Neutropenia and survival outcomes in metastatic colorectal cancer patients treated with trifluridine/tipiracil in the RECOURSE and J003 trials.

BACKGROUND: The phase II J003 (N = 169) and phase III RECOURSE (N = 800) trials demonstrated a significant improvement in survival with trifluridine (FTD)/tipiracil (TPI) versus placebo in patients with refractory metastatic colorectal cancer. This post hoc analysis investigated pharmacokinetic data of FTD/TPI exposure and pharmacodynamic markers, such as chemotherapy-induced neutropenia (CIN) and clinical outcomes. PATIENTS AND METHODS: A total of 210 patients from RECOURSE were enrolled in this substudy. A limited sampling approach was used, with three pharmacokinetic samples drawn on day 12 of cycle 1. Patients were categorized as being above or below the median area under the plasma concentration-time curve (AUC) for FTD and TPI. We conducted a post hoc analysis using the entire RECOURSE population to determine the correlations between CIN and clinical outcome. We then carried out a similar analysis on the J003 trial to validate the results. RESULTS: In the RECOURSE subset, patients in the high FTD AUC group had a significantly increased CIN risk. Analyses of the entire population demonstrated that FTD/TPI-treated patients with CIN of any grade in cycles 1 and 2 had significantly longer median overall survival (OS) and progression-free survival (PFS) than patients who did not develop CIN and patients in the placebo group. Patients who required an FTD/TPI treatment delay had increased OS and PFS versus those in the placebo group and those who did not develop CIN. Similar results were obtained in the J003 cohort. CONCLUSIONS: In RECOURSE, patients with higher FTD drug exposure had an increased CIN risk. FTD/TPI-treated patients who developed CIN had improved OS and PFS versus those in the placebo group and those who did not develop CIN. Similar findings were reported in the J003 cohort, thus validating the RECOURSE results. The occurrence of CIN may be a useful predictor of treatment outcomes for FTD/TPI-treated patients. CLINICALTRIALS. GOV IDENTIFIER: NCT01607957 (RECOURSE). JAPAN PHARMACEUTICAL INFORMATION CENTER NUMBER: JapicCTI-090880 (J003).

Crystallization and vitrification of electrons in a glass-forming charge liquid.

Charge ordering (CO) is a phenomenon in which electrons in solids crystallize into a periodic pattern of charge-rich and charge-poor sites owing to strong electron correlations. This usually results in long-range order. In geometrically frustrated systems, however, a glassy electronic state without long-range CO has been observed. We found that a charge-ordered organic material with an isosceles triangular lattice shows charge dynamics associated with crystallization and vitrification of electrons, which can be understood in the context of an energy landscape arising from the degeneracy of various CO patterns. The dynamics suggest that the same nucleation and growth processes that characterize conventional glass-forming liquids guide the crystallization of electrons. These similarities may provide insight into our understanding of the liquid-glass transition.

Randomized phase III study of bevacizumab plus FOLFIRI and bevacizumab plus mFOLFOX6 as first-line treatment for patients with metastatic colorectal cancer (WJOG4407G).

