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For quantification of histochemical reactions, color image analysis, as well as cytophotometry, is important. However, the extremely large amount of information, usually more than one million bits per image, involved in one microscopic picture of a colored histochemical specimen, when digitalized, has inhibited attempts to develop a more convenient system of color image analysis. Recent advances in microcomputer technology have now made it possible to build, at a reasonable cost, an intelligent color terminal equipped with array-processing hardware, a sufficiently large video random-access memory to represent 4096 colors in each pixel, and packages of machine language subroutines called “macro-commands” in the form of read-only memory that can process color images within a few seconds. This intelligent terminal can easily be controlled by a small personal computer via an IEEE-488 interface bus. The digitalized color image can be stored in and retrieved from a floppy disk. As the macro-commands in this system are controlled by BASIC program through CALLS with parameters, it is highly flexible and adapts readily to the varied needs of histochemists. Reported on herein will be the instrumentation of the microcomputer-based color image analyzer and a few examples of applications of this system to histochemistry: color intensification of cytochemical reactions, quantification of enzyme (e.g., horseradish peroxidase) reaction products, periodicacid-Schiff staining, collagen fibers visualized by Azan-Mallory stain, etc. Possible future applications of this color image analyzer will also be discussed.

Parkinson's disease (PD) is an age-related movement disorder that progresses over a period of 10 to 20 years. The existence of microglia in a long-lasting activated state, expressing MHC II, has been thought to play an important role in the progression of PD. PD mouse models, induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), exhibit only transient PD-like movement dysfunction in contrast to MPTP-intoxicated monkeys which show progressive and permanent movement dysfunction. To understand the reasons why the progression does not occur in MPTP-treated mice, we used immunohistochemical analyses to study whether activated microglia in the striatum and/or substantia nigra persist long after MPTP treatment. Microglial changes in the striatum and substantia nigra of mice at 2 days and 6 months after MPTP treatment (four intraperitoneal injections of 20 mg/kg MPTP at two hour intervals) were examined. C57BL/6 mice (which are highly sensitive to MPTP) displayed transient movement dysfunction and highly activated microglia were observed at day two. In contrast, BALB/c mice (which are less sensitive to MPTP) exhibited no movement dysfunction and only slightly activated microglia were observed at day two. After 6 months, the microglia in the striatum and substantia nigra pars compacta of the treated C57BL/6 mice were still more hypertrophic compared with the control, although less hypertrophic than those observed at day two. In the treated BALB/c mice, the microglia were also hypertrophic compared with the control after 6 months. MHC II-positive microglia were undetectable at any time after MPTP treatment in both mice. These data show that MPTP administration results in the existence of persistent activated microglia that are not MHC II-positive, and is independent of the MPTP sensitivity of the mouse strain. These results suggest that long lasting MHC II-positive microglia might be required for PD progression. In MPTP-intoxicated mice, the absence of MHC II-positive microglia might explain why there is no progression of PD-like dysfunctional symptoms.

The expression patterns of three microtubule-associated proteins (MAP1A, MAP1B, and MAP2A&B) were investigated in the developing optic tectum. Expression of MAP1B and middle-molecular-weight peptide of neurofilament (NF-M) was first observed in the same mesencephalic cells on day 3 of incubation, indicating that neuroblasts had been produced. At day 5, MAP1A and MAP2A&B expression appeared in the cellular layer containing the first neuroblasts that differentiate into large multipolar cells. The NF-M+ neurites in the striatum album centrale (SAC) and the striatum opticum (SO) were MAP1B+ up to day 19, but the intensity of MAP1B immunoreactivity decreased with development. All three MAPs were expressed in large multipolar neurons in the developing stratum griseum centrale from the beginning of maturation. Stratum griseum et fibrosum centrale cellular layers, containing radially arranged piriform neurons, were MAP1A−/MAP2A&B− on day 11 but became MAP1A+/MAP2A&B+ during later stages. These results suggest that the timing of MAP expression in neuronal maturation of large multipolar cells differs from that of piriform cells. The expression of MAPs has revealed specific cellular events in the developing optic tectum. Based on our observations, the development of the optic tectum can be divided into four periods.

The elevator movement of the matrix cell is synchronous with the mitotic cycle. Its mechanism has been thought to be due to cyclic changes in cell adhesion to the neighboring cells and the basement membrane. The present study aims to analyze the cytoplasmic changes by means of immunohistochemical investigations of the distributions of the adhe- sion molecules, N-cadherin, α-catenin, β-catenin, F-actin, etc. It was found that the adherent junctions and junctions toelevator ECM of the matrix cell disintegrate during mitosis, releasing the cell from the intercellular adhesions and the basement membrane. As a consequence, the mitotic matrix cell is rounded out and attracted to the ventricular surface where the junctional complexes remain unchanged during mitosis. After division, the daughter matrix cells recover their adhesion apparatuses and, extending their cytoplasmic processes, resume their downward movement.

