Bioinspired Total Synthesis of the Dimeric Indole Alkaloid (+)-Haplophytine by Direct Coupling and Late-Stage Oxidative Rearrangement.

A bioinspired convergent total synthesis of (+)-haplophytine, a dimeric indole alkaloid with diazabicyclo[3.3.1]nonane and hexacyclic aspidosperma segments, is described. This synthesis involves the direct coupling of the two segments in a AgNTf2 -mediated Friedel-Crafts reaction and construction of the diazabicyclo[3.3.1]nonane skeleton through late-stage chemoselective aerobic oxidation of the 1,2-diaminoethene moiety and a sequential semipinacol-type rearrangement.

Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment.

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds.

Isolation and characterization of mesenchymal stem cells from human umbilical cord blood: reevaluation of critical factors for successful isolation and high ability to proliferate and differentiate to chondrocytes as compared to mesenchymal stem cells from bone marrow and adipose tissue.

Human umbilical cord blood (CB) is a potential source for mesenchymal stem cells (MSC) capable of forming specific tissues, for example, bone, cartilage, or muscle. However, difficulty isolating MSC from CB (CB-MSC) has impeded their clinical application. Using more than 450 CB units donated to two public CB banks, we found that successful cell recovery fits a hyper-exponential function of time since birth with very high fidelity. Additionally, significant improvement in the isolation of CB-MSC was achieved by selecting cord blood units having a volume ≥90 ml and time ≤2 h after donor's birth. This resulted in 90% success in isolation of CB-MSC by density gradient purification and without a requirement for immunoaffinity methods as previously reported. Using MSC isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC) as reference controls, we observed that CB-MSC exhibited a higher proliferation rate and expanded to the order of the 1 × 10(9) cells required for cell therapies. CB-MSC showed karyotype stability after prolonged expansion. Functionally, CB-MSC could be more readily induced to differentiate into chondrocytes than could BM-MSC and AT-MSC. CB-MSC showed immunosuppressive activity equal to that of BM-MSC and AT-MSC. Collectively, our data indicate that viable CB-MSC could be obtained consistently and that CB should be reconsidered as a practical source of MSC for cell therapy and regenerative medicine using the well established CB banking system.

BLNK suppresses pre-B-cell leukemogenesis through inhibition of JAK3.

Pre-B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre-B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre-B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK(-/-) mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27(kip1) expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/STAT5 signaling, resulting in p27(kip1) induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7-dependent proliferation and survival of pre-B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/STAT5 pathway result in the generation of pre-B-cell leukemia.

Antioxidative activity and chemical constituents of edible terrestrial alga Nostoc commune Vauch.

The extract of terrestrial alga Nostoc commune Vauch. has high antioxidative activity. Our study on N. commune Vauch. resulted in the isolation of two ß-ionone derivatives, nostocionone and 3-oxo-ß-ionone, together with four indole alkaloids, scytonemin, reduced scytonemin, N-(p-coumaroyl)tryptamine, and N-acetyltryptamine. The structures of the isolated compounds were determined on the basis of 1D and 2D NMR and MS analyses. Among these isolates, nostocionone and reduced scytonemin demonstrated strong antioxidative activities which were assessed by using a ß-carotene oxidation assay.

The novel and independent association between single-point SNP of NPHP4 gene and renal function in non-diabetic Japanese population: the Takahata study.

