Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-B activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85 subunit of PI3-kinase interacted with tyrosine-phosphorylated IB- in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-B activation. The inhibition of NF-B activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-B p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-B activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-B activation through interaction with tyrosine-phosphorylated IB- and contributing to tyrosine phosphorylation of p65 NF-B.     

Localization of κ-casein on thin sections of casein micelles by the gold method

Summary The ultrastructural location of -casein in bovine casein micelles was investigated by the protein A-gold method. Casein micelles, fixed in glutaraldehyde, were embedded at low temperature to enhance immunocytochemical marking of thin sections. -Casein was found distributed throughout the micelles of all sizes with a higher concentration in the smaller micelles. No peripheral location of -casein was observed, even in the larger micelles. These results do not agree with coat-core structures proposed for casein micelles. However they favor models where -casein is distributed uniformly throughout the micelles.     

Expression of a bovine κ-CN cDNA in the mammary gland of transgenic mice utilizing a genomic milk protein gene as an expression cassette

Transgenic mice were produced by microinjection of a DNA construct composed of the bovine -casein (-CN) cDNA under the control of the goat -CN 5 promoter elements and 3 flanking regions into pronuclear-stage embryos. The gene construct targeted the expression of bovine -CN RNA to the mammary gland and secretion of bovine -CN in the milk. In the three lines studied (BC-7, BC-31 and BC-67) the transgene was stably integrated and propagated as a Mendelian locus. Expression of the bovine protein in lactating mice from the three transgenic lines was demonstrated by northern and western blots. In ten different tissues analysed by northern blotting, expression was confined to the mammary gland of lactating transgenic mice from line BC-7, with low-level expression also observed in the salivary gland of lines BC-31 and BC-67. Transgene expression in the mammary gland paralleled normal casein gene expression during lactation and was not observed in virgin females. The level of bovine -CN mRNA expression on day 10 of lactation in hemizygous transgenic females in relation to endogenous mRNA of whey acid protein (WAP) gene expression was 14%, 69% and 127% in lines BC-7, BC-31 and BC-67, respectively. No association between transgene copy number and expression was observed. The bovine -CN concentration in milk on day 10 of lactation ranged from 0.94 to 3.85 mg of protein per ml of milk. The bovine -CN expressed in mouse milk had the same molecular mass and immunoactivity with polyclonal antibodies as did -CN from bovine milk. A high degree of variation in the production of bovine -CN within each of the transgenic lines was observed.     

Interactions between NFκB and Its Inhibitor iκB: Biophysical Characterization of a NFκB/iκB-α Complex

The N-terminal domain (1–318 amino acids) of mouse NFB (p65) has been purified to homogeneity from the soluble fraction of Escherichia coli cells expressing this protein. Its complex with a full-length iB- (MAD3, 1–317 amino acids) molecule was generated by binding the E. coli-derived iB- to the purified NFB and purifying the complex by sequential chromatography. The stoichiometry of NFB to iB in the complex was determined to be 2 to 1 by light scattering and SDS–polyacrylamide gel electrophoresis. The secondary structure of the NFB (p65) determined by Fourier-transform infrared (FTIR) spectroscopy is in good agreement with that of the p50 in the crystal structure of the p50/DNA complex, indicating that no significant structural change in NFB occurs upon binding of DNA. The FTIR spectrum of the NFB/iB complex indicates that its secondary structure is composed of 17% -helix, 39% -strand, 18% irregular structures, and 26% -turns and loops. By comparing these data to the FTIR data for NFB alone, it is concluded that the iB (MAD3) in the complex contains 35% -helix, 27% -strand, 22% irregular structures, and 16% -turns and loops. Circular dichroism (CD) analysis of a shorter form of iB (pp40) indicates that it contains at least 20% -helix and that the iB subunit accounts for nearly all of the -helix present in the NFB/iB complex, consistent with the FTIR results. The stabilities of NFB, iB, and their complex against heat-induced denaturation were investigated by following changes in CD signal. The results indicate that the thermal stability of iB is enhanced upon the formation of the NFB/iB complex.     

