritic network rely on contact between the endoplasmic reticulum (ER) and mitochondria. Mitofusin 2 (Mfn2), a GTPase that tethers ER to mitochondria, predominantly mediates the transfer. A decline in Mfn2 expression with age occurs concomitantly with both impaired mitochondrial distribution and transfer in the osteocyte dendritic network. These data show a previously unknown function of ER-mitochondrial contact in mediating mitochondrial transfer and provide a mechanism to explain the homeostasis of osteocytes.

Exosomes-the enigmatic regulators of bone homeostasis.

Exosomes are a heterogeneous group of cell-derived membranous structures, which mediate crosstalk interaction between cells. Recent studies have revealed a close relationship between exosomes and bone homeostasis. It is suggested that bone cells can spontaneously secret exosomes containing proteins, lipids and nucleic acids, which then to regulate osteoclastogenesis and osteogenesis. However, the network of regulatory activities of exosomes in bone homeostasis as well as their therapeutic potential in bone injury remain largely unknown. This review will detail and discuss the characteristics of exosomes, the regulatory activities of exosomes in bone homeostasis as well as the clinical potential of exosomes in bone injury.

Morphologic and DNA autoradiographic studies of the microcirculation in a transplantable rat fibrosarcoma: growth phase.

The morphology and tritiated thymidine uptake of the vascular channels in a transplantable W rat fibrosarcoma sampled at various times during growth are documented. New vessels originated primarily from normal venules in the subcutaneous tissue surrounding the neoplastic implant and grew at least twofold faster than did wound-induced vessels. During the 4-week observation period, partial maturation of vascular channels newly induced by the neoplasm was seen. This partial maturation was evidenced by an increase in the concentration of micropinocytic vesicles, a reduction in concentration and localization of intraluminal processes at or near interendothelial cell junctions, changes in the endothelial cell nuclei, and partial deposition of basement membrane material. The development of smooth muscle and nerve tissue was not seen. The proportion (13%) of labeled endothelial cells in normal subcutaneous connective tissue surrounding the 3-day-old fibrosarcoma implant was significantly higher than that seen in controls, as was the labeling index (14-25%) for endothelial cells in the fibrosarcoma up to 2 weeks after implantation. Vascular channels in the established neoplasm were examined by scanning and transmission electron microscopy and freeze-fracture techniques, and a resemblance to venular morphology was detected.

Ependymal differentiation of a glioblastoma multiforme after heterotransplantation into nude mice.

A glioblastoma multiforme from a 64-year-old man was heterotransplanted sc into nude mice (nu/nu BALB/c out-bred). The subsequent growth of the transplanted tumor featured the appearance and eventual domination by cells with ependymal characteristics. This finding supports the view that the host's influence on heterotransplants may be substantial and should be considered when extrapolating the results of therapeutic trials on such animal models to the clinical situation. The view that ependymal cells and astrocytes share a common precursor is also partly supported by the observations.

Surface charge of macrophages and their interaction with charged particles.

The distribution and concentration of surface ionogenic groups on murine peritoneal macrophages and the responses of their plasmalemma to charged markers were investigated by electron microscopy. Quantitation of the distribution of cationized ferritin (CF) as a marker for anionic groups on fixed cells revealed surface anions on invaginated parts of the plasmalemma to be fewer than those on flat or projecting segments. Cationic groups on the surface membrane, however, could not be labeled with anionized ferritin (AF). Interaction of viable macrophages with cationic particles at 37 degrees C resulted in their "internalization" within vesicles and coated pits and a closer apposition between many segments of plasmalemma than with neutral or anionic substances. The process occurred unaltered at 4 degrees C and was unaffected by cytochalasin B and colchicine, suggesting that this close apposition between segments of plasmalemma resulted from neutralization of surface negative electrostatic charges by the cationic material and did not reflect endocytosis.

Morphological and functional changes during cytomegalovirus replication in murine macrophages.

