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H A de Boer

Publications and source records attributed to H A de Boer.

At least 19 recordsLinked to original sources
PubMed2001

Over-expression of the murine pIgR gene in the mammary gland of transgenic mice influences the milk composition and reduces its nutritional value.

The polymeric immunoglobulin receptor (pIgR) transports dimeric IgA (dIgA) across epithelial cells lining mucosal and glandular tissues, including the mammary gland. Four transgenic mouse lines were generated, overexpressing the murine pIgR gene in the epithelial cells of their mammary glands under control of the regulatory sequences of the bovine alphas1-casein gene. Ten to 270-fold over-expression of the IgA receptor was achieved. The pIgR transgenic line 3644, having the highest pIgR transgene expression, had a markedly altered milk composition compared to non-transgenic mice. In the other three transgenic lines the milk composition, other than SC levels, were not changed. In the milk of line 3644 a protein of 31 kD was lacking and a new protein of 11 kD appeared at relatively high levels. The 31 kD protein was identified as k-casein and the 11 kD protein as serum amyloid A-1 (SAA1). The nutritional value of the milk of females from transgenic line 3644 was dramatically impaired as shown by the retarded growth and development of the pups, leading to death two weeks after birth.

Age Factors↗
PubMed2001

Lymphocyte homing and Ig secretion in the murine mammary gland.

In mice the majority of the immunoglobulins (Ig) in milk belongs to the IgA class. Prior to its transepithelial transportation into the milk, dimeric IgA (dIgA) is bound to the transmembrane form of the secretory component or polymeric Ig receptor (SC/pIgR). The latter is synthesized in the epithelial cells lining the ducts and alveoli of the mammary gland. A candidate for playing the role of adhesion molecule to primed lymphocytes present in the murine mammary gland might be the mucosal addressin cell adhesion molecule-1 (MAdCAM-1). We studied the correlation between the levels of IgA in colostrum and milk, the number of IgA producing plasma cells in the mammary gland and the expression of MAdCAM-1 in mammary gland endothelial cells during pregnancy and lactation. The relation between the IgA levels in the milk and the expression levels of pIgR in mammary gland epithelial cells was also investigated. We found that the expression of MAdCAM-1 and pIgR starts in early-mid pregnancy; the number of IgA-producing plasma cells and the IgA concentration in milk increase from early lactation onwards. The MAdCAM-1 expression declines during lactation whereas the pIgR levels and IgA-producing plasma cell numbers rise until the end of lactation. Because the MAdCAM-1 level starts to rise several days before the rise of the IgA-producing plasma cell level, MAdCAM-1 cannot be the rate determining factor governing extravasation of primed B cells to the mammary gland. We also conclude that the pIgR is present in sufficient amounts to enable increasing S-IgA secretion into the milk during lactation.

Animals↗
PubMed2000

Expression of a functional mouse-human chimeric anti-CD19 antibody in the milk of transgenic mice.

Human B cell lymphomas are suitable targets for immunotherapy. Clinical trials with mouse-human chimeric B cell-specific monoclonal antibodies (mAbs) have already shown promising results. However, limitations for their use in clinical trials can be the lack of sufficient amounts and high production costs. Expression of mAbs in the mammary gland of transgenic animals provides an economically advantageous possibility for production of sufficient quantities of a promising antibody for clinical trials and beyond. In this paper, we show the feasibility of this approach, by generating transgenic mice expressing mouse-human chimeric anti-CD19 mAbs in their milk. Mouse anti-CD19 variable (V) region genes were combined with human IgG1 heavy (H) and kappa light (L) chain constant (C) region genes and fused to the bovine beta-lactoglobulin (BLG) promoter in two separate expression cassettes. Co-injection resulted in five transgenic lines. In one of these lines completely assembled chimeric mAbs were secreted into the milk, at an approximate level of 0.5 mg/ml. These mAbs were able to bind specifically to the CD19 surface antigen on human B cells.

Animals↗
PubMed1999

Over-expression of the murine polymeric immunoglobulin receptor gene in the mammary gland of transgenic mice.

