Objective tumor response to denosumab in patients with giant cell tumor of bone: a multicenter phase II trial.

BACKGROUND: Giant cell tumor of bone (GCTB) is a rare primary bone tumor, characterized by osteoclast-like giant cells that express receptor activator of nuclear factor-kappa B (RANK), and stromal cells that express RANK ligand (RANKL), a key mediator of osteoclast activation. A RANKL-specific inhibitor, denosumab, was predicted to reduce osteolysis and control disease progression in patients with GCTB. PATIENTS AND METHODS: Seventeen patients with GCTB were enrolled. Patients were treated with denosumab at 120 mg every 4 weeks, with a loading dose of 120 mg on days 8 and 15. To evaluate efficacy, objective tumor response was evaluated prospectively by an independent imaging facility on the basis of prespecified criteria. RESULTS: The proportion of patients with an objective tumor response was 88% based on best response using any tumor response criteria. The proportion of patients with an objective tumor response using individual response criteria was 35% based on the modified Response Evaluation Criteria in Solid Tumors (RECIST) criteria, 82% based on the modified European Organization for Research and Treatment of Cancer (EORTC) criteria, and 71% based on inverse Choi criteria. The median time of study treatment was 13.1 months. CONCLUSION: The findings demonstrate that denosumab has robust clinical efficacy in the treatment of GCTB.

Appropriateness of surgical antimicrobial prophylaxis in Japanese university hospitals.

BACKGROUND: Surgical antimicrobial prophylaxis (SAP) is one of the major purposes of antimicrobial use. AIM: To determine the adherence to the Japanese SAP guidelines in Japanese university hospitals. METHODS: This was a retrospective cohort study including 15 general hospitals and one dental university hospital. Up to three cases of 18 designated surgeries were evaluated regarding adherence to Japanese SAP guidelines: selection of antibiotics, timing of administration, re-dosing intervals, and duration of SAP. When all items were appropriate, surgery was defined as 'appropriate'. FINDINGS: In total, 688 cases (22-45 cases per surgery) were included. The overall appropriateness was 46.8% (322/688), and the appropriateness of each surgery ranged from 8.0% (2/25, cardiac implantable electronic device implantation) to 92.1% (35/38, distal gastrectomy). The appropriateness of each item was as follows: pre/intraoperative selections, 78.5% (540/688); timing of administrations, 96.0% (630/656); re-dosing intervals, 91.6% (601/656); postoperative selection, 78.9% (543/688); and duration of SAP, 61.4% (423/688). The overall appropriateness of hospitals ranged from 17.6% (9/51) to 73.3% (33/45). The common reasons for inappropriateness were the longer duration (38.5%, 265/688) and choice of antibiotics with a non-optimal antimicrobial spectrum before/during, and after surgery (19.0%, 131/688 and 16.9%, 116/688, respectively), compared to the guideline. CONCLUSIONS: Adherence to the guidelines differed greatly between the surgeries and hospitals. Large-scale multi-centre surveillance of SAP in Japanese hospitals is necessary to identify inappropriate surgeries, factors related to the appropriateness, and incidences of surgical site infections.

Non-D Rh antibodies appearing after apheresis platelet transfusion: stimulation by red cells or microparticles?

BACKGROUND: Apheresis platelets (APs) have gained favour over whole blood-derived platelets on the presumption that they are less likely to provoke alloimmunization to red-blood-cell antigens. CASE REPORTS: Non-D Rh antibodies appeared in three patients after apheresis platelet transfusion. Anti-C and anti-E arose in two female patients with previous antigen exposure. Both anti-c and anti-E arose in a male recipient with no prior transfusion history. MATERIALS AND METHODS: Fifty APs were analysed for residual RBCs and RBC-derived microparticles, using samples obtained from a local blood centre. Cells and microparticles were quantified with a flow cytometry gating scheme, using PE-labelled anti-CD235a (glycophorin A) and FITC-labelled anti-CD41a (platelet gp IIb/IIIa) to distinguish lineage. RESULTS: Apheresis platelets were found to contain a mean of 7·5×10(6) (95% C.I. [6·3-8·5×10(6) ]) RBCs on one manufacturer's device and 5·2×10(6) (95% C.I. [4·0-6·3×10(6) ]) RBCs on another's. RBC-derived microparticles averaged 210·7×10(6) (95% C.I. [166·2-254·2×10(6) ]) on one manufacturer's device and 232·3×10(6) (95% C.I. [194·3-272·9×10(6) ]) on another's. These counts all correspond to volumes of <1 µl. CONCLUSION: Despite RBC contamination of APs below commonly accepted thresholds for Rh immunogenicity, AP transfusion can provoke non-D Rh antibody formation. RBC-derived microparticles, smaller but more numerous than RBCs, are volumetrically comparable and may be a hitherto underappreciated antibody stimulus. Further microparticle research will guide considerations of extended phenotypic matching of platelet components.