BACKGROUND: FOLFIRI and FOLFOX have shown equivalent efficacy for metastatic colorectal cancer (mCRC), but their comparative effectiveness is unknown when combined with bevacizumab. PATIENTS AND METHODS: WJOG4407G was a randomized, open-label, phase III trial conducted in Japan. Patients with previously untreated mCRC were randomized 1:1 to receive either FOLFIRI plus bevacizumab (FOLFIRI + Bev) or mFOLFOX6 plus bevacizumab (mFOLFOX6 + Bev), stratified by institution, adjuvant chemotherapy, and liver-limited disease. The primary end point was non-inferiority of FOLFIRI + Bev to mFOLFOX6 + Bev in progression-free survival (PFS), with an expected hazard ratio (HR) of 0.9 and non-inferiority margin of 1.25 (power 0.85, one-sided α-error 0.025). The secondary end points were response rate (RR), overall survival (OS), safety, and quality of life (QoL) during 18 months. This trial is registered to the University Hospital Medical Information Network, number UMIN000001396. RESULTS: Among 402 patients enrolled from September 2008 to January 2012, 395 patients were eligible for efficacy analysis. The median PFS for FOLFIRI + Bev (n = 197) and mFOLFOX6 + Bev (n = 198) were 12.1 and 10.7 months, respectively [HR, 0.905; 95% confidence interval (CI) 0.723-1.133; P = 0.003 for non-inferiority]. The median OS for FOLFIRI + Bev and mFOLFOX6 + Bev were 31.4 and 30.1 months, respectively (HR, 0.990; 95% CI 0.785-1.249). The best overall RRs were 64% for FOLFIRI + Bev and 62% for mFOLFOX6 + Bev. The common grade 3 or higher adverse events were leukopenia (11% in FOLFIRI + Bev/5% in mFOLFOX6 + Bev), neutropenia (46%/35%), diarrhea (9%/5%), febrile neutropenia (5%/2%), peripheral neuropathy (0%/22%), and venous thromboembolism (6%/2%). The QoL assessed by FACT-C (TOI-PFC) and FACT/GOG-Ntx was favorable for FOLFIRI + Bev during 18 months. CONCLUSION: FOLFIRI plus bevacizumab was non-inferior for PFS, compared with mFOLFOX6 plus bevacizumab, as the first-line systemic treatment for mCRC. CLINICAL TRIALS NUMBER: UMIN000001396.

Characterization of Transporters in the Hepatic Uptake of TAK-475 M-I, a Squalene Synthase Inhibitor, in Rats and Humans.

TAK-475 (lapaquistat acetate) is a squalene synthase inhibitor and M-I is a pharmacologically active metabolite of TAK-475. Preclinical pharmacokinetic studies have demonstrated that most of the dosed TAK-475 was hydrolyzed to M-I during the absorption process and the concentrations of M-I in the liver, the main organ of cholesterol biosynthesis, were much higher than those in the plasma after oral administration to rats. In the present study, the mechanism of the hepatic uptake of M-I was investigated.The uptake studies of (14)C-labeled M-I into rat and human hepatocytes indicated that the uptakes of M-I were concentrative, temperature-dependent and saturable in both species with Km values of 4.7 and 2.8 µmol/L, respectively. M-I uptake was also inhibited by cyclosporin A, an inhibitor for hepatic uptake transporters including organic anion transporting polypeptide (OATP). In the human hepatocytes, M-I uptake was hardly inhibited by estrone 3-sulfate as an inhibitor for OATP1B1, and most of the M-I uptake was Na(+)-independent. Uptake studies using human transporter-expressing cells revealed the saturable uptake of M-I for OATP1B3 with a Km of 2.13 µmol/L. No obvious uptake of M-I was observed in the OATP1B1-expressing cells.These results indicated that M-I was taken up into hepatocytes via transporters in both rats and humans. OATP1B3 would be mainly involved in the hepatic uptake of M-I in humans. These findings suggested that hepatic uptake transporters might contribute to the liver-selective inhibition of cholesterol synthesis by TAK-475. This is the first to clarify a carrier-mediated hepatic uptake mechanism for squalene synthase inhibitors.

Pharmacokinetics of TAK-475, a Squalene Synthase Inhibitor, in Rats and Dogs.

