Biomedical subjects

L M Houdebine

Publications and source records attributed to L M Houdebine.

At least 55 records · Page 3Linked to original sources

PubMed1995

Protein kinase C is not necessary for beta-casein gene induction by prolactin in HC11 mouse mammary cells.

HC11 mouse mammary cells cultured in the presence of insulin, cortisol and prolactin for 24 h accumulated beta-casein mRNA. When the specific inhibitor of protein kinase C, GF 109203 X, was added to the medium with the hormones, the accumulation of beta-casein mRNA was unaltered, although the protein kinase C activity was almost completely suppressed. This suggests that protein kinase C is not strictly necessary for prolactin to induce milk protein gene expression.

Animals↗

PubMed1995

The production of pharmaceutical proteins from the milk of transgenic animals.

The preparation of recombinant proteins of pharmaceutical interest from the milk of transgenic animals is becoming a reality. No protein has reached the market yet but several have been prepared in large quantities not only from laboratory animals but also from ruminants (goat and sheep) and pigs. Rabbit appears more and more to be an intermediate animal well adapted for the preparation of limited amounts of proteins. Several problems remain to be solved to optimize the method. The expression level of genes of interest associated with milk protein gene control regions is usually unpredictable. The recombinant proteins secreted in milk are not always in a satisfactory biochemical form. Cleavage and glycosylation are not always carried out correctly. The problem of the possible presence of agents pathogenic for humans in proteins extracted from milk is not completely solved. Prions have not been found in mammary glands and other milk pathogens may be controlled using good practice in breeding.

Animals↗

PubMed1995

Human immunodeficiency virus type 1 infection of human CD4-transgenic rabbits.

Investigations of human immunodeficiency virus (HIV) infection of man have benefited from the study of relevant animal models of the infection and disease. However, the ultimate models use primate species which are either endangered, not generally available, or expensive to maintain. A transgenic rabbit specifically and stably expressing human CD4 protein on T lymphocytes was assessed as a new laboratory animal model for HIV-1 infection. In vitro studies demonstrate that lymphocytes derived from the transgenic rabbits are more susceptible to HIV-1IIIB infection than those from normal rabbits. In vivo infection of huCD4-transgenic rabbits using HIV-1IIIB-infected autologous lymphocytes was demonstrated by virus isolation, detection of HIV-1-specific DNA in peripheral blood lymphocytes and seroconversion to various HIV-1 proteins. Viral DNA was detected in the tissues of one rabbit sacrificed 7 weeks post-infection and virus was isolated from lymph node. Although these transgenic rabbits are less sensitive to HIV-1 infection than man, such a small and inexpensive animal model may be a useful tool.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Production of pharmaceutical proteins from transgenic animals.

Different systems are being studied and used to prepare recombinant proteins for pharmaceutical use. The blood, and still more the milk, from transgenic animals appear a very attractive source of pharmaceuticals. The cells from these animals are expected to produce well-matured proteins in potentially huge amounts. Several problems remain before this process becomes used in a large scale. Gene transfer remains a difficult and costly task for farm animals. The vectors carrying the genes coding for the proteins of interest are of unpredictable efficiency. Improvement of these vectors includes the choice of efficient promoters, introns and transcription terminators, the addition of matrix attached regions (MAR) and specialized chromatin sequences (SCS) to enhance the expression of the transgenes and to insulate them from the chromatin environment. Mice are routinely used to evaluate the gene constructs to be transferred into larger animals. Mice can also be utilized to prepare amounts as high as a few hundred mg of recombinant proteins from their milk. Rabbit appears adequate for amounts not higher than 1 kg per year. For larger quantities, goat, sheep, pig and cow are required. No recombinant proteins extracted from the blood or milk of transgenic animals are yet on the market. The relatively slow but real progress to improving the efficiency of this process inclines to be reasonably optimistic. Predictive reports suggest that 10% of the recombinant proteins, corresponding to a 100 million dollars annual market, will be prepared from the milk of transgenic animals by the end of the century.

Animals↗

PubMed1994

High level production of human growth hormone in the milk of transgenic mice: the upstream region of the rabbit whey acidic protein (WAP) gene targets transgene expression to the mammary gland.

