Expression of human lactoferrin in milk of transgenic mice

The expression of human lactoferrin (hLF) in the milk of transgenic mice is described. Regulatory sequences derived from the bovine S1-casein gene were fused to the coding sequence of the hLF cDNA and several lines of transgenic mice were generated. Human LF RNA was detected exclusively in the mammary gland of lactating females and only after the onset of lactation. No aberrant RNA products could be detected using northern blotting and primer extension analysis. The hLF concentrations in the milk ranged from less than 0.1 to 36 g ml^–1. Human LF thus expressed did not differ from human milk derived LF, with respect to molecular mass and immunoreactivity with monoclonal and polyclonal antibodies.     

Locus-control-region-coupled betaS Antilles- and alpha2-hemoglobin genes select for high alpha2-hemoglobin expression in adult transgenic mice

Two transgenic lines of mice were produced which contained the ^S _Antilles- and ₂-hemoglobin genes trandemly coupled to the micro locus control region (LCR). The LCR^S _Antilles2-hemoglobin transgenic mice expressed high levels of ₂-hemoglobin while ^S _Antilles-hemoglobin expression was virtually undetectable. Abundant ₂-hemoglobin protein was observed in the blood of transgenic mice, while ^S _Antilles-hemoglobin chains could not be detected. Transgenic red blood cells had substantially decreased sensitivity to osmotic lysis. Attempts to produce homozygotes containing the transgene were unsuccessful. The phenotype of these mice closely resembles that of -thalassemic mice. The LCR^S _Antilles2 transgenic mice demonstrate that if the LCR is coupled to the ^S _Antilles- and ₂-hemoglobin genes in tandem, only the distal ₂-hemoglobin gene is selected for expression to significant levels in adult mice. These results support a reciprocally competitive model for LCR-hemoglobin developmental switching.     

Models for glycosidic juvenogens: Enzymic formation of selected alkyl-β-D-glucopyranosides and alkyl-β-D-galactopyranosides under microwave irradiation

2-(4-Methoxybenzyl)-1-cyclohexyl--d-glucopyra nosides (1b and 2b) and 2-(4-methoxybenzyl)-1-cyclohexyl--d-galactopy ranosides (1c and 2c), models for glycosidic juvenogens, were synthesized using either D-glucose or D-galactose [in their natural form (3 and 5) or activated form (4 and 6)], and the respective racemic cis or trans isomers of 2-(4-methoxybenzyl)-1-cyclohexanol (1a and 2a) by either enzymic reverse hydrolysis or transglycosylation under both standard heating and microwave irradiation. Commercially available almond -glucosidase (EC 3.2.1.21) or -galactosidase (EC 3.2.1.23) from Escherichia coli were employed using different acetonitrile/water mixtures [9/1 (v/v) for the reverse hydrolysis, and 4/1 (v/v) for the transglycosylation].     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.     

Secondary structural changes in the intact and the disulfide bridges cleaved beta-lactoglobulin A and B in solutions of urea, guanidine hydrochloride, and sodium dodecyl sulfate

The relative proportions of -helix, -sheet, and unordered form in -lactoglobulin A and B were examined in solutions of urea, guanidine, and sodium dodecyl sulfate (SDS). In the curve-fitting method of circular dichroism (CD) spectra, the reference spectra of the corresponding structures determined by Chen et al. (1974) were modified essentially according to the secondary structure of -lactoglobulin B predicted by Creamer et al. (1983), i.e., that the protein has 17% -helix and 41% -sheet. The two variants showed no appreciable difference in structural changes. The reduction of disulfide bridges in the proteins increased -sheet up to 48% but did not affect the -helical proportion. The -helical proportions of nonreduced -lactoglobulin A and B were not affected below 2 M guanidine or below 3 M urea, but those of the reduced proteins began to decrease in much lower concentrations of these denaturants. By contrast, the -helical proportions of the nonreduced and reduced proteins increased to 40–44% in SDS. The -sheet proportions of both nonreduced and reduced proteins, which remained unaffected even in 6 M guanidine and 9 M urea, decreased to 24–25% in SDS.     

