Isolation and characterization of rabbit properdin of the alternative complement pathway

Rabbit factor P, which is involved in stabilizing the labile C3 convertase of the alternative complement pathway, was isolated from rabbit serum by a simple two-step procedure: polyethylene glycol precipitation and QAE-Sephadex A-50 column chromatography. The chemical properties, and functional activities of the rabbit P were very similar to those of human P, except that there were slight differences in the SDS-PAGE patterns of limited tryptic digests and in the amino terminal sequences. The molecular weights were estimated to be 58 000 for rabbit P and 59 000 for human P on SDS-PAGE under reducing conditions. The amino terminal 36 residues of rabbit P showed 78% homology to the equivalent region of human P.     

On the evaluation of muscle fiber conduction velocity considering waveform properties of an electromyogram in M. biceps brachii during voluntary isometric contraction

The surface myoelectric signal during 20% maximum voluntary contraction was measured in m. biceps brachii using array electrodes for ten subjects in order to evaluate the distribution of muscle fiber conduction velocity (MFCV) in a whole muscle. MFCV was estimated by two calculating methods of the peak maximum method and the cross-correlation method from the myoelectric signals which were processed by techniques of the averaging and the non-averaging. It was found that the values of MFCV depended on the location irrespective of the kind of calculating method used and the kind of processing technique of myoelectric signal. In both the motor end-plate zone and the tendon zone, the values of MFCV showed more than 7.0 m/s. In the regions other than the motor end-plate zone and the tendon zone, the values of MFCV showed about 3.90 m/s which were almost constant. The statistical differences of the values of MFCV were in the same locations measured not recognized between the two calculating methods nor between the two processing techniques. In the cross-correlation method, the relation between MFCV and the electrode location was evaluated by both the maximum correlation coefficient and the amplitude ratio between the different neighboring channels to evaluate the conductive waveform properties of action potentials. The changes of parameters (i.e., MFCV, maximum correlation coefficient and amplitude ratio) depended on the electrode location. The values of MFCV significantly increased in the regions of the motor end-plate zone and the tendon zone, where the maximum correlation coefficient and amplitude ratio significantly decreased. The values of the coefficient of variance (CV) of three parameters in those regions were larger than those in other regions, i.e., the regions other than the motor end-plate zone and the tendon zone. A high maximum correlation coefficient and a high amplitude ratio were necessary for a reliable measurement of the MFCV.     

Sequential change of the hypervariable region of the hepatitis C virus genome in acute infection

Hepatitis C virus (HCV) infection is characterized by persistence of liver inflammation that often leads to end-stage liver disease, although the mechanisms are not fully understood. A hyper-variable region (HVR) has been reported in the E2/NS1 region of the HCV genome, in which striking diversity is found among different HCV isolates. To investigate the association of the HVR alterations with the clinical courses of HCV infection, a longitudinal analysis of the HVR in patients with acute HCV infection was carried out. Plasma samples were obtained at several times in three patients with acute hepatitis C. Plasma RNA was extracted and reverse transcribed, and DNA fragments that included the HVR were amplified by PCR. The sequences of the HVR were directly determined from the PCR products by the dideoxy chain termination method, from which amino acid sequences were deduced. In all cases, plasma HCV-RNA disappeared with the improvement of the initial alanine aminotransferase (ALT) elevation, but HCV-RNA reappeared about 1 year later with or without deterioration of the hepatitis. In a case of sporadic acute hepatitis, the HCV in the recurrent phase had seven amino acid substitutions in the HVR compared with that in the acute phase, although no amino acid changes were noted during the initial acute phase. In a case of posttransfusion hepatitis, a marked difference was observed between the acute and the recurrent phases, with an amino acid homology of 30% (8/27). The mutation rate of the HVR had a tendency to accelerate as the HCV infection progressed to the chronic stage. In conclusion, the HVR changes serially during the course of acute HCV infection, and these HVR changes may play a part in the chronicity of HCV infection. © 1994 Wiiey-Liss, Inc.     