The 5' flanking region (6.3 kb) of the rabbit WAP (rWAP) gene possesses important regulatory elements. This region was linked to the human growth hormone (hGH) structural gene in order to target transgene expression to the mammary gland. Thirteen lines of transgenic mice were produced. Milk could be collected from six lines of transgenic mice. In five of them, hGH was present in the milk at high concentrations ranging from 4 to 22 mg ml-1. hGH produced by the mammary gland comigrated with hGH of human origin. It was biologically active, and through its prolactin-like activity induced lactogenesis when introduced into mammary culture media. Two of these mouse lines were studied further. hGH mRNA was only detected in the mammary gland during lactation. In the seven other transgenic lines, hGH was present in the blood of cyclic females. The prolactin-like effect of hGH in these mice probably induced female sterility, and milk could therefore not be obtained. In two lines studied in more detail, the mammary gland was the main organ producing hGH, even in cyclic mice. Low ectopic expression was detected in other organs which varied from one line to the other. This was probably due to the influence on the transgene of the site of integration into the mouse genome. In the 13 lines studied, high mammary-specific hGH expression was not correlated to the transgene copy number. The rWAP-hGH construct thus did not behave as an independent unit of transcription. However, it can be concluded that the 6.3 kb flanking region of the rWAP gene contains regulatory elements responsible for the strong mammary-specific expression of hGH transgene, and that it is a good candidate to control high levels of foreign protein gene expression in the mammary gland of lactating transgenic animals.

Animals↗

PubMed1994

Purification and characterization of casein kinase II from lactating rabbit mammary gland.

1. Highly purified 200 kDa casein kinase II from rabbit lactating mammary gland (MG-CK II) was obtained by means of a new purification procedure consisting of one phosphocellulose and three Monó Q steps. 2. Its Km for ATP was 2.22 microM and 0.57 mg/ml and 0.13 mg/ml for partially dephosphorylated casein and phosvitin respectively. Stathmine was also suitable as substrate. 2-aminopurine and 6-dimethylaminopurine inhibited efficiently MG-CK II (Ki = 5 and 1 mM respectively). 3. MG-CK II autophosphorylated on its alpha-, alpha'- and beta-subunits. The beta-subunit autophosphorylation was enhanced in presence of exogenous substrate. Its modulation was highly dependent on ATP concentration. 4. The effects of basic compounds which affected dramatically the phosphorylation of dephosphorylated casein in presence of various ATP concentrations were reported.

Adenosine Triphosphate↗

PubMed1994

Enhanced casein kinase II activity in metastatic melanoma.

The benign dermal nevus can be transformed into malignant melanoma. The possibility that the transformation process is accompanied with enhanced casein kinase II (CK II) activity was investigated. The tissue samples were obtained by incisional biopsy, homogenized and ultracentrifuged. The supernatant was injected onto a Mono Q column. CK II was monitored with [gamma-32P]GTP and its specific substrate RRREEETEEE. The CK II stimulators, spermine and polylysine, the inhibitors heparin, quercetin, poly (Glu-Tyr) 4:1 and 2,3-bisphosphoglycerate were used for identification. CK II activity in metastatic melanoma samples was about 2.5-fold higher than in dermal nevus. These results support our hypothesis that CK II takes a central role in the non-transformed and transformed skin proliferation.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of two casein kinase type II isozymes from bovine brain gray matter.

Highly purified casein kinase II (CK II) isozymes from bovine brain gray matter (BBGM) were obtained by means of a new purification procedure consisting of one phosphocellulose and three Mono-Q steps. The phosphocellulose eluate showed two BBGM-CK II activities. The first minor component (BBGM-CK IIa) was eluted with 0.9 M NaCl and the major component was eluted at 1.1 M NaCl (BBGM-CK IIb). The protein complexes responsible for these two activities were comprised of three subunits, i.e., alpha (40 kDa), alpha' (38 kDa), and beta (28 kDa), with various subunit ratios. The two isozymes displayed the same behavior on Superose 12 fast protein liquid chromatographic gel filtration and sucrose density centrifugation. BBGM-CK IIa and b showed chromatographic and biochemical differences including differing Km for ATP and GTP and Ki for heparin and 2,3-bisphosphoglycerate. The properties of the main peak (BBGM-CK IIb) were studied in detail. The stimulatory effect of Mg2+, Mn2+, and Co2+ was highly dependent both on the nature of the substrate and on ionic type and concentration. It is surprising that with phosvitin as substrate, BBGM-CK IIb was fully active even in the absence of Mg2+ and NaCl. The inhibitory effect of heparin and the stimulatory effects of NaCl, KCl, spermine, and polylysine were highly dependent on the ionic strength, buffer type, and substrate. BBGM-CK II isozymes phosphorylated stathmine in the presence of polylysine, but the requirement for polybasic compounds was not absolute, as is the case with calmodulin and clathrin beta-light chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

A combination of distal and proximal regions is required for efficient prolactin regulation of transfected rabbit alpha s1-casein chloramphenicol acetyltransferase constructs.