Granulysin has been identified as an effector molecule co-localized with perforin in the cytotoxic granules of cytotoxic T lymphocytes and natural killer (NK) cells, and has been reported to kill intracellular pathogens in infected cells in the presence of perforin and to induce a cytotoxic effect against tumor cells. The aim of the present study was to elucidate whether intracellular expression of granulysin and perforin by NK cells might be associated with progression of cancer. Flow cytometric analysis demonstrated high levels of perforin and granulysin expression by CD3(-) CD16(+) cells in healthy controls. In contrast, cancer patients exhibited significantly decreased levels of granulysin expression ( P

Ganglioside GD3, which has been used as “a marker for oligodendrocyte progenitor cells” in vitro, is present in a few cells under the ependymal lining of the ventricle in adult rat brains. However, recent studies using a monoclonal antibody against GD3(R24) suggested that GD3−positive cells in adult rat brains are microglia, thus making the cellular localization of GD3 controversial. In this immunohistochemical study, we compared images of immunostained sections of adult rat brains with a monoclonal antibody against GD3 (DSG−1) with those immunostained with microglia−specific (antibody against thymosin β4), astroglia−specific (antibodies against GFAP or S−100 protein) or with oligodendroglia−specific (antibodies against GalC or MBP) antibodies for the purpose of re−examining the identity of GD3−positive cells in adult rat brains. We observed co−localization of GD3 immunoreactivity with that of GFAP or S−100 protein, but not with that of thymosin β4, MBP or GalC. These results indicate that the GD3−positive cells in adult rat brains are astroglia, not microglia or oligodendroglia.

The relation between radical production and acute morphological changes of palisade cells of Saintpaulia induced by a rapid drop in leaf temperature was investigated. The drastic and apoptotic changes were observed immediately after the chilling treatment for 3 sec. OH radical production occurred immediately after the chilling treatment and then increased linearly depending on the time. The radical production might initiate and develop acute irreversible morphological changes resulting in apoptotic cell death of Saintpaulia palisade cells.

岐阜県における全身性強皮症(以下PSSとする)患者の実態を明らかにすることを目的とし岐阜県における全身性強皮症認定患者の「特定疾患対象患者認定申請書」に基づく調査を行った。1986年度および1990年度の2ヵ年間に強皮症, 多発性筋炎·皮膚筋炎として認定を受けた患者の中からPSS患者の申請書を選別し, これをもとに記載事項を集計した。PSS患者は1986年度で男性13名, 女性89名, 計102名であり, 一方1990年度の患者数は男性18名, 女性179名, 計197名と4年後にはほぼ倍増していた。男女比はほぼ1:7∼10で女性に多く, 年代別では両年度および男女とも40歳代, 50歳代, 60歳代に多かった。国保加入者数をもとにした強皮症の受給者率は人口10万対で1986年度では4.9であったが1990年度では11.7と2倍以上であった。主要臨床所見においては1990年度では1986年度に比べて「色素異常」がみられた症例の割合が少なかったことが特徴的であった。初発症状は両年度とも「レイノー現象」が男女とも多くみられた。抗核抗体の陽性率については68.9∼83.3%が陽性で, 1990年度に陽性率が高かった。新規受給者を対象にした場合の特徴および申請に至るまでの状況としては発病から申請までに5年以上の年数を経ているものが少なくないこと, その原因として発病から診断までにかなりの時間がかかっていることなどが考えられた。

An in vitro study employing dissociated matrix cells obtained from the rostral part of mouse neural tube or forebrain vesicle was done in an attempt to elucidate their cellular characteristics. We found a reaggregate formation with an organoid architecture. The organization of the reaggregate was studied by transmission as well as scanning electron microscopy, and by immunocytochemistry. After three days in a stationary culture, dissociated matrix cells formed a spherical, organoid reaggregate having a central lumen. The size of the reaggregate varied from approximately 100μm to 200μm. The transmission electron microscopy revealed that the reaggregate wall is composed of two types of cells; one has an electron-lucent cytoplasm with few organelles except for polysomes, whereas the other contains an electron-dense cytoplasm with many microtubules and mitochondria. The latter type of cell was located in the marginal portion of the wall intermingled with a large number of cellular processes. These cells were immunostained with anti-neurofilament antibodies and regarded as neuroblasts. The luminal surface was composed predominantly of the former cell type showing well-developed junctional complexes in between them and having cilia on its luminal side. Mitotic figures were frequently demonstrable adjacent to the lumen.

