Immunohistochemical localization of cell adhesion molecule epithelial cadherin in human arachnoid villi and meningiomas.

Cadherins are a family of intercellular glycoproteins responsible for calcium-dependent cell adhesion and are currently divided into four types: epithelial (E), neuronal (N), placental (P), and vascular (V). Since cadherins are known to be indispensable for not only morphogenesis in the embryo but also maintenance of tumor cell nest, we examined the expression of E-cadherin in 31 meningiomas (11 syncytial, 12 transitional, 8 fibroblastic) and 3 arachnoid villi by immunoblot and immunohistochemical analyses. In the immunoblot analysis, E-cadherin was detected at the main band of Mr 124,000 in all of the arachnoid villi, as well as syncytial and transitional types of meningiomas, but not in the fibroblastic type. The immunohistochemical examination showed that E-cadherin was expressed at the cell borders of syncytial and transitional types, but the expression was absent in the fibroblastic type. Immunoelectron microscopy showed that E-cadherin was localized at the intermediate junctions in arachnoid villi, while it was detected diffusely at the cell surface in meningiomas. It is suggested from these data that the expression of E-cadherin might be closely related to the differentiation and organogenesis of meningioma cells.

Up-regulation of Fas (APO-1/CD95) ligand and down-regulation of Fas expression in human esophageal cancer.

Fas (APO-1/CD95) is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or Fas antibody. In this study, we analyzed Fas and FasL expression in normal esophageal mucosa and esophageal squamous cell carcinomas. Reverse transcriptase-PCR revealed that Fas, soluble Fas, and FasL were expressed in all eight esophageal squamous carcinoma cell lines analyzed. Furthermore, it was demonstrated that FasL expressed in esophageal carcinoma cells is functional because coculture experiments using FasL-expressing TE-15 esophageal carcinoma cells resulted in apoptosis of Jurkat T leukemia cells, which are sensitive to Fas-mediated apoptosis. Immunohistochemistry of Fas and FasL showed that they are constitutively expressed in normal esophageal mucosa, FasL being predominantly in the basal and suprabasal layers, whereas Fas is in more differentiated layers, i.e., rows of polyhedral cells of the intermediate layers and squamous cells forming the outer layers. In 18 of 19 invasive esophageal squamous cell carcinomas, FasL expression was found in >50% of tumor cells. In contrast, most tumors (15 of 19, 79%) either showed no Fas expression or showed expression in <5% of tumor cells. These alterations were already detected in dysplasia and carcinoma in situ. These results suggest that up-regulation of FasL and down-regulation of Fas expression are early and frequent events associated with the evolution of esophageal squamous cell carcinomas.

Alterations of the INK4a/ARF locus in human intracranial germ cell tumors.

Little is known about the molecular mechanisms responsible for the development of intracranial germ cell tumors (ICGTs). Recently, we demonstrated that the balance of the p53-mdm2 interactions is disrupted in ICGTs. The p14ARF product, a tumor suppresser gene located on the INK4a/ARF locus, acts as one of the major factors affecting p53-mdm2 interactions via its binding to mdm2 and the stimulation of mdm2 degradation. To evaluate whether genetic alterations of the INK4a/ARF locus occur in the genesis of ICGTs, we analyzed the INK4a/ARF genes in 21 ICGTs-10 pure germinomas and 11 nongerminomatous germ cell tumors. Fifteen (71%) of the 21 ICGTs displayed genetic alterations, including 14 homozygous deletions and 1 frameshift mutation. Furthermore, the frequency of the alterations was higher in pure germinomas [9 (90%) of the 10] than in nongerminomatous germ cell tumors [6 (55%) of the 11; P = 0.09]. These data suggested that INK4a/ARF gene abnormalities could play an important role in the genesis of ICGTs, especially in pure germinoma.

Effects of cyclic tensile forces on the expression of vascular endothelial growth factor (VEGF) and macrophage-colony-stimulating factor (M-CSF) in murine osteoblastic MC3T3-E1 cells.