Induction of cellulose- and xylan-degrading enzyme complex in the yeast Trichosporon cutaneum

The specificity of induction of cellulose- and xylan-degrading enzymes was investigated on the yeast strain Trichosporon cutaneum CCY 30-5-4 using series of compounds structurally related to cellulose and xylan, including monosaccharides, glycosides, glucooligosaccharides and xylooligosaccharides. Determination of activities of secreted cellulase and -xylanase, intracellular, cell wall bound and extracellular -glucosidase and -xylosidase revealed that: (1) The synthesis of xylan-degrading enzymes is induced in the cell only by xylosaccharides, 1,3--xylobiose, 1,2--xylobiose, 1,4--xylosyl-L-arabinose, 1,4--xylobiose and thioxylobiose being the best inducers. The xylan-degrading enzymes show different pattern of development in time and discrete cellular localization, i.e. intracellular -xylosidase precedes extracellular -xylanase. (2) A true cellulase is not inducible by glucosaccharides and cellulose. Negligible constitutive cellulase activity was detected which was about two orders lower than an induced cellulase in the typical cellulolytic fungus Trichoderma reesei QM 9414. (3) The best inducer of intracellular -glucosidase splitting cellobiose was thiocellobiose in a wide range of concentration (0.1–10 mM), whereas xylosaccharides at high concentrations induced -xylosidase of xylobiose type and a non-specific aryl -D-glucosidase.The results were confirmed by growing cells on cellulose and xylan. T. cutaneum was found to be a xylan-voracious yeast, unable to grow on cellulose.     

Comparison of wild-type and UV-mutant β-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications

A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal -glucans. Activity against -(1, 3)(1, 4)-d-glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less -glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley -glucan (13.0–16.9%), but were more active against crude -glucan from barley (16.0–24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash -glucan. Finally, TC2 and TC5 produce more efficient -glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.     

APP Processing Enzymes (Secretases) as Therapeutic Targets: Insights from the Use of Transgenics (Tgs) and Transfected Cells

Secretases degrade amyloid precursor protein (APP) releasing fragments (-peptides A, A_x) that assemble to form hallmark extracellular deposits in Alzheimer's disease (AD) correlating with disease severity. As such, secretases supply targets for therapeutic intervention and form the focus of this overview. Progress in elucidating secretases and their modes of catalysis come from exploiting the use of transgenics or transfected cells. In addition to Ax, secretases also release C-terminal fragments with putative signaling properties (amyloid intracellular domain, AICD) similar in concept to those available for conversion of the Notch-r to release the nuclear transactivator NICD. The review considers lingering questions on APP fragmentation by secretase action, ancillary proteins such as presenilins (PS1/2), nicastrin, XII, or proteases (caspases), and the influence of familial mutations (mAPP, mPS) in terms of fibrillogenesis.     

Structural Changes of β-Lactoglobulin during Thermal Unfolding and Refolding – An FT-IR and Circular Dichroism Study

We have quantitatively characterized by FT-IR spectroscopy the contents of secondary structure of -lactoglobulin during thermal unfolding and subsequent refolding. Our data clearly indicate that considerable amount of secondary structure, particularly -sheet, still remained intact even at 90°C. Noticeable changes in secondary structure of -lactoglobulin were observed only above 70°C. The refolded protein regained, within limits of experimental error, all of the secondary structure lost during thermal unfolding. The data also indicate that the refolding mechanism operating at pH 7.0 and 2.0 are the same. Identical secondary structure of native and refolded -lactoglobulin was also indicated by far-UV circular dichroic spectra of the two forms of protein. Near UV circular dichroic spectra of the same two forms showed considerable differences indicating less tertiary structure of refolded -lactoglobulin. The combined CD and FT-IR data indicated that refolded form of -lactoglobulin could be characterized as a molten globule state as it had native-like secondary structure and compromised tertiary structure.     

Effects of the expression of bovine growth hormone on the testes and male accessory reproductive glands in transgenic mice

