Cytohistology of morule in cribriform-morular variant of papillary thyroid carcinoma.

INTRODUCTION: Cribriform-morular variant (CMV) is a rare variant of papillary thyroid carcinoma. It frequently occurs in association with familial adenomatous polyposis (FAP), although some cases are sporadic. Herein, we report a case of CMV and analyse morule cytohistology. CASE REPORT: The patient was a 47-year-old woman with no familial history of FAP. A 3.0-cm unifocal mass was identified in the left thyroidal lobe. Fine-needle aspiration cytology revealed papillary clusters of atypical cells with nuclear grooves, which was suspected to be conventional papillary thyroid carcinoma. Histologically, the tumour comprised a papillary and cribriform growth of atypical cells with cytoplasmic accumulation and nuclear translocation of b-catenin. In addition, frequent morule formation was identified. DISCUSSION: In this case, we performed morule analysis through correlative light and electron microscopy (CLEM), and revealed its ultrastructure. Although CMV is a rare form of thyroid carcinoma, it should be considered along with its distinct clinicopathological characteristics.

Randomised phase III study of neoadjuvant chemotherapy with methotrexate, doxorubicin, vinblastine and cisplatin followed by radical cystectomy compared with radical cystectomy alone for muscle-invasive bladder cancer: Japan Clinical Oncology Group Study JCOG0209.

BACKGROUND: This study aimed to determine the clinical benefit of neoadjuvant methotrexate, doxorubicin, vinblastine, and cisplatin (MVAC) in patients with muscle-invasive bladder cancer (MIBC) treated with radical cystectomy. PATIENTS AND METHODS: Patients with MIBC (T2-4aN0M0) were randomised to receive two cycles of neoadjuvant MVAC followed by radical cystectomy (NAC arm) or radical cystectomy alone (RC arm). The primary end point was overall survival (OS). Secondary end points were progression-free survival, surgery-related complications, adverse events during chemotherapy, proportion with no residual tumour in the cystectomy specimens, and quality of life. To detect an improvement in 5-year OS from 45% in the RC arm to 57% in the NAC arm with 80% power, 176 events were required per arm. RESULTS: Patients (N = 130) were randomly assigned to the RC arm (N = 66) and the NAC arm (N = 64). The patient registration was terminated before reaching the initially planned number of patients because of slow accrual. At the second interim analysis just after the early stoppage of patient accrual, the Data and Safety Monitoring Committee recommended early publication of the results because the trial did not have enough power to draw a confirmatory conclusion. OS of the NAC arm was better than that of the RC arm, although the difference was not statistically significant [hazard ratio 0.65, multiplicity adjusted 99.99% confidence interval 0.19-2.18, one-sided P = 0.07]. In the NAC arm and the RC arm, 34% and 9% of the patients had pT0, respectively (P < 0.01). In subgroup analyses, OS in almost all subgroups was in favour of NAC. CONCLUSIONS: This trial showed a significantly increased pT0 proportion and favourable OS of patients who received neoadjuvant MVAC. NAC with MVAC can still be considered promising as a standard treatment. UMIN CLINICAL TRIALS REGISTRY IDENTIFIER: C000000093.

The outcomes of surgical repairs of vesicovaginal fistula in 16 patients.

We retrospectively analysed 16 patients who underwent surgical repair for vesicovaginal fistula (VVF) in our department from 1995 to 2012. A total of 14 patients (88%) were cured after the primary repair and two patients were cured by reoperation. We compared the characteristics of the patients whose VVF occurred early and late after surgery. In univariate analysis, the estimated area of the fistula was significantly greater in the late-onset group (p = 0.011). There was a tendency for the maximum diameter of the fistula to be larger (p = 0.08) and a surgical energy device was used more frequently during surgery (p = 0.12) in the late-onset group than in the early-onset group. In conclusion, the outcomes of surgical VVF repair were acceptable. The characteristics of VVF that developed late postoperatively were different from those that developed early postoperatively.

Adrenal hemorrhagic pseudocyst as the differential diagnosis of pheochromocytoma--a review of the clinical features in cases with radiographically diagnosed pheochromocytoma.