The polymeric immunoglobulin receptor (pIgR), a transmembrane protein, transports dimeric IgA (dIgA) across the epithelial cells of the mucosal surfaces into the external secretions, for example milk from the mammary glands. The pIgR is consumed during the transcytosis of dIgA and is cleaved at the apical side of the epithelial cells, regardless of the binding to its ligand (dIgA), to form secretory component (SC). We hypothesize that the expression level of the endogenous murine pIgR gene in the epithelial cells is rate-limiting for the transport of dIgA across the epithelial cells into the secretions. We address this key issue by generating transgenic mice over-expressing the pIgR gene in their mammary glands in order to examine the effect on dIgA levels in the milk. Here we report on the generation of transgenic mice and analysis of the expression level of pIgR in their mammary glands. We cloned and characterized the murine pIgR gene and constructed an expression cassette bearing the pIgR gene under the control of the regulatory sequences of the bovine alpha s1-casein gene. Four transgenic lines were made, expressing the pIgR construct at RNA and protein level only in their mammary glands. The levels of the SC protein in the milk ranged from 0.1 to 2.7 mg/ml during mid-lactation. These levels are 10-270 times higher than wild-type SC levels (0.01 mg/ml).

Animals↗
PubMed1998

High-level expression of bovine alpha s1-casein in milk of transgenic mice.

The bovine alpha s1-casein gene, isolated from a cosmid library, was introduced into the murine germline. Transgene expression occurred in all transgenic mice, and was confined to the lactating mammary gland. Half of the mouse lines (five out of ten) expressed at relatively high expression levels (> 1 mg ml-1). The highest levels of expression were obtained with a transgene containing 14.2 kb of 5' flanking sequence, in two cases expression levels comparable to (10 mg ml-1) or well above (20 mg ml-1) alpha s1-casein levels in bovine milk were obtained. Transcription initiation occurred at the same site in the bovine alpha s1-casein gene in transgenic mouse as in the cow. A marked induction of expression occurred at parturition rather than at mid-pregnancy, and thus resembled the bovine rather than the murine developmental expression pattern. Bovine alpha s1-casein specific immunoblotting and RIA were developed for characterization and quantification of the recombinant protein. Using these assays, the properties of the recombinant protein could not be distinguished from those of the natural bovine protein. In spite of the high-level tissue-specific and correctly regulated developmental expression of the transgene, expression levels were integration-site dependent. This may indicate that not all cis-acting regulatory elements involved in bovine alpha s1-casein expression were included in the transgene.

Animals↗
PubMed1997

N-terminal stretch Arg2, Arg3, Arg4 and Arg5 of human lactoferrin is essential for binding to heparin, bacterial lipopolysaccharide, human lysozyme and DNA.

Human lactoferrin (hLF), a protein involved in host defence against infection and excessive inflammation, interacts with heparin, the lipid A moiety of bacterial lipopolysaccharide, human lysozyme (hLZ) and DNA. To determine which region of the molecule is important in these interactions, solid-phase ligand binding assays were performed with hLF from human milk (natural hLF) and N-terminally deleted hLF variants. Iron-saturated and natural hLF bound equally well to heparin, lipid A, hLZ and DNA. Natural hLF lacking the first two N-terminal amino acids (Gly1-Arg2) showed reactivities of one-half, two-thirds, one-third and one-third towards heparin, lipid A, hLZ and DNA respectively compared with N-terminally intact hLF. A lack of the first three residues (Gly1-Arg2-Arg3) decreased binding to the same ligands to one-eighth, one-quarter, one-twentieth and one-seventeenth respectively. No binding occurred with a mutant lacking the first five residues (Gly1-Arg2-Arg3-Arg4-Arg5). An anti-hLF monoclonal antibody (E11) that reacts to an N-lobe epitope including Arg5 completely blocked hLF-ligand interaction. These results show that the N-terminal stretch of four consecutive arginine residues, Arg2-Arg3-Arg4-Arg5, has a decisive role in the interaction of hLF with heparin, lipid A, hLZ and DNA. The role of limited N-terminal proteolysis of hLF in its anti-infective and anti-inflammatory properties is discussed.

Amino Acid Sequence↗
PubMed1997

Characterization of recombinant human lactoferrin secreted in milk of transgenic mice.