It has been reported that vascular endothelial growth factor (VEGF), expressed by osteoblasts, can induce osteoclast recruitment and thus affects bone remodeling. The purpose of this study was to investigate the effects of cyclic tensile forces on the expression of VEGF and macrophage-colony-stimulating factor (M-CSF) in osteoblastic MC3T3-E1 cells. VEGF and M-CSF gene expression and protein concentration were determined by real-time PCR and enzyme-linked immunoassay. The expression of VEGF and M-CSF mRNA in the experimental group was higher than in the control group. The increase in the concentration of VEGF and M-CSF protein in the experimental group was time-dependent. Moreover, gadolinium (an S-A channel inhibitor), but not nifedipine (L-Type Ca2+ channel blocker), treatment reduced the concentration of VEGF and M-CSF mRNA and protein in the experimental groups. These findings suggest that cyclic tensile forces increase the expression of VEGF and M-CSF in osteoblastic MC3T3-E1 cells via a stretch-activated channel (S-A channel).

Amyloid beta protein deposition in osteopetrotic (op/op) mice is reduced by injections of macrophage colony stimulating factor.

The deposition of amyloid beta (Abeta) protein is a neuropathological change that characterizes Alzheimer's disease. Animals with the osteopetrosis (op/op) mutation suffer from a general skeletal sclerosis, a significantly reduced number of macrophages and osteoclasts in various tissues, and have no systemic macrophage colony stimulating factor (M-CSF). This study examined the effect that M-CSF injections had on Abeta deposition and microglial cell distribution in the brains of normal and op/op mice. Abeta-positive plaques were detected in the cerebral cortex of op/op mice, but not in normal mice. M-CSF reduced the numbers of Abeta-positive plaques in op/op mice. The microglial cell population was reduced in op/op mice compared with normal mice, and M-CSF increased the numbers to 65.8% of that observed in normal mice. Our results suggest that a clearer understanding of the role that microglial cells play in Abeta deposition may help determine the mechanisms involved in the pathogenesis of Alzheimer's disease.

PTEN (MMAC1) mutations are frequent in primary glioblastomas (de novo) but not in secondary glioblastomas.

Loss of heterozygosity (LOH) on chromosome 10 is the most frequent genetic alteration associated with the evolution of malignant astrocytic tumors and it may involve several loci. The tumor suppressor gene PTEN (MMAC1) on chromosome 10q23 is mutated in approximately 30% of glioblastomas (WHO Grade IV). In this study, we assessed the frequency of PTEN mutations in primary glioblastomas, which developed clinically de novo, and in secondary glioblastomas, which evolved from low-grade (WHO Grade II) or anaplastic astrocytomas (WHO Grade III). Nine of 28 (32%) primary glioblastomas contained a PTEN mutation and an additional case showed a homozygous PTEN deletion. This indicates that after overexpression/amplification of the EGF receptor, loss of PTEN function is the most common alteration in primary glioblastomas. In this series, 5 of 28 (18%) primary glioblastomas showed both a PTEN mutation and EGFR amplification. In contrast, only 1 of 25 (4%) secondary glioblastomas contained a PTEN mutation, and none of them showed a homozygous PTEN deletion. The secondary glioblastoma with a PTEN mutation developed from an anaplastic astrocytoma that already carried the mutation. The observation that secondary glioblastomas have a p53 mutation as a genetic hallmark but rarely contain a PTEN mutation supports the concept that primary and secondary glioblastomas develop differently on a genetic level.

Necrogenesis and Fas/APO-1 (CD95) expression in primary (de novo) and secondary glioblastomas.

Glioblastomas may develop rapidly without clinical and histopathological evidence of a less malignant precursor lesion (de novo or primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). Primary glioblastomas typically show overexpression of EGFR, but rarely p53 mutations, while secondary glioblastomas frequently carry a p53 mutation, but usually lack overexpression of EGFR, suggesting that these glioblastoma subtypes develop through distinct genetic pathways. In the present study, we assessed the expression of Fas/APO-1 (CD95), an apoptosis-mediating cell membrane protein, and its relation to necrosis phenotype in primary and secondary glioblastomas. Large areas of ischemic necroses were observed in all 18 primary glioblastomas, but were significantly less frequent in secondary glioblastomas (10 of 19, 53%; p = 0.0004). Fas expression was predominantly observed in glioma cells surrounding large areas of necrosis and was thus significantly more frequent in primary glioblastomas (18 of 18, 100%) than in secondary glioblastomas (4 of 19, 21%; p < 0.0001), suggesting that these clinically and genetically defined subtypes of glioblastoma differ in the extent and mechanism of necrogenesis. Necrosis and microvascular proliferation are histologic hallmarks of the glioblastoma. Following incubation of glioblastoma cell lines under hypoxic/anoxic conditions for 24-48 hours, Fas mRNA levels remained unchanged, whereas VEGF expression was markedly upregulated. This suggests that in contrast to VEGF Fas expression is not induced by ischemia/hypoxia. Analysis of Fas mRNA levels in a glioblastoma cell line containing a p53 mutation and an inducible wild-type p53 gene showed little difference under induced and noninduced conditions, suggesting that in glioblastomas, Fas expression is not directly linked to the p53 status.