BACKGROUND: Clinical diagnosis of pheochromocytoma is difficult for some adrenal tumors. AIM: Herein, we review clinical and pathological findings of 31 cases with radiographically diagnosed pheochromocytoma, including three cases of hemorrhagic pseudocysts (HPC). MATERIALS/SUBJECTS AND METHODS: Between January 1992 and December 2010, 31 patients with adrenal tumors were pre-operatively diagnosed as having pheochromocytoma by radiographic imaging, and underwent adrenalectomy. Histological examination revealed HPC in 3 patients (9.7%), and pheochromocytoma in the remaining 28 patients. We reviewed and compared the clinical features, including the biochemical and radiographic features, of HPC and pheochromocytoma cases. RESULTS: Biochemical testing showed no definitive excessive catecholamine secretion in any of the three patients with HPC and four (14.3%) of those with histologically proven pheochromocytoma. (131)Imetaiodobenzylguanidine scintigraphy was negative in the three with HPC, but positive in all of the four with pheochromocytoma who did not have suggestive biochemical results. All HPC patients had concomitant disease or symptoms suggestive of pheochromocytoma, and two had received an anti-coaglant or anti-platelet agent. Laparoscopic surgery was completed in two cases of HPC uneventfully. CONCLUSIONS: Adrenal HPC may have radiographic characteristics similar to those of pheochromocytoma. Adrenal HPC should be considered as a differential diagnosis of pheochromocytoma.

Peroxisome assembly factor-2, a putative ATPase cloned by functional complementation on a peroxisome-deficient mammalian cell mutant.

Rat peroxisome assembly factor-2 (PAF-2) cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP92, using transient transfection assay. This cDNA encodes a 978-amino acid protein with two putative ATP-binding sites. PAF-2 is a member of a putative ATPase family, including two yeast gene products essential for peroxisome assembly. A stable transformant of ZP92 with the cDNA was morphologically and biochemically restored for peroxisome biogenesis. Fibroblasts derived from patients deficient in peroxisome biogenesis (complementation group C) were also complemented with PAF-2 cDNA, indicating that PAF-2 is a strong candidate for the pathogenic gene of group C peroxisome deficiency.

Visualization of gene activity in living cells.

Chromatin structure is thought to play a critical role in gene expression. Using the lac operator/repressor system and two colour variants of green fluorescent protein (GFP), we developed a system to visualize a gene and its protein product directly in living cells, allowing us to examine the spatial organization and timing of gene expression in vivo. Dynamic morphological changes in chromatin structure, from a condensed to an open structure, were observed upon gene activation, and targeting of the gene product, cyan fluorescent protein (CFP) reporter to peroxisomes was visualized directly in living cells. We found that the integrated gene locus was surrounded by a promyelocytic leukaemia (PML) nuclear body. The association was transcription independent but was dependent upon the direct in vivo binding of specific proteins (EYFP/lac repressor, tetracycline receptor/VP16 transactivator) to the locus. The ability to visualize gene expression directly in living cells provides a powerful system with which to study the dynamics of nuclear events such as transcription, RNA processing and DNA repair.

Excellent performance of aromatic polyguanamines induced by multiple hydrogen bondable tetraazacalix[2]arene[2]-triazine ring in their main chain.

A series of poly(guanamine) (c-PG)s containing tetraazacalix[2]arene[2]-triazine (mPDA2CyC2) were successfully prepared by solution polycondensation of mPDA2CyC2 with various aromatic diamines in an aprotic organic solvent with a lithium chloride additive (5 wt%) at 150 °C for 6 hours. The number-average molecular weights (M n)s of these c-PG polymers reached up to 31 500, with a relatively broad molecular weight distribution (M w/M n) of 5.3. They showed good solubility in aprotic organic solvents, such as N-methylpyrrolidone and N,N-dimethylacetamide at a concentration of 2 mg mL-1. The glass transition temperatures (T g) of the c-PG polymers were in the range 359 °C-392 °C, approximately 160 °C higher than those of counterpart polymers (i.e., with no aza-calixarene-based PG (l-PG)). The coefficients of thermal expansion (CTEs) of the c-PG polymers were 29.7-48.1 ppm K-1 (at 100 °C-150 °C), much lower than those of l-PG samples, i.e., 59.1-85.1 ppm K-1. Transparent and almost colorless c-PG films were successfully prepared by a solution casting method, showing maximum tensile strength (σ S), modulus (E γ), and elongation at break (E b) values of 151 MPa, 6.3 GPa, and 4.4%, respectively, for the c-PG polymer from mPDA2CyC2 and 4,4'-oxydianiline monomers. The corresponding l-PG film exhibited σ S, E γ, and E b values of just 76 MPa, 5.4 GPa, and 1.6%, respectively. These outstanding thermal and mechanical properties of the c-PG polymers can be attributed to their multiple hydrogen bonding interaction between mPDA2CyC2 residues in the polymer backbone. This interaction was identified by infrared spectroscopy measurements at the broad absorption band around 3000-3400 cm-1.