In the rabbit, alpha s1-casein is the major casein secreted in the milk. Transcription of the alpha s1-casein gene is induced by PRL. To define the positions of the cis-sequences involved in the control of rabbit alpha s1-casein gene expression by PRL, chimeric genes containing upstream regions of alpha s1-casein gene linked to the chloramphenicol acetyltransferase gene were cotransfected into Chinese hamster ovary cells with the plasmid expressing the rabbit mammary PRL receptor. It was observed that a distal fragment -3442/-3118 was responsible for a high induction of PRL sensitivity when linked in the 5'-position to a chimeric construct (-391/1774)-chloramphenicol acetyltransferase. A cooperation between distal and proximal regions of the alpha s1-casein gene is responsible for the PRL-dependent enhancer activity of the distal fragment. The mammary gland-specific nuclear factor-like binding sequence found around position -90 in the proximal promoter of the alpha s1-casein gene is involved in this cooperation. The distal fragment was further studied to determine the position of regulatory regions. A -3442/-3385 fragment was sufficient to induce a PRL sensitivity similar to that conferred by the larger -3442/-3118 distal fragment, but multiple interactions are likely to exist between other regulatory regions included in this distal fragment. Four DNA-binding regions (I-IV) have been identified within the reduced -3442/-3385 fragment by footprint experiments using rabbit mammary gland or liver nuclear extracts (NE). Protected area III is observed using both NE. Protected areas I, II, and IV are specific for lactating mammary gland NE. The sequences of areas I and IV share several homologies with the sequence of the mammary gland-specific nuclear factor-binding site.

Animals↗

PubMed1994

Variation of transferrin mRNA concentration in the rabbit mammary gland during the pregnancy-lactation-weaning cycle and in cultured mammary cells. A comparison with the other major milk protein mRNAs.

The concentration of transferrin mRNA was evaluated during pregnancy and lactation in rabbit mammary gland and liver using northern blot and dot blot assays. Transferrin mRNA was present in the virgin rabbit mammary gland and its concentration increased as pregnancy proceeded, with a major enhancement after day 15. A high concentration was reached 3 days after parturition, with no additional increase during lactation and with a marked decline after weaning. During the same period, the concentration of transferrin mRNA showed only a very weak variation in liver. This mRNA was six times more abundant in mammary gland than in liver of lactating rabbit. The accumulation of transferrin mRNA in the mammary gland was concomitant with the accumulation of alpha s1-, beta-, kappa-casein and WAP (whey acidic protein) mRNAs. The concentration of glyceraldehyde 3-phosphate dehydrogenase mRNA, taken as a non-inducible control mRNA, declined progressively during pregnancy to reach its lower level in lactation. These observations suggest that casein, WAP and transferrin mRNAs are subjected to a similar control mechanism in vivo, at least in the second half of pregnancy and during lactation. Experiments carried out in vitro using isolated rabbit epithelial mammary cells cultured on collagen I gel indicated that transferrin mRNA was abundant and only weakly inducible by the lactogenic hormones insulin, cortisol and prolactin, as opposed to caseins and WAP mRNAs. R5020, an analogue of progesterone, inhibited at most very slightly the accumulation of alpha s1-casein mRNA in the presence of prolactin and it did not reduce the expression of transferrin gene.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Effect of various protein kinase inhibitors on the induction of milk protein gene expression by prolactin.