Structural and functional changes were studied in murine peritoneal macrophages infected with murine cytomegalovirus by using centrifugal enhancement to achieve a high-level (greater than 90%) pulsed infection. During 3 d of culture the infected cells became enlarged and rounded with smooth surface contours. Transmission electron microscopy demonstrated various stages of viral maturation in the nucleus and cytoplasm. Intracellular organization was generally retained, apart from the development of large, irregular, intracytoplasmic vacuoles, in which enveloped virions and cell debris accumulated. The infected macrophages lost most surface markers tested (F4/80, Mac-1, FcR, and the receptor for gluteraldehyde-fixed sheep red blood cells), but H-2 expression was increased. Moreover, ingestion of colloidal gold or horseradish peroxidase was depressed, and the levels of acid phosphatase activity, lymphocytostatic activity, and interleukin 1 production were also decreased. The latter may explain the observed loss of accessory cell function.

Detection of mRNA for carbonic anhydrase II in human osteoclast-like cells by in situ hybridization.

Carbonic anhydrase II (CA II) plays an important role during osteoclastic bone resorption. Biochemical investigations of gene expression of CA II, however, have been hampered by difficulty in obtaining sufficient numbers of purified osteoclasts. In this study, we describe a nonradioactive, digoxigenin-labeled cDNA in situ hybridization technique capable of determining the pattern of CA II gene expression in human osteoclast-like cells (OC-like cells) at the single-cell level. The results showed that CA II mRNA was located in the cytoplasm of both imprinted and cultured OC-like cells from a giant cell tumor of bone. On the other hand, no evidence of CA II mRNA was found in either the mononuclear cells (tumor cells) of giant cell tumor of bone or osteosarcoma cells. There is a significant correlation between in situ hybridization and northern blot analysis for CA II mRNA in both the giant cell tumor of bone and the osteosarcoma. Our results also indicated that quantitation of in situ hybridization can be achieved by computed cytophotometry.

Cytokine regulation of mesothelial cell proliferation in vitro and in vivo.

Previous studies have demonstrated mitogenic effects of several mediators on mesothelial cells in vitro, but their effects in vivo have not been investigated. The aim of this study was to examine the effects of various cytokines on normal mesothelial cell proliferation in vitro and in vivo and correlate the findings in both assay systems. In vitro proliferation was assessed using a technique based on the uptake and subsequent release of methylene blue. Autoradiographic methods were applied in a murine model to assess mitogenic activity of these factors on mesothelium in vivo. In vitro data demonstrated a dose-dependent increase in human mesothelial cell proliferation by all mediators examined: at optimal concentrations, proliferation was enhanced between 26.53 +/- 3.77% standard deviation (SD), p < 0.001 for fibroblast growth factor-2 (FGF-2) and 114.58 +/- 6.97%, p < 0.001 for platelet-derived growth factor-AB (PDGF-AB) above control medium. In vivo, DNA synthesis in mesothelial cells was stimulated by FGF-2 (29.52 +/- 5.85% labeled cells, compared with 7.04 +/- 4.36% for control medium; p < 0.001), tumor necrosis factor-alpha (TNF-alpha; 13.14 +/- 4.55% compared with 7.23 +/- 2.85; p < 0.005) and PDGF-BB (11.53 +/- 4.74% compared with 4.67 +/- 3.48%; p < 0.005). Transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) had no effect on DNA synthesis in mesothelial cells in vivo. It is concluded that FGF2, TNF-alpha, and PDGF stimulate mesothelial cell proliferation in vitro and in vivo, whereas TGF-beta1 and EGF only had a mitogenic effect in vitro at the concentrations examined. The mitogenic potency of the different PDGF isoforms in vitro was consistent with PDGF-alpha and beta receptor expression.

Four permanent cell lines established from human malignant gliomas: three exhibiting striated muscle differentiation.

Twenty-two human gliomas were set up in tissue culture and inoculated into nude mice. Permanent cell lines were established from four of these and their growth characteristics, and morphological features defined and cytogenetic features described. All four failed to sustain evidence of glial differentiation while three of the four cell lines exhibited the characteristics of striated muscle, and were tumorigenic in nude mice. Nine gliomas showed some initial growth in nude mice but only two were successfully subpassaged.

Establishment of a human medulloblastoma cell line and its heterotransplantation into nude mice.

