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Over the last 2 decades, omalizumab is the only anti-IgE antibody that has been approved for asthma and chronic spontaneous urticaria (CSU). Ligelizumab, a higher-affinity anti-IgE mAb and the only rival viable candidate in late-stage clinical trials, showed anti-CSU efficacy superior to that of omalizumab in phase IIb but not in phase III. This report features the antigenic-functional characteristics of UB-221, an anti-IgE mAb of a newer class that is distinct from omalizumab and ligelizumab. UB-221, in free form, bound abundantly to CD23-occupied IgE and, in oligomeric mAb-IgE complex forms, freely engaged CD23, while ligelizumab reacted limitedly and omalizumab stayed inert toward CD23; these observations are consistent with UB-221 outperforming ligelizumab and omalizumab in CD23-mediated downregulation of IgE production. UB-221 bound IgE with a strong affinity to prevent FcԑRI-mediated basophil activation and degranulation, exhibiting superior IgE-neutralizing activity to that of omalizumab. UB-221 and ligelizumab bound cellular IgE and effectively neutralized IgE in sera of patients with atopic dermatitis with equal strength, while omalizumab lagged behind. A single UB-221 dose administered to cynomolgus macaques and human IgE (ε, κ)-knockin mice could induce rapid, pronounced serum-IgE reduction. A single UB-221 dose administered to patients with CSU in a first-in-human trial exhibited durable disease symptom relief in parallel with a rapid reduction in serum free-IgE level.

A continuous supply of O(2) is important for itaconic acid production in Aspergillus terreus. Any interruption of aeration significantly reduces itaconic acid production. To overcome this effect, A. terreus M8 was transformed with the Vitreoscilla hemoglobin gene (vgb) which, as shown by Southern hybridization, was integrated into the recipient chromosome. The activity of the expressed hemoglobin was confirmed by a CO-difference spectrum. During itaconic acid production, the effect of a break in aeration during cultivation in the transformant with the vgb gene is alleviated. Additionally, the transformant shows improved itaconic acid production.

Objective. Increased sialylation has been reported in various kinds of cancers, but to date, sialylation of cervical carcinoma has never been evaluated. This study of the changes in messenger ribonucleic acid (mRNA) expression of the four sialyltransferases (ST3Gal I, ST3Gal III, ST3Gal IV, and ST6Gal I) in a normal cervix and that with FIGO stage IB1 squamous cell carcinoma was undertaken to assess the extent of sialylation associated with establishment of the carcinoma. Methods. Alterations in ST mRNA expression in FIGO IB1 cervical cancer ( n = 30) and normal cervixes ( n = 30) were examined by semiquantitative reverse transcription–polymerase chain reaction (RT-PCR). Results. ST6Gal I expression was enhanced in squamous cell carcinoma of the cervix ( P = 0.026, Mann–Whitney U test), but mRNA expression from the other three STs (ST3Gal I, ST3Gal III, and ST3Gal IV) was significantly down-expressed in squamous cell carcinoma of the cervix compared to the normal cervix ( P = 0.003, P P = 0.001, respectively). High ST6Gal I expression was associated with more invasive properties of cervical cancer, such as deep stromal invasion, lymph or vascular space involvement, and poor differentiation ( P = 0.010, P P Conclusions. A combination of enhanced ST6Gal I mRNA expression and decreased mRNA expression from ST3Gal I, ST3Gal III, and ST3Gal IV might be important in cervical cancer. Future studies will investigate whether RT-PCR detection of the expression of these enzymes can be helpful for prognostic purposes.

