Thermostable xylanases, Xyn10A and Xyn11A, from the actinomycete Nonomuraea flexuosa: isolation of the genes and characterization of recombinant Xyn11A polypeptides produced in Trichoderma reesei.

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80 degrees C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80 degrees C.

Characterisation of steryl esterase activities in commercial lipase preparations.

Triglycerides, steryl esters, resin acids, free fatty acids and sterols are lipophilic extractives of wood (commonly referred to as pitch or wood resin) and have a negative impact on paper machine runnability and quality of paper. Thus, enzymes capable of modifying these compounds would be potential tools for reducing pitch problems during paper manufacture. In this work, 19 commercial lipase preparations were tested for their ability to degrade steryl esters, which may play a significant role in the formation and stabilisation of pitch particles. Six lipase preparations were shown to be able to degrade steryl esters. Lipase preparations of Pseudomonas sp., Chromobacterium viscosum and Candida rugosa were shown to have the highest steryl esterase activities. The enzymes were able to hydrolyse steryl esters totally in the presence of a surfactant (Thesit). Up to 80% of the steryl esters were degraded in aqueous dispersion. Preliminary characterisation of the enzymatic activities revealed that the lipase preparation of Pseudomonas sp. could be the most potential enzyme in industrial applications. The steryl esterase activity of this preparation was stable over a broad pH range and the enzyme was able to act efficiently at pH 6-10 and at temperatures up to 70 degrees C.

Lung cancer mortality in the Mayo Lung Project: impact of extended follow-up.

BACKGROUND: The Mayo Lung Project (MLP) was a randomized, controlled clinical trial of lung cancer screening that was conducted in 9211 male smokers between 1971 and 1983. The intervention arm was offered chest x-ray and sputum cytology every 4 months for 6 years; the usual-care arm was advised at trial entry to receive the same tests annually. No lung cancer mortality benefit was evident at the end of the study. We have extended follow-up through 1996. METHODS: A National Death Index-PLUS search was used to assign vital status and date and cause of death for 6523 participants with unknown information. The median survival for lung cancer patients diagnosed before July 1, 1983, was calculated by use of Kaplan-Meier estimates. Survival curves were compared with the log-rank test. RESULTS: The median follow-up time was 20.5 years. Lung cancer mortality was 4.4 (95% confidence interval [CI] = 3.9-4.9) deaths per 1000 person-years in the intervention arm and 3.9 (95% CI = 3.5-4.4) in the usual-care arm (two-sided P: for difference =.09). For participants diagnosed with lung cancer before July 1, 1983, survival was better in the intervention arm (two-sided P: =.0039). The median survival for patients with resected early-stage disease was 16.0 years in the intervention arm versus 5.0 years in the usual-care arm. CONCLUSIONS: Extended follow-up of MLP participants did not reveal a lung cancer mortality reduction for the intervention arm. Similar mortality but better survival for individuals in the intervention arm indicates that some lesions with limited clinical relevance may have been identified in the intervention arm.

Endoxylanase II from Trichoderma reesei has several isoforms with different isoelectric points.

Two minor xylanases present in Trichoderma reesei Rut C30 cultivation broth were purified as a mixture using ion-exchange, hydrophobic-interaction and gel chromatography. The purified enzyme preparation contained two active xylanases with pI values of 7.1 and 8.1. Both components had a molecular mass of 20 kDa. The purified xylanase preparation exhibited properties very similar to those of the previously isolated XYL II (pI 9.0) of T. reesei Rut C30. The activity and stability properties, apparent kinetic parameters as well as the titration curve forms were similar. The major difference in enzymic properties was the significantly lower specific activity of the pI-7.1+8.1 xylanase mixture (3350 nkat/mg) compared with the specific activity of XYL II (13500 nkat/mg). Amino acid sequences of tryptic peptides (34% of the total amino acid sequence was determined) were identical to the amino acid sequence of XYL II. Furthermore, in vitro modification of the pI-9.0 form of XYL II to pI-8.1 and pI-7.1 forms was demonstrated. Thus the purified xylanase preparation most probably contained two modified forms of XYL II. The primary amino acid sequence of XYL II contains 28 glutamine and asparagine residues and theoretically deamination of one of them lowers the pI to 8.06 and deamination of two amino acids lowers the pI to 7.02.

Design and evolution of the data management systems in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial.

This paper describes the design and evolution of the data management systems developed in support of the Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial. These systems span platforms from stand-alone computers to distributed systems on local area networks to mainframes. Allowing all of these systems to share appropriate information electronically introduces integration, synchronization, testing, and support challenges. For each platform, applications were developed to handle data entry, editing, trial management, reporting, telecommunications, and data sharing. Approaches to issues such as level of data access, integration with other, existing applications, and handling the expansion of the protocol are discussed.

Quality control of cancer screening examination procedures in the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial.