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Quercetin suppresses proinflammatory cytokines production through MAP kinases and NF-κB pathway in lipopolysaccharide-stimulated macrophage

Quercetin is a flavonoid molecule ubiquitous in nature and functions as an anti-oxidant and anti-inflammatory agent with little toxicity in vivo and in vitro. Dose- and time-dependent effect of quercetin has been investigated on proinflammatory cytokine expression and NO production, focusing on its effects on the MAP kinases and the NF-B signal transduction pathways in LPS-stimulated RAW 264.7 cells by using RT-PCR and immunoblotting. Quercetin strongly reduced activation of phosphorylated ERK kinase and p38 MAP kinase but not JNK MAP kinase by LPS treatment. In addition, quercetin treatment inhibited NF-B activation through stabilization of the NF-B/IB complex and IB degradation and proinflammatory cytokines and NO/iNOS expression. Quercetin may exert its anti-inflammatory and immunomodulatory properties in the effect molecules such as proinflammatory cytokines and NO/iNOS by suppressing the activation of ERK and p38 MAP kinase, and NF-B/IB signal transduction pathways.     

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that -, -, and -caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of -lactalbumin and -lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When -casein was digested with chymosin and fractionated top--casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but onlyp--casein fragment stimulated IgM production. These results indicate that -casein has at least two proliferation stimulating sites and an IgM production stimulating site in thep--casein region.     

Inhibition of NF-κ B activity and cFLIP expression contribute to viral-induced apoptosis

Virus-induced activation of nuclear factor-kappa B (NF-B) is required for Type 3 (T3) reovirus-induced apoptosis. We now show that NF-B is also activated by the prototypic Type 1 reovirus strain Lang (T1L), which induces significantly less apoptosis than T3 viruses, indicating that NF-B activation alone is not sufficient for apoptosis in reovirus-infected cells. A second phase of virus-induced NF-B regulation, where NF-B activation is inhibited at later times following infection with T3 Abney (T3A), is absent in T1L-infected cells. This suggests that inhibition of NF-B activation at later times post infection also contributes to reovirus-induced apoptosis. Reovirus-induced inhibition of stimulus-induced activation of NF-B is significantly associated with apoptosis following infection of HEK293 cells with reassortant reoviruses and is determined by the T3 S1 gene segment, which is also the primary determinant of reovirus-induced apoptosis. Inhibition of stimulus-induced activation of NF-B also occurs following infection of primary cardiac myocytes with apoptotic (8B) but not non-apoptotic (T1L) reoviruses. Expression levels of the NF-B-regulated cellular FLICE inhibitory protein (cFLIP) reflect NF-B activation in reovirus-infected cells. Further, inhibition of NF-B activity and cFLIP expression promote T1L-induced apoptosis. These results demonstrate that inhibition of stimulus-induced activation of NF-B and the resulting decrease in cFLIP expression promote reovirus-induced apoptosis.     

Monocyte-derived dendritic cells that capture dead tumor cells secrete IL-12 and TNF-α through IL-12/TNF-α/NF-κB autocrine loop

This study focused on the question of how monocyte-derived dendritic cells (Mo-DCs) that capture dead tumor cells (Mo-DCs-Tum) secrete interleukin 12 (IL-12) and tumor necrosis factor (TNF-). Mo-DCs-Tum showed higher secretions of IL-12 and TNF- than were shown by Mo-DCs. Enhanced nuclear factor-kappa B (NF-B) activation was also induced in Mo-DCs-Tum within 6 h. The NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), suppressed both IL-12 and TNF- secretions from Mo-DCs-Tum. Administration of recombinant TNF- or IL-12 enhanced IL-12 or TNF- secretion respectively in Mo-DCs-Tum. Addition of anti-TNF- or anti-IL-12 neutralizing antibody decreased NF-B activation and IL-12 or TNF- secretion in Mo-DCs-Tum. These results suggest that TNF- or IL-12 secretion induces NF-B activation, and it stimulates further TNF- and IL-12 secretions, i.e., an IL-12/TNF-/NF-B autocrine loop, in Mo-DCs-Tum. Thus, Mo-DCs-Tum secrete a large amount of IL-12 and TNF- through accelerated NF-B activation induced by the IL-12/TNF-/NF-B autocrine loop.     

Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells

Nitric oxide (NO), a reactive nitrogen species, plays an important role in inflammatory lung damage. In the present study, we investigated the role of NO in DNA-binding activity of NF-B in macrophages stimulated with silica or other inflammatory stimulants. Treatment of mouse macrophages (RAW264.7 cells) with a selective inhibitor of inducible nitric oxide synthase (iNOS), L-N⁶-(1-iminoethyl) lysine (L-NIL), or a nonselective iNOS inhibitor, N-nitro-L-arginine methylester (L-NAME), resulted in inhibition of silica-induced nitric oxide production as well as silica-induced NF-B activation. L-NIL also effectively inhibited NF-B activation induced by other inflammatory stimulants, such as lipopolysaccharide (LPS) or muramyl dipeptide (MDP). These inhibitory effects of L-NIL and L-NAME on silica- or LPS-induced NF-B activation were also observed in primary rat alveolar macrophages. Furthermore, NO generating compounds, such as sodium nitroprusside (SNP) and 3-morpholinosydnonimine (SIN-1), caused a dose-dependent increase in NF-B activation, which was positively correlated with the level of NO production. Specific inhibitors of protein tyrosine kinase, such as genistein and AG494, prevented NF-B activation in SNP- or SIN-1 treated cells, suggesting involvement of tyrosine kinase in the NO signaling pathway leading to NF-B activation. In contrast, inhibitors of protein kinase C or A, such as staurosporine or H89, had no inhibitory effect on SIN-1 induced NF-B activation. Metalloporphyrins, such as tetrakis (N-methyl-4-pyridyl) porphyrinato iron (III) (Fe-TMPyP) and Zn-TMPyP which are known to alter NO-dependent activity, markedly inhibited silica- and LPS-induced NF-B activation. The results suggest that NF-B activation in macrophages can be induced under certain conditions by nitric oxide and that nitric oxide produced by phagocytes exposed to inflammatory agents may up-regulate the activation of NF-B.     

Homocysteine stimulates inducible nitric oxide synthase expression in macrophages: Antagonizing effect of ginkgolides and bilobalide

Hyperhomocysteinemia is an independent risk factor for atherosclerotic diseases. Inducible nitric oxide synthase (iNOS) is mainly expressed in macrophages upon stimulation. Overproduction of nitric oxide (NO) by iNOS can exacerbate the development of atherosclerosis. Our previous studies demonstrated that the extract of ginkgo biloba leaves (EGb) inhibited the iNOS-mediated NO production in monocyte-derived macrophage. We also reported that homocysteine could stimulate monocyte chemoattractant protein-1 (MCP-1) expression in vascular cells causing enhanced monocyte chemotaxis. The objective of the present study was to investigate the effect of homocysteine on iNOS-mediated NO production in macrophages and the antagonizing effect of EGb. Human monocytic cell (THP-1)-derived macrophages were incubated with homocysteine for various time periods. Homocysteine at concentrations of 0.05–0.1 mM significantly stimulated NO production and iNOS activity in macrophages via increased expression of iNOS mRNA and protein. The increased iNOS expression was associated with activation of nuclear factor-kappa B (NF-B) arising from reduced expression of inhibitor protein (IB) mRNA as well as increased phosphorylation of IB protein in homocysteine-treated cells. EGb and its terpenoids (ginkgolide A, ginkgolide B and bilobalide) could antagonize the homocysteine effect on iNOS expression in macrophages via their antioxidant effect resulting in attenuation of NF-B activation. Taken together, our results have demonstrated that homocysteine, at pathophysiological concentrations, stimulates iNOS-mediated NO production in macrophages. EGb and its terpenoids can antagonize such stimulatory effect via antioxidation and attenuation of NF-B activation.     