Background: Germline BRCA1 mutations of sporadic ovarian cancers are presumed to be rare events, except among specific populations. To date, the status of germline BRCA1 mutations in Taiwanese with primary epithelial ovarian carcinoma (PEOC) is still unknown. In this study, we tried to answer part of this question. Methods: Sixty-four patients documented with PEOC, four patients with family history of breast and/or ovary cancer syndrome and five patients with sporadic primary serous peritoneal carcinoma (PSPC) were enrolled in this retrospective study from January 1994 through June 1999. At the same time, 50 normal healthy Taiwanese without family history were enrolled in this study. Germline DNA from these patients was screened for mutations in theBRCA1 gene using polymerase chain reaction-based single-stranded conformation polymorphism analysis (PCRSSCP). Shifting DNA bands were sequenced. Results: One of the 64 patients with PEOC (1.6%) exhibited germline BRCA1 heterozygous mutation which was exon11 single-base substitution at nucleotide1047 (CAG to TAG). One of the five patients with PSPC (20%) exhibited an exon11 single-base substitution at nucleotide 914 (TCT to TCC) with resultant silent mutation. One of the normal healthy Taiwanese (2%) was found to have an exon 2 single-base substitution at nucleotide 152 (A→ C) which was also a silent mutation. No mutations of BRCA1 were detected in four patients with a family history of breast and/or ovarian cancer. Conclusions: Based on this study, it was very difficult to obtain precise data to prove the value of applying genetic testing of BRCA1 mutations in Taiwanese patients with sporadic epithelial ovarian cancers or sporadic PSPC and even with a family history of breast and/or ovarian cancer because of its rare event and because of the too small number of cases available in this study.

Mutations in the Schizosaccharomyces pombe rec14 gene reduce meiotic recombination by as much as a factor of 1000 in the three intervals tested on chromosomes I and III. A DNA clone complementing the rec14 mutation was shown by genetic and physical analysis to contain the rec14 gene, which was functional in plasmid-borne inserts as small as 1.4 kb. The rec14 gene contains two exons separated by a 53-bp intron, which was confirmed by analysis of rec14 transcripts. The spliced transcript encodes a protein product of 302 amino acids, which contains six WD repeat motifs found in the G-beta transducin family of proteins and other proteins, including the Saccharomyces cerevisiae Ski8 (Rec103) protein. Although the rec14 transcripts were present in mitotically dividing cells, rec14 mutations had no detectable effect on mitotic recombination. The pattern of expression of rec14 differs from that of previously analyzed S. pombe rec genes. Based upon mutant phenotypes and amino acid sequence similarities, we propose that S. pombe Rec14 is a functional homologue of S. cerevisiae Rec103.

Photoreactivating enzyme (DNA photolyase; deoxyribocyclobutadipyrimidine pyrimidine-lyase, EC 4.1.99.3) repairs UV damage to DNA by utilizing the energy of near-UV/visible light to split pyrimidine dimers into monomers. The enzyme is widespread in nature but is absent in certain species in a seemingly unpredictable manner. Its presence in humans has been a source of considerable controversy. To help resolve the issue we used a very specific and sensitive assay to compare photoreactivation activity in human, rattlesnake, yeast, and Escherichia coli cells. Photolyase was easily detectable in E. coli, yeast, and rattlesnake cell-free extracts but none was detected in cell-free extracts from HeLa cells or human white blood cells with an assay capable of detecting 10 molecules per cell. We conclude that humans most likely do not have DNA photolyase.

Photolyases contain two chromophores, flavin plus either methenyltetrahydrofolate (MTHF) or 8-OH-5-deazaflavin (HDF). Amino acid sequence comparison reveals that all photolyases sequenced to date have extensive sequence homology in the carboxyl-terminal half; in the amino-terminal region the folate and deazaflavin class enzymes are more homologous to other members of the same class. This modular arrangement of sequence homologies suggests that the amino-terminal half of photolyase is involved in MTHF or HDF binding whereas the carboxyl-terminal half carries the flavin binding site. In this study we attempted to identify such structural domains of yeast photolyase by partial proteolysis and gene fusion techniques. Partial digestion with chymotrypsin yielded an amino-terminal 34-kDa fragment containing tightly bound MTHF and a carboxyl-terminal 20-kDa polypeptide which lacked chromophore or DNA binding activity. However, a fusion protein carrying the carboxyl-terminal 275 amino acids of yeast photolyase bound specifically to FAD but not to MTHF or DNA. We conclude that the amino-terminal half of yeast photolyase constitutes the folate binding domain and that the carboxyl-terminal half carries the flavin binding site.

Photolyases repair pyrimidine dimers in DNA by converting the light energy of 300- to 500-nm photons into chemical energy. Enzymes from various organisms contain two chromophore cofactors (FADH2 and either methenyltetrahydrofolate or 8-hydroxy-5-deazaflavin) that absorb the low-energy photons and initiate splitting of the cyclobutane ring by a radical mechanism. Here, we show that, in addition to these two chromophores, in the far UV range, direct excitation of one specific tryptophan residue (out of 15 total) in the polypeptide chain of Escherichia coli photolyase leads to splitting of the cyclobutane ring with high quantum yield (phi = 0.56), independent of the other chromophores. The specific tryptophan residue responsible for photosensitized repair was identified as Trp-277 by site-specific mutagenesis.

