Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.     

Effects of four trichothecene mycotoxins on activation marker expression and cell proliferation of human lymphocytes in culture

Four trichothecene mycotoxins – the type A trichothecenes T2-toxin and diacetoxyscirpenol and the type B trichothecenes nivalenol and deoxynivalenol – were studied. The effects of these mycotoxins on the expression of the sequentially expressed activation markers CD69, CD25, and CD71 and on proliferation of human lymphocytes were studied in culture with a duration of up to 72 h.All the examined toxins affected activation marker expression in a similar way. After 6 h, the CD69 expression was lower in exposed cultures compared to controls. After 24 and 48 h of exposure, an increased frequency of cells expressing CD69 was found in exposed cultures, indicating a delay in downregulation of CD69 expression. Stimulation of CD25 expression was observed for doses below the IC₅₀ value, while suppression was found for higher doses. The pattern was different from that detected for CD69 expression, in that an increased expression of CD25 never occurred after exposure to the highest concentration of the toxin, and in that no stimulatory effects were found after 48 h of exposure, indicating that the response was inhibited and not delayed. The effects of toxin exposure on CD71 expression were in many respects similar to the effects on CD25 expression.We conclude that the trichothecene mycotoxins investigated in this study inhibited the cell cycle in a similar way and exert their main antiproliferative action rather early in the cell cycle, before or in conjunction with CD25 expression.     

Surface plasmon resonance (SPR) analysis of coagulation in whole blood with application in prothrombin time assay

It is previously shown that surface plasmon resonance (SPR) can be used to study blood plasma coagulation. This work explores the use of this technique for the analysis of tissue factor induced coagulation, i.e. prothrombin time (PT) analysis, of whole blood and plasma. The reference method was nephelometry. The prothrombin time analysis by SPR was performed by mixing two volumes of blood/plasma, one volume of thromboplastin, and one volume of CaCl2 solution directly on a sensor surface. The measurements show good agreement between nephelometry and SPR plasma analysis and also between SPR plasma and whole blood analysis. The effect of anticoagulant treatment on the clotting times was significant both quantitatively and qualitatively. The impact on the SPR signal of different physiological events in the coagulation process is discussed, and tentative interpretations of the sensorgram features are given. The major advantage of the SPR method compared to nephelometry is the possibility to perform analysis on whole blood instead of plasma. In conclusion, SPR is a promising method for whole blood coagulation analysis.     

Lack of evidence for an immunosuppressive role for MUC1

The in vitro anti-proliferative properties of various supernatants from MUC1-expressing cell lines and of purified preparations of MUC1 were evaluated. We have observed that supernatants from the MUC1- and MUC3-positive cell line T47D, but not from the MUC1- and MUC4-positive cell line MCF7, were able to inhibit proliferation of cells from various haematopoietic cell lines. Although the activity of T47D supernatants could be abrogated by immunodepletion of MUC1, immunopurified MUC1 from T47D was unable to inhibit cell proliferation. Significantly, supernatants from mouse 3T3 cells transfected with a secreted form of MUC1 or from BHK-21 cells infected with a recombinant vaccinia virus coding for the secreted form of MUC1, as well as preparations of purified MUC1 from bile or urine, were likewise unable to inhibit T cell proliferation. Surprisingly, a crude mixture of bile mucins had a suppressive effect on T cell growth. Our results suggest that other molecules, such as amino sugars or other mucins, which can associate with MUC1, are likely to be responsible for the observed anti-proliferative effects of T47D cells. Received: 20 August 1998 / Accepted: 3 December 1998     

Comparison of PSA-specific CD8<Superscript>+</Superscript> CTL responses and antitumor immunity generated by plasmid DNA vaccines encoding PSA-HSP chimeric proteins

The ability of heat shock proteins (HSPs) to increase the potency of protein- and DNA-based vaccines has been previously reported. We have constructed several plasmid-based vectors encoding chimeric proteins containing prostate-specific antigen (PSA) fused to Mycobacterium tuberculosis hsp70, M. bovis hsp65, Escherichia coli DnaK (hsp70), or human hsp70. Immunizing mice with these plasmids induced CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human PSA and protected mice from a subsequent subcutaneous challenge with PSA-expressing tumors. We did not observe a significant difference either in the levels of PSA-specific CTLs or in protection against tumor challenge in mice immunized with plasmids expressing PSA-HSP chimeric proteins, as compared to mice receiving a conventional PSA-expressing DNA plasmid. Our data indicate that using HSPs as fusion partners for tumor-specific antigens does not always result in the enhancement of antigen-specific CTL responses when applied in the form of DNA vaccines.     

