Immunohistochemical Investigation of Vimentin, Neuron-specific Enolase, α1-antichymotrypsin and α1-antitrypsin in Adenoid Cystic Carcinoma of the Salivary Gland

Fifty-four adenoid cystic carcinomas (ACC) arising in major and minor salivary glands as well as in normal salivary glands were studied by immunohistochemistry for the presence of vimentin, neuron specific enolase (NSE), α₁-antichymotrypsin (α₁-ACT) and α₁-_- antitrypsin (α₁-_- AT). Five patterns of histological differentiation were found in ACC, and for the cellular components of each, it was possible to establish a special immunohistochemical profile. In ACC, vimentin-positive cells were observed in the outer tubular, cyst-lining and small angular cells. NSE was positive in the myoepithelial cells of normal salivary gland. Neoplastic cells of ACC showed NSE positivity mainly in the small angular cells and partly in the duct luminal cells. α₁-ACT was localized in the intercalated duct cells and serous acinar cells of normal salivary gland, and in the duct luminal cells of ACC. α₁-AT could not be detected in any of the epithelial cells of normal salivary gland. In ACC, eosinophilic hyaline material in the cribriform spaces was positive for α₁-AT, but no positivity was demonstrated in tumor cells. The present study showed that there are at least two populations of tumor cells in ACC: duct luminal cells that express α₁-ACT, thus indicating their ductal character, and small angular cells that express vimentin, characteristic of non-luminal cells. Moreover, our results indicate that α₁-AT is a useful marker of basement membrane-like material.     

Increase in the number of detectable preoptic glutamic acid decarboxylase 67-immunoreactive cells in immature male rats

Gonadotropin-releasing hormone (GnRH) neurons are mainly located in the anterior preoptic area (aPOA) and gamma-aminobutyric acid (GABA) is known as a potent regulator of the GnRH neurons. To examine the development of the GABAergic system in the aPOA, immunocytochemistry of glutamic acid decarboxylase 67 (GAD(67)) was performed in immature (postnatal d16, d25 and d30) and mature (postnatal 10 weeks) male rats. All immunocytochemical procedures were simultaneously performed. In the lateral part of the aPOA, the detectable number of GAD(67)-immunoreactive cells was small in the d16 group, but significantly increased in the d25, d30 and mature groups, up to 2.7, 4.8 and 5.7 times the number in the d16 group, respectively. In the diagonal band of Broca (DBB), the number was also small in the d16 group, and significantly increased in the d25, d30 and mature groups upto 1.8, 2.2 and 2.8 times the number in the d16 group, respectively. However, in the cingulate cortex, no significant developmental change was observed. These results suggest that the development of the GABAergic system in the lateral aPOA and the DBB occurs before sexual maturation of male rats.     

Clinical significance of measurement of resting energy expenditure in childhood

1. Biliary atresia (BA), as a common disease in Japan, and cystic fibrosis (CF), as an extremely uncommon disease in Japan, were selected to assess the clinical significance of measurement of energy expenditure (EE). 2. Energy expenditure was significantly higher in children with BA than in normal children. 3. Measurement of EE in BA lead to clues to resolving its mechanism by novel assessment of interleukin-6 and leptin. 4. Energy expenditure in children with CF is also higher, but this has been addressed by nutritional intervention with additional calories. 5. Individualization of EE measurement is necessary in the analysis of pathological mechanisms and nutritional management of patients with both common and uncommon diseases.     

Effects of estrogen and progesterone on the expression of connexin-36 mRNA in the suprachiasmatic nucleus of female rats

In rat, helium pressures induce locomotor and motor activity which requires dopaminergic and N-methyl-D-aspartate (NMDA) receptor activities at striatal level. However, biochemical studies have suggested that pressure exposure may increase striatal glutamate level. We used microdialysis technique to study the effects of pressure on glutamate level in the striatum and the effects of local administration of D1 (SCH23390) or D2 (sulpiride) on these changes. Pressures increase both glutamate and glutamine levels in striatal microdialysates. Administration of sulpiride (1 microM) or SCH23390 (1 microM) by reverse microdialysis did not affect significantly pressure induced glutamate increase. So, protective effects of D1 and D2 antagonists against locomotor and motor hyperactivity (LMA) are probably independent of the processes involved in the striatal glutamate increase evoked by pressure.     