Herpes simplex virus vector-mediated delivery of neurturin rescues erectile dysfunction of cavernous nerve injury.

Neurturin (NTN), a member of glial cell line-derived neurotrophic factor (GDNF) family, is known as an important neurotrophic factor for penis-projecting neurons. We recently demonstrated significant protection from erectile dysfunction (ED) following a replication-defective herpes simplex virus (HSV) vector-mediated GDNF delivery to the injured cavernous nerve. Herein, we applied HSV vector-mediated delivery of NTN to this ED model. Rat cavernous nerve was injured bilaterally using a clamp and dry ice. For HSV-treated groups, 20 microl of vector stock was administered directly to the damaged nerve. Delivery of an HSV vector expressing both green fluorescent protein and lacZ (HSV-LacZ) was used as a control. Intracavernous pressure along with systemic arterial pressure (ICP/AP) was measured 2 and 4 weeks after the nerve injury. Fluorogold (FG) was injected into the penile crus 7 days before being killed to assess neuronal survival. Four weeks after nerve injury, rats treated with HSV-NTN exhibited significantly higher ICP/AP compared with untreated or control vector-treated groups. The HSV-NTN group had more FG-positive major pelvic ganglion neurons than the control group following injury. HSV vector-mediated delivery of NTN could be a viable approach for the improvement of ED following cavernous nerve injury.

Isolation and characterization of Chinese hamster ovary cell mutants defective in assembly of peroxisomes.

We made use of autoradiographic screening to isolate two Chinese hamster ovary (CHO) cell mutants deficient in peroxisomal dihydroxyacetonephosphate acyltransferase, a key enzyme for the biosynthesis of ether glycerolipids such as plasmalogens. Morphological analysis revealed no evidence of peroxisome in these mutants. Catalase was as active as in the normal cells but was not sedimentable. Pulse-chase radiolabeling experiments and cell-free translation of RNA demonstrated that acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation system, was synthesized as the 75-kD form but was not converted to 53- and 22-kD mature components that were present in the wild-type CHO cells; rather, degradation was apparent. Peroxisomal thiolase was synthesized as in normal cells but remained as a larger, 44-kD precursor, whereas maturation to the 41-kD enzyme was detected in the wild-type cells. The peroxisomal 70-kD integral membrane protein was also equally synthesized, as in the wild-type cells, and was not degraded. These results suggest that assembly of the peroxisomes is defective in the mutants, whereas the synthesis of peroxisomal proteins appears to be normal. Cell-fusion studies revealed that the two mutants are recessive to the wild-type CHO cells and belong to different complementation groups. Thus, these mutants presumably contain different lesions in gene(s) encoding factor(s) required for peroxisome assembly.

A human gene responsible for Zellweger syndrome that affects peroxisome assembly.

The primary defect arising from Zellweger syndrome appears to be linked to impaired assembly of peroxisomes. A human complementary DNA has been cloned that complements the disease's symptoms (including defective peroxisome assembly) in fibroblasts from a patient with Zellweger syndrome. The cause of the syndrome in this patient was a point mutation that resulted in the premature termination of peroxisome assembly factor-1. The homozygous patient apparently inherited the mutation from her parents, each of whom was heterozygous for that mutation.

Direct observation of imploded core heating via fast electrons with super-penetration scheme.

Fast ignition (FI) is a promising approach for high-energy-gain inertial confinement fusion in the laboratory. To achieve ignition, the energy of a short-pulse laser is required to be delivered efficiently to the pre-compressed fuel core via a high-energy electron beam. Therefore, understanding the transport and energy deposition of this electron beam inside the pre-compressed core is the key for FI. Here we report on the direct observation of the electron beam transport and deposition in a compressed core through the stimulated Cu Kα emission in the super-penetration scheme. Simulations reproducing the experimental measurements indicate that, at the time of peak compression, about 1% of the short-pulse energy is coupled to a relatively low-density core with a radius of 70 µm. Analysis with the support of 2D particle-in-cell simulations uncovers the key factors improving this coupling efficiency. Our findings are of critical importance for optimizing FI experiments in a super-penetration scheme.