Prolactin has many known functions and one of them is to induce the expression of milk protein gene expression in the mammary gland. Specific membrane receptors have been recently characterized but the transduction mechanism involved in the transfer of the prolactin signal to milk protein genes remains unknown. In the present work, it is shown that several protein kinase inhibitors block prolactin action on milk protein genes. Primary rabbit mammary cells were cultured for several days on floating collagen gel in a serum-free medium. Prolactin and the inhibitors of protein kinase were then added to the culture medium. After 1 day, the concentration of alpha s1-casein in the culture medium was measured using a specific radioimmunoassay. The concentration of several mRNAs in cell extracts was also evaluated using Northern blot analysis. alpha s1-Casein secretion and alpha s1-casein mRNA accumulation were induced by prolactin. This induction was blocked by staurosporine, sphingosine, quercetin, genistein and to some extent by o-hydroxyphenyl acetate, but not by H7, polymyxin B, benzylsuccinate and lavendustin A. The concentration of the mRNA coding for transferrin, which is abundantly secreted in rabbit milk independently of prolactin action, was only moderately altered by the inhibitors. The concentration of two house-keeping mRNAs, beta-actin and glyceraldehyde 3-phosphate dehydrogenase, was lowered only by genistein after 1 day but not after 4 h of culture. These data show for the first time that a Ser/Thre kinase, which is not kinase C, and possibly a tyrosine kinase is involved in the transduction of the prolactin message from the receptor to the milk protein genes.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1993

[Expression of recombinant proteins in the milk of transgenic animals].

The bulky production of recombinant proteins can be achieved by procaryotes or eucaryotes cells. Cells from higher eucaryotes may be required when proteins have to be modified post-transcriptionally (glycosylation phosphorylation, cleavage, folding...). Cells from higher vertebrates in culture are used to prepare proteins like human factor VIII and erythropoietin. The use of transgenic organism has been suggested to reach the same goal. Indeed a whole living organism allows a very potent amplification, the number of cells involved in the biosynthesis of the recombinant proteins being very numerous and in the best metabolic conditions. Biological fluids (blood, milk, insect hemolymph, egg white...) and possibly organs from transgenic animals are a priori the best sources of recombinant proteins. Blood is abundant and it is a by-product of slaughter house. Its composition is relatively complex and the circulating recombinant proteins may heavily alter health of animals. Milk is very abundant, its composition is relatively simple, it is poor in proteolytic enzymes and it can be collected easily. Hemolymph from insects is relatively scarce. Egg white will be a possible source of recombinant proteins, when transgenesis has become more accessible in birds. Organs from transgenic animals should be solicited only when a particular cell type is required for the biosynthesis of the recombinant proteins. Milk appears therefore, presently, as the best source of recombinant proteins from transgenic animals. About 15 public and private laboratories try to use these techniques. They consist in preparing vectors containing regulatory regions of one of the milk proteins genes and the coding part (cDNA or gene) of the corresponding proteins to be produced. The transfer of these gene constructs to mouse, rabbit, sheep, goat, pig, shows that these techniques are indeed very promising. A single protein, human alpha 1-antitrypsin produced in milk of transgenic sheep, has presently reached the preparation at an industrial scale. This method has two theoretical limitations: 1) some of the proteins secreted in milk may be not matured as their native counterparts. Experiments carried out so far (about 20 proteins has been produced at an experimental scale) indicate that the mammary cell is able to achieve glycosylation in a correct way; 2) a significant proportion of the recombinant proteins migrate from the alveolar compartment of the mammary gland to blood circulation and they can alter health of lactating animals.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1993

Characterization of rabbit kappa-casein cDNA: control of kappa-casein gene expression in vivo and in vitro.

The rabbit kappa-casein cDNA was cloned and sequenced. One of the isolated clones included almost the entire 5' end, while another clone corresponded to the 3' end of the cDNA. No polyadenylation site was found and therefore this clone did not harbour the complete cDNA. The amino acid sequence of a full-length protein was deduced from the nucleotide sequence obtained for this partial cDNA. It revealed the presence of a chymosin cleavage site and five potential phosphorylation sites. Rabbit kappa-casein was compared with those already described in other species. The rabbit sequence is closer to the ovine than to the mouse sequence. This result supports the idea that Lagomorpha are not closer to Rodentia than to Artiodactyla. The cDNA described above was used to study kappa-casein gene expression in the rabbit mammary gland. This expression was induced primarily by prolactin in mammary gland organoids and was similar to alpha s1-casein gene expression in vivo. The kappa-casein gene present in the casein gene locus is thus subject to the same regulation as the alpha s1-casein gene, although it has evolved from a fibrinogen gene.

Amino Acid Sequence↗

PubMed1993

Transferrin gene expression in the mammary gland of the rat. The enhancing effect of 17 beta-oestradiol on the level of RNA is tissue-specific.