Investigators for the Prostate, Lung, Colorectal and Ovarian (PLCO) Cancer Screening Trial describe quality control procedures for the digital rectal examination, ovarian palpation examination, transvaginal ultrasound, chest X-ray, and flexible sigmoidoscopy. These cancer screening tests are subjective and difficult to standardize. PLCO quality control procedures aim to measure and, where possible, reduce variation, across examiner and screening center, with respect to cancer screening test performance. Initial protocols stressed examiner qualifications, experience, and training; equipment specifications; examination procedures; and definitions for positive tests. The PLCO quality assurance subcommittee developed a final quality assurance plan, which included central approval and registration of PLCO examiners, direct observation of screening test performance during periodic site visits by the National Cancer Institute and coordinating center auditors, periodic analysis of screening test data, and procedures for independently duplicating or reviewing selected examinations. For each modality, the periodic data analyses examine the test-positive and the test-inadequate proportions and aim to identify divergent centers or examiners. Procedures for duplicating examinations specify feasible sample sizes for precise estimates of agreement between examiners, at each center, for each screening test modality, and over a 1-year period. These quality control procedures will help characterize the consistency and reliability of the PLCO cancer screening tests.

Estimation of post-lead-time survival under dependence between lead-time and post-lead-time survival.

Early detection of cancer by screening advances the date of diagnosis, but may or may not alter time to death. Screening programme need to assess the true benefit of screening, that is, the length of time by which survival has been extended, beyond merely the time by which the diagnosis is advanced (lead-time). One method is to estimate the distribution of the time survived post-lead-time using total survival time data for screen-detected cancer cases, under the assumption of independence of the lead-time and the past-lead-time survival. However, it seems biologically reasonable that the lead-time and the post-lead-time survival are positively correlated. This paper investigates the consequences of departures from independence of lead-time and post-lead-time survival on estimation of post-lead-time survival. We introduce a new model that involves dependence between the lead-time and the post-lead-time survival. We show that the new model can be converted to the model discussed by Xu and Prorok. We consider the non-parametric maximum likelihood estimator of the post-lead-time survival under the new model. We apply the method to data from the HIP (Health Insurance Plan of Greater New York) breast cancer screening trial. We make comparisons with the survival of cancer cases not detected by screening, such as interval cases, cases among individuals who refused screening, and randomized control cases.

Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley.

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60 degrees C. It showed maximum activity over a broad pH range (7.0-9.0). The molecular mass of the monomer was about 50,000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53,620 Da. A relative mass estimation of 100,000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer. The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5'untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.

Characterization, subsite mapping and partial amino acid sequence of glucoamylase from the filamentous fungus Trichoderma reesei.

The pH optimum, temperature-dependence, thermal stability, substrate specificity and subsite affinities of the 66 kDa, pI 4.0 glucoamylase of the filamentous fungus Trichoderma reesei were determined. It had a pH optimum of 5.5 and a temperature optimum (5 min reaction time) of 70 degrees C with soluble starch as substrate. Thermal-inactivation studies revealed that the glucoamylase is relatively thermostable up to 60 degrees C. Metal ions and EDTA tested at 5 mM concentrations had no significant effect, and beta-cyclodextrin only slightly inhibitory effects, on the digestion of soluble starch. Estimated Km and kcat. values for soluble starch where 0.11 mg.ml-1 and 28.5 s-1 respectively. Hydrolysis of pullulan (Km 14 mg.ml-1 and kcat. = 6.6 s-1) indicated substantial activity towards 1,6-O-glucosidic bonds. From ratios of kinetic parameters of malto- and isomalto-oligosaccharides, it was apparent that the glucoamylase showed approx. 3-fold higher selectivity towards isomalto-oligosaccharides than most other reported fungal glucoamylases. Substrate binding affinities were calculated from kinetic data for the linear series of malto- and isomalto-oligosaccharides. The results were in good agreement with other reported glucoamylases. The main difference was that subsite 1 showed a slightly negative free energy of binding with malto-oligosaccharides, whereas most other glucoamylases show a positive free energy at this subsite. A set of peptides obtained from purified glucoamylase by tryptic digestion where sequenced. They covered approx. 17% of the total amino acid sequence as estimated from molecular mass on SDS/PAGE. Some of the sequences were tentatively aligned to known glucoamylase sequences. They showed about 60% identity with the extensively studied Aspergillus glucoamylase.

Evidence for a polysaccharide-binding domain in Hormoconis resinae glucoamylase P: effects of its proteolytic removal on substrate specificity and inhibition by beta-cyclodextrin.

