Pulmonary blastoma. Comparison between its epithelial components and fetal bronchial epithelium.

Three cases of pulmonary blastoma exhibiting biphasic epithelial and stromal patterns, and a case of fetal lung-type adenocarcinoma, were examined by immunohistochemistry and electron microscopy (EM) and compared with fetal bronchial epithelium in order to explore the multidirectional differentiation of their epithelial components. The glandular cells of all four tumors resembled fetal bronchial epithelial cells in the pseudoglandular stage. Neuroendocrine (NE) cells were also present; they were argyrophilic and expressed pan-NE markers, neurosecretory granules and peptide hormones. The neural cell adhesion molecule (NCAM) was strongly expressed on the cell membranes of glandular cells, as in the case of proximal bronchial epithelial cells at the pseudoglandular stage in fetal lung. Sialosylated Lewis X was also expressed, indicating that the epithelial cells were possibly of endodermal origin. Two of the four cases showed considerable immunoreactivity for alpha-fetoprotein (AFP). The epithelial cells of pulmonary blastomas may occasionally de-differentiate into cells functionally resembling fetal hepatic, foregut and yolk sac cells expressing AFP. Tumor examination by immunohistochemistry and EM suggested that the glandular cells of the tumors may differentiate to some extent like those of fetal large bronchi at the pseudoglandular stage, but there was concordance and discordance in the expression of neuroendocrine and oncofetal markers between blastomatous tumors and fetal bronchial epithelium.     

Modulation of a motor evoked response to transcranial magnetic stimulation by the activity level of the first dorsal interosseous muscle in humans when grasping a stationary object with different grip widths

A relation between corticospinal excitability and background voluntary muscular activity was investigated at low and high transcranial magnetic stimulation (TMS) intensities while grasping a stationary object with different widths (2 and 8 cm) using a precision grip. The muscle activity was recorded from the first dorsal interosseous muscle. Regression analysis revealed that there was a linear relation between the motor evoked potential (MEP) and background muscular activity for both grip widths at each of the TMS intensities. At the low TMS intensity, the slope of the regression lines was similar for the 2 and 8 cm grip widths. It was, however, different when the TMS intensity was high. The results suggested that sensitivity modulation of bias level (input) facilitation occurred with wider grip width. The results of this study would reflect to quantitative aspects of the relation between the synaptic drive to the motoneuron pool and the resulting efferent activity characterized, i.e. input-output relations of the human corticospinal pathway dependent on the occasion demand.     

Enhancement of lipopolysaccharide-stimulated cyclooxygenase-2 mRNA expression and prostaglandin E2 production in gingival fibroblasts from individuals with Down syndrome

It is well known that Down syndrome (DS) is a premature ageing syndrome. Periodontal disease in individuals with DS develops rapidly and extensively in a relatively younger age bracket compared with that in healthy controls. The mechanisms involved in the periodontal inflammatory processes in DS patients are not fully understood. In the present study, the non-inflamed gingival fibroblasts isolated from seven patients with DS (DGF) and seven healthy controls (NDGF) were stimulated with lipopolysaccharide (LPS) derived from Actinobacillus actinomycetemcomitans (A. a.). We measured the level of prostaglandin E2 (PGE2) production by DGF and NDGF by radioimmunoassay, and also measured the mRNA expression of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) by using the real-time PCR method. We found the higher levels of LPS-stimulated COX-2 mRNA expression and PGE2 production in DGF when compared with those in NDGF. This study may indicate that overexpression of LPS-stimulated COX-2 induced a greater ability of DGF to produce PGE2, and that these phenomena may be responsible for the severer periodontal disease in DS patients.     

Development of mitochondrial helical sheath in the middle piece of the mouse spermatid tail: Regular dispositions and synchronized changes

Morphologic changes in the development of the mitochondrial helical sheath in the mouse spermatid tail were examined with the scanning electron microscope (SEM) using the osmium-DMSO-osmium method and classified into several stages. During late spermiogenesis, spherical mitochondria gathered around the forming spermatid tail. The shape of these mitochondria gradually changed from spheroid to long and rod-like. Mitochondria first were arranged in four longitudinal rows (stage 1) that twisted dextrally, and the mitochondria began to stagger (stage 2). They became elongated and arranged into a staggered pattern; they then attached to each other in an end-to-end fashion to form a sinistral double helix around the core of the axoneme (stage 3). These end-to-end contacts were observed in every second gyre on the four lines surrounding the core of the axoneme at stage 3. Mitochondria further elongated and end-on touching appeared with every third gyre on the five longitudinal lines that surround the core of the axoneme (stage 4). The direction of the helix, always sinistral, was clearly discernible only in the later stages. Disposition of the mitochondria in the spermatid tail was regular throughout development, which indicates that these mitochondria elongate simultaneously and also at the same rate. On any given cracked surface of the seminiferous tubule, spermatid tails with the same stage of mitochondria predominantly were observed. This ultrastructural finding appears compatible with the histologic synchronism, (termed the “wave”) in differentiating germ cells.     