We have investigated the physiological factors which regulate transferrin gene expression in the mammary gland of the rat. Our studies by dot blot analysis have demonstrated that multiple doses of 17 beta-oestradiol (OE2; 0.5 mg/kg per day for 3 days) elicit a specific 3.5-fold increase in the transferrin mRNA levels in the mammary glands of virgin rats. The hormonal action of OE2 in mammary tissue was specific for the transferrin gene, as judged by hybridization with beta-actin cDNA. The accumulation of transferrin mRNA induced by OE2 treatment was similar to the developmentally regulated expression of the gene observed during the reproductive cycle. The steady-state level of mammary transferrin mRNA increased by up to 4.5-fold at day 21 of lactation, when compared with virgin and pregnant rats. Our results show that the pattern of transferrin gene expression is different in mouse and rat mammary glands. The specific response of the transferrin gene to OE2 was not found in the liver or in the uterus. In the uterus alone, OE2 produced a significant increase in the content of nucleic acids and also induced the accumulation of transferrin and beta-actin mRNAs. We have detected for the first time an induction of transferrin gene expression in the mammary gland in response to OE2, and these results support the view that the pattern of transferrin gene multi-modulated expression is tissue- and species-specific.

Animals↗

PubMed1993

Efficiency of introns from various origins in fish cells.

Several vectors containing (1) regulatory regions from Rous sarcoma virus (RSV), human cytomegalovirus (CMV), and herpes simplex thymidine kinase (TK); (2) introns from early or late SV40 genes and from trout growth hormone gene (tGH); (3) chloramphenicol acetyltransferase gene (CAT); and (4) transcription terminators from SV40 were transfected into carp EPC cells, salmon CHSE cells, tilapia TO2 cells, quail QT6 cells, and hamster CHO cells. CAT activity was measured in extracts from several cell lines 3 days after transfection and in the fish EPC stable clones. The CMV and RSV promoters were the most potent in all cell types. The intron from late SV40 genes (VP1 intron) worked properly in QT6 and CHO cells but not in EPC and very weakly in TO2 cells. The tGH intron was efficient in all cell types but preferentially in fish cells. The small t intron from SV40 was processed in all cell types. The small t and, to a lesser extent, the tGH introns amplified expression of cat gene in stable clones, in comparison to the transiently transfected cells. These results indicate that elements from mammalian genes may not be properly recognized by the fish cellular machinery and in an unpredictable manner. This finding suggests that vectors prepared to express foreign genes in transfected cultured fish cells and transgenic fish should preferably contain DNA sequences from fish genes or, alternatively, those sequences from mammalian genes that have been previously proved to be compatible with the fish cellular machinery.

Animals↗

PubMed1992

A 17.6 kbp region located upstream of the rabbit WAP gene directs high level expression of a functional human protein variant in transgenic mouse milk.

We have investigated whether DNA regions present in the rabbit whey acidic protein (WAP) promoter/5' flanking sequence could potentially confer, in vivo, high level expression of reporter genes. Transgenic mice were generated expressing a variant of human alpha 1-antitrypsin, which has inhibitory activity against plasma kallikrein under the control of a 17.6 kbp DNA fragment located upstream of the rabbit WAP gene. Up to 10 mg/ml of active and correctly processed recombinant protein were detected in mouse milk, thus suggesting that the far upstream DNA sequences from the rabbit WAP gene might be useful for engineering efficient protein production in the mammary glands of transgenic animals.

Animals↗

PubMed1992

Structure of the gene encoding rabbit alpha s1-casein.

The complete nucleotide sequence of the entire rabbit alpha s1-casein-encoding gene Aslca and its flanking regions was determined. These data represent the first complete primary sequence of an Aslca gene. The gene consists of 19 exons spread over 16 kb. Highly conserved sequences were found between this gene and other casein-encoding genes mainly upstream from the gene from position -180 to -10. Several repeated interspersed elements of unknown function were also identified within introns.

Amino Acid Sequence↗

PubMed1992

6-Dimethyl amino purine and 2-amino purine inhibit the induction of expression of milk protein genes by prolactin.

Two protein kinase-inhibitors, 6-dimethyl amino purine and 2-amino purine inhibited induction of beta-casein synthesis by prolactin when added to the culture medium of rabbit mammary explant and cells. The accumulation of the mRNA for alpha s1- and beta-caseins and for whey acidic protein did not take place in the presence of the inhibitors whereas beta-actin mRNA concentration was not altered. In the same experimental conditions, H7, an inhibitor of protein kinase C and, to a lower extent, of protein kinase A did not prevent prolactin from acting. These data suggest for the first time that specific protein kinases are involved in the transduction of the prolactin signal to milk protein genes.

2-Aminopurine↗