Quantitative analysis for soluble elastin in circulation and cell culture fluids using monoclonal antibody-based sandwich immunoassay

We have newly established 3 distinct murine monoclonal antibodies (MoAbs) against human soluble elastin by using chemically denatured immunogen isolated from human aorta; they are designated as HASG-2, HASG-30, and HASG-61-1. All of these MoAbs were highly reactive with soluble forms of native elastin in normal human serum. HASG-2 and HASG-61-1 MoAbs can recognize soluble bovine elastin as well as human antigen, but HASG-30 cannot. The sandwich enzyme-linked immunosorbent assay (ELISA) for human soluble elastin was developed with HASG-61-1 labeled with peroxidase and HASG-30 immobilized on the microplates. The circulating levels of soluble elastin in human healthy subjects (mean +/- SD; 42.9 +/- 19.9ng/mL; n = 85) could be measured with full accuracy and reproducibility, and gradually increased with aging. The positive correlation between the levels and ages was statistically significant (r = 0.581, p < 0.0001). In addition, we could also determine the concentration of tropoelastin secreted from cultured human dermal fibroblasts accurately by this ELISA. This simple assay can be utilized for the routine clinical laboratory screening of patients with arteriosclerotic vascular diseases or to accurately determine the concentrations of tropoelastin secreted from cultured human cells.     

Evidence of allosteric conformational changes in the antibody constant region upon antigen binding

We have addressed the question of whether antigen binding induces a conformational change in the heavy chain constant (C(H)) domain of antibodies using staphylococcal protein A or streptococcal protein G as probes, since these proteins are known to bind to IgG domains such as C(H)1 and C(H)2-C(H)3 domains. Biosensor assays on interactions between these proteins and mouse IgG specific to (4-hydroxy-3-nitrophenyl)acetyl (NP) or their enzymatic fragments conducted in the presence or absence of the hapten, NP-epsilon-aminocaproic acid (NP-Cap), showed that the binding of IgG to these proteins was inhibited by the binding of NP-Cap. The results of isothermal titration calorimetry also revealed that the association constant for the interaction of protein A with IgG2b decreased by the addition of NP-Cap. These results suggested that antigen binding induced conformational changes in binding sites for protein G or protein A located at C(H)1 and C(H)2-C(H)3 domains, respectively.     

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F‐positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell‐by‐cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21. © 2001 Wiley‐Liss, Inc.     

The absence of DNA polymerase kappa does not affect somatic hypermutation of the mouse immunoglobulin heavy chain gene

During the immune response to T cell-dependent antigen, somatic hypermutation (SHM) is introduced into immunoglobulin (Ig) genes. The variable region is the target for SHM and it is here that DNA lesions are introduced and mutations are generated. It has been suggested that error-prone DNA polymerase(s) may play an important role in this mutagenesis phase. Recently, DNA polymerase kappa (Polkappa), which belongs to the Y-family of DNA polymerases, was identified. Since a hot spot of SHMs (RGYW motif) is also a hot spot of mutations by human Polkappa, this enzyme was suggested to be an SHM instigator. In order to address the question whether Polkappa is involved in SHM, we immunized Polkappa-deficient mice and analyzed the SHM of the Ig heavy chain gene. We found that the SHM frequency and spectrum were indistinguishable between the Polkappa knockout mice and control mice. These results suggested that Polkappa is not essential for this process.     

Mechanomyography of the human quadriceps muscle during incremental cycle ergometry

The mechanical activity of the human quadriceps muscle during maximal incremental cycle ergometry was investigated by mechanomyography (MMG). MMG and surface electromyography (EMG) recordings of vastus lateralis muscle activity were obtained from nine males. Cycle ergometry was performed at 60?rev/min and work load was incremented step wise by 20?W (3.2?Nm) every minute until volitional fatigue. The mean amplitudes of MMG (mMMG) and EMG (mEMG) during the contraction phase were calculated from the last six contractions in each load. The duration, load and work rate of exercise at exhaustion were 13.3 (1.6)?min, 44.1 (5.5)?Nm, 276.7 (34.7)?W, respectively. A linear relationship between mMMG and load was evident in each subject (r?=?0.868–0.995), while mEMG seemed to dissociate as the load became greater. In the grouped mean data, mMMG was linearly related to load whether aligned to the absolute (r?=?0.995) or maximal (r?=?0.995) load. Involvement of the noise component was further investigated by studying passive cycling by four subjects. Pedals were rotated passively for the first half of each stage (PAS) and the subject then pushed the pedals for the second half (ACT). In the lighter load region, the mMMG of ACT was as small as that of PAS. However, the change in the mMMG of PAS was very small compared with that of ACT. In conclusion, this study demonstrates a linear relationship between the mMMG of the quadriceps muscle and work load during maximal incremental cycle ergometry. The effect of movement noise was thought to be small and stable.