Identification of neural genes using Xenopus DNA microarrays.

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant-negative receptor. RNAs were isolated from control animal cap explants and from dominant-negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high-density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole-mount in situ hybridization analysis. Thirty-two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning.     

Allergenicity of the osmophilic fungus Aspergillus restrictus evaluated by skin prick test and radioallergosorbent test

Recently large amounts of Aspergillus restrictus, a species of osmophilic fungi, have been detected in house dust using culture media with low water activity. But little attention has been paid to this fungus as an allergen. In the present study, the authors examined the allergenic activity of A. restrictus by skin prick tests and radioallergosorbent tests (RAST) on 94 asthmatic patients (mean age 12.0, range 3-18). Aspergillus fumigatus, Alternaria alternata and house dust were used for comparison. In the skin prick tests, A. restrictus, A. fumigatus, A. alternata and house dust elicited positive reactions in 8 (8.5%), 8 (8.5%), 15 (16.0%) and 69 (73.4%) patients, respectively. RAST showed positive reactions in 27 (28.7%) subjects for A. restrictus, 22 (23.4%) for A. fumigatus, 35 (37.2%) for A. alternata, and 75 (79.8%) for house dust. These results indicated that some asthmatic individuals showed immediate-type hypersensitivity to A. restrictus, and the prevalence of hypersensitivity of A. restrictus determined by skin prick tests and RAST was comparable with that of A. fumigatus but lower than that of A. alternata or house dust. This indicates that this fungal species may be of importance as a causative agent in atopic diseases.     

Loss of cell-cell contact is induced by integrin-mediated cell-substratum adhesion in highly-motile and highly-metastatic hepatocellular carcinoma cells

The cadherin-mediated cell-cell adhesion system plays a critical role in normal development and morphogenesis. Inactivation of this system is thought to be responsible for cancer invasion and metastasis. A human hepatocellular carcinoma (HCC) cell line, KYN-2, was observed to have great potential for intrahepatic metastasis when orthotopically implanted into the liver of SCID mice. In vitro cultures of KYN-2 cells showed that they formed trabecular structures in suspension but lost tight cell-cell adhesion and became scattered when attached to a substratum such as collagen or fibronectin. In response to adhesion to the substratum, subcellular colocalization of E-cadherin and actin filaments were shown to be reduced, and a significant amount of alpha-catenin was dissociated from the E-cadherin-catenin complex in KYN-2 cells. These changes of cell-cell adhesion were blocked by inhibitory monoclonal antibodies against beta1 and beta5 integrins. We found that c-Src was coimmunoprecipitated with E-cadherin-catenin complex and was tyrosine-dephosphorylated and activated in the adherent cells. The tyrosine dephosphorylation of c-Src was induced by cell adhesion to the substratum and inhibited by addition of inhibitory monoclonal antibodies against beta1 and beta5 integrins. These findings indicate that integrin-mediated cell-substratum adhesion inhibits cadherin-mediated cell-cell adhesion, possibly through c-Src activation, and suggest that this cross-talk mediates transient inactivation of the cadherin system and plays an important role in intrahepatic metastasis of human HCC. Modulation of this interaction might provide a new approach to prevent metastasis and recurrence of HCC.     

A promiscuous T cell hybridoma restricted to various I-A molecules

In a previous study, we identified T cell receptor and major histocompatibility complex (MHC) contact sites on the pigeon cytochrome c p43-58 peptide. Positions 46 and 54 of p43-58 were shown to be the MHC-binding sites. Specific amino acids were identified on the MHC-binding sites which bound to the relevant I-A molecule. In the present study, using NOD (I-A^g7) mice, we established a T cell hybridoma, NOE33-1-2, specific for a p43-58 analog 46R50E54A with arginine (R) and alanine (A) at positions 46 and 54, respectively. Interestingly, NOE 33-1-2 recognized 46R50E54A in the presence of not only I-A^g7, but also I-A^{d, s, u and v}. In contrast to previous reports that promiscuous T cells were able to recognize peptide antigens with various HLA-DR or I-E molecules consist of monomorphic α and polymorphic β chains, the promiscuous T cell clone NOE33-1-2 recognized peptides with various I-A molecules lacking the monomorphic chain.     