The hydrolysis of soluble starch, raw starch and pullulan with recombinant glucoamylase P from Hormoconis resinae was competitively inhibited by beta-cyclodextrin with apparent Ki values of 190 microM, 13 microM and 1.4 microM, respectively. Inhibition of dextran hydrolysis was partial: a maximum inhibition of 22% was achieved with a dextran concentration of 0.3 x Km and up to 4 mM beta-cyclodextrin. Hydrolysis of short oligosaccharides was not inhibited by beta-cyclodextrin at levels up to 20 mM. The enzyme bound to raw starch at pH 4.3 and 4 degrees C with an association constant of 3.4 x 10(5) M-1. Sequence alignment studies showed raw-starch-binding consensus amino acids in the C-terminal part of glucoamylase P. Partial hydrolysis with papain resulted in degradation of deglycosylated glucoamylase P into three fragments of 53, 51 and 14 kDa, respectively, as estimated by SDS-PAGE. The amino-terminal sequences of the 51 and 53 kDa fragments were identical with that of native glucoamylase P. The amino terminus of the 14 kDa fragment (Ser-Ser-X-Gln-Val-Ser-), corresponded to the sequence starting at residue 474 of intact glucoamylase P. Kinetic measurements of truncated glucoamylase P showed changes in the Km values of larger polysaccharides, but no changes in kcat values compared to the intact enzyme. It was concluded that glucoamylase P contains a catalytic core domain and a raw-starch-binding domain involved in inhibition of polysaccharide hydrolysis by beta-cyclodextrin.

Correlation between depression and vision in aged patients before and after cataract operations.

To investigate the association between depression and vision, 100 cataract operation patients (25 were men and 75 women) aged 71 to 76 years were studied. One day before the operation and 3 mo. after, the patients' depression was tested with the short form of the Beck Depression Inventory and their personalities with Mini-Mult MMPI. The cataract operation restored visual acuity sufficient for reading (minimum E-test value 0.40) to 79% of the subjects. The analysis indicated that their depression was significantly correlated with vision only after the cataract operation. Depression increased with weakened visual acuity and diminished with improved visual acuity. Part of the postoperation depression was, however, associated with glaucoma and serious somatic diseases (asthma, cerebrovascular disorders, and heart diseases).

Purification and specificity of recombinant Hormoconis resinae glucoamylase P and endogenous glucoamylase from Trichoderma reesei.

Hormoconis resinae glucoamylase P of high debranching activity was purified from a recombinant Trichoderma reesei strain. Four different purified fractions were obtained. Three had the same amino terminal sequence as the wild-type enzyme and about the same specific activity, and yielded the same single band on SDS-PAGE after deglycosylation. Presumably they resulted from different glycosylation patterns of the recombinant glucoamylase P. One fraction had a much lower specific activity and yielded tryptic peptides that identified it as the host cellobiohydrolase I contaminated with glucoamylase P. The different glycosylation patterns of recombinant glucoamylase P had only minor effects on its thermal inactivation. During purification of the recombinant glucoamylase, a protein with lower debranching activity was found and purified by chromatofocusing to homogeneity as assessed by SDS-PAGE. It had a pI of about 4.0 and a ratio of pullulan- to starch-degrading activity of 15%. Its amino terminal sequence showed 60% identity to the amino terminal sequence of glucoamylases P and S from Hormoconis resinae. Presumably this enzyme is the endogenous glucoamylase of Trichoderma reesei.

Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2.

The Trichoderma reesei xln2 gene coding for the pI9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed by targeting a construct composed of the xln2 gene, including its promoter, to the endogenous cbh1 locus. Highest overall production levels of xylanase were obtained using T. reesei ALKO2721, a genetically engineered strain, as a host. Integration into the cbh1 locus was not required for enhanced expression under control of the xln2 promoter.

Fear of a cataract operation in aged persons.

Self-reports of fear from 100 patients (25 men and 75 women 71 to 76 years old) having two cataract operations were investigated. 33% of the patients reported having fear and 32% felt tension about the operation performed on the first eye. Women feared the operation significantly more than men. Fears were significantly associated with hypochondriasis, hysteria, and hypomania (unadjusted) as indicated by correlations with scores on the Mini-Mult MMPI. The cataract operation restored sufficient visual acuity for reading (minimum E-test value 0.40 or 1.8-cm high letters at a distance of 6 meters) to 79% of the subjects. The experience of a good operation result on the first eye significantly reduced the fear of the cataract operation on the second eye and at the same time the fear of becoming blind. Other factors reducing fear included positive experiences of a safe and painless cataract operation.

Correlations of memory and learning with vision in aged patients before and after a cataract operation.