Kappa opioid agonists inhibit transmitter release from guinea pig hippocampal mossy fiber synaptosomes

Opioid agonists specific for the , , and opioid receptor subtypes were tested for their ability to modulate potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity from guinea pig hippocampal mossy fiber synaptosomes. The opioid agonists U-62,066E and (–) ethylketocyclazocine, but not the agonist [D-Ala²,N-MePhe⁴,Gly⁵-ol]-enkephalin (DAGO) nor the agonist [D-Pen^2,5]enkephalin (DPDE), inhibited the potassium-evoked release of L-glutamate and dynorphin B-like immunoreactivity. U-62,066E, but not DAGO or DPDE, also inhibited the potassium-evoked rise in mossy fiber synaptosomal cytosolic Ca²⁺ levels, indicating a possible mechanism for agonist inhibition of transmitter release. DAGO and DPDE were found to be without any effect on cytosolic Ca²⁺ levels or transmitter release in this preparation. The U-62,066E inhibition of the potassium-evoked rise in synaptosomal cytosolic Ca²⁺ levels was partially attenuated by the opioid antagonist quadazocine and insensitive to the -opioid specific antagonist ICI 174,864 and the opioid-preferring antagonists naloxone and naltrexone. Quadazocine also reversed U-62,066E inhibition of the potassium-evoked release of L-glutamate, but not dynorphin B-like immunoreactivity. These results suggest that opioid agonists inhibit transmitter release from mossy fiber terminals through both opioid and non- opioid receptor mediated mechanisms.     

Endogenous regulation of the acute inflammatory response

The acute inflammatory response has been triggered in rat lungs by deposition of IgG immune complexes. The inflammatory reaction triggered is highly tissue damaging and requires activation of NF-B with ensuing generation of chemokines and cytokines. Endogenous generation of IL-10 and IL-13 as well as secretory leukocyte protease inhibitor (SLPI), significantly regulates this inflammatory response. IL-10 and IL-13 attenuate NF-B activation by interfering with breakdown of IB, while SLPI likewise suppresses NF-B activation, but by interfering with breakdown of IB. Antibody induced blockade of IL-10, IL-13 or SLPI enhances NF-B activation in lung and exacerbates the lung inflammatory response and injury. These data indicate that endogenous IL-10, IL-13 and SLPI are important regulators of the inflammatory response by reducing gene activation with resultant generation of peptide mediators/cytokines and chemokines.     

Second-derivative Fourier transform infrared spectra of seaweed galactans

The Fourier transform infrared spectra of agar, agarose, -, -, and -carrageenan, and ofChondrus canaliculatus, Iridaea ciliata, I. membranacea, I. laminarioides andGracilaria chilensis polysaccharides were recorded in the 4000–400 cm^-1 region. The bands in the second derivative mode are sharper and more bands are resolved than in the normal spectra.Agar, agarose andG. chilensis phycocolloids exhibit diagnostic bands at 790 and 713 cm^-1. -, - and -carrageenans, and native carrageenan-type polysaccharides fromC. canaliculatus andIridaea species exhibit bands at around 1160, 1140, 1100, 1070, 1040, 1008, 610, and 580 cm^-1. Therefore, FT-IR spectroscopy in the second-derivative mode may be applied to differentiate between agar- and carrageenan-types seaweed galactans.     

Involvement of nuclear factor-kappaB in endothelin-A-receptor-induced proliferation and inhibition of apoptosis

Endothelins have been implicated in the regulation of cell proliferation, differentiation, and apoptosis, but the mechanisms of these complex events are not yet fully understood. Although the nuclear factor-B (NF-B) was shown to play a prominent role in the above processes, its participation in endothelin receptor A (ET_AR) signaling has not been previously demonstrated. This study provides evidence that NF-B is involved in ET_AR-induced proliferation and inhibition of apoptosis. Endothelin (ET)-1, ET-3, and sarafotoxin b induce cell proliferation and prevent apoptosis induced by serum deprivation in a Chinese hamster lung (CCL39) cell line that stably expresses ET_AR (CCL39ET_A). Activation of ET_AR resulted in enhanced DNA-binding activity of NF-B and degradation of IB-. Expression of the dominant negative form of IB- (IBN) inhibited the proliferative activities mediated by ET_AR as well as its antiapoptotic activities. Treatment of the cells with prostaglandin A₁, an inhibitor of IB kinase-, reduced ET-1-induced proliferation and its antiapoptotic effect. These findings indicate that the regulation of cell proliferation and apoptosis by ET_AR is mediated by the ET_AR-activated NF-B.     