In eight groups of subjects operating various hand-held vibrating tools and aged from 30 to 59 years, the prevalence rates of vibration-induced white finger (VWF) and numbness, pain, or stiffness in the upper and lower extremities were investigated. Hand-transmitted vibration levels (HTVLs) were measured on the back of the hand, by means of unidirectional (x-axis) vibration dosimeters, and the frequency-weighted acceleration levels [(Lh,w)eq,t] were determined as the vibration levels. The prevalence rates of VWF and numbness of the hands in these subjects were compared to the prevalence rates of Raynaud's phenomenon (RP) and numbness of the hands in 1027 males and 1301 females not occupationally exposed to vibration (age range: 30-59 years). It was observed that in subjects exposed to HTVLs of between 1.1 and 2.5 m/s2, the prevalence of VWF was between 0.0% and 4.8%. The prevalence of VWF reached 9.6% in a group of workers exposed to HTVLs of 2.7-5.1 m/s2. The latter group showed a significant difference (P < 0.05) in the prevalence of VWF compared to the 2.7% prevalence of RP in male subjects of the general population. The prevalence of VWF in female subjects exposed to vibration (4.3%) was not significantly different from the prevalence of RP in females of the general population (3.4%). The prevalence rates of numbness of the hands were in the range of 6.5%-30.4% in the exposed groups and in the range of 13.4%-29.5% in the general population. Among the subjective symptoms, only VWF showed a significant positive correlation with HTVLs (R2 = 0.5, P < 0.05). It was concluded that in decisions concerning quantitative recommendations for vibration exposure, the prevalence of VWF should be employed. With a view to decreasing the risk of developing VWF, estimated vibration safety values for 4 h and 2 h daily exposures are discussed.

To evaluate the factors relating to traffic accidents resulting in injury or death of drivers, questionnaire survey was performed on seat belt use in 136 official workers (101 males and 35 females) driving cars. In data analyses, multiple logistic regression analysis was applied and the factors relating to seat belt use were detected. The results obtained were as follows. 1. The percentage of males using seat belt at any time was 82.6% . This value decreased to 68.6% in females, but the difference was not significant. 2. Seat belt unuse was significantly related to Body Mass Index (Odds ratio 0.52), nights of drive per week (Odds ratio 4.29) and experiences of traffic accidents resulting in injury or death (Odds ratio 109.29) and resulting in property dameges (Odds ratio 14.15).

The applications of a confocal laser scanning microscope for imaging fast dynamic changes of the intracellular calcium ion concentration ([Ca2+]i) in the living cell are described. Confocal images enable us to determine the cell-to-cell junction between clustered heart muscle cells of guinea pig and reveal that the high [Ca2+]i can evoke calcium-induced calcium release from the sarcoplasmic reticulum in the neighboring heart muscle cells through the gap junction. In rat peritoneal mast cells, secretagogue-induced calcium signaling initiates at the periphery of the cell and propagates to the nucleus as calcium wave. Nuclear [Ca2+]i is upheld at higher level than cytoplasmic [Ca2+]i for 2min. X-t scan mode with high-temporal resolution provides comprehensive images of the phenomenon. The confocal laser scanning microscope with depth-discriminating ability is a valuable tool to know a sequence of biological event within the living cell.

Noise levels were evaluated before and during aerobic sessions in 3 aerobic studios. In addition, hearing threshold levels (HTLs) of 10 female aerobic instructors with the mean (+/- SD) ages of 25.4 (+/- 2.4) years were measured before and 2 min after an aerobic session. The mean (+/- SD) of total instruction time of these subjects was 1274.0 (+/- 704.9) h. Before the aerobic session, noise level was 63 dB(A) and increased to 87 dB(A) during a warm-up period. The highest noise levels of 93-96 dB(A) were recorded during a peak exercise period and decreased to 73 dB(A) during a cool-down period. The dominant spectra of sound pressure levels (SPLs) during the aerobic session were in the frequency range of 1-2 kHz at levels of 75-88 dB(C). The mean values of HTL obtained for each ear of these subjects were between 5 and 15 dB in the frequency range of 1-8 kHz. No significant differences in the mean HTLs of pre- and post-aerobic sessions could be obtained. The mean values of HTL of two groups of subjects divided by their total instruction time (n = 5 in each group, and p < 0.01 in their total instruction time) were quite comparable.

To clarify the mechanism of secretagogue (compound 48/80)-induced calcium signaling in rat peritoneal mast cells, we analyzed serial confocal calcium images with high spatial and temporal resolution using different Ca(2+)-probes. The Ca(2+)-wave began at the periphery of the cytoplasm, and then spread to the center of the nucleus. Nuclear [Ca2+]i was clearly higher than cytoplasmic [Ca2+]i. The heterogeneity of [Ca2+]i continued until about 2 min after degranulation. These results suggest the existence of an intranuclear Ca(2+)-store which possesses a Ca(2+)-releasing mechanism similar to that in the cytoplasm.

To evaluate the actual condition of the traffic accidents resulting in injury or death of drivers and to assess the factors relating to them in Japan, the data concerning traffic accidents in 47 prefectures in Japan were surveyed and analyzed using multiple regression analysis. The results obtained were as follows. 1) In general, the numbers of traffic accidents resulting in injury or death of drivers/100, 000 persons having driver's licence and the ratio of traffic accident resulting in death of drivers to those resulting in injury and death of drivers in Great Tokyo were the lowest in Japan. 2) The numbers of traffic accidents resulting in injury and death of drivers/100, 000 persons having driver's license in each prefecture had significant positive relation to the total length of national highway and also to the proportion of workers in the secondary industries in males, and to the number of cars retained/total number of persons having driver's license in females. 3) The numbers of traffic accidents resulting in death of drivers/100, 000 persons having driver's license in each prefecture had significant positive relation to the total length of national highway and negative relation to the percentages of drivers with a seat-belt in persons driving a car and the population density in males . In females, they had significant postive relation to the number of cars retained/total number of persons having driver's licence. 4) The ratio of traffic accidents resulting in death of drivers to those resulting in injury and death of drivers had no relation to any considered factors in males or females.