Human lactoferrin (hLF) is an iron-binding protein involved in host defense against infection and severe inflammation. Transgenic mice were produced harboring either hLF cDNA or genomic hLF sequences fused to regulatory elements of the bovine alphaS1 casein gene. Recombinant hLF expressed in the milk of transgenic mice (transgenic hLF) was compared with natural (human milk-derived) hLF. Immunological identity of the two forms was shown by double antibody immunoassays and the absence of an anti-hLF antibody response in transgenic mice on hyperimmunization with natural hLF. Mono S cation-exchange chromatography and N-terminal protein sequencing of transgenic and natural hLF revealed identical cationicity and N-terminal sequences. SDS-polyacrylamide gel electrophoresis and absorbance measurements of purified transgenic hLF showed this protein was 90% saturated with iron, whereas natural hLF is only 3% saturated. The pH-mediated release of iron from transgenic hLF was not different from that of iron-saturated natural hLF. Unsaturated transgenic hLF could be completely resaturated upon addition of iron. Slight differences in mobility between transgenic and natural hLF on SDS-polyacrylamide gel electrophoresis were abolished by enzymatic deglycosylation. Binding of transgenic and natural hLF to a range of ligands, including bacterial lipopolysaccharide, heparin, single-stranded DNA, Cibacron blue FG 3A, and lectins, was not different. Based on these observations, we anticipate that (unsaturated) rhLF and natural hLF will exert similar, if not identical, antibacterial and anti-inflammatory activity in vivo.

Animals↗
PubMed1997

Structure and expression of the mouse casein gene locus.

The analysis of yeast artificial chromosomes (YACs) containing the complete mouse casein gene locus revealed the presence of five casein genes, alpha-, beta-, gamma-, delta-, and kappa-casein, in this order, in the locus. The alpha- and beta-casein genes are only 10 kb apart and have convergent transcriptional orientations. The distance between the beta-casein gene and the alpha s2-like gamma-casein gene is about 70 kb, and these genes have divergent transcriptional orientations. The gamma- and delta-casein genes, both encoding a alpha s2-like casein, are linked within 60 kb and convergently transcribed. The kappa-casein gene is located about 100 kb from the delta-gene. Except for the presence of the delta-casein gene, the organization of the mouse casein locus resembles that of the bovine locus, including the transcriptional orientation of the genes. In contrast to the other casein genes, which are strongly induced at mid-lactation, expression of the delta-casein gene is abruptly induced upon parturition. Comparative analysis of alpha s2-like sequences from various species suggests that the ancestral alpha s2-like gene duplicated around the time of radiation of the rodent and artiodactylid ancestors.

Amino Acid Sequence↗
PubMed1996

Heterogeneity in utilization of N-glycosylation sites Asn624 and Asn138 in human lactoferrin: a study with glycosylation-site mutants.

Human lactoferrin (hLF) is a glycoprotein involved in the host defence against infection and excessive inflammation. Our objective was to determine to what extent each of the three sequons for N-linked glycosylation in hLF is actually used. Human kidney-derived 293(S) cell lines expressing recombinant hLF (rhLF) or glycosylation-site mutants were produced. The mutations involved replacement of asparagine residues with glutamine at one or more sequons for N-glycosylation (Asn138, Asn479 and Asn624). Comparative SDS/PAGE analyses of rhLF, mutated rhLF and human-milk-derived (natural) hLF led us to propose that glycosylation of hLF occurs at two sites (at Asn138 and Asn479) in approx. 85% of all hLF molecules. Glycosylation at a single site (Asn479) or at all three sites occurs in approx, 5% and 9% of hLF respectively. The extent of glycosylation at Asn624 was increased to approx. 29% and 40% of Asn479 and Asn138/479 mutant molecules respectively, which indicates that glycosylation at Asn624 in natural hLF might be limited by glycosylation at Asn479. The presence in supernatant of unglycosylated hLF (approx. 60% of the total) after mutations of Asn138 and Asn479 suggests that glycosylation of hLF is not an absolute requirement for its secretion. The pronounced degradation of unglycosylated hLF in supernatant after mutation at all three glycosylation sites (Asn138/479/624 mutant) but not after mutation at both Asn138 and Asn479 suggests that an altered conformation rather than the lack of glycosylation has rendered the Asn138/479/624 mutant susceptible to intra- and/or extra-cellular degradation.

