Construction of engineered bifunctional enzymes and their overproduction in Aspergillus niger for improved enzymatic tools to degrade agricultural by-products

Two chimeric enzymes, FLX and FLXLC, were designed and successfully overproduced in Aspergillus niger. FLX construct is composed of the sequences encoding the feruloyl esterase A (FAEA) fused to the endoxylanase B (XYNB) of A. niger. A C-terminal carbohydrate-binding module (CBM family 1) was grafted to FLX, generating the second hybrid enzyme, FLXLC. Between each partner, a hyperglycosylated linker was included to stabilize the constructs. Hybrid proteins were purified to homogeneity, and molecular masses were estimated to be 72 and 97 kDa for FLX and FLXLC, respectively. Integrity of hybrid enzymes was checked by immunodetection that showed a single form by using antibodies raised against FAEA and polyhistidine tag. Physicochemical properties of each catalytic module of the bifunctional enzymes corresponded to those of the free enzymes. In addition, we verified that FLXLC exhibited an affinity for microcrystalline cellulose (Avicel) with binding parameters corresponding to a Kd of 9.9 x 10(-8) M for the dissociation constant and 0.98 micromol/g Avicel for the binding capacity. Both bifunctional enzymes were investigated for their capacity to release ferulic acid from natural substrates: corn and wheat brans. Compared to free enzymes FAEA and XYNB, a higher synergistic effect was obtained by using FLX and FLXLC for both substrates. Moreover, the release of ferulic acid from corn bran was increased by using FLXLC rather than FLX. This result confirms a positive role of the CBM. In conclusion, these results demonstrated that the fusion of naturally free cell wall hydrolases and an A. niger-derived CBM onto bifunctional enzymes enables the increase of the synergistic effect on the degradation of complex substrates.     

Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos

The Bex1/Rex3 gene was recently identified as an X-linked gene that is differentially expressed between parthenogenetic and normal fertilized, preimplantation stage mouse embryos. The Bex1/Rex3 gene appears to be expressed preferentially from the maternal X chromosome in blastocysts, but from either X chromosome in later stage embryonic tissues and adult tissues. To investigate whether differential expression of the Bex1/Rex3 gene between normal and parthenogenetic blastocyst stage embryos reflects genomic imprinting at the Bex1/Rex3 locus itself, or instead is the result of preferential inactivation of the paternal X chromosome or differences in timing of cellular differentiation, we examined in detail the expression pattern of the Bex1/Rex3 mRNA in normal preimplantation stage embryos, and compared its expression between androgenetic, gynogenetic, and normal fertilized embryos. Expression data reveal that the Bex1/Rex3 gene is initially transcribed at the 2-cell stage, transiently induced at the 8-cell stage, and then increases in expression again at the blastocyst stage. Very little expression is observed in isolated inner cell masses, indicating selective expression in the trophectoderm. Comparisons of Bex1/Rex3 mRNA expression between male and female androgenetic and control embryos and gynogenetic embros failed to reveal any significant difference in expression between the different classes of embryos at the 8-cell stage, or the expanding blastocyst stage (121 hr post-hCG). At the late blastocyst stage (141 hr post-hCG), expression was significantly lower in XY control embryos as compared with XX controls. Bex1/Rex3 mRNA expression did not differ between XX and XY androgenones at the blastocyst stage or between gynogenones and XX control embryos. Thus, the Bex1/Rex3 gene does not appear to be regulated directly by genomic imprinting during the preimplantation period, just as it is not regulated by imprinting at later stages. Apparent differences in gene expression may arise through the effects of trophectoderm-specific expression coupled with differences in timing of trophectoderm differentiation between the different classes of embryos and effects of preferential paternal X chromosome inactivation (XCI).     

Inhibition of HIV-1 nuclear import via schiff base formation with arylene bis(methylketone) compounds

Arylene bis(methylketone) compounds specifically block nuclear translocation of the HIV-1 pre-integration complex by forming Schiff-base adducts with contiguous lysines within nuclear localization signal.     

Rejection of tumors of the B cell lineage by idiotype-vaccinated mice

Idiotypic determinants of immunoglobulins of malignant B lymphocytes and plasma cells are tumor-specific antigens and have been used extensively in immunotherapy studies. The mechanisms involved in resistance to tumor challenge following idiotype vaccination are poorly understood. Although a predominant role has been attributed to anti-idiotype antibodies, both humoral and cellular immune responses are probably involved. Cell-mediated responses may be particularly effective against tumor cell variants that do not express the idiotype on the cell surface and are therefore resistant to anti-idiotype antibodies but continue to produce one of the original immunoglobulin polypeptides that may be processed and presented to T cells. In this report we describe two experimental models of idiotype vaccination in which antibodies are unlikely to play a role, and hence tumor immunity is attributed to cell-mediated responses. One model consists of the murine B lymphocyte tumor 38C-13 and its idiotype-negative variant DB2, which has lost the idiotypic specificity of the parental 38C-13 cell line through the production of a different light chain but expresses the original heavy chain. Vaccination of mice with the purified IgM of 38C-13 induced resistance to 38C-13 tumor cells as well as to the variant cells. Although immunized mice produced high levels of anti-idiotype antibodies that bound to 38C-13 cells, no binding of antibodies to DB2 cells occurred. The finding that idiotype-vaccinated mice were resistant to idiotype-negative DB2 cells suggested that cellular mechanisms are involved in mediating resistance. The second model consists of the two plasma cell line JLμs and JLμm, which produce IgM with an identical specificity. Whereas one of them (JLμs) secretes the IgM, the other one(JLμm) can neither secrete nor deposit it on the cell surface. Immunization against JLμs IgM followed by tumor challenge resulted in prolonged survival of both JLμs- and JLμm-challenged mice. Although sera of immunized mice contained high levels of anti-idiotype antibodies, they did not react with the plasmacytoma cells. Similarly to the results obtained in the 38C-13 experimental model, these results suggest that a non-antibody-mediated mechanism was involved in the resistance of mice to tumor growth. Received: 11 June 1998 / Accepted: 26 November 1998     