The effects of physiological and excessive levels of growth hormone (GH) on reproductive functions are poorly understood, and impairment of fertility is frequently observed in transgenic animals overexpressing GH genes. The present study was undertaken to determine the effects of chronic exposure to heterologous bovine GH (bGH) on the testes and accessory reproductive glands in transgenic mice. Endocrine function of the testes was evaluated by measuring the activities of two steroidogenic enzymes, 5-3-hydroxysteroid dehydrogenase (5-3-HSD) and 17-hydroxysteroid dehydrogenase (17-HSD). The activities of acid phosphatase, alkaline phosphatase and -glucuronidase, important hydrolytic enzymes of lysosomal origin, were measured in testes, seminal vesicles and ventral prostates in normal and transgenic mice. Testicular 5-3-HSD activity was higher in transgenic than in normal mice, while testicular 17-HSD activity in transgenic mice was not altered. Acid phosphatase activity was elevated in both seminal vesicles and ventral prostates of transgenic mice, while alkaline phosphatase activity was increased only in the prostate. The activity of -glucuronidase was elevated in the testes, seminal vesicles and ventral prostate gland of transgenic mice. These results suggest that chronic exposure to bGH is associated with significant stimulation of some hydrolytic enzymes in the testes and in the accessory reproductive glands of transgenic mice.     

Role of the pituitary gland in experimental hormonal induction and prevention of benign prostatic hyperplasia in the dog

Summary The antiandrogen, cyproterone acetate (CPA), prevents development of prostatic hyperplasia, induced in castrated dogs by a 6 month-treatment with 5-androstane-3,l 7-diol (A)alone or in combination with 17-oestradiol (E ₂). The immunoperoxidase technique was used to study functional cell types in the pars distalis of the pituitary gland and to detect growth hormone (GH) and prolactin (PRL) target sites in the prostate gland. Homologous radioimmunoassays for estimation of serum canine GH and PRL concentrations were also performed. Treatment with the combinations A + E ₂ and A + E ₂ + CPA resulted in morphological indications of stimulated GH and PRL cells and depressed gonadotrophs. This correlates well with an increase in PRL-dependent staining in glandular epithelium and fibromuscular tissue of the prostate gland. However, basal serum PRL and GH levels were not significantly affected. Treatment with A and A + E₂ stimulated, while additional treatment with CPA clearly suppressed adrenocorticotrophin/melanotrophin (ACTH/MSH) cells. These findings indicate that an endocrine imbalance in hypothalamic-pituitary-adrenal function may be involved in induction and prevention of prostatic hyperplasia in the dog.     

Effects of Lovastatin and Pravastatin on Amyloid Processing and Inflammatory Response in TgCRND8 Brain

Previous studies suggest that treatment with statins reduce beta amyloid (Abeta) deposition in brains of mouse models of Alzheimer's disease (AD) and may reduce the prevalence of AD in humans. Since lipophilicity influences the biological efficacy of statins, we compared the effects of lovastatin, a lipophilic statin, to effects of the hydrophilic pravastatin on amyloid processing and inflammatory responses in brain. Three-month old TgCRND8 mice expressing mutant human amyloid precursor protein (mHuAPP) were treated daily with various doses of either statin. After 1 month, levels of cerebral soluble and fibrillar Abeta peptides, soluble sAPPalpha, and inflammatory cytokines were measured. Both statins caused dose-dependent reductions in total Abeta peptides with parallel increases in total sAPPalpha. At all doses, slightly greater effects were observed with lovastatin than with pravastatin. In contrast, only lovastatin significantly increased levels of IL-1beta and of TNFalpha in a dose-dependent manner. Lovastatin, but not pravastatin, decreased succinic dehydrogenase and increased lactate dehydrogenase activities in skeletal muscle and increased TUNEL staining in liver. Our data demonstrate that both statins shift the balance of APP processing from excessive beta-toward the normal alpha-cleavage while reducing the total amyloid burden in TgCRND8 brain and that lovastatin, but not pravastatin, potentiates cerebral inflammation and is associated with liver and muscle histotoxicity in these animals. These data show that pravastatin can reduce amyloid burden without potentiating inflammatory responses in brain and, therefore, may have a wider dose-range of safety than have lipophilic statins in the treatment or prevention of AD.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Effect of hyperprolinemia on acetylcholinesterase and butyrylcholinesterase activities in rat

Summary. We observed here that acute proline (Pro) administration provoked a decrease (32%) of acetylcholinesterase (AChE) activity in cerebral cortex and an increase (22%) of butyrylcholinesterase (BuChE) activity in the serum of 29-day-old rats. In contrast, chronic administration of Pro did not alter AChE or BuChE activities. Furthermore, pretreatment of rats with vitamins E and C combined or alone, N-nitro-L-arginine methyl ester or melatonin prevented the reduction of AChE activity caused by acute Pro administration, suggesting the participation of oxidative stress in such effects.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside