NG2 expression regulates vascular morphology and function in human brain tumours.

Tumour angiogenesis is a tightly regulated process involving cross-talk between tumour cells and the host tissue. The underlying mechanisms that regulate such interactions remain largely unknown. NG2 is a transmembrane proteoglycan whose presence on transformed cells has been demonstrated to increase proliferation in vitro and angiogenesis in vivo. To study the effects of NG2 during tumour growth and progression, we engineered an NG2 positive human glioma cell line (U251-NG2) from parental NG2 negative cells (U251-WT) and implanted both cell types stereotactically into immunodeficient nude rat brains. The tumours were longitudinally monitored in vivo using multispectral MRI employing two differently sized contrast agents (Gd-DTPA-BMA and Gadomer) to assess vascular leakiness, vasogenic oedema, tumour volumes and necrosis. Comparisons of Gd-DTPA-BMA and Gadomer revealed differences in their spatial distribution in the U251-NG2 and U251-WT tumours. The U251-NG2 tumours exhibited a higher leakiness of the larger molecular weight Gadomer and displayed a stronger vasogenic oedema (69.9 +/- 15.2, P = 0.018, compared to the controls (10.7 +/- 7.7). Moreover, immunohistochemistry and electron microscopy revealed that the U251-NG2 tumours had a higher microvascular density (11.81 +/- 0.54; P = 0.0010) compared to controls (5.76 +/- 0.87), with vessels that displayed larger gaps between the endothelial cells. Thus, tumour cells can regulate both the function and structure of the host-derived tumour vasculature through NG2 expression, suggesting a role for NG2 in the cross-talk between tumour-host compartments.

Mast cells and multiple sclerosis: a quantitative analysis.

The average number of mast cells observed in multiple sclerosis (MS) plaques from four patients, was 0.35 mast cells per mm2. This number represents 1/100 of the amount found in normal human skin. Most mast cells were observed in the border zones of the MS plaques and were clustered in restricted areas along venules and capillaries, which represent the main area of oedema formation in the brain. This cell type may be considered as a contributor to the pathogenesis of oedema formation and subsequent myelin destruction in MS.

The C-terminal part of the surface-associated protein MopE of the methanotroph Methylococcus capsulatus (Bath) is secreted into the growth medium.

A protein with an apparent molecular mass of 46 kDa was detected as the major polypeptide in the culture medium of the biotechnologically important methanotrophic bacterium Methylococcus capsulatus (Bath). The protein cross-reacted with polyclonal antibodies raised against the outer-membrane-associated protein MopE. The antiserum was used to identify a positive clone from a lambda gt11 library. The nucleotide sequence determined for the clone demonstrated that MopE and the secreted protein are encoded by the same gene, and that the secreted protein represents an N-terminally truncated form of MopE. By using monospecific antibodies against MopE in immunogold electron microscopy, the protein was localized at the cell surface and cell periphery. The mopE gene was expressed in Escherichia coli. The MopE protein synthesized was found in the periplasmic space of E. coli. No protein with sequence similarity over the entire length of MopE was detected in the databases, but some sequence similarity to the copper-repressible CorA protein of the methanotroph Methylomicrobium albus (Berson and Lidstrom 1997) was observed for the C-terminal region of MopE.

Mast cell and mast cell granule phenotypes in normal and Nippostrongylus-infected rats. A qualitative laser confocal microscopic study.

In the rat, the individual mast cell secretory granules may be divided into three subpopulations based on the presence of the specific proteases RMCP-1, RMCP-2, or a variable combination of these two proteases. Mast cells in the tongue only express RMCP-1, both in normal and infected animals, whereas in the other tissue locations studied (lung, intestinal mucosa and submucosa, tracheal epithelium and submucosa) the mast cells contain all three granule subtypes in a wide variation of combinations. These studies demonstrate that there is wide heterogeneity in protease expression in rat mast cells, which may be influenced by local stimulation with environmental tissue factors.

The role of mast cells and diet in the onset and maintenance of multiple sclerosis: a hypothesis.

Mast cells may invade the brain as a consequence of a childhood infection or predisposition, and it is proposed that multiple sclerosis arises due to the effect of various mediators (histamine and protease) released from the perivascular mast cells after stimulation by some diet factor.

Role of the endothelium in regulation of vascular functions in two teleosts.

Functional and structural aspects of the vascular endothelium were studied in major blood vessels from two distantly related species, the Atlantic salmon (S. salar) and the cod (G. morhua). The ventral aorta (VA) of both teleosts and the dorsal aorta (DA) and the coeliaco mesenteric artery (CMA) of the cod and the salmon respectively were examined for endothelium dependent and independent responses to acetylcholine (ACh), adrenaline (A) and endothelin-1 (ET-1). In the salmon, endothelial probing resulted in reduced contractile responses to high K+ in both VA and CMA while the responses to ACh and A were reduced only in CMA. Indomethacin, but not L-NMMA, enhanced vasoconstrictions to high K+, ACh and A in the unprobed CMA. In the cod vessels the endothelial probing caused reduced contractile responses to the two effective vasoconstrictors in both vessels, to high K+ and A in VA and to high K+ and ET-1 in DA. Both indomethacin and L-NMMA enhanced contractile tension to A in VA, while indomethacin, but not L-NMMA, enhanced the constrictions by high K+ in VA and by ET-1 in DA. These experiments have revealed heterogeneous patterns of endothelial function in blood vessels of two teleosts, reflecting differences in endothelial morphology and in production of potent endothelial derived contracting factors as well as prostanoic and non-prostanoic endothelium-derived dilating factors.

Mast cells in brains during experimental allergic encephalomyelitis in Lewis rats.

We studied the number of mast cells and their extent of degranulation in brains of Lewis rats with acute experimental allergic encephalomyelitis (EAE), activity induced with guinea pig spinal cord and Freund's complete adjuvant. Non-immunized controls and EAE rats were killed on days 10, 11, 12, and 16 post-immunization (p.i.). The percentage of degranulated mast cells was significantly increased in EAE brains. Signs of degranulation were observed as early as day 10 p.i. Clinical EAE signs appeared from day 10 p.i. A significant change in mast cell number was not observed. The percentage of degranulated cells was largest at day 16 p.i., at a time when the inflammation had reached the thalamus. This indicates that mast cell degranulation may occur as a result of the inflammation. Collectively, the data suggest that mast cells may play a role in the pathogenesis of EAE.

The mast cells derived from mouse bone marrow grow in close apposition with the reticular cells.

Cultures of mast cells of more than 95% purity were grown from bone marrow of BALB/c mice, and examined with various morphological methods. The presence of elongated, reticular cells was documented in the adherent layer on day 7 of the culture. The committed stem cells as well as immature bone marrow-derived mast cells (BMMCs) growing in clusters over the reticular cells were observed. After 14 days of cultivation BMMC harvested from the medium showed extensive plasma membrane ridges and numerous immature granules in their cytoplasm. These BMMCs increased their histamine to 0.7-1.1 pg/cell as compared to 0.1-0.2 pg/cell on the day 7. In the adherent layer BMMCs were seen in close apposition to the reticular cells. Their microvilli interdigitated with one another, forming end-to-end contracts. Our findings provide the evidence that for differentiation and proliferation of BMMCs in vitro close contacts with reticular cells in the adherent layer are necessary.

Response of rat myocardial mast cells to experimental ischemia.

The effects of ischemia on mast cells in the rat heart were studied. After 5 hours morphological signs of mediator release: granule dissolution, were observed. After 4 weeks the number of mast cells was significantly increased in comparison with nonischemic. Furthermore, 4 weeks after ischemia there was an increased number of regenerating mast cells. An increase in the number of mast cells and morphological signs of granule synthesis in the ventricular or septal muscle of the sham operated controls indicates that the surgical procedure itself can affect the mast cells of the heart generally. Morphological signs of granule synthesis were observed within mast cells of all experimental groups, and the figure was highly increased after 4 weeks of ischemia.

Mast cells and multiple sclerosis: a light and electron microscopic study of mast cells in multiple sclerosis emphasizing staining procedures.

In the brains of 7 patients with multiple sclerosis, mast cells were observed within the demyelinated plaques, in the border zone of the plaques as well as in seemingly normal white matter. The cells were mostly located in close connection with small vessels. The routine staining with toluidine blue for the demonstration of mast cells is not adequate as compared with staining of similar sections in pinacyanol erythrosine. Mast cells may be a hitherto underestimated contributor to the demyelinating process of multiple sclerosis.

Recovery of rat mast cells after secretion: a morphometric study.

Granule reconstitution in rat peritoneal mast cells following massive secretion was studied by morphometric techniques. Immediately following secretion, the earliest identifiable mast cells showed a substantial decrease in cell volume associated with granule loss. Cell volume then increased almost to the original level over a period of a month. The size of the Golgi apparatus increased markedly in the week following secretion and then returned to its original size. The total volume of granules increased slowly after the secretory depletion and by 34 days had not returned to the original value although the number of granules had recovered fully. The reconstitution of mast cells after secretion is a prolonged process with several phases resulting in mast cells of varying appearance and content. This heterogeneity generated by reconstitution post secretion must be considered in studies of populations of mast cells in vivo.

Studies on the growth of mast cells in rats. Changes in granule size between 1 and 6 months.

Mast cells from rats aged 1 month, 3 months and 5 to 7 months were compared with respect to cell size and secretory granule size and number. With increasing age there was an increase in mean total cell volume accounted for almost entirely by an increase in mean total granule volume/cell even though there was no apparent increase in the number of granules. An increase in histamine content closely paralleled the increase in total granule volume. In each age group, the distribution of equivalent volumes of granule profiles exhibited periodic multimodality consistent with a Poisson distribution of granule volume. We have interpreted these observations in terms of a model in which total granule volume/cell increases through the production of unit granules of uniform size; the increasing size of individual granules we attribute to the fusion of unit granules with each other and with larger granules.