Chromosomal instability by beta-catenin/TCF transcription in APC or beta-catenin mutant cells.

Adenomatous polyposis coli (APC/Apc) gene encodes a key tumor suppressor whose mutations activate beta-catenin/T-cell factor (TCF)-mediated transcription (canonical Wnt signaling). Here, we show that Wnt signaling can cause chromosomal instability (CIN). As an indicator of CIN, we scored anaphase bridge index (ABI) in mouse polyps and ES cells where Wnt signaling was activated by Apc or beta-catenin mutations. We found three to nine times higher ABI than in wild-type controls. Furthermore, karyotype analysis confirmed that the Wnt signal-activated ES cells produced new chromosomal aberrations at higher rates; hence CIN. Consistently, expression of dominant-negative TCFs in these cells reduced their ABI. We also found that Wnt signal activation increased phosphorylation of Cdc2 (Cdk1) that inhibited its activity, and suppressed apoptosis upon exposure of the cells to nocodazole or colcemid. The data suggest that Wnt signaling stimulates the cells to escape from mitotic arrest and apoptosis, resulting in CIN. In human gastric cancer tissues with nuclear beta-catenin, ABI was significantly higher than in those without. These results collectively indicate that beta-catenin/TCF-mediated transcription itself increases CIN through dysregulation of G2/M progression.

Docetaxel plus prednisolone for the treatment of metastatic hormone-refractory prostate cancer: a multicenter Phase II trial in Japan.

BACKGROUND: Docetaxel-based chemotherapy has been shown to be effective and well tolerated by Western patients with metastatic hormone-refractory prostate cancer (HRPC). This study was undertaken to assess the feasibility of docetaxel in combination with prednisolone in Japanese patients with HRPC. METHODS: Patients aged 50-74 years with measurable metastatic HRPC were included in this non-comparative Phase II study. Treatment consisted of docetaxel 70 mg/m(2) once every 3 weeks plus prednisolone 5 mg twice daily, for a maximum of 10 cycles. The primary endpoint was overall tumor response rate, assessed by Response Evaluation Criteria in Solid Tumors; secondary endpoints included prostate-specific antigen (PSA) response and toxicity. RESULTS: A total of 43 patients were evaluable for efficacy and toxicity. The response rate was 44.2% (90% CI, 31.2-57.8%), with partial responses in 19/43 patients. The median duration of response was 19.3 weeks. PSA responses were recorded in 44.4% of patients (95% CI, 27.9-61.9%). The most common non-hematological adverse events (of any grade) possibly related to treatment were alopecia (88.4%), anorexia (65.1%) and fatigue (53.5%). Grade 3/4 leukopenia and neutropenia occurred in 81.4 and 93.0% of patients, respectively; however, the grade 3/4 rates of febrile neutropenia (16.3%) and infection without fever (14.0%) were lower. CONCLUSION: The combination of docetaxel and prednisolone was feasible and active in Japanese patients with HRPC, with a manageable adverse-event profile similar to that observed in Western patients.

[Non-chondromatous hamartoma].

A 69-year-old female visited our hospital because of dyspnea. Chest X-ray showed an abnormal shadow in the right lower lung field. Chest computed tomography (CT) showed a round, well-circumscribed, homogenous subpleural nodle of 8 mm in diameter in the right lower lobe, which had no calcification and no pleural indentation. Bronchofiber scope, abdominal CT, brain magnetic resonance imaging (MRI), bone scintigraphy could not establish definitive diagnosis. Scince the possibility of malignancy could not be excluded throughout, video-assisted thoracoscopic surgery was performed to obtain confirmed diagnosis. Pathological examination revealed non-chondromatous hamartoma of the lung. Non-chondromatous hamartoma should be considered in the differential diagnosis of pulmonary nodles. We report a rare case of non-chondromatous hamartoma.

Catecholamine regulation of human erythrocyte membrane protein kinase.