Fas ligand expression in glioblastoma cell lines and primary astrocytic brain tumors.

Fas/APO-1 (CD95) is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or Fas antibody. We previously reported that Fas expression is predominantly induced in perinecrotic glioma cells, suggesting that Fas induction is associated with apoptosis and necrosis formation, a histological hallmark of glioblastomas. In this study, we assessed the expression of FasL in 10 glioblastoma cell lines and in 14 astrocytic brain tumors (three low-grade astrocytomas and 11 glioblastomas). Reverse transcriptase (RT)-PCR revealed that all glioblastoma cell lines and primary astrocytic brain tumors express FasL. Immunohistochemically, FasL was predominantly expressed on the plasma membrane of glioma cells. These results suggest that FasL expression is common in human astrocytic brain tumors and may cause apoptosis of glioma cells if Fas expression is induced.

Analysis of digital subtraction coronary angiography for estimation of flow reserve in critical coronary stenosis.

To examine the accuracy of digital subtraction angiographic assessment of coronary flow reserve in critical coronary stenosis, time-density curves were obtained from digital angiograms for a myocardial region of interest. Time-to-peak contrast (TPC) and contrast washout rate (T) were measured in 11 patients with critical 1-vessel lesions before and after percutaneous transluminal coronary angioplasty (PTCA). Collectively, the values of TPC and T were significantly shortened, from 5.8 +/- 1.1 to 4.4 +/- 1.0 seconds (p less than 0.01) and from 11.3 +/- 4.0 to 5.2 +/- 1.2 seconds (p less than 0.001) after PTCA, respectively. All 11 patients except 1 showed shortened T after PTCA; however, in 5 of the 11 patients, TPC after PTCA had approximately the same values as those before PTCA. In experiments in dogs with critical circumflex stenosis, coronary flow and posterior wall thickening at rest were not different from control; however, contrast media-induced hyperemia was markedly attenuated, accompanied by a significant prolongation of T (7.7 +/- 4.5 vs 15.8 +/- 1.9 seconds, p less than 0.01) and completely unchanged TPC (both 6.8 seconds). With simultaneous tracings of coronary flow and time-density curves, TPC and the washout phase on the curve corresponded with contrast-induced transient flow reduction and hyperemic phases, respectively. It is concluded that T appears more sensitive than TPC when basal coronary flow is maintained to almost normal levels, as in patients with stable effort angina pectoris having critical coronary stenosis.

Diagnosis and quantitative evaluation of secundum-type atrial septal defect by transesophageal Doppler echocardiography.

Transesophageal echocardiography (horizontal sector scan) was performed in 11 patients with secundum atrial septal defect (ASD). In all 11 patients, transesophageal echocardiography presented the definite visualization of the defect and a clear laminar shunt flow that showed its 2 peaks in late systole and late diastole. We estimated the size of ASD and a shunt volume across the defect by using transesophageal echocardiography. The defect size determined by transesophageal echocardiography was correlated with the surgical measurement (horizontal width, r = 0.92, p less than 0.001; vertical length, r = 0.85, p less than 0.01). A significant high correlation was shown between the shunt volume measured by transesophageal echocardiography and that by Fick's method (r = 0.87, p less than 0.01). There was no significant correlation between the pulmonary to systemic flow volume (ratio) and the mean shunt flow velocity across ASD, although a high linear correlation was observed between the pulmonary to systemic flow ratio and the defect size in horizontal direction (r = 0.82, p less than 0.01). Transesophageal echocardiography used for diagnosis and quantitative evaluation of ASD could be performed easily and satisfactorily within 10 minutes. Thus, transesophageal echocardiography is a useful method in evaluation of the defect size and the shunt flow volume of ASD. The mean shunt flow velocity was not a reliable index for estimating the shunt flow volume. The defect size might be a valuable determinant of left-to-right shunt volume in ASD.

Expression of E-cadherin-catenin complex in human benign schwannomas.