Caseins of various origins and biologically active casein peptides and oligosaccharides: Structural and physiological aspects

Summary The first part of the present review is focused on structural aspects concerning the so far studied casein fractions of various origins: they are compared to the four classical major bovine caseins (_sl-, _s2- - and ). The calcium-sensitive casein fractions are always phosphorylated whereas -caseins are glycosylated. The study of the casein genes showed that the calcium-sensitive caseins diverged from a common ancestral gene and during the evolution, intergenic and intragenic duplications occurred. The considerable conservation of the phosphorylation sites emphasizes the importance of phosphorylated residues for the function of caseins, i.e. the formation of micelles and the binding of Ca²⁺. In -caseins all the prosthetic sugar groups are linked by O-glycosidic linkages: their number varies from 0 to 5 in bovine -casein and up to 10 in human -casein. The structures of the known -casein carbohydate moieties are described. Finally the milk clotting process (interaction -casein/chymosin) is compared to the blood clotting process (interaction fibrinogen/thrombin): a large number of similarities could be noted between both clotting phenomena.The second part of the review is devoted to the study of short casein peptides endowed with various biological activities. Some of them behaved as immunomodulators or casomorphins or angiotensin I converting enzyme inhibitors; others demonstrated an effect on platelet functions. A strategic zone containing immunostimulating and opioid peptides could be located in cow and human -caseins. Furthermore bitter peptides, emulsifying peptides, calcium absorption enhancing peptides, chymosin-inhibiting peptides, have also been described and several further properties have been attributed to the -caseinoglycopeptide; two tetrasaccharides isolated from the latter possess blood group activities.In conclusion caseins, the main milk proteins, should not only be considered as a nutriment but as a possible source of biologically active components.If, in the future, some of the discussed active peptides cannot be characterized in vivo, they can all, nevertheless, be synthesized and used either as food additives or in pharmacology.     

Calcium-induced associations of the caseins: Thermodynamic linkage of calcium binding to colloidal stability of casein micelles

The caseins occur in milk as colloidal complexes of protein aggregates, calcium, and inorganic phosphate. As determined by electron microscopy, these particles are spherical and have approximately a 650 Å radius (casein micelles). In the absence of calcium, the protein aggregates themselves (submicelles) have been shown to result from mainly hydrophobic interactions. The fractional concentration of stable colloidal casein micelles can be obtained in a calcium caseinate solution by centrifugation at 1500g. Thus, the amount of stable colloid present with varying Ca²⁺ concentrations can be determined and then analyzed by application of equations derived from Wyman's Thermodynamic Linkage Theory. Ca²⁺-induced colloid stability profiles were obtained experimentally for model micelles consisting of only _s1- (a calcium insoluble casein) and the stabilizing protein -casein, eliminating the complications arising from - and minor casein forms. Two distinct genetic variants _s1-A andB were used. Analysis of _s1-A colloid stability profiles yielded a precipitation (salting-out) constantk ₁, as well as colloid stability (salting-in) parameterk ₂. No variations ofk ₁ ork ₂ were found with increasing amounts of -casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added -casein an association constant of 4 L/g could be calculated for the complexation of _s1-A and -casein. For the _s1-B and -casein micelles, an additional Ca²⁺-dependent colloidal destabilization parameter,k ₃, was added to the existingk ₁ andk ₂ parameters in order to fully describe this more complex system. Furthermore, the value ofk ₃ decreased with increasing concentration of -casein. These results were analyzed with respect to the specific deletion which occurs in _s1-caseinA in order to determine the sites responsible for these Ca²⁺-induced quaternary structural effects.     