Subcellular localization of human immunodeficiency virus type 1 (HIV-1) Tat and Rev was examined using a confocal laser scanning microscope (CLSM). In transfected COS-7 cells, Tat resided exclusively in the perinocleolar region, while Rev infiltrated fully into the nucleoli. The chimeric Tat in which the nucleolar targeting signal was replaced by that of Rev, which retains trans-acting activity of Tat, remained still in the perinucleolar region as wild-type Tat. Perinucleolar distribution of Tat protein suggests the existence of a novel nucleolar architecture that affects transcription.

Glial fibrillary acidic protein (GFAP) was purified from normal bovine brain by a modification of the procedure used to isolate vimentin in order to avoid contamination by other cytoskeletal components: vimentin, neurofilament triplet proteins, tubulin and actin. GFAP is thought to be separated from vimentin in the DE cellulose column chromatography step. The three other major proteins were also separable through ion exchange and gel filtration column chromatographies. A purified 49 kDa polypeptide was estimated to be GFAP from peptide mapping and subsequent immunoblotting analysis. We obtained 4.4 mg GFAP/1 g bovine brain white matter in less than 3 days. The polyclonal antibody raised against purified GFAP was able to detect 49 kDa GFAP by immunoblotting analysis. This isolation method is simpler and more rapid than previous methods.

We applied laser microtomography, a direct optical section technique using a confocal laser scanning microscope, to the mapping of subcellular three-dimensional morphology. A novel model of laser microscope in combination with a fine focus control device and a digital image processor enables us to readily reconstruct high resolution, extended-focus, three-dimensional images at any viewing angle. This system facilitates recognition of the in situ morphology of such subcellular components as nuclear chromosomes or cytoskeletons.

SUMMARY Morphogenesis of the brain is analysed by 3-D computer graphics simulation. The initial form of the brain, i.e. a neural tube, is created in the computer as a small tubular figure in 3-D Cartesian coordinates which are subsequently transformed into curvilinear coordinates by a set of growth functions. By this method the morphological development of the human brain is simulated. The same computer program can be applied to simulate the development of the brains of mouse, rat and monkey simply by changing the parameters slightly. The result of the analysis reveals that the smaller brains are produced by faster growth of the matrix cell population at the onset of development, coupled with faster rate of retardation of the growth during ontogeny. Thus, smaller brains in smaller animals are produced in a shorter period during their ontogeny.

The thickness of Brunner's glands was measured with an ocular micrometer in 75 cases of surgically resected duodenal ulcer and in 75 autopsy cases (control group). Endoscopic findings before operation were also studied. Maximum mean thickness of Brunner's glands in the control group was 1.54 +/- 0.38 mm (mean +/- SD), and no regional difference in thickness was noted. The thickness of Brunner's glands in the duodenal ulcer group was widely distributed, from 0.5 mm to 5.0 mm, and the average value was 3.0 +/- 1.0 mm, with a statistically significant difference from that of the control group. In most duodenal ulcer cases Brunner's glands were thickest within 1 cm from the center of an ulcer. Only six cases of duodenal ulcer (8.0%) showed a diffusely thin layer of Brunner's glands, less than 1.5 mm thick. These results show that the Brunner's glands become hyperplastic in duodenal ulcer patients, especially near the ulcer. In the healed ulcer Brunner's glands were thin at the center of an ulcer scar, and the average thickness was 0.42 +/- 0.26 mm. This histologic finding corresponded to the depressed scarred area observed endoscopically, suggesting a decreased mucosal resistance at this area.

Regional ploidy variations within individual tumors were analyzed by in-situ cytofluorometry of metaphase cells in Feulgen-stained paraffin sections, using 45 resected stomachs with early and advanced signet ring cell carcinomas. Aneuploid cells were found in one of 30 early cancers and in eight of 15 advanced cancers, and were almost always accompanied by diploid cancer cells in the mucosal part of the cancers. The diploid and the aneuploid cells were generally found to be distributed in different territories in the mucosa, and aneuploid foci were often included in the diploid area. These findings suggest the diploid origin of signet ring cell carcinomas and the occurrence of aneuploidy during the tumor development. Moreover, the aneuploid cells appeared to infiltrate beyond the mucosa more readily than the diploid cells; most of the aneuploid populations already invaded the extramucosal tissue, and the cancer cells infiltrating in the extramucosal tissue were predominantly aneuploid in six of the nine cancers with aneuploidy. Thus, it appears that the occurrence of aneuploid clones may accelerate the progression of signet ring cell carcinomas from early to advanced stages.