The morphologic feature of mucus leakage appearing in low papillary carcinoma of the breast.

This report summarizes the clinicopathologic findings in 11 cases of low papillary carcinoma of the breast accompanied by the morphologic feature of mucus leakage into the mammary stroma. These cases were characterized by two morphologic findings. First, abundant mucus produced by the tumor cells filled the intraductal spaces where neoplastic epithelium formed very low papillary projections, ie, a feature of mucinous-producing low papillary carcinoma in situ. Second, there was expansive leakage of mucus into the mammary stroma occasionally accompanied by a few epithelial cells. All the cases showed a high level of mucus production and contained no elements of invasive ductal carcinoma or ordinary invasive mucinous carcinoma. These cases have no evidence of direct invasion of the mammary stroma by malignant cells. The average age of the 11 patients was 41 years. Foci of microcalcification were seen in some tumors (seven cases; 64%). There were no cases with lymph node metastases. All the patients underwent mastectomy with no adjuvant therapy, and they are currently alive and well.     

Complete Nucleotide Sequence and Phylogenetic Analyses of Hepatitis B Virus Isolated from Two Pileated Gibbons

Virus-like particles (VLPs, named HmTV1-17), about 40nm in diameter were found in the violet root rot fungus Helicobasidium mompa Tanaka strain No. 17, which had been isolated from an apple tree. Purified preparations of HmTV1-17 contained two species of double-stranded RNA (dsRNA), designated 17L and 17S. cDNAs were constructed from HmTV1-17 genomic dsRNAs purified using CF-11 cellulose column chromatography. The sequences of 17L and 17S cDNA comprised 5207 and 2096bp, respectively. Although 17S has no large open reading flame (ORF) on either strand, 17L has two large overlapping ORFs. The 5 located ORF1 encodes the coat protein (CP, 788 amino acids), whereas the gene product of ORF2, which is in the –1 frame relative to ORF1, shows the typical features of a RNA dependent RNA polymerase (RDRP, 845 amino acids). Phylogenetic analysis based on RDRP showed that HmTV1-17 is closely related to Sphaeropsis sapinea SsRV1, a member of the genus Totivirus from filamentous fungus S. sapinea.     

Detection of genes expressed in primary colon cancers by in situ hybridisation: overexpression of RACK 1.

AIMS: The isolation of various genes that are expressed in a region specific manner is considered useful for research in molecular pathology. In situ hybridisation (ISH) was used in a screening procedure to isolate these genes efficiently, using colon cancer as a model. METHODS: Suppression subtractive hybridisation (SSH) between colon cancer tissue samples and corresponding non-cancerous tissues was performed. Genes showing high expression in the cancers were selected using macro-DNA array analysis. As a final screening procedure, conventional ISH was performed to isolate genes expressed specifically in colon cancers. RESULTS: Sixty nine clones were selected by SSH and macro-DNA array analyses. These clones were then analysed by ISH to examine their expression patterns. ISH screening revealed that all the clones screened showed more intense signals in colon cancers than in non-cancerous tissues. Among them, RACK 1, which is a protein kinase C receptor and a homologue of the G protein beta subunit, was expressed intensely in colon cancer cells. RACK 1 expression was evaluated in multiple samples by ISH, and the results confirmed that RACK 1 was universally overexpressed in cells of all 11 colon cancers examined. CONCLUSIONS: Many genes, including RACK 1, expressed in colon cancer cells can be isolated efficiently by this method, and their precise expression pattern can be evaluated. These results indicate that ISH is an excellent technique for systemic screening of genes expressed in a region specific manner.