Langerhans' cells in seborrheic keratosis. A clinical and ultrastructural study.

Skin biopsies from 10 patients with seborrheic keratoses were examined by electron microscopy for the presence of Langerhans' cells. Comparing seborrheic keratoses with normal skin of the same patient and with normal skin from controls, neither an increased number of Langerhans' cells nor an increased number of specific granules, Birbeck granules, nor abnormal Langerhans' cells, was found. Melanosomes in a Langerhans' cell were observed in 2 seborrheic keratoses, suggesting phagocytic activity of the Langerhans' cell.

Phagocytosis by mast cells in urticaria pigmentosa.

Mast cells with endocytic and autophagic vacuoles were observed in a biopsy from a lesion of urticaria pigmentosa. In several areas of the specimen the mast cells revealed the typical morphology of mast cells which have released low molecular weight mediators, but this phenomenon did not seem to be correlated morphologically to the phagocytic activity of the mast cells. Some mast cells contained giant granules with a normal as well as an abnormal substructure.

Mitomycin-C-induced changes in the nucleoid of Escherichia coli K12.

The influence of low concentrations of mitomycin-C on the structure of the envelope-free nucleoid was studied in several strains of Escherichia coli K12. The wild-type strain AB1157 uvr+ rec+ and 3 mitomycin-C-sensitive derivatives carrying mutations in the uvrA, uvrB and recA genes, were used. Treatment of the control strain with mitomycin-C, 0.5 microgram/ml, followed by incubation in drug-free medium resulted in the formation of a transient fast-sedimenting nucleoid with a sedimentation coefficient of 2200 S. A fraction of 25% of the nucleoids had attained the normal sedimentation coefficient of 1570 S 3 h after removal of mitomycin-C. With the uvr- strains, mitomycin-C induced a slow, almost linear increase in the S value of the envelope-free nucleoid. In these cases the S value continued to increase during post-incubation and was 2050 S 3 h after removal of the drug. Post-incubation of recA- cells resulted in loss of supercoiling, decrease in S value of the nucleoid and degradation of DNA. Results obtained with phase-contrast and electron microscopy were in good agreement with the hydrodynamic data.

Isolation, properties and nucleolytic degradation of chromatin from Escherichia coli.

A new procedure has been developed for the isolation of the chromosome complex, termed chromatin, from Escherichia coli. The bacteria were subjected to low ionic strength and T4 lysozyme, followed by detergent treatment analogous to that employed for the isolation of eukaryotic chromosomes. The chromatin was an insoluble viscous material which contained approximately equal amounts of DNA and RNA. The protein content of the chromatin was almost three times greater than the nucleic acid content. Electron microscopy revealed that the chromatin was highly condensed, having multiple loops and beaded structures with various diameters. The chromatin could be completely solubilized by both micrococcal nuclease and DNAase I, whereas RNAase had no effect. The initial degradation by micrococcal nuclease resulted in the production of a DNA-protein particle, sedimentation coefficient 10S, and an RNA-protein complex of 24S. Further degradation led to a decrease in sedimentation coefficient of the DNA-protein complex, but not of the RNA-protein particle. The peak size of the DNA of the initial DNA-protein particle was approximately 2400 bp. The action of micrococcal nuclease also resulted in the production of several discrete RNA species of various sizes. Several low molecular weight proteins (12000-27000) were found in the DNA-protein complex. The DNA-binding protein HU was present in the undigested chromatin; varying amounts of HU were, however, detected in the DNA-protein and RNA-protein particles.

Effect of methyl methanesulphonate on the nucleoid structure of Escherichia coli.

Incubation of a strain of Escherichia coli K12 with 25 mM-methyl methanesulphonate (MMS) for 1 h changed the sedimentation coefficient of the nucleoids from 1600S to 850S. When isolated nucleoids were treated with MMS under identical conditions in vitro there was no change in the sedimentation coefficient. Alkaline sucrose-gradient centrifugation of DNA from cells treated with 25 mM-MMS for 1 h indicated that there were approximately 100 breaks plus apurinic sites per chromosome. Titration with ethidium bromide of nucleoids from MMS-treated cells showed that almost all supercoiling had been lost, suggesting that the breaks plus apurinic sites consisted mostly of breaks. Further experiments showed that the apurinic sites were probably created by non-enzymic depurination and that little non-enzymic strand breakage had occurred. The depurinated sites thus created could then serve as substrates for the apurinic-specific endonucleases of the cell, with the result that strand breakage occurred. MMS treatment did not cause any changes in the DNA:RNA ratio of the nucleoids. Removal of MMS followed by a period of incubation resulted in a decrease in the number of breaks plus apurinic sites and an increase in the sedimentation coefficient of the nucleoids. After 2 h incubation in MMS-free medium the sedimentation coefficient of the nucleoids from MMS-treated cells was the same as that of the control; the supercoiling was also partially restored. The effect of MMS on two MMS-sensitive mutants of E. coli, one a polA and the other a recA mutant, was also studied. In both cases MMS caused complete collapse of the nucleoid structure.