A permanent cell line arising from a cerebellar medulloblastoma was established and its growth characteristics were investigated. Although the original tumor inoculum failed to take, the cultured cells were readily tumorigenic in nude mice and gave rise to rapidly growing tumors which could be easily subpassaged. The primary tumor showed evidence of both glial and neuronal differentiation, and retention of neuronal differentiation, albeit minimal, occurred in both the cultured neoplastic cells and the nude mouse tumors. Glial differentiation, on the other hand, could not be demonstrated. G-banding analysis of the chromosomes present in the cell line showed that they were exclusively human.

Pax7 includes two polymorphic homeoboxes which contain rearrangements associated with differences in the ability to regenerate damaged skeletal muscle in adult mice.

Pax7 is a paired-type homeobox gene which has previously been shown to play an important role in skeletal muscle formation. It is expressed in skeletal muscle of the limbs during embryogenesis and in adulthood. The aims of this study were firstly to determine the degree of polymorphism of Pax7 amongst inbred laboratory mice using Southern blotting and Pax7 regional specific sub-probes. Secondly, functional studies were performed on mice with each of the different structural forms of Pax7 to determine whether they were associated with differences in the ability to regenerate damaged skeletal muscle. Four different allelic forms of Pax7 have now been identified in laboratory mice indicating that the previously reported DNA sequence of Pax7 is not applicable to all laboratory mice. Hybridisation patterns of TaqI digested DNA representing each of the different Pax7 alleles with the Pax7 specific sub-probes suggested that in contrast to previous findings, Pax7 is associated with two highly polymorphic homeoboxes. The presence of two homeoboxes in BALB/c mice has been confirmed by DNA sequencing. Results of functional studies have also shown that the ability to regenerate damaged skeletal muscle in adult mice is strongly associated with the presence of a 0.15-kb TaqI fragment derived from one of the homeoboxes.

Polymorphism of the murine inhibitor of differentiation-controlling gene, Id.

The structure of Id was examined by Southern analysis in inbred mouse lines which included five subspecies of Mus musculus. No variation was detected within this species. The species Mus cookii shares the same form found in mice of M. musculus derivation, indicative of a long evolutionary history and a common origin. However, five TaqI polymorphisms were found among several Mus species, suggesting that Id has been modified throughout species diversification. Whether these variants impart functional changes is yet to be determined.

Association of an unusual form of a Pax7-like gene with increased efficiency of skeletal muscle regeneration.

Efficiency of regeneration of mechanically injured skeletal muscle is more pronounced in SJL/J mice, as compared to other laboratory strains in which regenerative properties of skeletal muscle are uniformly poor. Previously, we postulated that a small number of genes might differ between SJL/J and other mouse strains, and would be responsible for this variation in the efficiency of skeletal muscle regeneration. The results of initial experiments demonstrated that SJL/J mice have a unique form of the myogenic gene, Myo-D1, which partly influences efficiency of skeletal muscle repair, and that other genes were also involved. To identify other candidate genes, differences were sought within the myogenic paired box/homeobox-containing gene Pax7 between SJL/J and other laboratory mouse strains. Southern blotting indicated that SJL/J, Quackenbush and DDO mice share a Pax7/TaqI RFLP which differs from all other laboratory strains tested. This RFLP is most likely due to sequence differences within the homeobox of a Pax7-like gene. In vivo studies revealed that Quackenbush and DDO mice also share the same regenerative properties of mechanically damaged skeletal muscle as SJL/J mice. Since Quackenbush and DDO mice lack the SJL/J type of Myo-D1, and DDO belong to a different mouse sub-species, these studies suggest that structural alterations in the homeobox of a Pax7-like gene may be implicated in the effectiveness of renewal of damaged skeletal muscle of the limb in the mature animal.

Polymorphism of the mouse E2A gene due to an intronic deletion of 536 bp.