Five-year point prevalence survey of healthcare-associated infections and antimicrobial use in a Japanese university hospital.

BACKGROUND: Periodic point prevalence surveys (PPSs) provide a method for assessing changes in healthcare-associated infections (HAIs) and antimicrobial use over time. Following the introduction of an antimicrobial stewardship programme at Nagoya University Hospital (Aichi, Japan) a five-year PPS study was performed to highlight any epidemiological changes. METHODS: One-day PPSs were performed annually in July at Nagoya University Hospital. Data on patient characteristics, medical devices, active HAIs and antimicrobial use were collected using a standard data-collection form. RESULTS: A total of 4339 patients were included. Over the five-year study period the median patient age was 62 years, median duration of hospital admission was nine days, 9% of patients had an HAI and 35.2% received at least one antimicrobial. Overall there were 406 HAIs (95% confidence interval, 369-447) with surgical site infection, pneumonia and febrile neutropenia occurring most frequently. Enterobacterales were the most common pathogens (N = 78, 28.6%) and 32.1% were third-generation cephalosporin-resistant. Meropenem was the most frequently prescribed antimicrobial for HAIs. Surgical antimicrobial prophylaxis changed drastically, with shorter durations and a marked reduction in oral cephalosporin use. However, antimicrobials for medical prophylaxis gradually increased. CONCLUSIONS: This five-year PPS study shows consistent data for patient background, HAIs and causative pathogens and highlights changes in antimicrobial use during the era of the National Action Plan on Antimicrobial Resistance. To describe the epidemiology of Japanese hospitals by PPS, multicentre PPSs including in community hospitals should be performed annually.

Curettage and allograft reconstruction for giant cell tumours.

PURPOSE: To evaluate treatment outcomes in patients with giant cell tumours after curettage and allograft reconstruction and to identify the risk factors for poor oncological and functional outcome. METHODS: 29 patients with giant cell tumours of bone who underwent curettage and allograft reconstruction were retrospectively reviewed. The adjuvants used were heat treatment by electrocautery and hot water. Types of allograft used, time to bone union, complications, functional outcomes, and risk factors for poor function were analysed. RESULTS: The mean time to bone union was 2.8 (range, 1-5) months. In 7 patients the tumours recurred (6 within 2 years); the 5-year recurrence-free survival rate was 77%. Three recurrences were classified as grade III and 4 as grade II; recurrence and the Campanacci grade showed a trend towards association (p=0.06). Tumour in the distal femur was a risk factor for postoperative fracture (p=0.02). Functional outcomes were excellent in 20 patients, good in 6, fair in 2, and a failure in one. The risk factors for poor function were recurrence (p=0.002) and joint instability (p=0.008) but not the Campanacci grade (p=0.10) or postoperative fracture (p=0.76). Lung metastasis, infection, and non-union were not encountered. CONCLUSION: Despite a relatively high recurrence rate (24%), 26 (90%) of the 29 patients had excellent/good functional outcomes. We recommend the use of adjuvants and allografts for the management of giant cell tumours.

The first multi-centre point-prevalence survey in four Japanese university hospitals.