The effect of catecholamines on membrane-associated protein kinase in the mature human erythrocyte was investigated. Protein kinase activity was assayed after isolation of membranes from intact erythrocytes incubated with and without catecholamines. Activation of the enzyme is expressed as the ratio of the extent of phosphorylation of exogenous protein substrate in the absence to that in the presence of 2.5 microM cyclic AMP (cAMP). The potent beta-adrenergic agonist, (-)isoproterenol (2 microM), (-)epinephrine (10 microM) and (-)norepinephrine (10 microM) stimulated the cAMP-dependent protein kinase in membranes, 38 +/- 7%, 31 +/- 6%, and 30 +/- 6%, respectively. Maximal stimulation of membrane protein kinase by 10 microM (-)epinephrine was obtained approximately equal to 30 min after initiation of the incubation of erythrocytes with the hormone. The concentrations of (-)catecholamines that gave half-maximal stimulation of the membrane protein kinase were 0.17 microM for isoproterenol, 0.35 microM for epinephrine, and 0.63 microM for norepinephrine. The membrane protein kinase response to beta-adrenergic agonists was found to be stereospecific. The stimulation of membrane protein kinase by 10 microM (-)epinephrine was inhibited by the beta-adrenergic antagonist, (-)propranolol with EC50 = 0.60 microM, and the inhibition of agonist stimulation of the cAMP-dependent protein kinase by propranolol was stereospecific. These studies suggest that a functional beta-adrenergic receptor exists in the mature human erythrocyte.

Animal cell mutants represent two complementation groups of peroxisome-defective Zellweger syndrome.

Generalized peroxisome-deficient disorders including cerebro-hepato-renal Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease are autosomal recessive diseases, where catalase-containing particles (peroxisomes) are morphologically absent. We previously isolated two Chinese hamster ovary (CHO) cell mutants (Z24 and Z65) that resemble the fibroblasts from patients with such diseases, in their defective peroxisome assembly (Tsukamoto, T., S. Yokota, and Y. Fujiki. 1990. J. Cell Biol. 110:651-660). Here we report isolation by the P9OH/UV method of a peroxisome-deficient CHO mutant, ZP92, of the third complementation group distinct from those of Z24 and Z65. Peroxisomal membrane ghosts were noted by immunochemical staining in all of the CHO mutants. Complementation analysis by cell fusion of the CHO mutants with cultured fibroblasts from patients with generalized peroxisomal disorders revealed that two CHO mutants (Z24 and ZP92) represent the human complementation groups, E (the same as group 1 in the U.S.) and C (the same as group 4), respectively. These CHO cell mutants are an apparently relevant animal cell model for studies on the molecular bases and primary defects of human peroxisome-deficient diseases.

Human neuroblastoma cells express alpha and beta platelet-derived growth factor receptors coupling with neurotrophic and chemotactic signaling.

Both platelet-derived growth factor (PDGF) A- and B-chains are expressed in mammalian neurons, but their precise roles still remain to be clarified. In the present studies, we examined the expression of two PDGF receptor genes in human tumor cell lines derived from neural crest. The expression of alpha and/or beta PDGF receptors was detected in a wide variety of neural crest-derived human tumor cell lines such as neuroblastoma, primitive neuroectodermal tumor, and Ewing's sarcoma by RNA blot analysis, and confirmed by immunoblot analysis. We have also demonstrated that PDGF receptors on the human neuroblastoma cell lines were biologically functional. Accordingly, chemotactic and mitogenic activities were induced by either PDGF-AA or PDGF-BB in serum-free medium. PDGF isoforms as well as nerve growth factor induced morphological changes showing neuronal cell maturation. Moreover, PDGF coordinately increased the levels of the transcript of the midsize neurofilament gene. The neuroblastoma cell lines also expressed the transcripts of PDGF A- and B-chains. These findings suggest that PDGF isoforms are involved not only in the promotion of the neuroblastoma cell growth, but also in neuronal cell migration, growth, and differentiation in human brain development.

Peroxisome assembly factor 1: nonsense mutation in a peroxisome-deficient Chinese hamster ovary cell mutant and deletion analysis.

A cDNA encoding 35-kDa peroxisome assembly factor 1 (PAF-1), a peroxisomal integral membrane protein, was cloned from Chinese hamster ovary (CHO) cells and sequenced. The CHO PAF-1 comprised 304 amino acids, one residue shorter than rat or human PAF-1, and showed high homology to rat and human PAF-1: 90 and 86% at the nucleotide sequence level and 92 and 90% in amino acid sequence, respectively. PAF-1 from these three species contains a conserved cysteine-rich sequence at the C-terminal region which is exactly the same as that of a novel cysteine-rich RING finger motif family. PAF-1 cDNA from a peroxisome-deficient CHO cell mutant, Z65 (T. Tsukamoto, S. Yokota, and Y. Fujiki, J. Cell Biol. 110:651-660, 1990), contained a nonsense mutation at the codon for Trp-114, resulting in premature termination. Truncation in PAF-1 of either 19 amino acids from the N terminus or 92 residues from the C terminus maintained the peroxisome assembly-restoring activity when tested in both the Z65 mutant and the fibroblasts from a Zellweger patient. In contrast, deletion of 27 or 102 residues from the N or C terminus eliminated the activity. PAF-1 is encoded by free polysomal RNA, consistent with a general rule for biogenesis of peroxisomal proteins, including membrane polypeptides, implying the posttranslational transport and integration of PAF-1 into peroxisomal membrane.