The pharmacokinetics of TAK-475 (lapaquistat acetate), a squalene synthase inhibitor, was investigated in rats and dogs. After oral administration of (14)C-labeled TAK-475 ([(14)C]TAK-475) to rats and dogs at a dose of 10 mg/kg, the bioavailability (BA) was relatively low at 3.5 and 8.2%, respectively. The main component of the radioactivity in the plasma was M-I, which has a comparable pharmacological activity to TAK-475 in vitro. The radioactivity in the portal plasma after intraduodenal administration of [(14)C]TAK-475 to portal vein-cannulated rat was also mainly M-I, suggesting that most of the TAK-475 was hydrolyzed to M-I during the permeable process in the intestine. The concentrations of M-I in the liver, the main organ of cholesterol biosynthesis, were much higher than those in the plasma after oral administration of [(14)C]TAK-475 to rats. The main elimination route of the radioactivity was fecal excretion after oral administration of [(14)C]TAK-475 to rats and dogs, and the absorbed radioactivity was mainly excreted via the bile as M-I in rats. M-I excreted into the bile was partially subjected to enterohepatic circulation. These results suggest that although the BA values of TAK-475 are low, M-I can exert compensatory pharmacological effects in the animals. These pharmacokinetic characteristics in animals were also confirmed in the clinical studies. The evaluation of M-I disposition is important for the pharmacokinetics, pharmacodynamics and toxicity of TAK-475 in animals and humans.

An internally and externally validated nomogram for predicting the risk of irinotecan-induced severe neutropenia in advanced colorectal cancer patients.

BACKGROUND: In Asians, the risk of irinotecan-induced severe toxicities is related in part to UGT1A1*6 (UGT, UDP glucuronosyltransferase) and UGT1A1*28, variant alleles that reduce the elimination of SN-38, the active metabolite of irinotecan. We prospectively studied the relation between the UGT1A1 genotype and the safety of irinotecan-based regimens in Japanese patients with advanced colorectal cancer, and then constructed a nomogram for predicting the risk of severe neutropenia in the first treatment cycle. METHODS: Safety data were obtained from 1312 patients monitored during the first 3 cycles of irinotecan-based regimen in a prospective observational study. In development of the nomogram, multivariable logistic regression analysis was used to test the associations of candidate factors to severe neutropenia in the first cycle. The final nomogram based on the results of multivariable analysis was constructed and validated internally using a bootstrapping technique and externally in an independent data set (n=350). RESULTS: The UGT1A1 genotype was confirmed to be associated with increased risks of irinotecan-induced grade 3 or 4 neutropenia and diarrhoea. The final nomogram included type of regimen, administered dose of irinotecan, gender, age, UGT1A1 genotype, Eastern Cooperative Oncology Group performance status, pre-treatment absolute neutrophil count, and total bilirubin level. The model was validated both internally (bootstrap-adjusted concordance index, 0.69) and externally (concordance index, 0.70). CONCLUSIONS: Our nomogram can be used before treatment to accurately predict the probability of irinotecan-induced severe neutropenia in the first cycle of therapy. Additional studies should evaluate the effect of nomogram-guided dosing on efficacy in patients receiving irinotecan.

Bonding and electronic states of boron in silicon nanowires characterized by an infrared synchrotron radiation beam.

The infrared synchrotron radiation (IR-SR) beamline of SPring-8 as an IR light source was applied to characterize boron (B) atoms in silicon nanowires (SiNWs). The use of an IR-SR beam with much higher brilliance than conventional IR light sources and a wide range of wavenumbers from visible to far IR regions made it possible to detect a local vibrational mode of B in SiNWs. The use of this technique has also made it possible to detect other IR peaks related to transitions of a bound hole from the ground state of a B acceptor atom to excited states, clarifying the electronic state of B acceptors in SiNWs.

Survival analysis of platinum-refractory patients with advanced esophageal cancer treated with docetaxel or best supportive care alone: a retrospective study.