Nephronophthisis (NPHP) 4 gene coding nephrocystin-4 is involved in the development of renal tubules and its congenital mutations cause juvenile end-stage renal disease, NPHP. To investigate the association between single-point single-nucleotide polymorphism (SNP) of NPHP4 gene and renal function, we conducted a cross-sectional study in Japanese population. The subjects of this study were non-diabetic general population consisting of 2604 individuals >40 years in Takahata town, Japan. We genotyped 11 SNPs within NPHP4 gene that displayed frequent minor allele frequencies (>0.1) in Japanese general population. Among 11 SNPs in NPHP4 gene, only rs1287637 that induces amino acid substitution (A (Gln)/T (Leu)), located in the acceptor site of exon 21, showed a significant association with estimated glomerular filtration rate (eGFR; T/T: 81.3±15.6 (n=1886), A/T: 82.0±15.5 (n=652) and A/A: 87.4±21.4 ml min(-1) per 1.73m(2) (n=66); mean±s.d., P=0.006). This SNP was not in linkage disequilibrium with the surrounding SNPs. The multivariate analysis adjusted with possible confounders showed that the A/T+T/T genotype of rs1287637 was independently associated with reduced renal function (eGFR <90 ml min(-1) per 1.73m(2); odds ratio (OR) 1.75, 95% confidence interval (CI) 1.05-2.94, P=0.033). These results indicate the novel and independent association between single-point SNP rs1287637 in NPHP4 gene and renal function in non-diabetic Japanese population.

Histology of fibrovascular membranes of proliferative diabetic retinopathy after intravitreal injection of bevacizumab.

PURPOSE: The purpose was to study the histology of the fibrovascular membranes in proliferative diabetic retinopathy (PDR) with an intravitreal injection of bevacizumab. METHODS: Light and electron microscopic studies were performed on surgical specimens obtained during a pars plana vitrectomy from 6 PDR eyes after intravitreal injection of bevacizumab. The patients had preoperatively received no or scant retinal photocoagulations. The presence and distribution of CD34 was assessed as a marker of vascular endothelium using immunostaining. The presence of vascular endothelial growth factor was stained with a method of immunostaining. As controls, we examined 7 surgical specimens from 7 PDR eyes obtained during pars plana vitrectomy without bevacizumab therapy. All control patients had preoperatively received full or nearly full pan retinal photocoagulations. RESULTS: Light microscopy showed that the CD34-positive vascular endothelial cells formed capillarylike structures in the fibrovascular membranes of all 13 PDR eyes. Vascular endothelial growth factor was positively stained in the vascular endothelium of both groups; however, the number of vascular endothelial growth factor-positive vascular endothelial cells significantly decreased in the fibrovascular membranes with intravitreal injection of bevacizumab. Electron microscopy showed the newly formed vascular endothelial cells with junctional complex in both groups. CONCLUSION: The vascular endothelial cells with decreased expression of vascular endothelial growth factor are still present in the fibrovascular membranes of patients with PDR after intravitreal injection of bevacizumab.

Acute effect of poly-gamma-glutamic acid on calcium absorption in post-menopausal women.

OBJECTIVE: Poly-gamma-glutamic acid (PGA) increases calcium (Ca) solubility in vitro and in vivo, and is associated with reduced bone loss in post-menopausal Japanese women. This study is the first to examine the effect of PGA on Ca absorption in humans. METHODS: A single-blind, randomized, crossover study with a 3-4 week wash-out was performed to determine the effect of PGA (80.6% glutamic acids) on Ca absorption measured by the double stable isotope method. Twenty-four healthy, non-smoking, postmenopausal women (mean age: 56.4 +/- SE 0.9) were given 200 g of orange juice containing 200 mg Ca as Ca-44 enriched CaCO(3), with or without 60 mg of PGA, after an overnight fast. The two tests were separated by 3-4 weeks. An intravenous injection of Ca-42 (CaCl(2) solution) was given 30 min after consuming the drink and a complete urine collection carried out from 24-48 h post-dosing. Ca absorption was calculated from the Ca isotope ratios measured by thermal ionization quadrupole mass spectrometry (TIQMS). RESULTS: Mean Ca absorption with PGA was significantly higher (P < 0.01) than without PGA, 39.1 (SE 1.6) % and 34.6 (SE 1.9) %, respectively. The effect of PGA on increasing Ca absorption was more marked in a sub-group of subjects whose baseline Ca absorption (without PGA) was lower than the population mean value. CONCLUSION: Postmenopausal women who received a single dose of PGA increased their intestinal Ca absorption particularly those individuals with lower basal absorptive capacity.