Environmental influences on bovine kappa-casein: reduction and conversion to fibrillar (amyloid) structures

The caseins of milk form a unique calcium–phosphate transport complex that provides these necessary nutrients to the neonate. The colloidal stability of these particles is primarily the result of -casein. As purified from milk, this protein occurs as spherical particles with a weight average molecular weight of 1.18 million. The protein exhibits a unique disulfide bonding pattern, which (in the absence of reducing agents) ranges from monomer to octamers and above on SDS-PAGE. Severe heat treatment of the -casein (90°C) in the absence of SDS, before electrophoresis, caused an increase in the polymeric distribution: up to 40% randomly aggregated high–molecular weight polymers, presumably promoted by free sulfhydryl groups (J. Protein Chem. 17: 73–84, 1998). To ascertain the role of the sulfhydryl groups, the protein was reduced and carboxymethylated (RCM-). Surprisingly, at only 37°C, the RCM--casein exhibited an increase in weight average molecular weight and tendency to self-association when studied at 3000 rpm by analytical ultracentrifugation. Electron microscopy (EM) of the 37°C RCM sample showed that, in addition to the spherical particles found in the native protein, there was a high proportion of fibrillar structures. The fibrillar structures were up to 600 nm in length. Circular dichroism (CD) spectroscopy was used to investigate the temperature-induced changes in the secondary structure of the native and RCM--caseins. These studies indicate that there was little change in the distribution of secondary structural elements during this transition, with extended strand and turns predominating. On the basis of three-dimensional molecular modeling predictions, there may exist a tyrosine-rich repeated sheet-turnsheet motif in -casein (residues 15–65), which may allow for the stacking of the molecules into fibrillar structures. Previous studies on amyloid proteins have suggested that such motifs promote fibril formation, and near-ultraviolet CD and thioflavin-T binding studies on RCM--casein support this concept. The results are discussed with respect to the role that such fibrils may play in the synthesis and secretion of casein micelles in lactating mammary gland.     

Production of citric acid with immobilized Yarrowia lipolytica

Summary Citric acid was produced with immobilized Yarrowia lipolytica yeast in repeated batch-shake-flask and air-lift fermentations. In active and passive immobilization methods calcium alginate, -carrageenan, polyurethane gel, nylon web and polyurethane foams were tested as carriers in repeated-batch fermentations. The highest citric acid productivity of 155 mg l^–1 h^–1 was reached with alginate-bead-immobilized cells in the first batch. A decrease in bead diameter from 5–6 mm to 2–3 mm increased the volumetric citric acid productivity threefold. In an air-lift bioreactor the highest citric acid productivity of 120 mg l^–1 h^–1 with a product concentration of 16.4 g l^–1 was obtained with cells immobilized in -carrageenan beads. Offprint requests to: H. Kautola     

Variants within the 5′-flanking regions of bovine milk protein genes: I. κ-casein-encoding gene

In order to identify DNA variants within the 5-flanking region of the bovine -casein (Cn)-encoding gene, this area of the gene from 13 cows belonging to seven breeds (Holstein Friesian, Brown Swiss, German Simmental, Jersey, Galloway, Scottish Highland and Ceylon Dwarf Zebu) was analysed. For each individual, about 1 kb of the 5-flanking region including exon I was amplified by polymerase chain reaction (PCR). The biotinylated PCR product was immobilized on magnetic beads followed by direct bidirectional sequencing using an automated DNA sequencer. Fifteen DNA variants were identified, some of which are located within potential regulatory sites and possibly involved in the expression of the -casein encoding gene.Abbreviations AP2 activator protein 2 - bp base pair(s) - GRE/RC glucocorticoid response element/reverse complement - HNF3 hepatocyte nuclear factor 3 Cn -casein - MGF mammary gland-specific nuclear factor - nt nucleotid(s), OCT1 octamer-binding site 1 - PA polyacrylamide - PCR polymerase chain reaction - PMF pregnancy-specific mammary nuclear factor - kb kilobase(s) or 1000 bp