We have cloned the phr gene that encodes DNA photolyase from Salmonella typhimurium by in vivo complementation of Escherichia coli phr gene defect. The S.typhimurium phr gene is 1419 base pairs long and the deduced amino acid sequence has 80% identity with that of E. coli photolyase. We expressed the S.typhimurium phr gene in E.coli by ligating the E.coli trc promoter 5' to the gene, and purified the enzyme to near homogeneity. The apparent molecular weight of S.typhimurium photolyase is 54,000 dalton as determined by SDS-polyacrylamide gel electrophoresis, which is consistent with the calculated molecular weight of 53,932 dalton from the deduced phr gene product. S.typhimurium photolyase is purple-blue in color with near UV-visible absorption peaks at 384, 480, 580, and 625 nm and a fluorescence peak at 470 nm. From the characteristic absorption and fluorescence spectra and reconstitution experiments, S.typhimurium photolyase appears to contain flavin and methenyltetrahydrofolate as chromophore-cofactors as do the E.coli and yeast photolyases. Thus, S.typhimurium protein is the third folate class photolyase to be cloned and characterized to date. The binding constant of S.typhimurium photolyase to thymine dimer in DNA is kD = 1.6 x 10(-9) M, and the quantum yield of photorepair at 384 nm is 0.5.

Replacement of Gly124 on the S1 pocket of subtilisin YaB with Ala changed the cleavage pattern on oxidized insulin B-chain from the subtilisin type to the elastase type. The initial cleavage site in the B-chain shifted from L15-Y16 for wild-type YaB to A14-L15 for the G124A mutant. Upon complete hydrolysis with the G124A mutant, four of the six major cleavage sites on the B-chain were identical to porcine pancreatic elastase cleavage sites.

Objective. Altered messenger ribonucleic acid (mRNA) expression of the four sialyltransferases (STs including ST3Gal I, ST3Gal III, ST3Gal IV, and ST6Gal I) is important in squamous cell carcinoma of the cervix. This study further investigates their changes in mRNA expression of the four STs in FIGO stage IB1 squamous cell carcinoma to assess the extent of sialylation associated with lymph node metastases. Methods. Alterations in ST mRNA expression in FIGO IB1 cervical squamous cell carcinomas ( n = 79) were examined by semiquantitative reverse transcription–polymerase chain reaction. Results. Both ST6Gal I mRNA and ST3Gal III mRNA expressions were significantly increased in patients with lymph node metastases compared to those without lymph node metastases ( P = 0.002 and P = 0.001, respectively, Mann–Whitney U test). Using receiver operating characteristic curves of ST ratio index for accuracy comparison of lymph node metastases, ST3Gal III and ST6Gal I were observed to be fairly interchangeable (area under the curve (AUC) of 3Gal I=0.810; AUC of 6Gal I=0.786, significance of difference between AUC=0.810). High ST6Gal I expression was associated with other invasive properties of cervical cancer, such as deep stromal invasion and presence of lymph–vascular space involvement. ST6Gal I expression seemed to be more enhanced in bigger tumors. Conclusions. Our results suggested that ST3Gal III and ST6Gal I were of importance for the lymph node metastases in FIGO IB1 cervical cancer patients; more specifically, overexpression of ST6Gal I was of crucial relevance for the presence of poor prognostic factors, such as deep stromal invasion and lymph–vascular space involvement and lymph node metastases.

Objective. The aim of this study was to study the presence of cytokeratin 19 (CK19)-expressing cancer cells in the blood of preoperative patients with FIGO stage Ib and IIb cervical cancers who received radical hysterectomy and to investigate the cells' clinical significance. Methods. CK19 mRNA in the blood cells of the patients was detected preoperatively by a newly designed nested reverse transcriptase–polymerase chain reaction, which excluded pseudogenes a and b, performed on 84 patients with stage Ib and IIb cervical carcinoma. Possible correlations between clinicopathological factors were then analyzed. Results. The sensitivity of this assay was 1 CK19-mRNA-positive cell per 10 6 peripheral blood mononuclear cells. Results showed that 21.4% of the 84 patients with cervical carcinoma had CK19-mRNA-positive cells in the blood, in comparison with 5.7% of the 35 patients with benign gynecological tumors and 0% of the 28 healthy controls ( P = 0.037 and 0.006, respectively). The positive tests in the cervical cancer patients were not associated with prognostic factors including stage, pelvic lymph node metastasis, pathological types, bulky tumor size (≥4 cm), differentiation, parametrial extension, lymphovascular space involvement, deep stromal invasion, or age. Conclusions. This study revealed the presence of circulating CK19-expressing cancer cells in the blood of patients with untreated early-stage cervical carcinomas, indicating that cervical cancer disseminated early. The survival effect of this phenomenon must be clarified. This detection assay provides an early checkpoint in the multistep process for developing metastasis in cervical cancer patients.