Clinical characteristics and genotype-phenotype correlation of hearing loss patients with SLC26A4 mutations.

CONCLUSIONS: The present study confirmed the clinical characteristics of patients with SLC26A4 mutations: congenital, fluctuating, and progressive hearing loss usually associated with vertigo and/or goiter during long-term follow-up. This clarification should help to facilitate appropriate genetic counseling and proper medical management for patients with these mutations, but there was no particular genotype-phenotype correlation among them, suggesting that other factors may contribute to such variability. OBJECTIVES: Due to the wide range of phenotypes caused by SLC26A4 mutations, there is controversy with regard to genotype-phenotype correlation. The present study was performed: (1) to determine phenotypic range in patients with biallelic SLC26A4 mutations, and (2) to evaluate whether possible genotype-phenotype correlation exists. SUBJECTS AND METHODS: Phenotypes in 39 hearing loss patients with SLC26A4 mutations were summarized and genotype-phenotype correlation was analyzed. RESULTS: Hearing level varied in the individuals from mild to profound severity. Most of the patients had fluctuating and progressive hearing loss that may have been of prelingual onset. Twenty-four (70.6%) patients had episodes of vertigo, and 10 (27.8%) patients had goiter, which had appeared at age 12 or older. In contrast to such phenotypic variabilities, no apparent correlation was found between these phenotypes and their genotypes.

Transforming growth factor beta expression during an inner ear immune response.

OBJECTIVES: The involvement of transforming growth factor beta (TGF-beta), a strong mediator of fibrogenesis, during cochlear immune responses was investigated. METHODS: An inner ear adaptive immune response to antigen was created in mice that were painlessly sacrificed 3 to 48 hours and 7 days after initiation of the immune response. The cochleas were assayed by immunocytochemistry for TGF-beta and latency-associated peptide (LAP). RESULTS: We found LAP expressed in normal cochleas and the endolymphatic sac, in the small round cells in the cochlear scalae and the mesothelial cells under the basilar membrane, and in the endolymphatic sac perisaccular area. We found TGF-beta expressed in infiltrated, inflammatory cells in the scalae and the endolymphatic sac lumen 3 hours after cochlear antigen challenge. At this time, LAP immunoreactivity was decreased. This rapid shift in immunoreactivity provides evidence for activation of TGF-beta during an immune response. This reversal of expression persisted for 48 hours, but conditions reverted to normal after 7 days. Surgical controls did not show TGF-beta expression. CONCLUSIONS: We conclude that TGF-beta activation occurs in the early phase of a cochlear adaptive immune response and is down-regulated as the response resolves. This finding suggests that the process of cochlear fibrosis starts early and that proper treatment could prevent cochlear fibrosis.

Endolymphatic sac tumor located around semicircular canals.

We report a case of endolymphatic sac tumor (ELST). A 48-year-old female had recurrent and slowly progressive hearing loss, accompanied with dizziness like Meniere's disease. A tumor was located around the semicircular canals, and was detected on CT and MRI. The patient underwent total removal of the tumor using a transmastoid approach. Histopathological examinations agreed with features of an ELST. The tumor was highly suspected to have originated from the rugose portion of the endolymphatic sac or the endolymphatic duct, based on surgical and imaging studies. Structure of the membranous labyrinth was preserved regardless of the existence of the tumor around semicircular canals with bone destruction. ELSTs seem to have an osteolytic or osteophilic nature, by examining patterns of tumor infiltration.

Insect-vertebrate chimeric nicotinic acetylcholine receptors identify a region, loop B to the N-terminus of the Drosophila Dalpha2 subunit, which contributes to neonicotinoid sensitivity.