Thrombin inhibitor reduces myocardial infarction in apoE-/- x LDLR-/- mice

We have previously shown that atherosclerotic apolipoprotein E-deficient (apoE(-/-)) x LDL receptor-deficient (LDLR(-/-)) mice develop myocardial infarction when exposed to hypoxic stress. This study was performed to assess the role of thrombin and thrombosis in this process. ApoE(-/-) x LDLR(-/-) mice were fed a cholesterol-rich diet for 8 mo and were then subjected to hypoxic stress while receiving isoflurane anesthesia. One group received a bolus dose (5.6 micromol/kg) of the thrombin inhibitor melagatran, and control animals received PBS 10 min before the hypoxic stress. The mice were exposed to 10 min of hypoxia followed by normoxia. Ten minutes after the stress, Alzet pumps delivering melagatran (20 nmol x kg x (-1)min(-1)) or PBS were implanted, and the mice were allowed to recover for 48 h. The cardiac response was analyzed by histology, immunohistochemistry, and serum troponin T assay. All animals showed reversible ECG changes as a sign of ischemia during hypoxic stress, and 50% developed infarctions afterward as judged by troponin T levels. The group that received thrombin inhibitor had significantly lower troponin T and smaller myocardial infarctions than the PBS-treated group. These data show that thrombin generation is an important pathogenetic factor and suggest that coronary thrombosis is involved in myocardial infarction in atherosclerotic mice. Exposure of atherosclerotic mice to hypoxia leads to myocardial infarction through a two-phase pathway in which acute transient ischemia is followed by thrombin-dependent, irreversible, myocardial ischemia and myocardial cell death.     

Electromagnetic field exposure and health among RF plastic sealer operators

Operators of RF plastic sealers (RF operators) are an occupational category highly exposed to radiofrequency electromagnetic fields. The aim of the present study was to make an appropriate exposure assessment of RF welding and examine the health status of the operators. In total, 35 RF operators and 37 controls were included. The leakage fields (electric and magnetic field strength) were measured, as well as induced and contact current. Information about welding time and productivity was used to calculate time integrated exposure. A neurophysiological examination and 24 h ECG were also carried out. The participants also had to answer a questionnaire about subjective symptoms. The measurements showed that RF operators were exposed to rather intense electric and magnetic fields. The mean values of the calculated 6 min, spatially averaged E and H field strengths, in line with ICNIRP reference levels, are 107 V/m and 0.24 A/m, respectively. The maximum measured field strengths were 2 kV/m and 1.5 A/m, respectively. The induced current in ankles and wrists varied, depending on the work situation, with a mean value of 101 mA and a maximum measured value of 1 A. In total, 11 out of 46 measured RF plastic sealers exceeded the ICNIRP reference levels. RF operators, especially the ready made clothing workers had a slightly disturbed two-point discrimination ability compared to a control group. A nonsignificant difference between RF operators and controls was found in the prevalence of subjective symptoms, but the time integrated exposure parameters seem to be of importance to the prevalence of some subjective symptoms: fatigue, headaches, and warmth sensations in the hands. Further, RF operators had a significantly lower heart rate (24 h registration) and more episodes of bradycardia compared to controls.     

Observation of a ZZW female in a natural population: implications for avian sex determination

Avian sex determination is chromosomal; however, the underlying mechanisms are not yet understood. There is no conclusive evidence for either of two proposed mechanisms: a dominant genetic switch or a dosage mechanism. No dominant sex-determining gene on the female-specific W chromosome has been found. Birds lack inactivation of one of the Z chromosomes in males, but seem to compensate for a double dose of Z-linked genes by other mechanisms. Recent studies showing female-specific expression of two genes may support an active role of the W chromosome. To resolve the question of avian sex determination the investigation of birds with a 2A: ZZW or 2A: ZO genotype would be decisive. Here, we report the case of an apparent 2A: ZZW great reed warbler (Acrocephalus arundinaceus) female breeding in a natural population, which was detected using Z-linked microsatellites. Our data strongly suggest a role of W-linked genes in avian sex determination.     

Between‐year variation of MHC allele frequencies in great reed warblers: selection or drift?

The major histocompatibility complex (MHC) genes are extremely polymorphic and this variation is assumed to be maintained by balancing selection. Cyclic interactions between pathogens and their hosts could generate such selection, and specific MHC alleles or heterozygosity at certain MHC loci have been shown to confer resistance against particular pathogens. Here we compare the temporal variation in allele frequencies of 23 MHC class I alleles with that of 23 neutral microsatellite markers in adult great reed warblers (a passerine bird) in nine successive cohorts. Overall, the MHC alleles showed a significantly higher variation in allele frequencies between cohorts than the microsatellite alleles, using a multi-variate genetic analysis (amova). The frequency of two specific MHC alleles, A3e (P = 0.046) and B4b (P = 0.0018), varied more between cohorts than expected from random, whereas none of the microsatellite alleles showed fluctuations exceeding the expectation from stochastic variation. These results imply that the variation in MHC allele frequencies between cohorts is not a result of demographic events, but rather an effect of selection favouring different MHC alleles in different years.     

A new approach to study dispersal: immigration of novel alleles reveals female-biased dispersal in great reed warblers

We use the assignment technique and a new approach, the 'novel allele technique', to detect sex-biased dispersal in great reed warblers Acrocephalus arundinaceus. The data set consisted of immigrants and philopatric birds in a semi-isolated population in Sweden scored at 21 microsatellite loci. Fourteen cohorts were represented of which the four earliest were used to define a reference population. Female immigrants had lower assignment probability than males (i.e. were less likely to have been sampled in the reference population), and carried the majority of 'novel alleles' (i.e. alleles observed in the population for the first time). The difference in number of novel alleles between sexes was caused by a strong over-representation of females among the few individuals that carried several novel alleles, and there was a tendency for a corresponding female bias among individuals with low assignment probabilities. Immigrant males had similar or lower reproductive success than females. These results lead us to conclude that important interregional gene flow in great reed warblers depends on relatively few dispersing females, and that the novel allele technique may be a useful complement to the assignment technique when evaluating dispersal patterns from temporally structured data.