BACKGROUND: The Japanese government adopted a national action plan on antimicrobial resistance, which aims to reduce drug-resistant pathogens and antimicrobial use. A point-prevalence survey (PPS) is a useful surveillance method to gain information about hospital epidemiology; however, no multi-centre PPS has previously been performed in Japan. AIM: To investigate general information about hospital epidemiology, healthcare-associated infections (HCAIs), and antimicrobial use in multiple Japanese university hospitals. METHODS: In July 2016, a multi-centre PPS was conducted using a standardized protocol at four university hospitals in Japan. FINDINGS: A total of 3199 patients were included. Median age and duration of hospital stay were 64 years and 10 days, respectively. A total of 246 (7.7%; 95% confidence interval (CI): 6.8-8.7) patients had 256 active HCAIs, and 933 (29.2%; 95% CI: 27.6-30.8) patients received 1318 antimicrobials. Pneumonia and gastrointestinal system infection were the most common HCAIs (N = 42, 16.4%), and Enterobacteriaceae (N = 49, 30.8%) were the predominant causative organisms. Carbapenems (N = 52, 17.8%), anti-MRSA medications, and cephems with antipseudomonal activity were the most frequently prescribed antimicrobials for HCAIs. As surgical prophylaxis, 46 of 278 antimicrobials (16.5%) were administered orally. Proportions of HCAI and antimicrobial use in each hospital ranged from 4.8% to 9.5% and 19.3%-35.0%, respectively. CONCLUSION: This multi-centre PPS recorded detailed HCAI data and distinct antimicrobial use in Japanese university hospitals. Further surveillance is necessary to reduce HCAIs and formulate feasible plans to achieve the national action plan on antimicrobial resistance.

Structural and functional analysis of the rat malic enzyme gene promoter.

We have characterized sequences of genomic DNA 5' to the coding region of the rat malic enzyme gene. This sequence possesses neither TATA nor CCAAT sequences in their usual positions but is rich in GC residues. Sequences similar to those found in the regulatory regions of other genes are discussed. Deletion analyses have revealed that sequences +1 to -41 are sufficient to initiate expression, although inclusion of information up to -177 is necessary for maximal promoter activity.

A role for MHR1, a gene required for mitochondrial genetic recombination, in the repair of damage spontaneously introduced in yeast mtDNA.

A nuclear recessive mutant in Saccharomyces cerevisiae, mhr1-1, is defective in mitochondrial genetic recombination at 30 degrees C and shows extensive vegetative petite induction by UV irradiation at 30 degrees C or when cultivated at a higher temperature (37 degrees C). It has been postulated that mitochondrial DNA (mtDNA) is oxidatively damaged by by-products of oxidative respiration. Since genetic recombination plays a critical role in DNA repair in various organisms, we tested the possibility that MHR1 plays a role in the repair of oxidatively damaged mtDNA using an enzyme assay. mtDNA isolated from cells grown under standard (aerobic) conditions contained a much higher level of DNA lesions compared with mtDNA isolated from anaerobically grown cells. Soon after a temperature shift from 30 to 37 degrees C the number of mtDNA lesions increased 2-fold in mhr1-1 mutant cells but not in MHR1 cells. Malonic acid, which decreased the oxidative stress in mitochondria, partially suppressed both petite induction and the temperature-induced increase in the amount of mtDNA damage in mhr1-1 cells at 37 degrees C. Thus, functional mitochondria require active MHR1, which keeps the extent of spontaneous oxidative damage in mtDNA within a tolerable level. These observations are consistent with MHR1 having a possible role in mtDNA repair.

DNA binding mode of the Fab fragment of a monoclonal antibody specific for cyclobutane pyrimidine dimer.

