Takafumi Noma

Construction of chimaeric processed immunoglobulin genes containing mouse variable and human constant region sequences

The specificity of monoclonal antibodies provides a powerful diagnostic and therapeutic tool in investigating human neoplasia. Radiological scanning and immunotherapy with mouse tumour-specific monoclonal antibodies have been applied to patients with some success1–3, but a major problem is the neutralization of the mouse antibody induced by repeated administration of heterologous antibodies. To avoid or reduce such immune reactions, chimaeric immunoglobulins consisting of mouse variable (V) and human constant (C) regions can be synthesized. We have constructed a recombinant retrovirus DNA carrying genomic heavy-chain (H) variable–diversity joining (VH−D−JH) and Cγ1 genes from different species and show here that the chimaeric intervening sequences are spliced out precisely. This procedure provides a useful method to construct the chimaeric mouse–human immunoglobulin gene to be expressed in Escherichia coli, yeast and animal cells. Unexpectedly, a hidden splice donor site in the 5′-flanking region of a human VH gene is used in place of the donor site of the leader sequence exon, resulting in the formation of the V region without the leader sequence.

Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.

Enhancement of the interleukin 2 receptor expression on T cells by multiple B-lymphotropic lymphokines

Three new human lymphokines, interleukin-5, BSF-2 and BSF-MP6, were shown to be active in the enhancement of the IL-2 receptor expression on T cells, although they do not stimulate growth of the T cells.

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis‐inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix‐associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.

Genomic structure of HTLV (human T-cell leukemia virus): detection of defective genome and its amplification in MT-2 cells.

We studied the genomic structure of human T‐cell leukemia virus (HTLV) in the HTLV producer cell line MT‐2. Southern blotting revealed that at least eight HTLV proviruses were integrated in the chromosomes of MT‐2 cells. The genomic structure of these proviruses was analyzed using fragments of cloned HTLV that were specific to gag, pol, env, pXs and U3R genes as probes. We have identified a complete genome of HTLV in MT‐2 (non‐defective type). However, seven of the eight proviruses had defective genomes. Provirus T2‐a contains only the U3R (LTR) of HTLV and T2‐b corresponds to the non‐defective genome. T2‐c possesses only a portion of env, and pXs and U3R. T2‐d consists of gag, pol, part of env and U3R. On the other hand, T2‐e, f, g and h consist of gag, pXs and U3R. Northern blotting experiments with mRNA from MT‐2 cells supported the evidence of amplification of the gag‐pXs gene of HTLV. 26S mRNA is considered to be a subgenomic species of 35S RNA. 32S mRNA may represent the T2‐d provirus which lacks a portion of env and pXs, while 20S mRNA was a subgenomic species. The gag‐pXs gene may correspond to 24S mRNA, the amount which was amplified in MT‐2 cells.

Enhanced Expression of Matrix Metalloproteinase-9 in Abdominal Aortic Aneurysms

Abstract. Abdominal aortic aneurysms (AAAs) are characterized by structural alterations of the aortic wall resulting from degradation of collagen and elastin. Matrix metalloproteinases (MMPs), particularly MMP-2 and MMP-9, show strong elastinolytic activity. We examined the levels of mRNA for MMP-2, MMP-9, membrane type (MT)-MMP-1, tissue inhibitor of metalloproteinase-1 (TIMP-1), and TIMP-2 in AAAs (n= 8), atherosclerotic occlusive diseases (AOD) (n= 8), and normal subjects (n= 8) using the reverse transcription-polymerase chain reaction (RT-PCR). We also analyzed the gelatinolytic activity of these metalloproteinases using gelatin zymography. The levels of MMP-2 and MMP-9 mRNA were increased in the AAA group compared with those in the AOD group and normal subjects. The levels for TIMP-1 and TIMP-2 mRNA in the AAA group were also higher than those in the AOD and normal groups. Only in the case of MT-MMP-1 was the difference between AAA and AOD not statistically significant. By gelatin zymography with the same samples used for RT-PCR, gelatinolytic activity of MMP-9 was elevated in all AAA tissues. The 62-kDa form of MMP-2 was elevated in both the AAA and AOD groups and did not differ significantly between them. Linear regression analysis demonstrated a significant positive correlation between mRNA levels of MMPs and those of TIMPs. These observations suggest that aneurysm formation in patients with atherosclerosis is related to the degree of MMP-9 expression.

Generation of human induced pluripotent stem cells from oral mucosa.

Induced pluripotent stem (iPS) cells are one of the most promising sources for cell therapy in regenerative medicine. Using a patients own genetically identical and histocompatible cells is the ideal way to practice personalized regenerative medicine. For personalized iPS cell therapy, the prerequisites for cell source preparation are a simple and safe procedure, no aesthetic or functional damage, and quick wound healing. Oral mucosa fibroblasts (OFs) may have high potential to fulfill these requirements. In this study, biopsy was performed in a dental chair; no significant incisional damage was recognized and rapid wound healing (within a week) was observed. We generated human iPS cells from the isolated OFs via the retroviral gene transfer of OCT4, SOX2, c-MYC, and KLF4. Reprogrammed cells showed ES-like morphology and expressed undifferentiated markers such as OCT4, NANOG, SSEA4, TRA-1-60, and TRA-1-81. Subsequent in vitro and in vivo analyses confirmed the pluripotency of resultant iPS cells, which matched the criteria for iPS cells. In addition, we found that the endogenous expression levels of c-MYC and KLF4 in OFs were similar to those in dermal fibroblasts. Taken together, we propose that OFs could be a practical source for preparing iPS cells to achieve personalized regenerative medicine in the near future.