Asparagine↗
PubMed1996

Expression of cDNA-encoded human acid alpha-glucosidase in milk of transgenic mice.

Enzyme replacement therapy is at present the option of choice for treatment of lysosomal storage diseases. To explore the feasibility of lysosomal enzyme production in milk of transgenic animals, the human acid alpha-glucosidase cDNA was placed under control of the alpha S1-casein promoter and expressed in mice. The milk contained recombinant enzyme at a concentration up to 1.5 micrograms/ml. Enzyme purified from milk of transgenic mice was internalized via the mannose 6-phosphate receptor and corrected enzyme deficiency in fibroblasts from patients. We conclude that transgenically produced human acid alpha-glucosidase meets the criteria for therapeutic application.

Animals↗
PubMed1995

Glycosylated and unglycosylated human lactoferrins both bind iron and show identical affinities towards human lysozyme and bacterial lipopolysaccharide, but differ in their susceptibilities towards tryptic proteolysis.

We studied the role of N-glycosylation of human lactoferrin (hLF) with respect to properties that are relevant to its antibacterial and anti-inflammatory activities. A human kidney-derived 293(S) cell line that constitutively expresses recombinant hLF (rhLF) was produced. The reactivity towards various antibodies of rhLF that had been expressed in the absence or presence of tunicamycin (which blocks N-linked glycosylation) did not differ from that of natural (human milk-derived) hLF. Cation-exchange chromatography and N-terminal protein sequencing showed identical cationic properties and an intact N-terminal sequence for rhLF and natural hLF. SDS/PAGE of rhLF expressed in the presence of tunicamycin revealed a protein with the same M(r) as that of enzymically deglycosylated natural hLF. Both glycosylated and unglycosylated rhLF appeared to be completely saturated with iron. The affinity of natural hLF, glycosylated and non-glycosylated rhLF for both human lysozyme (Kd 4.5 x 10(-8) M) and bacterial lipopolysaccharide did not differ. SDS/PAGE of hLF species subjected to trypsin indicated that unglycosylated rhLF was much more susceptible to degradation. Furthermore, this analysis suggests that N-glycosylation heterogeneity in natural hLF and rhLF resides in the C-lobe. Thus our results provide no argument for differential antibacterial and/or anti-inflammatory activity of natural and (glycosylated) rhLF and suggest that a major function of glycosylation in hLF is to protect it against proteolysis.

Amino Acid Sequence↗
PubMed1995

Expression analysis of the individual bovine beta-, alpha s2- and kappa-casein genes in transgenic mice.

To identify cis-acting regulatory elements involved in the regulation of expression of the casein genes, the bovine beta-, alpha s2- and kappa-casein genes were isolated from cosmid libraries and introduced into the murine germline. Bovine casein expression was analysed at the RNA and protein level. The bovine beta-casein gene, including 16 kb of 5'- and 8 kb of 3'-flanking region, appeared to be expressed in all 12 transgenic mouse lines analysed. In 50% of these lines expression levels in milk exceeded 1 mg/ml. Three lines displayed expression levels comparable with or well above (20 mg/ml) the beta-casein levels in bovine milk. Transgene expression was restricted to the mammary gland. Strong induction of expression occurred at parturition and thus resembled the bovine rather than the murine pattern. In spite of this high-level tissue-specific and developmentally regulated expression, beta-casein expression levels were integration-site-dependent, suggesting that not all elements involved in regulation of expression were included in this beta-casein clone. Neither the bovine alpha s2- nor the kappa-casein gene, including 8 kb and 5 kb of 5'- and 1.5 kb and 19 kb of 3'-flanking sequences respectively, were properly expressed in transgenic mice. However, they were transcribed in stably transfected mouse mammary epithelial cells. This indicates that regulatory elements required for high-level, mammary gland-specific expression are not present in the alpha s2- and kappa-casein clones used in this study and are probably located elsewhere in the casein gene locus.

Animals↗
PubMed1995

Cloning and characterization of the bovine polymeric immunoglobulin receptor-encoding cDNA.