Monoclonal antibodies specific for the cyclobutane pyrimidine dimer (CPD) are widely used for detection and quantification of DNA photolesions. However, the mechanisms of antigen binding by anti-CPD antibodies are little understood. Here we report NMR analyses of antigen recognition by TDM-2, which is a mouse monoclonal antibody specific for the cis - syn -cyclobutane thymine dimer (T[ c, s ]T). (31)P NMR and surface plasmon resonance data indicated that the epitope recognized by TDM-2 comprises hexadeoxynucleotides centered on the CPD. Chemical shift perturbations observed for TDM-2 Fab upon binding to d(T[ c, s ]T) and d(TAT[ c, s ]TAT) were examined in order to identify the binding sites for these antigen analogs. It was revealed that d(T[ c, s ]T) binds to the central part of the antibody-combining site, while the CPD-flanking nucleotides bind to the positively charged area of the V(H)domain via electrostatic interactions. By applying a novel NMR method utilizing a pair of spin-labeled DNA analogs, the orientation of DNA with respect to the antigen-binding site was determined: CPD-containing oligonucleotides bind to TDM-2 in a crooked form, draping the 3'-side of the nucleotides onto the H1 and H3 segments, with the 5'-side on the H2 and L3 segments. These data provide valuable information for antibody engineering of TDM-2.

Establishment and characterization of the human cholangiocarcinoma cell line HChol-Y1 in a serum-free, chemically defined medium.

A human cholangiocarcinoma cell line, designated as HChol-Y1, was established in a protein-free, chemically defined medium after a very short period of primary culture in 0.1% fetal bovine serum (FBS)-containing medium. The cell line has been propagated in this medium for 2 years. The cells grew as a monolayer and the doubling time was about 52 hours. Addition of FBS did not stimulate cell growth (population-doubling time = 50 hr) or increase saturation density. The cells grown in a protein-free medium secreted small amounts of carcinoembryonic antigen (CEA) and large amounts of carbohydrate antigen (CA) 19/9 (CEA: 12.5 +/- 2.1 ng/10(6) cells/48 hr; CA 19/9: 760 +/- 52 IU/10(6) cells/48 hr); these tumor markers were immunohistochemically demonstrated in HChol-Y1 cells. Addition of FBS slightly stimulated the production of CEA and CA 19/9. The HChol-Y1 cell line was xenotransplantable in athymic nude mice and increased the serum CEA and CA 19/9 levels in the tumor-bearing nude mice. For determination as to whether a human carcinoma cell line can proliferate and secrete CEA and CA 19/9 in synthetic medium without any protein supplements, the cells were cultivated long term (2 yr) in a protein-free, chemically defined medium. When this method of cultivation is used, it is easy to purify these substances from spent medium, because contaminating antigens such as FBS or other substances usually added to cultures are absent.

Schwannian cell differentiation of human neuroblastoma cell lines in vitro induced by bromodeoxyuridine.

In normal development the neural crest gives rise to sympathetic neuroblasts, sensory and autonomic ganglia, as well as Schwann cells. One tumor arising from this tissue is the neuroblastoma (NB), a malignancy of the adrenergic component of the sympathetic nervous system. Recent histological studies have shown that neuroblastomas can present with a schwannian cell component, rich in S100 protein. We have investigated the differentiation of NB cell lines, GOTO and RT-LN-1, into a schwannian cell phenotype using bromodeoxyuridine (BrdU). This agent induced morphological changes in these cell lines. Flat-epithelial cells were identified in the GOTO cell line and both flat-epithelial and neuronal phenotypes were found in the RT-LN-1 cell line. S100 protein (beta-Subunit) was induced in both cell lines after 18-25 days of BrdU treatment as determined by enzyme-linked immunoassay. In addition increase in the beta-subunit of S100 protein was identified in BrdU-treated flat-epithelial cells by indirect immunofluorescence using a monoclonal antibody specific for the beta-subunit of the protein. Cyclic nucleotide phosphodiesterase activity significantly increased in both BrdU-treated NB cell lines, as compared with nontreated cells. However no significant increase of glial fibrillary acidic protein in BrdU-treated cells was found either by enzyme-linked immunoassay or indirect immunofluorescence using a monoclonal antibody to glial fibrillary acidic protein. Thus, cells with Schwann cell characteristics can clearly be identified in the neuroblastoma cell lines after BrdU treatment. Fluorescence-activated cell sorting analysis revealed no quantitative changes in cell membrane antigens recognized by monoclonal antibodies UJ-13A (neuroectodermal associated antigen) and anti-Thy-1 (Thy-1) on BrdU treatment. In contrast, UJ-127-11 (neuroectodermal associated) decreased, and W6/32 and BB7.7 (HLA-ABC) and BBM.1 (beta 2-microglobulin) markedly increased in both BrdU-treated cell lines. No induction of L243 (HLA-DR), B7/21 (HLA-DP), and Genox 3.55 (HLA-DQ) was noted. The increased HLA-ABC (HLA class I) antigen may enable BrdU-treated NB cells to be recognized by cytotoxic T-cells. This may be related to the pathological evidence that NB patients whose tumors are rich in S100 protein have a better prognosis. Further studies on the potential of differentiation agents to induce a phenotypic change, that is associated with an improved prognosis for NB patients, are required.