A chimera based on the chicken alpha4 nicotinic acetylcholine receptor (nAChR) subunit containing an insert from loop B to the N-terminus of the Drosophila melanogaster Dalpha2 (=SAD) subunit was constructed and co-expressed with the chicken beta2 nAChR subunit in Xenopus laevis oocytes. The actions of the neonicotinoid insecticide imidacloprid were examined. Replacement of the region loop B to the N-terminus of the alpha4 subunit by the corresponding region of the Dalpha2 subunit had little effect on the concentration-response curve for imidacloprid. However, replacement of Glu219 by proline in the YXCC motif in loop C of the chimeric alpha4 subunit resulted in a marked displacement to the left of the concentration-response curve for imidacloprid not seen when an equivalent mutation was made in the alpha4beta2 nAChR. The results suggest that the region loop B to the N-terminus in the Dalpha2 subunit contributes to the high imidacloprid sensitivity of the hybrid Dalpha2beta2 nAChR.

Efficient inhibition of tumor angiogenesis and growth by a synthetic peptide blocking S100A4-methionine aminopeptidase 2 interaction.

The prometastatic calcium-binding protein, S100A4, is expressed in endothelial cells, and its downregulation markedly suppresses tumor angiogenesis in a xenograft cancer model. Given that endothelial S100A4 can be a molecular target for inhibiting tumor angiogenesis, we addressed here whether synthetic peptide capable of blocking S100A4-effector protein interaction could be a novel antiangiogenic agent. To examine this hypothesis, we focused on the S100A4-binding domain of methionine aminopeptidase 2, an effector protein, which plays a role in endothelial cell growth. Overexpression of the domain in mouse endothelial MSS31 cells reduced DNA synthesis, and the corresponding synthetic peptide (named NBD) indeed interacted with S100A4 and inhibited capillary formation in vitro and new blood vessel formation in vivo. Intriguingly, a single intra-tumor administration of the NBD peptide in human prostate cancer xenografts significantly reduced vascularity, resulting in tumor regression. Mechanistically, the NBD peptide enhanced assembly of nonmuscle myosin IIA filaments along with Ser1943 phosphorylation, stimulated formation of focal adhesions without phosphorylation of focal adhesion kinase, and provoked G1/S arrest of the cell cycle. Altogether, the NBD peptide is a potent inhibitor for tumor angiogenesis, and is the first example of an anticancer peptide drug developed on the basis of an endothelial S100A4-targeted strategy.

Establishment of a novel dwarf rat strain: cartilage calcification insufficient (CCI) rats.

Rats with dwarfism accompanied by skeletal abnormalities, such as shortness of the limbs, tail, and body (dwarf rats), emerged in a Jcl-derived Sprague-Dawley rat colony maintained at the Institute for Animal Experimentation, St. Marianna University Graduate School of Medicine. Since the dwarfism was assumed to be due to a genetic mutation based on its frequency, we bred the dwarf rats and investigated their characteristics in order to identify the causative factors of their phenotypes and whether they could be used as a human disease model. One male and female that produced dwarf progeny were selected, and reproduction was initiated by mating the pair. The incidence of dwarfism was 25.8% among the resultant litter, and dwarfism occurred in both genders, suggesting that it was inherited in an autosomal recessive manner. At 12 weeks of age, the body weights of the male and female dwarf rats were 40% and 57% of those of the normal rats, respectively. In soft X-ray radiographic and histological examinations, shortening and hypoplasia of the long bones, such as the tibia and femur, were observed, which were suggestive of endochondral ossification abnormalities. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led to delayed calcification and increased growth plate thickening in the dwarf rats. We hypothesized that the principal characteristics of the dwarf rats were systemically induced by insufficient cartilage calcification in their long bones; thus, we named them cartilage calcification insufficient (CCI) rats.

Identification of the Tslc1 gene, a mouse orthologue of the human tumor suppressor TSLC1 gene.