The expression of astrocyte marker proteins (S100beta and GFAP) during infarction and glial scar formation after transient middle cerebral artery (MCA) occlusion was examined using double immunostaining. S100beta immunoreactivity markedly decreased in the core of the injured area when observed immediately after reperfusion and did not increase again. In the periphery, however, S100beta expression increased, showing that S100beta synthesis was up-regulated. S100beta+/iNOS+ astrocytes in the periphery were observed from day 1, when small infarct areas were detectable, up to day 5, when infarct expansion had almost ended. TUNEL+ cells in the periphery were present from days 1 to 5. S100beta+/TUNEL+ cells were observed centrally and around the periphery of the injured area, predicting that cell death contributes to the increase of S100beta concentration in the injured area. Our results suggest that (1) higher concentration of S100beta in the extracellular space due to S100beta leakage from damaged astrocytes leads to up-regulation of S100beta synthesis and induction of inducible nitric oxide synthase (iNOS) synthesis in astrocytes, contributing to infarct expansion that results in DNA damage or cell death via NO and ROS production, and (2) GFAP, but not S100beta, is a main contributor to glial scar formation. On day 1 postreperfusion, the microdiascopic images of the injured areas from the unstained thick sections or the areas detected by S100beta immunoreactivity were larger than those of the infarct areas detected by hematoxylin--eosin (HE)-staining. The difference between these sizes might be useful to predict infarct expansion.

In the early 1960s I applied 3H-thymidine autoradiography to the study of the cells constituting the neural tube, and found that its wall was composed solely of one kind of single-layered epithelial cell, which perform an elevator movement between the mitotic and DNA-synthetic zones in the wall in accord with the cell cycle. They were identified as multipotent stem cells of the central nervous sytem (CNS) to which I gave the name of matrix cells. (3)H-thymidine autoradiography also revealed the chronology of development of these matrix cells: At first they proliferate only to expand the population (stage I), then switch to differentiate specific neuroblasts in given sequences (stage II), and finally change themselves into ependymoglioblasts, common progenitors of ependymal cells and neuroglia (stage III). Based on these findings, I proposed a monophyletic view of cytogenesis of the central nervous sytem. This matrix cell theory claiming the existence of multipotent stem cells has long been the target of severe criticism and not been accepted among neuro-embryologists for a long time. Recent findings by experimental and clinical neuroscientists on the importance of stem cells have renewed interest in the nature and biology of the multipotent neural stem cells. The present paper describes how the concept of the matrix cell (multipotent neural stem cells in vivo) emerged and what has come out from this view over the last 45 years, and how the basic concept of the matrix cell theory has recently been reconfirmed after a long period of controversy and neglect.

Mitosis is a highly coordinated process that assures the fidelity of chromosome segregation. Errors in this process result in aneuploidy which can lead to cell death or oncogenesis. In this paper we describe a putative mammalian protein kinase, AIM-1 (Aurora and Ipl1-like midbody-associated protein), related to Drosophila Aurora and Saccharomyces cerevisiae Ipl1, both of which are required for chromosome segregation. AIM-1 message and protein accumulate at G2/M phase. The protein localizes at the equator of central spindles during late anaphase and at the midbody during telophase and cytokinesis. Overexpression of kinase-inactive AIM-1 disrupts cleavage furrow formation without affecting nuclear division. Furthermore, cytokinesis frequently fails, resulting in cell polyploidy and subsequent cell death. These results strongly suggest that AIM-1 is required for proper progression of cytokinesis in mammalian cells.

To investigate the possible role of phosphorylation of protein tyrosine during myofibrillogenesis (6- to 13-somite stages) of the chicken embryonic heart tube, immunolocalization of phosphotyrosine (P-Tyr) and the relationship between P-Tyr and developing myofibrils were studied by means of confocal scanning laser microscopy and immuno-electron microscopy. The staining pattern of P-Tyr varied in different sites of myocytes at different stages of embryonic development: At the cell-cell boundaries, P-Tyr was localized at the adhesion belt of outer myocardial layer cells (6- to 13-somite stages), non-junctional cell-cell contacts (6- to 13-somite stages) and early intercalated disks of both the outer and inner myocardial layer cells (8- to 13-somite stages). At the cell-extracellular matrix boundaries of inner layer cells, the first stages of myofibril formation appeared as serially aligned areas of P-Tyr localization closely associated with circumferentially aligned thick actin bundles (8- to 9-somite stages). This P-Tyr immunostaining decreased when the thick actin bundles developed into mature striated myofibrils at the 10- to 13-somite stages. These findings suggest that the phosphorylation of protein tyrosine residues is primarily concentrated at the modulating cell-cell and cell-matrix adhesion sites of developing myocytes and myofibrils.