Maternal estrogen and progesterone levels after hypophysectomy in early pregnancy and in term fetuses or newborn monkeys

Pregnant rhesus monkeys ($\text{Macaca}$$\text{mulatta}$) were hypophysectomized at 8–10 weeks gestation to determine effects on plasma levels of estrone (E₁), estradiol-17β (E₂) and progesterone (P). The first group of monkeys was subsequently fetectomized At 107–114 days. After hypophysectomy there was an initial decrease in maternal peripheral plasma E₂ followed by a rise to preoperative levels within 4–5 weeks. Plasma levels of e₁ and P were not markedly altered. After fetectomy, peripheral estrogen concentrations, especially E₂, declined markedly. In the second experimental series, we have examined the effects of maternal hypophysectomy on levels of E₁, E₂ and P either (1) in both mother and newborn baby or (2) in mother, term fetus and umbilical vein. Groups of hypophysectomized and intact pregnant monkeys (3 each) were delivered by cesarean section at the expected time of parturition. Other hypophysectomized and intact monkeys (2 each) delivered normally. E₂ levels were elevated significantly in plasma of hypophysectomized monkeys at the time of cesarean delivery and in newborn babies of hypophysectomized mothers shortly after parturition. Except for these differences, the maternal hypophysis apparently is not a major factor in the control of E₁, E₂ and P concentrations in pregnant rhesus monkeys.     

Two Interlinked Bistable Switches Govern Mitotic Control in Mammalian Cells

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Microcalorimetric study of glutamine fixation on the glutamine-binding protein of Escherichia coli.

The enthalpy variation (ΔH) induced by addition of glutamine to glutamine binding protein isolated from $\text{E. coli}$ has been studied by microcalorimetry. The reaction was very exothermic. The free energy variation (ΔG) was calculated from the dissociation constant (K_D) measured by dialysis techniques. The entropic variation (ΔS) was deduced from ΔG and ΔH values; it was found highly negative, indicating that an important conformational change is occuring. Comparison with others binding proteins and possible significance of such a phenomenon is discussed.     

Responses of atrial natriuretic peptide and other fluid regulating hormones to long distance swimming in the sea

The plasma hormonal response following a swimming competition in the sea (18 km) was evaluated in 12 top level male endurance swimmers. At the end of the effort, while plasma renin activity (PRA) and aldosterone concentration (ALDO) were unchanged, a significant increase in plasma atrial natriuretic peptide (ANP) and antidiuretic hormone (ADH) concentrations were recorded. These changes were associated with a decrease in haematocrit and an increase in Na⁺ and Cl^– plasma concentrations. The individual variations of ANP (difference between the final and initial concentrations) were inversely correlated with the corresponding individual variations of PRA and ADH. The results suggest that, during prolonged swimming, ANP may exert an inhibitory effect on the PRA-ALDO axis and have a modulatory role with regard to ADH secretion.     

Effects of different doses of atorvastatin on human apolipoprotein B-100, B-48, and A-I metabolism

Nine hypercholesterolemic and hypertriglyceridemic subjects were enrolled in a randomized, placebo-controlled, double-blind, crossover study to test the effect of atorvastatin 20 mg/day and 80 mg/day on the kinetics of apolipoprotein B-100 (apoB-100) in triglyceride-rich lipoprotein (TRL), intermediate density lipoprotein (IDL), and LDL, of apoB-48 in TRL, and of apoA-I in HDL. Compared with placebo, atorvastatin 20 mg/day was associated with significant reductions in TRL, IDL, and LDL apoB-100 pool size as a result of significant increases in fractional catabolic rate (FCR) without changes in production rate (PR). Compared with the 20 mg/day dose, atorvastatin 80 mg/day caused a further significant reduction in the LDL apoB-100 pool size as a result of a further increase in FCR. ApoB-48 pool size was reduced significantly by both atorvastatin doses, and this reduction was associated with nonsignificant increases in FCR. The lathosterol-campesterol ratio was decreased by atorvastatin treatment, and changes in this ratio were inversely correlated with changes in TRL apoB-100 and apoB-48 PR. No significant effect on apoA-I kinetics was observed at either dose of atorvastatin. Our data indicate that atorvastatin reduces apoB-100- and apoB-48-containing lipoproteins by increasing their catabolism and has a dose-dependent effect on LDL apoB-100 kinetics. Atorvastatin-mediated changes in cholesterol homeostasis may contribute to apoB PR regulation.