Differential susceptibility of HLA class II antigens induced by gamma-interferon in human neuroblastoma cell lines.

Five neuroblastoma cell lines have been examined for the induction of HLA class II antigens by a recombinant gamma-interferon. The expression of HLA-DR and -DP was induced on up to 80% of cells in two of five neuroblastoma cell lines examined (KP-N-SI and KP-N-RT). Low expression of HLA-DR and -DP was induced on the SK-N-DZ line in the presence of recombinant gamma-interferon for 10 days. In contrast HLA-DQ was not induced on any of five neuroblastoma cell lines studied. HLA-DR and -DP antigen induction was reversible, falling to nondetectable levels when interferon was removed from the culture medium. The reinduction of interferon to the culture medium again induced HLA-DR and -DP antigen expression in a fashion similar to that originally observed. These results were confirmed by Northern blot analysis using a probe to HLA-DR alpha mRNA. Recombinant interferon appears to induce HLA class II expression at the level of gene transcription or posttranscription. The results also indicate that HLA-DR, -DP, and -DQ antigens are independently regulated. Treatment of neuroblastoma cell lines with gamma-interferon results in the induction of a differentiated phenotype. Although the cytokine gamma-interferon induces neurofilament expression in some of the cell lines, this was not the case for all lines studied. Thus no correlation could be established between the morphological differentiation and either HLA class II or neurofilament expression. In addition, no correlation between response to recombinant interferon and N-myc amplification was noted. The biological significance of HLA class II expression on neuroblastoma cell lines by gamma-interferon may be related to the differentiation stage of neuroblastoma cells or may enable gamma-interferon-treated neuroblastoma cells to be recognized by cytotoxic T-cells.

Identical expression of cell surface membrane antigens on two parent and eighteen cloned cell lines derived from two different neuroblastoma metastases of the same patient.

Two neuroblastoma cell lines established from tumor tissue taken from one individual are described. The first of these was established from a bone marrow aspirate (RT-BM) and the other from a right axillary lymph node (RT-LN) of a 1-yr 2-mo-old patient with Stage IV disease. The original lines were cloned in soft agar to yield six clones of the bone marrow-derived line (RT-BM 1-6) and 12 of the lymph node line (RT-LN 1-12). Chromosomal analysis of the original lines and clones showed they all have either identical or very similar karyotypes, with a deletion of chromosome 1p. Transmission electron microscopy indicates all contain neurosecretory (dense core) granules and neurotubules. In addition catecholamine metabolites of dopamine and noradrenaline have been identified. Different growth characteristics of the lymph node and bone marrow lines have been identified. RT-LN lines grow in a single cell layer with neurite processes, whereas bone marrow-derived lines form focal aggregates with neurite processes. In addition the colony-plating efficiency of the lymph node-derived lines is higher than those derived from bone marrow. Comparison of the cell surface antigen profile of the original tumor tissue, parent lines, and clones demonstrates they all bind seven of a panel of nine monoclonal antibodies. The expression of these antigens has remained stable in vitro for 25 passages undertaken over a 2-yr period. The definition of antigens that are expressed on the membranes of neuroblastoma cells in a stable form can aid in the differential diagnosis of neuroblastoma from other "small round cell tumors of childhood" and hopefully contribute to a greater understanding of the biology of this highly malignant tumor.

Altered expression of cell surface membrane antigens in a common acute lymphoblastic leukemia-associated antigen-expressing neuroblastoma cell line (SJ-N-CG) with morphological differentiation.