The Ca2+-dependent cell adhesion molecule E-cadherin has been known to express in normal and reactive Schwann cells in rodents, and to play an important role in Schwann cell-Schwann cell adhesion and maintenance of peripheral nervous tissue architecture. However, little is known about expression of E-cadherin in schwannomas. The aim of the present study was to investigate the cellular expression and localization of E-cadherin, and its associated protein, alpha E-, alpha N- and beta-catenins in human schwannomas, which are supposed to derive from Schwann cells. We tested the hypothesis that these proteins might show an altered expression/distribution in schwannoma cells which correlates with their neoplastic behavior, including sparse cell-cell contact, as seen those in meningiomas and various carcinomas. In human schwannomas, however, E-cadherin, alpha E-catenin, and beta-catenin were detected by western blotting and immunohistochemistry, whereas alpha N-catenin was not. Immunoprecipitation using anti-E-cadherin antibody resulted in alpha E-catenin forming a complex with E-cadherin. SSCP analysis revealed no mutations in the transmembrane domain or in intracellular catenin-binding site of E-cadherin. These data suggest that the E-cadherin-alpha E-catenin complex is well preserved in human schwannoma cells, which is compatible with its benign behavior, and these molecules might be used as additional cell markers of Schwann cell-derived tumors.

Expression of vascular endothelial growth factor and the effects on bone remodeling during experimental tooth movement.

Vascular endothelial growth factor (VEGF) has an ability to induce functional osteoclasts as well as neovascularization. We recently reported that the number of osteoclasts was enhanced by the injection of recombinant human VEGF (rhVEGF) with the application of mechanical force for experimental tooth movement. In this study, the expression of VEGF was detected in osteoblasts on the tension side of the alveolar bone. Moreover, the rate of tooth movement was significantly increased in the rhVEGF injection groups compared with the controls. These results suggested that VEGF, highly expressed by mechanical stimuli, enhances the number of osteoclasts as a paracrine factor, and that the amount of tooth movement is accelerated by both endogenous VEGF and injected rhVEGF.

Effects of sex hormone disturbances on craniofacial growth in newborn mice.

It is well-known that sex hormones influence bone metabolism. However, it remains unclear as to how sex hormones affect bone growth in newborn mice. In this study, we performed orchiectomy (ORX) and ovariectomy (OVX) on newborn mice, and examined the effects on craniofacial growth morphometrically. ORX and OVX were performed on five-day-old C57BL/6J mice. Four weeks after surgery, lateral cephalograms were taken of all of the mice, with the use of a rat and mouse cephalometer. Cephalometric analysis of the craniofacial skeleton was performed by means of a personal computer. Inhibition of craniofacial growth was found in the experimental groups but not in the sham-operated groups. In the nasomaxillary bone and mandible, the amount of growth was significantly reduced. These results suggest that craniofacial growth is inhibited by sex hormone disturbances not only in puberty but also immediately after birth.

Expression of cell adhesion molecule E-cadherin in human arachnoid villi.

Calcium-dependent epithelial cell adhesion molecules designated as E-cadherin (also known as uvomorulin or L-CAM) were identified in human arachnoid villi by immunoblotting and immunocytochemical analyses using a monoclonal antibody HECD-1 raised against human mammary carcinoma MCF-7 cells. Immunoblot analysis showed that HECD-1 recognizes E-cadherin with a molecular weight of 124 kD. In all arachnoid cells of an arachnoid villus, E-cadherin was detected by immunolight microscopy within the cytoplasm rather than the cellular boundaries as seen in the control group. Furthermore, the extent of expression by immunolight microscopy varied from portion to portion. The expression was usually weak in the syncytial cluster which was ultrastructurally composed of tightly juxtaposed cells characterized by few extracellular cisterns and numerous cell junctions, while it was intense in the reticular cluster and the surface layer which were ultrastructurally characterized by abundant extracellular cisterns and smaller numbers of cell junctions. The cells of the reticular cluster and the surface layer contained more free ribosomes than those of the syncytial cluster. Immunoelectron microscopy showed that E-cadherin was localized not only to the opposing plasma membranes and the cytoplasm around the free ribosomes or the rough endoplasmic reticulum but also to the extracellular cisterns. As the expression of E-cadherin was closely related to the arachnoid cells adjacent to the cerebrospinal fluid pathway, it is suggested that, instead of the cell junctions, E-cadherin may play an important role in the flexible adhesion of arachnoid cells even in the presence of the cerebrospinal fluid.

[A case of glioblastoma multiforme which indicated the early stage on brain MRI].