The connection between memory and learning with vision was investigated by studying 100 cataract operation patients, aged 71 to 76 years, 25 of them being men and 75 women. The cataract operation restored sufficient acuity of vision for reading (minimum E-test value 0.40) to 79% of the subjects. Short-term memory was studied with series of numbers, homogenic and heterogenic inhibition, and long sentences. Learning was tested with paired-associate learning and word learning. Psychological symptoms were measured on the Brief Psychiatric Rating Scale and personality on the Mini-Mult MMPI. Memory and learning improved significantly when vision was normalized after the cataract operation. Poor memory and learning scores correlated with monocular vision before the operation and with defects in the field of vision, due to glaucoma and exceeding 20%, postsurgery. Monocular vision and defects in the visual field caused a continuous sense of abnormalness, which impaired old people's ability to concentrate on tasks of memory and learning. Cerebrovascular disturbances, beginning dementia, and moderate psychological symptoms obstructed memory and learning on both test rounds. Depression was the most important psychological symptom contributing to poor memory and learning scores after the cataract operation. The memory and learning defects mainly reflected disturbances in memorizing.

Correlation between psychic and somatic symptoms and vision in aged patients before and after a cataract operation.

The connection between psychic and somatic symptoms with vision was investigated by studying 100 cataract operation patients, aged 71 to 76 years, 25 of them being men and 75 women. The investigations were conducted one day before the operation and three months afterwards. The cataract operation restored sufficient acuity of vision for reading (minimum E-test value 0.40) to 79% of the old people. Psychic symptoms were tested with the Brief Psychiatric Rating Scale, Mini-Mult MMPI, and direct questions. Somatic symptoms were studied through questionnaires. Psychic symptoms showed a statistically significant correlation with vision before the cataract operation but not afterwards. Psychic symptoms increased with deterioration of the acuity of vision and diminished when the acuity of vision improved. Somatic symptoms did not show similar association with vision but the symptoms were significantly alleviated after the cataract operation. Restoration of vision through the cataract operation normalized the old people's psychic condition and reduced their somatic symptoms to correspond with their prior chronic diseases.

Comparison of two glucoamylases from Hormoconis resinae.

Two extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its 1,4-glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes.

High-density lipoprotein cholesterol is reduced in patients with sarcoidosis.

PURPOSE: Sarcoidosis is a disease in which the proliferation of monocyte-macrophage-derived cells is observed. In other diseases characterized by expansion of the monocyte-macrophage system, such as Gaucher's disease and myeloid metaplasia, abnormalities of lipoprotein metabolism have been demonstrated. To determine whether similar abnormalities in lipoprotein cholesterol concentrations could be identified in patients with sarcoidosis, we studied total cholesterol, low-density lipoprotein (LDL) cholesterol, and high-density lipoprotein (HDL) cholesterol as well as triglyceride levels in 52 patients with biopsy-proven sarcoidosis. PATIENTS AND METHODS: Patients had no other medical disorders and were not being treated with corticosteroids or antimalarial agents. Blood samples were collected by venipuncture after an overnight fast. Plasma total cholesterol and triglyceride levels were measured using enzymatic techniques. Lipoprotein cholesterol was quantified by lipoprotein fractionation. HDL cholesterol was measured as cholesterol remaining in the supernatant after precipitation of LDL and very-low-density lipoprotein from whole plasma by the heparin-maganese chloride method. Computation was used to determine the level of LDL cholesterol. RESULTS: We found significantly reduced levels of total cholesterol (183.9 +/- 27.6 versus 194.3 +/- 16.5 mg/dl, mean +/- SD, p = 0.021) and HDL cholesterol (41.2 +/- 13.0 versus 51.9 +/- 6.1 mg/dl, p = 0.0001) in sarcoid patients versus an age-, sex-, and race-matched reference group. Differences were not observed in triglyceride or LDL cholesterol levels (p greater than 0.05). CONCLUSION: These findings are similar to those observed in the myeloproliferative diseases, Gaucher's disease, and rheumatoid arthritis and suggest a functional role for monocytes-macrophages in the regulation of serum lipoprotein cholesterol levels.

Cardiovascular effects of a nifedipine infusion during fentanyl-nitrous oxide anesthesia in dogs.

The hemodynamic effects of a nifedipine infusion were investigated in eight dogs given fentanyl/pancuronium/nitrous oxide/oxygen anesthesia. Nifedipine (20 micrograms/kg) was given intravenously over two minutes immediately prior to each 30-minute infusion at 2 micrograms/kg/min, 4 micrograms/kg/min, and 6 micrograms/kg/min. The range of plasma nifedipine levels obtained was 52.1 to 113.7 ng/mL. The predominant hemodynamic effects were significant reductions in systemic vascular resistance (SVR) and mean aortic pressure (MAP), accompanied by a rise in cardiac index and heart rate (HR). Administration of calcium chloride (20 mg/kg) after the nifedipine infusion had no effect on SVR or MAP, but HR was significantly reduced. Serum epinephrine and norepinephrine levels increased after the infusion of nifedipine and suggested that fentanyl did not completely overcome the sympathetic response to the profound vasodilatation. The resulting tachycardia in combination with diastolic hypotension from nifedipine could have a detrimental effect on the myocardial oxygen balance.