To use comparative genomic hybridization (CGH) to analyzefrozen tissuesfrom adenomyosis cases to discover and map genomic regions for chromosomal gains and losses.In a retrospective study, upper and lower threshold values of 1.20 and 0.80, respectively, were used to define positive findings.No positive recurrent gene copy number alterations were detected in the 25 cases of pathologically proven adenomyosis.Although CGH is extremely useful in investigating candidate genes in the development of adenomyosis, CGH was not useful in this study. Genetic changes might be indeed extremely rare in adenomyosis, or CGH was not sensitive enough to detect candidate genes.

Transforming growth factor beta-1 (TGF-beta1) is a multifunctional growth factor and known to inhibit the proliferation of epithelial cell. The relationship between serum TGF-beta1 level and the presence of cervical cancer was investigated in this study. The patients with cervical squamous cell carcinoma (SCC) had significantly lower level of serum TGF-beta1 (39.14 +/- 9.03 ng/mL; mean +/- SD) than those with myoma (49.17 +/- 9.38 ng/mL) and normal subjects (49.13 +/- 8.81 ng/mL), (p < 0.007 and p < 0.001, respectively). TGF-beta1 level was also lower in the patients with cervical intraepithelial neoplasia (CIN3) (42.07 +/- 9.38 ng/mL) than in normal controls (49.13 +/- 8.81 ng/mL) (p < 0.04). It suggested that diminution of the production of TGF-beta1 has close association with the neoplastic transformation of cervical epithelium.

Objective. Because of the limited sensitivity and specificity of conventional tools such as computerized tomography (CT) or magnetic resonance imaging (MRI) for detecting persistent or recurrent primary serous peritoneal carcinoma (PSPC), a reliable means of diagnosis remains elusive. Positron emission tomography (PET) scanning may offer another approach to this problem. Methods. A prospective study of three patients requiring surgical exploration for suspected recurrence of PSPC received a whole-body PET (fluorine-18)-2-deoxyglucose (FDG) scanning in a teaching hospital from July 1995 to December 1998. The suspected recurrence was based upon clinical findings including a detailed physical examination, serum CA-125 marker ultrasound, CT, and MRI. Three patients were enrolled in this study. Results. In all three patients, PET images demonstrated increased FDG uptake in a distribution that correlated with surgical–pathologic findings (100%); on the contrary, CT can detect 33.3% of these patients with malignant diseases and MRI can detect two-thirds of cases. Serum CA-125 was also elevated in all three patients, although one patient showed an equivocal elevation of 25.7 IU/ml. Conclusions. Conventional imaging studies are neither sensitive nor specific for detecting recurrent PSPC. In contrast, besides CA-125, PET might offer a relatively effective tool for detecting recurrent primary serous peritoneal carcinoma. Due to the very small number of patients available in this study, considerable research must be performed to clarify the impact of PET on detecting recurrence of PSPC.

Previously isolated meiotic recombination (rec) mutants of Schizosaccharomyces pombe define 16 complementation groups. The rec genes cloned and sequenced to date reveal little amino acid sequence identity to other reported proteins. We examined the rec mutants for alterations in meiotic events other than recombination to gain insight into the rec gene functions and to assess whether they affect recombination directly or indirectly. While mutations in the rec6–12, 14, 15 and 19 genes appeared to affect only meiotic recombination, a mutation in rec16 delayed meiotic DNA synthesis and, in some instances, reduced its amount; mitotic DNA synthesis was not detectably altered, indicating that the rec16 effect is limited to meiosis. In the rec16 mutant some meiotically induced transcripts (e.g., rec7 and 15) were significantly reduced in abundance, whereas others (e.g., rec10 and exo1) were induced and degraded with normal timing and extent during meiosis, indicating that the rec16 mutation leaves the basic meiotic program intact. These results indicate that the rec genes other than rec16 have their primary effect on meiotic recombination. In contrast, the rec16 gene product is essential for normal meiotic replication, recombination, and induction of some transcripts. These meiotic events may be coupled via a dependence of recombination and transcription on replication or via a cascade of gene expression.

