The developmental pattern of Brca1 expression implies a role in differentiation of the breast and other tissues.

We have examined the developmental expression of the murine breast and ovarian cancer susceptibility gene, Brca1, to investigate its role in the control of cell growth and differentiation. Specifically, we have analysed Brca1 expression during embryonic development, in adult tissues, and during postnatal mammary gland development, particularly in response to ovarian hormones. Our results suggest that Brca1 is expressed in rapidly proliferating cell types undergoing differentiation. In the mammary gland, Brca1 expression is induced during puberty, pregnancy, and following treatment of ovariectomized animals with 17 beta-estradiol and progesterone. These observations imply that Brca1 is involved in the processes of proliferation and differentiation in multiple tissues, notably in the mammary gland in response to ovarian hormones.

Evaluation in vitro of synthetic curcumins as agents promoting monocytic gene expression related to ß-amyloid clearance.

Accumulation of amyloid-beta (Aß) is one of the hallmarks of Alzheimer's disease (AD), and efficient clearance of Aß by cells of the innate immune system may be an important mechanism for controlling or preventing disease onset. It was reported that peripheral blood mononuclear cells (PBMCs) of most AD patients are defective in the phagocytosis of soluble Aß. Natural curcumins were shown to restore Aß phagocytosis by AD PBMCs and to up-regulate the expression of key genes including MGAT3 and those encoding Toll-like receptors (TLRs). Bisdemethoxycurcumin (BDC), a minor component of natural curcumin, was shown to have the greatest potency for stimulating AD PBMCs. Because natural curcumins have inherent limitations with regard to physicochemical properties, synthetic curcumin analogues were developed that showed improved solubility, stability, and bioavailability. An in vitro system using human monocytic cell lines (U-937, THP-1) was used to evaluate analogues for the potency of innate immune cell stimulation. These cell lines showed responses to curcuminoids and to 1α,25-dihydroxyvitamin D3 (VD3) resembling those seen in human PBMCs. From more than 45 curcuminoids analyzed, the most potent compounds possessing enhanced pharmaceutical properties were identified. The most promising candidates included prodrug versions containing water solubility-enhancing amino acids and stability-increasing modifications near the central diketone. In vivo studies showed compound (5) substantially increased bioavailability by combining several promising structural modifications. Studies examining ex vivo phagocytosis of Aß and bead particles in mouse microglia showed that BDC and several water-soluble analogues were quite effective compared to curcumin or an unnatural analogue. In vitro studies using monocytic cell lines reported herein complement those using human PBMCs and represent a routinely accessible and uniform cellular resource allowing direct comparisons between compounds.

Curcumins promote monocytic gene expression related to ß-amyloid and superoxide dismutase clearance.

Neurodegenerative diseases are associated with accumulation of modified proteins or peptides including amyloid-ß (Aß) in Alzheimer's disease (AD), and misfolded superoxide dismutase-1 (SOD-1) in amyotrophic lateral sclerosis (ALS). Clearance of Aß or SOD-1 by the innate immune system may be important for controlling or preventing disease onset. Curcumins restore Aß phagocytosis by peripheral blood mononuclear cells (PBMCs) from AD patients and Aß clearance with upregulation of key genes including MGAT3, vitamin D receptor (VDR) and Toll-like receptors (TLRs). Certain curcumins inhibit inflammatory processes of PBMCs from ALS patients. We developed an in vitro system using human monocytes from patients and monocytic cell lines (i.e. U-937, THP-1) for evaluating curcuminoid potency of innate immune cell stimulation. Bisdemethoxycurcumin and certain analogs potentiated MGAT3,VDR and TLR gene expression 3- to 300-fold in U-937 cells. The effect of curcumins on inflammation in monocytes from patients with ALS was examined. Recursive medicinal chemistry was applied to identify compounds that stimulate the innate immune system for use in the clearance of Aß in AD and the reversal of neuroinflammation and defective SOD-1 accumulation in ALS.

Thymic lymphomas in mice with a truncating mutation in Brca2.

Inherited mutations in the BRCA2 gene predispose women to breast and ovarian cancer. We created a mutation in the mouse Brca2 gene that terminates translation in exon 11 at 45% of the normal transcript length. Ninety % of Brca2(tm1Cam) homozygous mutant mice die prenatally or perinatally. The location of the Brca2(tm1Cam) mutation differs from those reported previously, and this phenotype suggests a correlation with genotype analogous to that previously reported in humans. Although heterozygote mice have remained free of tumors for 10 months, Brca2(tm1Cam) homozygous mutants that survived to adulthood died with thymic lymphomas between 12 and 14 weeks of age.