Analysis of 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) as a reliable marker of cellular oxidative DNA damage after gamma-irradiation

In order to improve 8-hydroxyguanine (8-OH-Gua) detection in DNA, we digested isolated DNA with nuclease P1 and analyzed for 8-hydroxydeoxyguanosine 5'-monophosphate (8-OH-dGMP) using a high-performance liquid chromatography system equipped with an electrochemical detector (HPLC-ECD). The amount of 8-OH-Gua in the DNA was expressed as the ratio of 8-OH-dGMP to deoxycytidine monophosphate (dCMP). Using this analysis, the background level of 8-OH-Gua in DNA from human lung carcinoma cells (A549) was several-fold lower than that obtained by a previous method. A549 cells were exposed to 20-60 Gy of gamma-radiation and an increase in 8-OH-Gua concentration was observed with increasing gamma-ray dose (0.3 residues per 10(7) dCMP per Gy). Moreover, by an immunohistochemical procedure using a commercial FITC-kit, 8-OH-Gua was clearly detected in A549 cells and the fluorescence intensity of cells with oxidative DNA damage increased with the doses of gamma-irradiation. Using an endonuclease nicking assay, we also found that gamma-rays decreased 8-OH-Gua repair activity. The results indicate that 8-OH-dGMP is a useful and sensitive marker for estimating oxidative damage in DNA.     

Further evidence for excitability changes in human primary motor cortex during ipsilateral voluntary contractions

The present study aimed to further investigate whether the intracortical neural circuits within the primary motor cortex (M1) are modulated during ipsilateral voluntary finger movements. Single- and paired-pulse (interstimulus intervals, ISIs; 3 ms and 12 ms) transcranial magnetic stimulations of the left M1 were applied to elicit motor evoked potential (MEP) in the right first dorsal interosseous (Rt-FDI) muscle during voluntary contractions (10% and 30% maximum voluntary contraction) of the left FDI (Lt-FDI) muscle. F-waves of Rt-FDI muscle were recorded under these left index-finger conditions for ensuring that the excitability changes occur at the supraspinal level. MEPs were also recorded during motor imagery of the left index-finger abduction instead of overt movement. The results showed that, in single-pulse transcranial magnetic stimulation (TMS) paradigm, MEPs in Rt-FDI muscle were markedly enhanced during voluntary contractions of Lt-FDI muscle compared with the complete resting state. In paired-pulse TMS paradigm, the short intracortical inhibition was significantly reduced in proportion to increments of the ipsilateral muscle contraction, whereas the intracortical facilitation had no change. F-wave of Rt-FDI muscle was unchanged under these conditions, while MEP in Rt-FDI muscle was also enhanced during motor imagery of the left index-finger abduction. Based on the present results, it is suggested that the intracortical inhibitory neural circuits may be modulated in the transition from rest to activity of the ipsilateral homonymous muscle. The excitability changes in M1 might be induced by overflows of voluntary drive given to the ipsilateral limb, probably via the transcallosal pathway.     

In vivo administration of cytokine in allogeneic bone marrow chimeras.

When we analyzed the in vivo efficacy of cytokine administration in murine allogeneic bone marrow chimeras, mitogen-induced responses to ConA, PHA, LPS, or PWM were increased by the in vivo administration of human recombinant granulocyte colony-stimulating factor (rG-CSF), human recombinant interleukin 2 (rIL-2), or WEHI-3B conditioned medium (CM). Furthermore, we found increased alloreactive mixed lymphocyte reactions (MLRs) against donor and/or host type alloantigens in spleen cells from (BALB/c----C3H/He) chimeras, although cytotoxic activity against BALB/c or C3H/He target cells was not detected in spleen cells from these chimeras. Since no significant increase of T cells or Ia positive cells was observed, some functional activation, rather than changes in the cell count, appeared to relate to increase immunoreactivity. An increased IL-2 production in spleen cells from chimeras injected with cytokine was observed shortly after the cessation of cytokine administration. Thereafter, an IL-2 production in these chimeras decreased around 45 days after bone marrow transplantation and then recovered nearly to the control level. An increased IL-2 responsiveness was also observed in spleen cells from these chimeras. These findings suggest that the in vivo administration of rG-CSF as well as rIL-2 or WEHI-3B CM (IL-3) can modulate the immunoreactivity in chimeras via the network of immune systems.     

pH-dependent fusion of synaptosomal membrane studied by fluorescence quenching method

We have found that both the synaptic vesicles (SV) and synaptic plasma membrane vesicles (SPM) have an activity to fuse with phosphatidylcoline/phosphatidylserine liposomes in a pH-dependent manner. The activity increases with decreases in extravesicular pH. At a pH lower than 4.0, the activity is almost steady at its maximum value, and there was a rapid drop around pH 5.5. The pH-dependent fusion was inhibited by proteolysis with trypsin; hence, at least in part, some membrane proteins play an important role in these pH-dependent fusion processes. To find specific markers, we screened various protein modifiers and found that anion channel blockers, stilbene derivatives (DIDS and SITS) and glibenclamide, affected the fusion process. DIDS and SITS decreased the fusion activity with an IC50 of 180 and 300 microM, respectively, whereas glibenclamide, on the contrary, increased it. From the results of an autoradiogram using 3H-tagged DIDS, a 30 kDa DIDS-binding protein was identified in the synaptic plasma membrane, which is possible to be responsible for the pH-dependent fusion.