DNA photolyases repair cyclobutadipyrimidines (Pyr()Pyr) in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains methenyltetrahydrofolate (MTHF), which functions as photoantenna, and FADH2, which is the redox-active cofactor. During purification, FADH2 is oxidized to the blue neutral radical form, FADH., which has greatly diminished activity. Previous nanosecond flash photolysis studies [Heelis, P.F., Okamura, T., & Sancar, A. (1990) Biochemistry 29, 5694-5698] indicated that excitation of FADH. either directly by absorbing a photon or indirectly by electronic energy transfer from MTHF excited singlet state yielded an FADH. quartet which abstracted a hydrogen atom from a nearby tryptophan to generate the catalytically competent FADH2 from of the enzyme. Using site-directed mutagenesis, we replaced all 15 photolyase tryptophan residues by phenylalanine, individually, in order to identify the internal hydrogen atom donor responsible for photoreduction. We found that W306F mutation abolished photoreduction of FADH. without affecting the excited-state properties of FADH. or the substrate binding (KA approximately 10(9) M-1) of the enzyme. The specificity constant (kcat/km) was approximately 0 for the mutant enzyme in the absence of reducing agents in the reaction mixture, indicating that photoreduction of FADH. is an essential step for photorepair by photolyase in vitro. Chemical reduction of FADH. of the mutant enzyme restored the specificity constant to the wild-type level.

Escherichia coli DNA photolyase repairs pyrimidine dimers by a photoinduced electron-transfer reaction. The enzyme binds to UV-damaged DNA independent of light (the dark reaction) and upon absorbing a 300-500-nm photon breaks the cyclobutane ring of the dimer (the light reaction) and thus restores the DNA. No structural information on the enzyme is available at present. However, comparison of the sequences of photolyases from five different organisms has identified highly conserved regions of homology. These regions are presumably involved in chromophore (flavin and folate) and substrate binding or catalysis. Trp277 (W277) in E. coli photolyase is conserved in all photolyases sequenced to date. We replaced this residue with Arg, Glu, Gln, His, and Phe by site-specific mutagenesis. Properties of the mutant proteins indicate that W277 is involved in binding to DNA but not in chromophore binding or catalysis. Of particular significance is the finding that compared to wild type W277R and W277E mutants have about 300- and 1000-fold lower affinity, respectively, for substrate but were indistinguishable from wild-type enzyme in their photochemical and photocatalytic properties.

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.

The alkaline elastase produced by alkalophilic Bacillus Ya-B was a new type of proteinase which had a very high optimum pH and high elastolytic activity. It also had a high hydrolyzing activity against keratin and collagen. The molecular weight was determined to be 23 700 and 25 000 by ultracentrifugation analysis and SDS-polycrylamide gel electrophoresis, respectively. The isoelectric point was 10.6. The optimum reaction temperature was 60°C. Like many alkaline proteinases, this enzyme required Ca 2+ for stability. The optimum reaction pH was 11.75 toward casein and elastin-orcein. The K cat / K m values of the enzyme to synthetic substrates were constant from pH 8.5 up to 12.75. The enzyme was stable in the pH range 5.0–10.0. The enzyme was inhibited by alkaline proteinase inhibitors Streptomyces subtilisin inhibitor and microbial alkaline proteinase inhibitor, but not by elastatinal or the metalloproteinase inhibitor metalloproteinase inhibitor. Sodium chloride inhibited the elastolytic activity but not the caseinolytic activity at a concentration below 0.2 M. The inhibitory effect of sodium chloride to elastolytic activity was much more prominent at pH 9.0 than at pH 11.5. More than 50% of the enzyme bound onto elastin in the pH range below the isoelectric point of this enzyme. The amino-terminal sequence of the enzyme was determined, and compared with those of subtilisin BPN′ and subtilisin Carlsberg. Extensive sequence homology was noted among these three enzymes.