The expression of common acute lymphoblastic leukemia antigen (CALLA) on a human neuroblastoma cell line, SJ-N-CG, was demonstrated by indirect membrane immunofluorescence, complement-dependent cytotoxicity, and quantitative absorption, using two monoclonal antibodies (J-5 and BA-3) directed against CALLA. Immunoprecipitation of solubilized 125I-labeled membrane proteins from SJ-N-CG cells with J-5 antibody revealed a protein with a molecular weight of 100,000 as determined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Morphological differentiation of SJ-N-CG cells could be induced in the presence of 2.0 mM dibutyryl adenosine 3'-5'-cyclic monophosphoric acid for 10 days of culture. Changes in cell surface membrane antigens associated with morphological differentiation were studied by indirect immunofluorescence and complement-dependent cytotoxicity using a panel of seven monoclonal antibodies. Increases in the antigens recognized by BA-2 (detecting leukemia-associated antigen), anti-Thy-1, and antibody 390 (Thy-1 antigen) were found in "differentiated cells," while those detected by BA-1 (B-cell-associated antigen) and J-5 (CALLA) were unchanged. In contrast, the antitransferrin receptor defined by B3/25 was inhibited, and expression of B7/21-defined la-like antigen was not induced. Kinetic studies on antigenic alterations showed that the expression of BA-2-defined antigen rose on Day 2 and remained at the same level until Day 10. The expression of CALLA was not changed from Days 2 to 10. The augmentation of Thy-1 antigen was noted on Day 4 and reached the maximum on Day 10. These results show that dibutyryl adenosine 3'-5'-cyclic monophosphoric acid is capable of inducing phenotypic changes in SJ-N-CG cells. The changes of expression of some antigens on exposure of cells to dibutyryl adenosine 3'-5'-cyclic monophosphoric acid may enable us to have a greater understanding of the differentiation of neuroblastoma to a more mature ganglioneuroblastoma phenotype.

Diagnosis and treatment of bone metastasis: comprehensive guideline of the Japanese Society of Medical Oncology, Japanese Orthopedic Association, Japanese Urological Association, and Japanese Society for Radiation Oncology.

Diagnosis and treatment of bone metastasis requires various types of measures, specialists and caregivers. To provide better diagnosis and treatment, a multidisciplinary team approach is required. The members of this multidisciplinary team include doctors of primary cancers, radiologists, pathologists, orthopaedists, radiotherapists, clinical oncologists, palliative caregivers, rehabilitation doctors, dentists, nurses, pharmacists, physical therapists, occupational therapists, medical social workers, etc. Medical evidence was extracted from published articles describing meta-analyses or randomised controlled trials concerning patients with bone metastases mainly from 2003 to 2013, and a guideline was developed according to the Medical Information Network Distribution Service Handbook for Clinical Practice Guideline Development 2014. Multidisciplinary team meetings are helpful in diagnosis and treatment. Clinical benefits such as physical or psychological palliation obtained using the multidisciplinary team approaches are apparent. We established a guideline describing each specialty field, to improve understanding of the different fields among the specialists, who can further provide appropriate treatment, and to improve patients' outcomes.

Effect of acriflavine on the hexokinase isozyme pattern of a yeast, Hansenula jadinii.

Dried cells of a yeast, Hansenula jadinii, that had been cultured aerobically with acriflavine, contained three hexokinase isozymes and metabolized glucose at 0.6 M to produce ATP to phosphorylate nucleotides in the presence of a high concentration of phosphate. Dried cells cultured aerobically without acriflavine contained two hexokinase isozymes and could not metabolize glucose under the same conditions. Two of the isozymes of the yeast cultured with acriflavine were similar to isozymes of the yeast cultured without acriflavine. However, the third isozyme was resistant to a high phosphate concentration and caused regeneration of ATP through glycolysis and phosphorylation of nucleotides.

Pioglitazone attenuates the inhibitory effect of phorbol ester on epidermal growth factor receptor autophosphorylation and tyrosine kinase activity.