Examination of genetic polymorphism of the transcription factor-encoding gene E2A in laboratory and wild mice by Southern blotting has revealed the presence of two alleles. The most frequent allele is found in Mus musculus (Mm) musculus, as well as Mm domesticus. The less common allele is restricted to the Mm domesticus subspecies. Characterisation of these alleles has shown that the less common allele contains a deletion of approx. 500 bp located within a 1.8-kb intron immediately upstream from the E12 basic helix-loop-helix exon. DNA sequencing determined the deletion to span 536 bp including nucleotides 1045-1580 of the intron within the common allele. The deleted region includes several sequences with similarity to gene regulatory motifs; however, expression of E12 and intron splicing appeat unaltered. The occurrence of an identical deletion in mice from different geographical regions suggests that the uncommon allele may have a long evolutionary history.

Polymorphism of the mouse transcription factor-encoding gene E2A.

Southern blotting analysis using a cDNA probe consisting of the central portion of the E12 coding region has revealed two distinct forms of E2A, one which is common to all inbred and wild mouse strains derived from Mus musculus musculus and Mus musculus domesticus, whereas the other is less common and has only been found in the wild mouse population of Mus musculus domesticus.

Studies on the evolution and function of different forms of the mouse myogenic gene Myo-D1 and upstream flanking region.

The product of the murine Myo-D1 gene is able to initiate the complete sequence of genetic events required for formation of skeletal muscle. Because efficiency of regeneration of skeletal muscle is more pronounced in SJL/J mice, as compared to other strains, differences in the structure of Myo-D1 and the upstream regulatory region were sought to determine whether efficiency of tissue repair was influenced by the structure of the gene itself. Analysis of the restriction-fragment length polymorphism (RFLP) of genomic DNA from SJL/J and different sub-strains of mouse indicated that there are at least three different structural forms of Myo-D1, one of which is unique to SJL/J mice and may have been derived from a double recombinational event involving founder forms of Myo-D1. The unique form of Myo-D1 in SJL/J mice also exhibits a PvuII RFLP upstream from the gene, which may reflect some form of rearrangement or variation in methylation of a potential Myo-D1-binding region. Reference to the size of fragments hybridising with the Myo-D1 probe, following digestion of genomic DNA with TaqI, suggests that in most tissues, adenine residues within Myo-D1 may be extensively methylated. Segregation of Myo-D1 allotypes with response to mechanical injury to skeletal muscle in F2 offspring derived from SJL/J and BALB/c parental strains reveals that increased efficiency of tissue repair is associated with the SJL/J type of Myo-D1 gene. These observations provide new approaches to investigation of genetic control of tissue regeneration and cellular differentiation and proliferation in general.

Variation in the methylation profile and structure of Pax3 and Pax7 among different mouse strains and during expression.

Structural alterations within the myogenic and neurogenic developmental gene Pax7 which involve TaqI recognition sequences have previously been reported. These alterations are associated with differences in the efficiency of regrowth of damaged skeletal muscle. To identify other structural features of Pax genes which may influence skeletal muscle regrowth, variation in the structure and methylation status of Pax7 and the closely related gene Pax3 has been sought among different mouse strains and during gene expression using the restriction endonucleases MspI and HpaII. Following MspI digestion, RFLPs within Pax7 have been found which most likely reflect intron size variability within the paired box. Differences in the size of MspI and HpaII fragments hybridising with Pax7 and Pax3 region specific sub-probes indicate that the paired boxes are hypomethylated, whereas the region encoding the homeodomain of each gene is highly methylated in the spleen and other tissues from adult mice. In the skeletal muscle precursor cell line C2C12, which expresses Pax7 but not Pax3, the homeodomain encoding region of Pax7 is hypomethylated. In spleen cells, the Pax7 paired box is transcribed but the homeodomain encoding region is not. By contrast, both the paired box and the homeobox of Pax3 are hypermethylated in C2C12 cells indicating that generation of alternate transcripts from Pax genes may be controlled by DNA methylation. In contrast to Pax3, reference to the size of fragments hybridising with a Pax7 homeobox specific probe provides evidence for CpNpG methylation within and immediately downstream from the region encoding the homeodomain. Interestingly, CpNpG methylation remains when the Pax7 homeobox is expressed. Structural variation recognised by MspI digestion and differences in the methylation profile of Pax7 are not associated with the ability to regrow damaged skeletal muscle.