Structure and expression of human mitochondrial adenylate kinase targeted to the mitochondrial matrix

The previously isolated cDNA encoding human adenylate kinase (AK) isozyme 3 was recently renamed AK4. Consequently, human AK3 cDNA remains to be identified and we have little information about the functional relationship between human AK3 and AK4. In pursuit of the physiological roles of both the AK3 and AK4 proteins, we first isolated an authentic human AK3 cDNA and compared their expression. Nucleotide sequencing revealed that the cDNA encoded a 227-amino-acid protein, with a deduced molecular mass of 25.6 kDa, that shares greater homology with the AK3 cDNAs isolated from bovine and rat than that from human. We named the isolated cDNA AK3. Northern-blot analysis revealed that AK3 mRNA was present in all tissues examined, and was highly expressed in heart, skeletal muscle and liver, moderately expressed in pancreas and kidney, and weakly expressed in placenta, brain and lung. On the other hand, we found that human AK4 mRNA was highly expressed in kidney, moderately expressed in heart and liver and weakly expressed in brain. Western-blot analysis demonstrated expression profiles of AK3 and AK4 that were similar to their mRNA expression patterns in each tissue. Over expression of AK3, but not AK4, in both Escherichia coli CV2, a temperature-sensitive AK mutant, and a human embryonic kidney-derived cell line, HEK-293, not only produced significant GTP:AMP phosphotransferase (AK3) activity, but also complemented the CV2 cells at 42 degrees C. Subcellular and submitochondrial fractionation analysis demonstrated that both AK3 and AK4 are localized in the mitochondrial matrix.

Transcriptional Control of Expression of the TGF‐βs

In recent years, there has been an exponential increase in understanding of the chemistry and biology of the family of peptides called transforming growth factor-S (TGFp). The discovery and characterization of five distinct, highly conserved, yet functionally similar TGF-Ps has added an unexpected level of complexity to the problems of defining the roles of the different TGF-Ps in normal and pathological physiology! Furthermore, understanding of the biology of the TGF-ps, once narrowly defined by their ability to confer a transformed phenotype on nonneoplastic NRK fibroblasts, has now expanded to include effects on almost every cell type and to range from processes such as control of steroidogenesis or control of epithelial cell growth and differentiation, to more complex processes involving multiple cell types such as wound healing, bone remodeling, hemopoiesis, or specific morphogenetic and histogenetic events in embryonic development ! Given the many cell types sensitive to TGF-p action, it is clear that multiple levels of control of its activity must exist. Though control of TGF-P receptor expression, receptor signalling, and activation of the latent forms of the various TGF-Ps are without doubt important, this review will be limited to a discussion of transcriptional regulation of the activity of mF-0 in a variety of in vitro and in vivo systems and of evidence suggesting that expression of the five TGF-ps is under different celland tissue-specific transcriptional control.

The nm23-H1 gene as a predictor of sensitivity to chemotherapeutic agents in oesophageal squamous cell carcinoma

SummaryRecently, nm23-H1, an anti-metastasis gene, has been reported to correlate with sensitivity to chemotherapeutic agents including cisplatin in human breast and ovarian carcinoma cells. The aim of this study was to evaluate a role for nm23-H1 in responsiveness to cisplatin-based chemotherapy in patients with oesophageal squamous cell carcinoma (OSCC). The expression of nm23-H1 protein was examined immunohistochemically in 32 eligible patients with OSCC who underwent adjuvant chemotherapy with cisplatin, etoposide, and 5-fluorouracil after tumour resection. Fifteen (46.9%) of 32 patients were positive for nm23-H1 staining and 17 (53.1%) were negative. Both disease-free survival and overall survival rates of nm23-H1-negative patients were significantly shorter than in nm23-H1-positive patients (P < 0.01 for both). There was no significant difference in clinicopathologic characteristics between nm23-H1-positive and nm23-H1-negative groups. Multivariate analysis also showed that nm23-H1 expression was the most significant factor for overall survival of OSCC patients included in this study (P = 0.0007). To further study the role of nm23-H1, a human OSCC cell line (YES-2) was transfected with a plasmid containing a fragment of the nm23-H1 cDNA in an antisense orientation. Reduced expression of nm23-H1 protein in the antisense-transfected (AS) clones was found by Western blot analysis as compared to wild-type YES-2 and YES-2/Neo (clone transfected with the neomycin resistance gene alone). MTT (3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide) assay showed that reduced expression of the nm23-H1 protein in AS clones was consistent with the degree of increased resistance to cisplatin but not etoposide or 5-fluorouracil. These data support the conclusion that reduced expression of nm23-H1 may be associated with resistance to cisplatin, suggesting the value of nm23-H1 expression as a prognostic marker for OSCC patients who are to undergo cisplatin-based chemotherapy.