A new anti-diabetic drug, pioglitazone, was tested as to whether it could ameliorate the decreased kinase activity of epidermal growth factor (EGF) receptor induced by phorbol ester (PMA) in A431 cells. The treatment of A431 cells with PMA decreased the tyrosine kinase activity of EGF receptors to 37% of normal in autophosphorylation and to 24% in tyrosine kinase activity toward Glu/Tyre synthetic polymers. Co-incubation of the cells with pioglitazone and PMA improved the receptor tyrosine kinase activity to 81% of control. Pioglitazone treatment alone did not change the kinase activity of EGF receptors. Pioglitazone did not decrease the PMA-activated protein kinase C activity and did not affect the protein tyrosine phosphatases activity in A431 cells. These results suggest that pioglitazone may act as a specific antagonist to the inhibitory effect by protein kinase C on the EGF receptor tyrosine kinase.

A new Drosophila ultraviolet light-damaged DNA recognition endonuclease that selectively nicks a (6-4) photoproduct site.

We have previously described the purification of an ultraviolet light (UV) damage-specific DNA-binding protein from Drosophila melanogaster, designated D-DDB P1 [Nucleic Acids Res., 23 (1995) 2600-2607]. Here, we obtained highly purified D-DDB P1 from Drosophila Kc cells, and we found that D-DDB P1 is also a nuclease. D-DDB P1 can selectively bind to pyrimidine (6-4) pyrimidone photoproducts, and in the presence of Mg++, D-DDB P1 can catalyze an incision immediately on the 3' and 5' sides of the (6-4) photoproduct site.

Antibodies specific for (6-4) DNA photoproducts: cloning, antibody modeling and construction of a single-chain Fv derivative.

We have investigated a series of four monoclonal antibodies that specifically recognize pyrimidine (6-4) pyrimidone photoproducts. One of these antibodies (64M4), bound all four possible pyrimidine-pyrimidone photoadducts with equal affinities whereas the others (64M2, 64M3 and 64M5) were selective for TC and TT sequences. In addition, 64M5 had the highest binding affinity for photodamaged DNA of the four [T. Mori et al., Photochem. Photobiol. 54 (1991) 225-232]. To help understand the differences between these antibodies, we have cloned and sequenced the variable region genes from all four. Comparing these sequences revealed that all four were highly similar to one another, although there were some differences in potential antigen-contact regions. To assess the influences of these sequence differences at the structural level, computer models were constructed for all four antibodies. Most of the sequence differences occurred in potential antigen contact regions, suggesting specific positions that might account for the observed differences in binding affinities and selectivities. A single-chain Fv derivative of 64M5 was therefore constructed and characterized to provide an experimental system in which structure-function relationships can be tested. This derivative could be isolated from Escherichia coli using two chromatographic steps and possessed the same binding specificity as the parent monoclonal antibody.

Crystallization and preliminary X-ray investigation of non-specific complexes of a mutant ribonuclease T1 (Y45W) with 2'AMP and 2'UMP.

We have succeeded in crystallizing complexes of a mutant ribonuclease T1 (Y45W) with the non-cognizable ribonucleotides 2'AMP and 2'UMP by macroscopic seeding of microcrystals of the mutant enzyme complexed with 2'GMP, which is the cognizable nucleotide inhibitor. The mutant enzyme has a tryptophan residue instead of Tyr45 of the wild-type enzyme and thus this mutation enhances the binding of ribonucleotides to the enzyme. The space group is P212121 with unit cell dimensions a = 49.40 A, b = 46.71 A, c = 41.02 A for the complex with 2'AMP and a = 48.97, b = 46.58 A, c = 40.97 A for the complex with 2'UMP, both of which are poorly isomorphous to the mother crystals. Diffraction data for the complexes with 2'AMP and 2'UMP were collected on a diffractometer at 1.7 A and 2.4 A resolution, respectively. The present studies show that crystallization of non-specific complexes of other protein-ligand systems with the dissociation constants around 10(-3) M, or even larger, could be feasible by application of the seeding technique. A comparison of the crystal structures of the complexes with that with 2'GMP may serve as a structural basis for the determination of differences between the specific and non-specific interactions of the enzyme.