PEX12, the pathogenic gene of group III Zellweger syndrome: cDNA cloning by functional complementation on a CHO cell mutant, patient analysis, and characterization of PEX12p.

Rat PEX12 cDNA was isolated by functional complementation of peroxisome deficiency of a mutant CHO cell line, ZP109 (K. Okumoto, A. Bogaki, K. Tateishi, T. Tsukamoto, T. Osumi, N. Shimozawa, Y. Suzuki, T. Orii, and Y. Fujiki, Exp. Cell Res. 233:11-20, 1997), using a transient transfection assay and an ectopic, readily visible marker, green fluorescent protein. This cDNA encodes a 359-amino-acid membrane protein of peroxisomes with two transmembrane segments and a cysteine-rich zinc finger, the RING motif. A stable transformant of ZP109 with the PEX12 was morphologically and biochemically restored for peroxisome biogenesis. Pex12p was shown by expression of bona fide as well as epitope-tagged Pex12p to expose both N- and C-terminal regions to the cytosol. Fibroblasts derived from patients with the peroxisome deficiency Zellweger syndrome of complementation group III (CG-III) were also complemented for peroxisome biogenesis with PEX12. Two unrelated patients of this group manifesting peroxisome deficiency disorders possessed homozygous, inactivating PEX12 mutations: in one, Arg180Thr by one point mutation, and in the other, deletion of two nucleotides in codons for 291Asn and 292Ser, creating an apparently unchanged codon for Asn and a codon 292 for termination. These results indicate that the gene encoding peroxisome assembly factor Pex12p is a pathogenic gene of CG-III peroxisome deficiency. Moreover, truncation and site mutation studies, including patient PEX12 analysis, demonstrated that the cytoplasmically oriented N- and C-terminal parts of Pex12p are essential for biological function.

Peroxisome targeting signal type 1 (PTS1) receptor is involved in import of both PTS1 and PTS2: studies with PEX5-defective CHO cell mutants.

To investigate the mechanisms of peroxisome assembly and the molecular basis of peroxisome assembly disorders, we isolated and characterized a peroxisome-deficient CHO cell mutant, ZP139, which was found to belong to human complementation group II, the same group as that of our earlier mutant, ZP105. These mutants had a phenotypic deficiency in the import of peroxisomal targeting signal type 1 (PTS1) proteins. Amino-terminal extension signal (PTS2)-mediated transport, including that of 3-ketoacyl coenzyme A thiolase, was also defective in ZP105 but not in ZP139. PEX5 cDNA, encoding the PTS1 receptor (PTS1R), was isolated from wild-type CHO-K1 cells. PTS1R's deduced primary sequence comprised 595 amino acids, 7 amino acids less than the human homolog, and contained seven tetratricopeptide repeat (TPR) motifs at the C-terminal region. Chinese hamster PTS1R showed 94, 28, and 24% amino acid identity with PTS1Rs from humans, Pichia pastoris, and Saccharomyces cerevisiae, respectively. A PTS1R isoform (PTS1RL) with 632 amino acid residues was identified in CHO cells; for PTS1R, 37 amino acids were inserted between residues at positions 215 and 216 of a shorter isoform (PTS1RS). Southern blot analysis of CHO cell genomic DNA suggested that these two isoforms are derived from a single gene. Both types of PEX5 complemented impaired import of PTS1 in mutants ZP105 and ZP139. PTS2 import in ZP105 was rescued only by PTS1RL. This finding strongly suggests that PTS1RL is also involved in the transport of PTS2. Mutations in PEX5 were determined by reverse transcription-PCR: a G-to-A transition resulted in one amino acid substitution: Gly298Glu of PTS1RS (G335E of PTS1RL) in ZP105 and Gly485Glu of PTS1RS (G522E of PTS1RL) in ZP139. Both mutations were in the TPR domains (TPR1 and TPR6), suggesting the functional consequence of these domains in protein translocation. The implications of these mutations are discussed.