The survival benefit of second-line chemotherapy with docetaxel in platinum-refractory patients with advanced esophageal cancer (AEC) remains unclear. A retrospective analysis of AEC patients with Eastern Cooperative Oncology Group performance status (PS)≤2 was performed, and major organ functions were preserved, who determined to receive docetaxel or best supportive care (BSC) alone after failure of platinum-based chemotherapy. The post-progression survival (PPS), defined as survival time after disease progression following platinum-based chemotherapy, was analyzed by multivariate Cox regression analysis using factors identified as significant in univariate analysis of various 20 characteristics (age, sex, PS, primary tumor location, etc) including Glasgow prognostic score (GPS), which is a well-known prognostic factor in many malignant tumors. Sixty-six and 45 patients were determined to receive docetaxel and BSC between January 2007 and December 2011, respectively. The median PPS was 5.4 months (95% confidence interval [CI] 4.8-6.0) in the docetaxel group and 3.3 months (95% CI 2.5-4.0) in the BSC group (hazard ratio [HR] 0.56, 95% CI 0.38-0.84, P=0.005). Univariate analysis revealed six significant factors: treatment, PS, GPS, number of metastatic organs, liver metastasis, and bone metastasis. Multivariate analysis including these significant factors revealed three independent prognostic factors: docetaxel treatment (HR 0.62, 95% CI 0.39-0.99, P=0.043), better GPS (HR 0.61, 95% CI 0.46-0.81, P=0.001), and no bone metastasis (HR 0.31, 95% CI 0.15-0.68, P=0.003). There was a trend for PPS in favor of the docetaxel group compared with patients who refused docetaxel treatment in the BSC group (adjusted HR 0.61, 95% CI 0.29-1.29, P=0.20). Docetaxel treatment may have prolonged survival in platinum-refractory patients with AEC.

Oxidation resistance 1 is essential for protection against oxidative stress and participates in the regulation of aging in Caenorhabditis elegans.

Human oxidation resistance 1 (OXR1) functions in protection against oxidative damage and its homologs are highly conserved in eukaryotes examined so far, but its function still remains uncertain. In this study, we identified a homolog (LMD-3) of human OXR1 in the nematode Caenorhabditis elegans (C. elegans). The expressed LMD-3 was able to suppress the mutator phenotypes of E. coli mutMmutY and mutT mutants. Purified LMD-3 did not have enzymatic activity against 8-oxoG, superoxide dismutase (SOD), or catalase activities. Interestingly, the expression of LMD-3 was able to suppress the methyl viologen or menadione sodium bisulfite-induced expression of soxS and sodA genes in E. coli. The sensitivity of the C. elegans lmd-3 mutant to oxidative and heat stress was markedly higher than that of the wild-type strain N2. These results suggest that LMD-3 protects cells against oxidative stress. Furthermore, we found that the lifespan of the C. elegans lmd-3 mutant was significantly reduced compared with that of the N2, which was resulted from the acceleration of aging. We further examined the effects of deletions in other oxidative defense genes on the properties of the lmd-3 mutant. The deletion of sod-2 and sod-3, which are mitochondrial SODs, extended the lifespan of the lmd-3 mutant. These results indicate that, in cooperation with mitochondrial SODs, LMD-3 contributes to the protection against oxidative stress and aging in C. elegans.

Determination of the absolute chirality of tellurium using resonant diffraction with circularly polarized x-rays.

Many proteins, sugars and pharmaceuticals crystallize into two forms that are mirror images of each other (enantiomers) like our right and left hands. Tellurium is one enantiomer having a space group pair, P3(1)21 (right-handed screw) and P3(2)21 (left-handed screw). X-ray diffraction with dispersion correction terms has been playing an important role in determining the handedness of enantiomers for a long time. However, this approach is not applicable for an elemental crystal such as tellurium or selenium. We have demonstrated that positive and negative circularly polarized x-rays at the resonant energy of tellurium can be used to absolutely distinguish right from left tellurium. This method is applicable to chiral motifs that occur in biomolecules, liquid crystals, ferroelectrics and antiferroelectrics, multiferroics, etc.

Optical probe of carrier doping by X-ray irradiation in the organic dimer Mott insulator kappa-(BEDT-TTF)_{2}Cu[N(CN)_{2}]Cl.