A 57-year-old male was urgently carried to our hospital because of sudden loss of consciousness, lasting about 10 minutes. He had resumed consciousness before he arrived at our hospital. Neurologically, he had mild muscle weakness of the right arm. Deep tendon reflexes in the right upper extremity were reduced. In high level functions, speech disturbance, dysgraphia (disturbed ability to write Hiragana), and constructive apraxia were noted. A brain MRI upon admission showed a poorly demarcated, high signal intensity area in the cortical and subcortical layers of the left temporal and parietal lobes. This was visible on T 2 weighted images(T 2 WI), although no abnormalities were visible on T 1 weighted images(T 1 WI). No contrast enhancement was effected by Gd-DTPA. The patient was therefore suspected of having a tumor or degenerative disease and was monitored closely. About 4 months later after onset, his symptoms became aggravated, and brain MRI disclosed a marked low signal intensity area on T 1 WI and a heterogeneous high signal intensity area on T 2 WI. The abnormal signal intensity area was surrounded by extensive edema and mass effect. Ring-shaped, irregular, contrast enhanced areas were also visible. Cerebral angiography revealed a poorly demarcated tumor stain in the area supplied by the middle cerebral artery. The tumor was removed surgically and was histopathologically rated as glioblastoma multiforme(GBM). Because this case represents a valuable example of early stage of GBM, it will be discussed in this paper, along with differential diagnoses.

[Tarantulas bite: two case reports of finger bite from Haplopelma lividum].

The risk of envenomations by venomous exotic spiders have not been well recognized in Japan. Two cases of finger bite from Asian pet tarantula, Haplopelma lividum (Cobalt blue), are reported. In both cases, initial severe pain and inflammatory signs were completely healed with only symptomatic treatments within several hours. In one case, arthritic stiffness lasted for a few weeks following bite but resulted no permanent deficit. Bites from Haplopelma lividum seemed relatively harmless like other various tarantulas, although the composition of the venom has been unknown.

Influence of sex hormone disturbances on the internal structure of the mandible in newborn mice.

It has not yet been clarified how sex hormones affect craniofacial bone development immediately after birth. The purpose of this study was to examine the effects of sex hormone deficiency on craniofacial bone development immediately after birth, in terms of the internal structure of the mandible in newborn mice with orchiectomy (ORX) and ovariectomy (OVX). ORX, OVX and a sham-operation were performed on 40 five-day-old C57BL/6J mice. Eight weeks after surgery, each mandible was subjected to histomorphometric analysis of trabecular (Tr) and cortical (Ct) bone mineral density (BMD) by peripheral quantitative computed tomography (pQCT). In the experimental groups, a significant reduction in BMD was found in comparison with the control groups. In histomorphometric analysis, the number of tartrate-resistant acid phosphatase (TRAP)-positive cells in the condyle and the thickness of the condylar cartilage layer was significantly greater in the experimental mice than in the controls. Trabecular bone volume of the condyle measured on azocarmine-aniline blue (AZAN) sections was significantly less in the experimental mice than in the controls. These results indicate that mandibular growth is inhibited by sex hormone disturbances and the relevant internal structures changed. The findings show that sex hormones are one of the key determinants of mandibular growth and development immediately after birth.

Biochemical significance of 19-hydroxytestosterone in the process of aromatization in human corpus luteum.

19-Hydroxyandrogens are known to be an intermediary metabolite in the aromatizing reaction, though the physiological role of this compound has not yet been clarified. In this study, microsomes obtained from human corpus luteum were incubated with testosterone or 19-hydroxytestosterone (19-OHT) as the substrate to investigate the biochemical significance of 19-OHT in the process of aromatization in the ovary. The inhibitory effects of 4-hydroxyandrostenedione (4-OHA) on the formation of estradiol from testosterone and 19-OHT in human ovary were also investigated. When testosterone was incubated with human ovarian microsomes, 19-OHT and estradiol were identified. When 19-OHT was used as the substrate, the formation of estradiol was demonstrated. To our knowledge, this is the first report to demonstrate the formation of estradiol from 19-OHT in human ovarian tissue. The Km value of aromatase for testosterone on human corpus luteum microsomes was 0.21 microM. 4-OHA exhibited inhibition with a Ki of 35 nM. With testosterone and 19-OHT as the substrate, the formation of estradiol was also equally inhibited by 4-OHA. A dose dependent inhibition of estradiol formation was observed, with no apparent accumulation of 19-OHT. These results suggest that 19-OHT may not only be an intermediary metabolite in the aromatization of testosterone by human ovary but could be a product of the microsomal enzyme.