Objective To track changes in interferon (IFN) production in healthy individuals to shed light on the effect these changes have on the course of healthy ageing. Design Study is based on data that were collected over 24 years from a cohort of individuals whose IFN-α production was quantified as a part of their annual routine health check-up. Setting All individuals in this study underwent regular health check-ups at Louis Pasteur Center for Medical Research. Participants 295 healthy individuals (159 males and 136 females) without a history of cancer, autoimmune diseases and hepatitis C virus (HCV) whose IFN-α production was quantified more than five times within 24 years were selected. Finally, 29 males and 4 females whose IFN-α production was quantified more than 25 times were selected and their data were analysed using a mixed model. Main outcome measures HVJ stimulated IFN-α production was quantified. Healthy individual9s periodical log transformed IFN-α values (y) were plotted versus age (x) and fitted to linear (y=mx+n) and quadratic formula (y=ax 2 +bx+c) expressions to reveal changes in the IFN-α production in these healthy individuals. Results The linear expression showed that log (IFN-α) had a slight tendency to decline (3% over 10 years). However, the quadratic formula analysis showed the quadratic expression to be more positive than negative (a concave U-shaped pattern) which means that individuals’ once declining IFN production recovered as they aged. Conclusions Although we observed a marginal decline in IFN-α production, we also observed that IFN production recovered even in individuals in their mid50s to early 60s. These results combined with our previous cross-sectional studies of patients with various diseases suggest that in healthy individuals, the impairment of IFN production is triggered more by the onset of disease (notwithstanding the cause) rather than by ageing.

To trace the sequence of numerical chromosomal aberrations during tumor progression of colorectal tumors, we studied intratumoral heterogeneity of chromosomal copy number by a combined fluorescence in situ hybridization (FISH) and ploidy analysis. We used six formalin-fixed paraffin-embedded tumors of which the mucosal lesions were preserved. Nuclear suspensions were made from the tumor tissues that were scraped from several small regions in 100 μm thick sections. Copy number of chromosomes 1, 7, 17, and 18 were examined by FISH with centromeric repetitive probes. DNA ploidy was monitored by cytofluorometry, and was correlated to the chromosomal copy number on the smear slides of identical nuclear suspension from each tumor portion. All the tumors examined included the DNA-diploid regions in the mucosa, where cancer cells commonly showed monosomy 18 and/or trisomy 7. These chromosomal changes may be quite common early events before the occurrence of DNA-aneuploidy in the development of colorectal tumors. Three out of the 6 tumors included neartetraploid (3.6–4.1C) cells in deeper invasive regions. Chromosomal constitution of DNA-aneuploid cells was suggested to derive from that of DNA-diploid cells through ploidy duplication with or without additional loss or gain of chromosomes.

Human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, including two essential trans-activators for viral replication, Rev and Tat. Both Rev and Tat have a nucleolar targeting signal and are actually located predominantly in the nucleoli. Within the nucleoli, Rev is localized to the combined regions of the dense fibrillar (DFC) and the granular (GC) components. Tat does not colocalize precisely with any nucleolar component tested, but partly overlaps regions of the DFC and the GC. Regions of both Rev and Tat are overlapped by the distribution of the major nucleolar protein B23. Overexpression of Rev causes nucleolar ballooning and general structural deformity with aberrant accumulation of rRNAs, whereas Tat does not have that effect. B23 is markedly accumulated in those nucleoli deformed by Rev. Components of the nucleolar DFC, GC, and fibrillar center domains are not accumulated but dispersed in few small spots or larger patches within the enlarged nucleoli. Cytophotometric DNA determinations revealed that transient expression of Rev results in accumulation of G2, prophase, and mitotic cells which have failed cytokinesis, suggesting that Rev is capable of preventing or slowing the progression through mitosis. Tat, in contrast, does not affect the cell cycle. We speculate, based on these results, that Rev represses cell growth by inhibiting the transport of ribosomal proteins and preribosomal particles across the nuclear envelope and affecting the cell cycle, both of which may be related to proposed functions of B23.

The rex-encoded protein (Rex) of human T-cell leukemia virus type I (HTLV-I) is responsible for the cytoplasmic accumulation of incompletely spliced mRNAs that encode the virion structural proteins. Rex is known to be located predominantly in the cell nucleoli in transient transfections or in the isolated nuclei of HTLV-I-infected cells. However, precise location of Rex under physiological conditions has not been determined unequivocally. Here we report that Rex is primarily located as intranucleolar speckles in HTLV-I-infected cells, except for a few nucleoplasmic speckles. This is in contrast to the more diffuse nucleolar distribution of the rev-encoded protein (Rev) of human immunodeficiency virus type 1 (HIV-1), the functional homologue to Rex, in HIV-1-infected cells. Accumulation of Rev is associated with disruption of nucleolar structure and cell death, whereas Rex does not have these effects. The difference in distribution of Rex and Rev within the nucleoli may reflect the difference of toxicity toward the host cells. Involvement of the nucleolus in processing of certain mRNAs is also discussed.