We investigated the infrared optical spectra of an organic dimer Mott insulator kappa-(BEDT-TTF)_{2}Cu[N(CN)_{2}]Cl, which was irradiated with x rays. We observed that the irradiation caused a large spectral weight transfer from the midinfrared region, where interband transitions in the dimer and Mott-Hubbard bands take place, to a Drude part in a low-energy region; this caused the Mott gap to collapse. The increase of the Drude part indicates a carrier doping into the Mott insulator due to irradiation defects. The strong redistribution of the spectral weight demonstrates that the organic Mott insulator is very close to the phase border of the bandwidth-controlled Mott-insulator-metal transition.

Impacts of excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase, and epidermal growth factor receptor on the outcomes of patients with advanced gastric cancer.

Using laser-captured microdissection and a real-time RT-PCR assay, we quantitatively evaluated mRNA levels of the following biomarkers in paraffin-embedded gastric cancer (GC) specimens obtained by surgical resection or biopsy: excision repair cross-complementing gene 1 (ERCC1), dihydropyrimidine dehydrogenase (DPD), methylenetetrahydrofolate reductase (MTHFR), epidermal growth factor receptor (EGFR), and five other biomarkers related to anticancer drug sensitivity. The study group comprised 140 patients who received first-line chemotherapy for advanced GC. All cancer specimens were obtained before chemotherapy. In patients who received first-line S-1 monotherapy (69 patients), low MTHFR expression correlated with a higher response rate (low: 44.9% vs high: 6.3%; P=0.006). In patients given first-line cisplatin-based regimens (combined with S-1 or irinotecan) (43 patients), low ERCC1 correlated with a higher response rate (low: 55.6% vs high: 18.8%; P=0.008). Multivariate survival analysis of all patients demonstrated that high ERCC1 (hazard ratio (HR): 2.38 (95% CI: 1.55-3.67)), high DPD (HR: 2.04 (1.37-3.02)), low EGFR (HR: 0.34 (0.20-0.56)), and an elevated serum alkaline phosphatase level (HR: 1.00 (1.001-1.002)) were significant predictors of poor survival. Our results suggest that these biomarkers are useful predictors of clinical outcomes in patients with advanced GC.

A phase I study of S-1 combined with weekly cisplatin for metastatic gastric cancer in an outpatient setting.

A dose-escalation study was conducted for patients with metastatic gastric cancer to determine the recommended dose of weekly intravenous (i.v.) cisplatin combined with a fixed dose of a new oral dihydropyrimidine dehydrogenase-inhibitory fluoropyrimidine, S-1, on an outpatient basis. Secondary endpoints were to define the toxicity profile and to determine tumour responses. S-1 was fixed at a dose of 70 mg/m(2)/day and was administered for 2 weeks followed by a 1-week rest. Three dose levels of cisplatin (10, 15 and 20 mg/m(2)) were studied. Cisplatin was infused over 30 min on days 1 and 8. 20 patients were enrolled. No dose-limiting toxicities (DLTs) were recorded during the administration of cisplatin up to 20 mg/m(2), except for grade 3 diarrhoea and stomatitis in one patient at dose level 3. No grade 4 adverse events occurred. However, grade 2 gastrointestinal adverse reactions, such as nausea and anorexia, were seen in 7 of 13 patients at dose level 3 within the first two treatment cycles. This was determined to be the maximum acceptable level that would not negate the advantages observed with use of an oral drug such as S-1. An objective tumour response was seen at all dose levels, and the overall response rate in the 18 patients evaluated was 61%. A higher response rate of 78% was observed in 9 patients who had received no prior chemotherapy. Oral S-1 with weekly cisplatin is a feasible and promising combination regimen that is appropriate for an outpatient setting. A randomised phase II study comparing this combination with S-1 alone in chemo-nai;ve patients is warranted.

[Two cases of pyogenic cervical discitis presenting tetraparesis].