Physical characterization of genetic rearrangements at the mouse renin loci.

Many inbred strains of mice have a single locus encoding renin, Ren-1, whereas other inbred strains have two tandemly linked loci, Ren-1 and Ren-2. Each of these renin genes in inbred mice exhibits a unique pattern of tissue-specific expression. As a prerequisite to understanding the structural basis for the expression differences, we have physically characterized the sequence organization of this chromosomal region in both types of strains. Pulsed field gel electrophoresis was initially used to compare the long-range structure of this region in C57BL/6 (Ren-1) and DBA/2 (Ren-1 + Ren-2) mice. The structure in both inbred strains is extremely similar, except for an additional 30 kb containing Ren-2 in DBA/2 mice. The boundaries of the extra 30-kb segment were sequenced and compared to homologous sequences flanking the Ren-1 alleles. This analysis identified the precise recombination site, and also the presence of a large insertion, between the renin loci in DBA/2. The renin gene duplication apparently resulted from recombination between sequences sharing little homology, suggesting that nonhomologous chromosomal breakage and rejoining may have been involved mechanistically in the event.

Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli.

A series of new plasmid expression vectors (the pTrc series) has been constructed for the regulated expression of genes in Escherichia coli. Based on pKK233-2 [Amann and Brosius, Gene 40 (1985) 183-190], the vectors carry a strong hybrid trp/lac promoter, the lacZ ribosome-binding site (RBS), the multiple cloning site of pUC18 and the rrnB transcription terminators. With the aid of synthetic oligodeoxynucleotides, the multiple cloning site has been inserted behind an NcoI site in three reading frames. Thus, the vectors are equally useful for the expression of proteins in their authentic, non-fused form (by using the NcoI site) and for the expression of fusion proteins (by choosing any of the cloning sites in the correct translational frame). To ensure complete repression of the hybrid trp/lac promoter during construction and growth in any host strain, the lacIq allele of the lac repressor gene was added to some of the vectors. The complete vector nucleotide sequence and examples of heterologous gene expression (human coagulation factor XIIIa and human placental anticoagulant protein PP4) with the new vectors are presented.

Determination of the clearance factor for transmissible spongiform encephalopathy agents during the manufacturing process of polygeline.

OBJECTIVE: To determine the safety of polygeline, a gelatine-derived plasma substitute produced from bovine bones, in terms of safety for bovine spongiform encephalopathy (BSE) by evaluating the ability of the manufacturing process of polygeline to eliminate agents related to transmissible spongiform encephalopathy (TSE) through the validation of three main production steps. DESIGN: Laboratory scale experimental process (in duplicate) using 20% hamster-adapted 263K scrapie-infected brain homogenate as infective titrated source (10(9) LD50/2 ml), added to each material before being processed and titrated in hamsters. Experiment 1: time/temperature dependency of gelatine autoclaving. Experiment 2: cross-linking and distillation. Experiment 3: final sterilization. Monitoring period: 1 year with daily animal clinical observation. Histology of all brains. SETTING: LCG-RBM laboratories, Italy; strict GLP compliance. MEASUREMENTS AND RESULTS: Heating the gelatine (at conditions lower than those used in production process) was very effective in inactivating the infectivity of TSE agents. Clearance factors were reproducible, dependent upon time and temperature, reaching a total theoretical process clearance in the range of 9.2-13.8 [6.9 + 2.3 (+ 4.6)] log10 LD50. CONCLUSIONS: These experimental results provide further important data confirming the safety of the procedural steps; this complements the safety due to the careful sourcing of the raw material. There is high assurance that there is no significant risk of TSE transmission to humans by the therapeutic administration of polygeline.

Close physical linkage of the murine Ren-1 and Ren-2 loci.

In addition to the Ren-1 gene common to all mice, some inbred strains carry a second copy of the renin structural gene, Ren-2. These two loci are tightly linked genetically on mouse chromosome one. We have used pulsed field gel electrophoresis (PFGE) to study the physical arrangement of the two renin genes in the inbred strain DBA/2. PFGE mapping permitted the construction of a restriction map of the Ren loci spanning roughly 120 Kb. The results indicate that the genes are transcribed in the same relative direction, that Ren-2 lies upstream relative to Ren-1, and that the respective coding sequences are separated by approximately 20 Kb.

DNA insertions distinguish the duplicated renin genes of DBA/2 and M. hortulanus mice.