Background. Systemic lupus erythematosus (SLE) is designated by the Japanese government as one of the intractable diseases and all patients, who suffer from these diseases, are registered to get financial aid for treatment. Using newly registered SLE patients, a case-control study was conducted to evaluate potential risk factors. Methods. Two-hundred and eighty-two women SLE patients, newly registered to receive financial aid for treatment, and 292 randomly selected health examination participants at public health centers (controls) were surveyed from April 1988 through March 1990. By means of a self-administered questionnaire, data concerning demographic variables, smoking and drinking habits, past medical and reproductive history, and family history were collected. Results. Based on unconditional logistic regression analysis, the risk of SLE was significantly increased for current smokers (age-adjusted odds ratio (OR) = 2.31, 95% confidence interval (CI) (1.34–3.97). Alcohol and milk intake were inversely associated with risk. Family histories of asthma and collagen diseases, including SLE, were associated with significantly elevated risk of SLE (OR = 2.07, 95% ci 1.14–3.77; OR = 5.20, 95% CI 1.08–24.95, respectively). Regarding reproductive function, women with menarche at age 15 or later had significantly higher risk than those, who started menstruating before age 12 (OR = 3.82, 95% CI 1.66–8.81 for menarche at > 15 years and OR = 2.90, 95% a 1.14–7.39 for menarche at 16y). Conclusions. Our study suggests several risk factors, including smoking, family history, and reproductive history that may increase the risk of SLE.

From the beginning of cardiac myofibrillogenesis in the chick embryo, developing myofibrils at the bottom of the inner myocardial cell layer facing the cardiac jelly are already aligned circumferentially in the direction of the heart tube. To elucidate the mechanism of this alignment, we investigated the temporal and spatial expression of fibronectin and its relationship to actin filaments before and during looping (4- to 13-somite stages) by using a confocal scanning laser microscope. Serial optical tomograms were obtained from whole-mounted heart tubes stained with fluorescein-conjugated antibody against cellular fibronectin and rhodamine-conjugated phalloidin. Before looping (4- to 7-somite stages), particulate and speckled fibronectin formed loose networks. At the onset of looping (8- to 9-somite stages), fine fibrils of fibronectin appeared. They became dense and were arranged circumferentially in the direction of the heart tube. They were aligned parallel with the thick actin bundles that appeared as an initial stage of developing myofibrils. During looping, (10- to 13-somite stages), fibronectin fibrils were fragmented and showed a speckled pattern, while the number of circumferentially aligned mature striated myofibrils increased. These observations suggest that the temporal arrangement of fibronectin fibrils at the beginning of looping plays a role in the circumferential alignment of developing myofibrils.

The rev protein (Rev) of the human immunodeficiency virus type 1 (HIV-1) is known as a post-transcriptional regulator of viral gene expression. It is located in the cell nucleolus. Transiently expressed Rev caused nucleolar ballooning and deformity with aberrant accumulation of rRNAs, and de novo synthesis of rRNAs decreased dramatically in these cells. However, similarly expressed rex protein (Rex) of the human T-cell leukemia virus type I, which is a functional homologue to Rev, did not affect nucleolar structure and function. Rev expression resulted in cell death with nucleolar destruction in an inducible cell line. Analysis of Rev routants revealed that both the nucleolar targeting signal of Rev and the multimerization domain are prerequisites to the nucleolar disintegration by Rev. Human T-cells acutely infected with HIV-1 contained nucleoli which were deformed and filled with Rev, but chronically infected cells had intact nucleoli. Involvement of Rev in cytopathic effects in HIV-1 infection is discussed.

Background. Stomach cancers show various growth patterns. It remains to be clarified how this variability is related to the genetic changes that occur during tumor progression. Methods. To estimate the genetic changes from tumor ploidy, maps were made (using DNA cytofluorometry of metaphase cells in histologic sections) of 39 advanced signet ring cell carcinomas of the human stomach and correlated with tumor stage and the size of the primary mucosal lesion. Results. Aneuploid area and multipattern aneuploidy were particularly common in advanced cancers with primary mucosal lesions smaller than 2 cm in diameter, of which a large portion were predominantly aneuploid and already diffusely infiltrating. As the tumor stage advanced, the incidence of aneuploidy in the mucosal lesion increased, whereas predominantly aneuploid tumors were less common as primary mucosal lesions became larger. Purely diploid areas with an incidence of polyploidy as low as in early cancers were common in advanced cancers. In addition, there were diploid-appearing cancer cells that infiltrated diffusely and were accompanied by polyploid as often as aneuploid cells. Some of these were aneuploid at the chromosomal level. Conclusions. In signet ring cell carcinoma, aneuploid cells show higher invasive activity toward the extramucosal part and may occur incidentally in originally diploid tumors, depending on the degree of genetic instability. An analysis of polyploidy is useful for differentiating cytometrically diploid (but actually, aneuploid) cells from diploid cells with minor genetic abnormalities. Cancer 1993; 71:1938-47.