Trans-epithelial transport of polymeric immunoglobulins (pIg) into mucosal and glandular secretions is carried out by the pIg receptor (pIgR). Therefore, expression of the pIgR gene in epithelial cells of mucosal and glandular tissues is an absolute requirement for achieving mucosal immunity. We report the cloning and characterization of the bovine pIgR cDNA. Three overlapping cDNA clones with a total length of 3608 bp yielded an open reading frame encoding a 757-amino-acid (aa) transmembrane (TM) glycoprotein. Although polymorphism was found in two separate clones, Northern blot analysis showed a single pIgR mRNA (approx. 3.8 kb) to be present in the mammary gland, liver, lung, kidney and intestine of a lactating cow. There was no detectable expression of pIgR in the spleen of the same animal. Comparison of the deduced bovine pIgR as sequence with those of rat, mouse, man and rabbit shows that this receptor is highly conserved both in aa sequence and structural organization. The degree of conservation in the TM sequence and the C-terminal cytoplasmic tail, which contains the various signals for intracellular trafficking of the receptor, is 65-73%. We also find a high degree of conservation (61-66%) in the ectoplasmic part of the receptor, known as the secretory component (SC), with an exception for that of the rabbit SC, which is much lower (47%). Among the five Ig-like domains in the SC, the N-terminal domain I, where the primary pIg-binding site is located, showed the highest (72-83%) aa sequence conservation.

Amino Acid Sequence↗
PubMed1995

Specialized ribosomes: highly specific translation in vivo of a single targetted mRNA species.

In previous studies [Hui and de Boer, Proc. Natl. Acad. Sci. USA 84 (1987) (1987) 4762-4766; Hui et al., Methods Enzymol. 153 (1987) 432-452], it was shown that efficient translation of the human growth hormone mRNA (hGH) species having an altered Shine-Dalgarno (SD) sequence, 5'-GUGUG-3', depends on the presence of specialized (spc) ribosomes containing the modified anti-SD (ASD) sequence, 5'-CACAC-3', near the 3' end of their 16S rRNA. In spite of the altered ASD sequence, spc ribosomes were not found to be committed exclusively to the translation of the hGH mRNA; no more than 30% of the total amount of protein synthesized by such ribosomes was hGH. Once we replace the coding sequence of the hGH mRNA with that of chloramphenicol acetyltransferase (CAT), the specificity of spc ribosomes for translation of a single targetted mRNA, relative to the endogenous mRNAs, is greatly enhanced; an estimated 80% of the total amount of protein synthesized by spc ribosomes is CAT. Using the inducible spc ribosome system containing the cat gene, we show that, upon induction, spc ribosomes accumulate in large excess over the number needed for optimal translation of the targetted cat mRNA. Despite the excess, only few spc ribosomes initiate translation on a limited number of endogenous mRNAs. The excessive accumulation of spc ribosomes, which are predominantly present as free 30S subunits, is neither deleterious to the cells, nor does it lead to a feedback inhibition of the synthesis of wild-type ribosomes.

Chloramphenicol O-Acetyltransferase↗
PubMed1994

Kinetics of first polar body extrusion and the effect of time of stripping of the cumulus and time of insemination on developmental competence of bovine oocytes.

Kinetics of extrusion of the first polar body was examined as well as the effect of the time of stripping of the cumulus cells on this kinetics. In addition, the effects of time of stripping and time of insemination on developmental competence of the oocytes, as evaluated by the percentage of morulae and blastocysts, were studied. Polar body extrusion occurred in 80% of the oocytes between 12 and 18 h after the onset of maturation. The remainder of the oocytes did not extrude a polar body at all. Stripping of the cumulus at 12 h after the onset of maturation delayed polar body extrusion significantly by about 1 h. No significant differences were found in the percentage of oocytes that could be fertilized, and the percentage of oocytes that cleaved and developed to the morula and blastocyst stages, between oocytes that were stripped free of cumulus and inseminated at either 16 or 20 h after onset maturation. Oocytes that had extruded a polar body at either 16 or 20 h after onset maturation showed significantly higher percentages of cleavage and development than oocytes that had not extruded a polar body at those time points. However, the percentage of oocytes that could be fertilized was not affected.

Journal Article↗