The authors reported two cases of pyogenic cervical discitis presenting tetraparesis. Case 1: A 66-year-old male patient entered the hospital because of tetraparesis. Two weeks before the hospitalization, he had become feverish and awakened with motor weakness in all extremities. Magnetic Resonance Imaging (MRI) study revealed a lesion filling the anterior epidural space from C4 to C6 levels and posterior displacement of the spinal cord. Findings suggesting discitis of C5/6 and osteomyelitis of C5 and C6 were also obtained on MRI. These findings suggested that the tetraparesis was caused by cord compression by the epidural abscess as the acute stage of pyogenic spinal infection. On the day following admission, surgical removal of the epidural abscess and of the infected bodies was performed. Spinal fusion through C4 to C7 was also carried out with iliac bone graft. Antibiotic administration and Halo-vest application were performed after the operation. The postoperative course was good and the tetraparesis had completely disappeared within 12 months after the operation. Case 2: A 60-year-old male patient entered the hospital because of tetraparesis. Since 6 weeks before the hospitalization, he had become feverish and suffered from pain in the neck. He had also awakened with motor weakness of all extremities. The tetraparesis was progressive. Plain X-ray films of the cervical spine showed destructive change of C5 and C6 and kyphotic displacement. An epidural abscess of the cervical spine at the level of C4 to C6, discitis of C5/6 and osteomyelitis of C5 and C6 were diagnosed on MRI findings. The disarranged kyphotic vertebral bodies and the epidural abscess caused posterior displacement of the spinal cord. Based on these findings, it was concluded that the abscess and the kyphotic change of the bodies had been induced by spinal infection in the subacute stage. On the 8th hospital day, surgical removal of the anterior portion of the infected bodies as well as fusion of the vertebral column from C4 to C7 was performed. Iliac bone was used for the fusion graft. Postoperative administration of antibiotics and Halo-vest application for external fixation were carried out. On the 7th postoperative day, symptoms caused by radiculopathy of the left C5 appeared, but gradually ameliorated. The patient was free from motor weakness in the 8th month after the surgical treatment. Surgical intervention is a useful treatment for pyogenic cervical discitis with symptoms due to compression of the spinal cord both in the acute and subacute stages.

Continuous measurement of tympanic temperature with a new infrared method using an optical fiber.

The purpose of this study was to investigate the utility of an infrared tympanic thermometry by using an optical fiber for measuring tympanic temperature (Tty). In the head cooling and facial fanning tests during normothermia, right Tty measured by this method (infrared-Tty) and esophageal temperature (Tes) were not affected by decreased temple and forehead skin temperatures, suggesting that the infrared sensor in this system measured the infrared radiation from the tympanic membrane selectively. Eight male subjects took part in passive-heat-stress and progressive-exercise tests. No significant differences among infrared-Tty, the left Tty measured by thermistor (contact-Tty), and Tes were observed at rest or at the end of each experiment, and there was no significant difference in the increase in these core temperatures from rest to the end. Furthermore, there were no significant differences in the core temperature threshold at the onset of sweating and slope (the relationship of sweating rate vs. infrared-Tty and vs. contact-Tty). These results suggest that this method makes it possible to measure Tty accurately, continuously, and more safely.

Six-month repeated oral toxicity study of NK-104 in rats.

NK-104 is a novel potent inhibitor of the rate-limiting enzyme in cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and has been shown to be a highly effective agent in lowering blood cholesterol. In the present study, NK-104 was orally administered to Wistar rats at a dose of 0.3, 1, 3 or 010 mg/kg for 6 months for examination of toxicity. Additional recovery groups of 8 rats each of both sexes receiving 0 and 10 mg/kg were maintained without treatment for 1 month in order to assess recovery. As a result, no toxicological changes were observed in general signs, body weight, food intake, ophthalmological examination, urinalysis, hematological and blood chemical examinations for organ weights. An autopsy revealed thickening of the forestomach mucosa in both sexes at a dose of 1 mg/kg or more. This change was microscopically recognized as hyperkeratosis and hypertrophy of the spinous layer associated with both cell infiltration of the mucosal propria and edema of sub-mucosa in the forestomach in both sexes at doses of 3 and 10 mg/kg. Forestomach changes were not observed in any cases after 1 month cessation of drug treatment. The non-toxic dose of NK-104 in the 6-month repeated oral toxicity study in rats is estimated to be 1 mg/kg/day.