It is not yet understood whether cell adhesion molecules play an active role in early cardiac morphogenesis or not. We present here the spatial and temporal expressions of N-cadherin and its relationships to actin filaments during looping (7- to 13-somite stages) of the chick embryonic heart tube observed by means of a confocal laser scanning microscope. Serial optical tomograms were obtained from the whole-mounted heart tubes stained with antibody to N-cadherin (fluorescein-conjugated) and phalloidin (rhodamine-conjugated). Three patterns of N-cadherin expression were observed during looping; a belt-like pattern, speckled pattern, and clumped pattern, corresponding to adhesion belt, nonjunctional cell contact and early intercalated disks, respectively. At the 7-somite stage, myocytes expressed N-cadherin as adhesion belt and nonjunctional cell contact. At the 8- to 10-somite stages, the clumped pattern of N-cadherin was detected before striated myofibrils appeared. Myofibrils began to develop across the clumps to form transcellular networks in the outer layer, and to form circumferential alignments in the inner layer. These results suggest that N-cadherin is responsible for the connection of myofibrils between the neighboring myocytes, and the alignment of the two layers in the developing heart tube.

Dermatological tests and examinations of the hand(s) were carried out in vibration-exposed and unexposed males. The subjects were 179 chain-saw workers in private forestry companies and 205 local inhabitants who had never used vibrating tools. The prevalences of Raynaud's phenomenon (RP), sclerodactylia, and edema of the hands were estimated in both groups, and associations between these cutaneous signs and vibration exposure were evaluated. The prevalences of RP and edema in the exposed group were 9.5% and 1.7%, respectively, and in the unexposed group, 2.9% and 1.5%, respectively. Sclerodactylia was seen in 31.8% of the chain-saw workers but in only 6.4% of the unexposed individuals. In statistical analyses based on unconditional logistic regression models with adjustment for age, RP was associated with long-term (or = 20 years) vibration exposure [odds ratio (OR) = 7.06; 95% confidence interval (CI) = 2.51-19.87]. Sclerodactylia was associated with both short- and long-term vibration exposure (OR = 6.54, CI = 3.30-13.36; OR = 7.05; CI = 3.41-14.60, respectively). There were significant dose-response relationships between RP and duration of exposure and between sclerodactylia and duration of exposure. Results of function tests indicated a longer recovery time and a higher vibration threshold for the workers with RP. The presence of sclerodactylia, however, did not have any significant influence on function test results. It is possible to conclude that not only RP but also sclerodactylia could be induced by vibration exposure. However, most cases of sclerodactylia were not so serious as to involve disturbances of peripheral circulatory and nerve function.

Using a confocal laser scanning microscope (CLSM), we observed subcellular three-dimensional (3-D) arrangements of actin filaments stained with fluorescein-labeled phalloidin during myofibrinogenesis of chick embryonic heart (7- to 13-somite stages). Serial optical tomograms were obtained from whole-mounted heart tubes and reconstructed into stereoscopic images. Development of myofibrils in myocardial differentiation considerably differed in inner and outer myocardial cell layers. In the outer layer, initial myofibrils appeared along cell membranes at the 8-somite stage. They increased rapidly and constituted network structures with spatial extension over cell-cell junctions. In the inner layer, myofibrils appeared at the bottom, facing the cardiac jelly, at the 10-somite stage, and, when the straight heart tube began to bend, they were already aligned circumferentially in the direction of the heart tube. Double staining of fluorescein-phalloidin and DiI [1,1'-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate; DiI-C18-(3)] of the looped heart revealed that while myocytes in the outer layer were round, those of the inner layer were spindle-shaped, and their long axes coincided with the circumferential direction. These results suggest that the circumferentially arranged myofibrils at the bottom of the inner layer may play an important role in the looping of the heart tube.

To investigate the biological load of aerobic dance instruction on the body, a blood biological study was performed on 13 female instructors. The values were compared with 10 female healthy controls. The results obtained were as follows. 1. A significant negative correlation was observed between the total experience of aerobic dancing (in years) and the levels of serum phospholipids. In addition, the numbers of white blood cells and the levels of serum phospholipids, lipoperoxide, uric acid and creatinine were significantly related to aerobic dance instruction time (years). 2. The levels of serum CPK, aldolase, GOT, BUN and creatinine in the aerobic dance instructors were significantly higher than those in the controls. 3. The levels of serum CPK, uric acid, BUN, creatinine and Ca increased significantly after an intermediate class lesson (about 60 minutes). In addition, the numbers of white blood cells also increased after the lesson, but not significantly. Moreover, Significant decreases of the mean corpuscular volume and serum Mg were observed after the lesson.

A case of right frontal pleomorphic xanthoastrocytoma that occurred in a 7-year-old boy is reported clinicopathologically. The patient underwent surgery on September 29, 1988. Histologic diagnosis of pleomorphic xanthoastrocytoma was made because, in addition to the unique pleomorphic histologic features, positive glial fibrillary acidic protein in immunohistochemical staining and characteristic ultrastructural features, i.e., cytoplasmic intermediate fibrils and lipid vacuoles, basal lamina, and abundant reticulin networks were demonstrated. The DNA cytofluorometric analysis of the nuclei of the tumor cells disclosed the main mode to be diploid with polyploid classes (4, 8, 16, and 32C) without any aneuploidy. Despite the presence of many pleomorphic nuclei, DNA histogram of the tumor disclosed very few DNA synthetic cells indicating a biologically inactive nature of the tumor. The patient is still alive and totally asymptomatic 20 months postoperatively.