We have recently identified the TSLC1 gene as a novel tumor suppressor in human non-small lung cancer on chromosome 11q23.2. TSLC1 encodes a membrane glycoprotein showing significant homology with immunoglobulin superfamily molecules. Here, we report the isolation of a mouse orthologous gene, Tslc1. The Tslc1 cDNA contains a single open reading frame of 1335 bp encoding a putative protein of 445 amino acids, and its expression was detected in all tissues examined. The Tslc1 gene is mapped on mouse chromosome 9, a synteny of human chromosome 11q, and is composed of ten exons, the exon-intron junctions being highly conserved between human and mouse. The predicted amino acids of mouse Tslc1 display 98% identity with that of human TSLC1. Furthermore, data base analysis indicates that the amino acid sequences corresponding to the cytoplasmic domain of Tslc1 are identical in five mammals and highly conserved in vertebrates, suggesting an important role of Tslc1 in normal cell-cell interaction.

Isolation of the mouse Tsll1 and Tsll2 genes, orthologues of the human TSLC1-like genes 1 and 2 (TSLL1 and TSLL2).

We have recently identified the human TSLL1 and TSLL2 genes, which are highly homologous to the human lung tumor suppressor gene, TSLC1. Loss of expression of the TSLL1 or TSLL2 in several cancers suggests that these genes could also act as tumor suppressors. Here, we report the isolation of the mouse orthologous genes, Tsll1 and Tsll2. The Tsll1 and Tsll2 cDNAs contain a single open reading frame of 1188 and 1164 bp encoding a putative immunoglobulin-like cell adhesion molecules of 396 and 388 amino acids, which display 95% and 98% identity with those of human TSLL1 and TSLL2, respectively. The Tsll1 and Tsll2 genes are both composed of nine exons and mapped on mouse chromosome 1q H2-H4 and on 7q A3-B2, respectively, both of which conserve syntenies with human chromosomes 1q and 19q. Like the human TSLL1, the mouse Tsll1 was expressed exclusively in the brain and neurogenic cells, while Tsll1 expression was lost in one of four rodent neuroblastoma cell lines. Tsll2 was expressed in the brain and several organs including the kidney and liver, whereas loss of Tsll2 expression was detected in some rodent cancer cells derived from these tissues. Furthermore, both murine TSLL1 and TSLL2 proteins were expressed on the plasma membrane, especially at the cell-cell attached site. These data, together with their strong conservation during the vertebrate evolution, suggest that TSLL1and TSLL2 could play an important role in cell-cell interaction as well as in tumor suppression.

Inhibitory efficacy of pitavastatin on the early inflammatory response and neointimal thickening in a porcine coronary after stenting.

Neointimal hyperplasia plays a crucial role in restenosis after stenting. The severity of neointimal thickness correlates with inflammatory reactions in the injured vessel and statins can inhibit inflammation. Pitavastatin has favorable effects on plasma lipoproteins and inflammation. Thus, we hypothesized that pitavastatin might inhibit the early inflammatory response, resulting in prevention of neointimal hyperplasia in porcine coronary arteries after stenting. Pitavastatin (18 coronaries, 40 mg per day) or placebo (20 coronaries) was administered orally from 7 days before stenting until the time of euthanasia at 3 or 28 days after stenting. The coronary artery of the animals was injured with an oversized metallic coil stent. Inflammatory cell infiltration was evaluated by scanning electron microscopy and was significantly reduced in the treated vessels compared to controls. On Day 28, intravascular ultrasound analysis revealed the neointimal area was significantly less at the stent site in the pitavastatin group than in the placebo. Histopathologic assessment showed significantly decreased in neointimal area in the pitavastatin group compared to the placebo (2.16 +/- 0.13 mm(2) versus 2.88 +/- 0.25 mm(2), p = 0.029), whereas the mean injury score in the pitavastatin group was larger than in the placebo group. In conclusion, Pitavastatin inhibited neointimal hyperplasia after stenting through a reduction of inflammatory reactions.