[A family with facioscapulohumeral muscular dystrophy and hereditary long QT syndrome].

We describe a family with facioscapulohumeral muscular dystrophy (FSHD) and hereditary long QT syndrome (LQT) for three generations. The proband, a 50-year-old woman, had noticed difficulty in raising the upper extremities since the age of 40. At the age of 48, she was admitted to our hospital because of arrhythmia attack. She, her mother, and one of her three children were diagnosed as having LQT. These three individuals and the proband's two siblings were clinically diagnosed as having FSHD which was confirmed by genetic analysis using EcoR1. FSHD is an autosomal dominant disorder and the gene locus is mapped to chromosome 4q35-ter, but the gene has not been isolated. LQT is a group of disorders which cause syncope and sudden death from ventricular arrhythmia in an autosomal dominant fashion. Four loci for this syndrome (LQT1-4) have been known, and three of the genes have been shown to encode ion-channels. Genetic analysis of the proband failed to detect any of previously known mutations in the LQT1, LQT2, and LQT3 genes. The locus for LQT4 has been mapped to chromosome 4q25-7. There have so far been no report of FSHD associated with LQT. Although the pathogenesis is unclear, we speculate that these two diseases are linked each other on chromosome 4q.

Alteration of N-linked oligosaccharide structures of human chorionic gonadotropin beta-subunit by disruption of disulfide bonds.

The human chorionic gonadotropin beta-subunit (hCGbeta) is a glycoprotein in which 12 cysteine residues pair to form six intramolecular disulfide bonds. In order to elucidate the effect of each disulfide bond on glycosylation of the molecule, we analysed structures of asparagine-linked oligosaccharides of various recombinant hCGbeta produced in Chinese hamster ovary (CHO) cells: wild-type hCGbeta (betaWT) and mutants in which any one of the six intramolecular disulfide bonds had been disrupted by site-directed mutagenesis. SDS-PAGE analysis of betaWT and these mutants before and after digestion with endoglycosidase F and H revealed structural changes in the oligosaccharide moieties of some mutants. In addition, structural analysis of oligosaccharides obtained from metabolically labeled betaWT and a mutant showed that the mutant contained additional high mannose type oligosaccharides. These results suggest that elimination of a specific disulfide bond, resulting in a change in the protein conformation, disturbs the normal assembly of the mature complex type oligosaccharides in the hCGbeta molecule.

Successful pregnancy after intracytoplasmic injection of a single testicular spermatozoon into a human oocyte: a case report.

Testicular sperm was retrieved from a man with an epididymal obstruction, and an intracytoplasmic sperm injection was made into his wife's oocytes. In 4 mature oocytes treated, 2 fertilized eggs were obtained, and a clinical pregnancy was established with embryo transfers. Our experience demonstrated the possibility of fertilization and conception with testicular sperm.

Nucleotide sequence of the cDNA encoding nucleoside diphosphate kinase II from spinach leaves.

The primary structure of nucleoside diphosphate (NDP) kinase II, one of the two isozymes found in spinach leaves, has been deduced from its cDNA sequence. NDP kinase II comprises 233 amino acid residues and has a molecular mass of 26,107 Da, which is larger than that of the purified NDP kinase II subunits (18 kDa) by about 8 kDa, suggesting that NDP kinase II might be post-translationally processed. Homology was found between the sequence of spinach NDP kinase II, and the sequences of spinach NDP kinase I, rat NDP kinases alpha and beta, Dictyostelium discoideum NDP kinase, the human Nm23-H1 and Nm23-H2 proteins and the awd protein of Drosophila melanogaster.