Proinflammatory cytokine expression in the endolymphatic sac during inner ear inflammation.

The inner ear is capable of rapidly mounting an immune response that can ultimately lead to cochlear degeneration and permanent hearing loss. The role of the endolymphatic sac in this immune process is not clear. In order to investigate the cytokine expression of cells within the endolymphatic sac, a secondary inner ear immune response to keyhole limpet hemocyanin (KLH) was created in mice. The animals were sacrificed 3-48 h and 7 days following initiation of the immune response. The cochleas and endolymphatic sacs were assayed by immunocytochemistry for IL-1beta, TNFalpha, and IL-6. Three hours after KLH challenge of the scala tympani, the perisaccular tissue of the endolymphatic sac contained more inflammatory cells than the scala tympani or endolymphatic sac lumen. Only a few of these cells, however, expressed the proinflammatory cytokines IL-1beta and TNFalpha between 3 and 12 h after KLH injection. On the other hand, TNFalpha, which plays an important role in the cochlear secondary immune response, was expressed in cells in the endolymphatic sac lumen. The maximum percentage of cells expressing TNFalpha was seen later than in the scala tympani. Animals treated with systemic injection of the TNF blocker, etanercept, showed a reduction in the number of cells in the endolymphatic sac lumen. It is concluded that the cells in the endolymphatic sac lumen contribute to the amplification of the adaptive immune response by expressing TNFalpha, while the infiltration of cells into the perisaccular connective tissue is part of the nonspecific, innate, cochlear immune response.

Receptor occupancy theory-based analysis of antiemetic effects and standard doses of 5-HT3 receptor antagonists in cancer patients.

PURPOSE: The aim of this study was to determine the usefulness of receptor occupancy theory-based analysis using pharmacokinetic and pharmacodynamic parameters for predicting the average receptor occupancy (PhiB) in humans of each of five 5-HT3 antagonists administered at standard doses. METHODS: The relationship between the PhiB value and the complete vomiting inhibition rate after a single intravenous administration of cisplatin (not less than 50 mg/m2) was analyzed. RESULTS: The predicted PhiB values after intravenous administration and oral administration of 5-HT3 antagonists were more than 65% and 50%, respectively, suggesting that relatively high receptor occupancy is required to elicit sufficient antiemetic effects of 5-HT3 antagonists. Moreover, significant ( P<0.05) linear relationships were found between PhiB values and complete vomiting inhibition rates of 5-HT3 antagonists in preventive cisplatin therapy, with correlation coefficients higher than 0.9, suggesting that the 5-HT3 receptor occupancy is an appropriate index of clinical efficacy of 5-HT3 antagonists, with higher receptor occupancy indicating more extensive antiemetic action. CONCLUSION: The receptor occupancy theory-based analysis of the antiemetic effect of a 5-HT3 receptor antagonist used in this study should be very useful for not only estimating a rational dosage regimen but also determining the standard dose of a new drug using experimental data obtained in a preclinical study.

Characterization of a newly established nonproducer lymphoma cell line for feline leukemia virus.

A feline lymphoblastoid cell line (KO-1) was established from a 5-year-old neutered female cat with naturally occurring thymic lymphoma. KO-1 cells had a rearrangement of T-cell receptor beta-chain gene and a germ-line configuration of immunoglobulin heavy chain gene, however, they were devoid of T-cell-specific surface phenotype. Cytogenetically, KO-1 cells showed a hyperploidy (2n = 41) due to the trisomy of B2, F2 and X chromosomes. Although KO-1 cells were shown to be clonally expanded cells integrated with feline leukemia virus (FeLV) proviruses and expressed its structural proteins in their cytoplasm, they did not produce virus particles as shown by transmission electron microscopy and the absence of the viral protein and reverse transcriptase activity in the culture supernatant. The present study showed that the KO-1 cell line established here was a feline T-cell lymphoma cell line having a unique characteristic as an FeLV nonproducer.