In a survey of inbred and wild mouse DNAs for genetic variation at the duplicate renin loci, Ren-1 and Ren-2, a variant Not I hybridization pattern was observed in the wild mouse M. hortulanus. To determine the basis for this variation, the structure of the M. hortulanus renin loci has been examined in detail and compared to that of the inbred strain DBA/2. Overall, the gross features of structure in this chromosomal region are conserved in both Mus species. In particular, the sequence at the recombination site between the linked Ren-1 and Ren-2 loci was found to be identical in both DBA/2 and M. hortulanus, indicating that the renin gene duplication occurred prior to the divergence of ancestors of these mice. Renin flanking sequences in M. hortulanus, however, were found to lack four DNA insertions totaling approximately 10.5 kb which reside near the DBA/2 loci. The postduplication evolution of the mouse renin genes is thus characterized by a number of insertion and/or deletion events within nearby flanking sequences. Analysis of renin expression showed little or no difference between these mice in steady state renin RNA levels in most tissues examined, suggesting that these insertions do not influence expression at those sites. A notable exception is the adrenal gland, in which DBA/2 and M. hortulanus mice exhibit different patterns of developmentally regulated renin expression.

Escherichia coli-derived envelope protein gD but not gC antigens of herpes simplex virus protect mice against a lethal challenge with HSV-1 and HSV-2.

Immunization studies with HSV-1 and HSV-2 envelope proteins expressed in Escherichia coli were performed. After active immunization of mice with a gD-1 antigen (Leu53-Ala312) expressed as a fusion protein, the animals were protected from a lethal challenge with HSV-1 and HSV-2. In addition, antisera from rabbits immunized with the same gD-1 antigen also conferred passive immunity to mice against a challenge infection with either HSV-1 or HSV-2. In contrast to these successful gD-1 protection experiments, various gC-1 and gC-2 fusion proteins from E. coli failed to induce protective immunity. Moreover, the mice sera from immunized animals were not able to react with the authentic, glycosylated gC-1 and gC-2 envelope proteins, whereas sera raised against authentic gC-1 and gC-2 glycoproteins do recognize the gC fusion proteins from E. coli. These results indicate, that E. coli might represent an ideal system for expressing gD antigens as a possible component of a HSV vaccine, whereas gC antigen cannot be produced in an immunocompetent form in E. coli.

Characterization of cDNA encoding human placental anticoagulant protein (PP4): homology with the lipocortin family.

A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10(6)independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA we identified 9 other recombinants encoding a protein with considerable similarity (74%) TO PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I.

Synthesis of human factor XIIIa in bacterial cells.

The coding sequence for human factor XIIIa (FXIIIa) was introduced into Escherichia coli expression vectors. Bacterial cells transformed with the recombinant plasmids synthesized fusion proteins of the expected molecular weights and the proteins were shown to be immunoreactive with anti-FXIII antibodies. Furthermore, with the help of oligodeoxynucleotide synthesis, we constructed a plasmid which directs the synthesis of the human FXIIIa protein in the unfused form. Sequence determination at the aminoterminus of this protein revealed the identical sequence compared to placental FXIIIa. The protein is expressed intracellularly in a denatured and biologically inactive form. It constitutes approximately 2% of total cellular protein and can easily be purified by standard methods.

Expression of the mouse Ren-1 gene in the coagulating gland: localization and regulation.

The components of the renin-angiotensin system have been detected in various reproductive tissues of mammals including the testis, epididymis, ovary, and placenta. Using Northern blot and in situ hybridization analyses, we detected abundant levels of renin mRNA in the coagulating gland (anterior prostate) of mice with one (Ren-1) but not two (Ren-1/Ren-2) copies of the renin gene. In contrast to mice, the single renin gene of the rat (R. norvegicus) was silent in the coagulating gland. The results of this survey suggest that the ability of the renin gene to express in the coagulating gland was acquired during the speciation of mice and subsequently lost as a result of the duplication event at the renin locus. In the coagulating gland, we found that renin mRNA transcripts initiated at a series of upstream start sites, some of which map within a 0.5-kb transposable-like element previously identified in the promoter region of the mouse, but not the rat, renin gene. Furthermore, renin gene expression in the coagulating gland was positively regulated by testosterone. The coagulating gland thus represents a male reproductive tissue that demonstrates high-level, species-specific, and differential expression of renin mRNA.

Isolation and expression of cDNA coding for a new member of the phospholipase A2 inhibitor family.

The placental protein PP41,2 was shown to have thromboplastin-inhibitor activity. We used partial amino acid sequence information from PP4 cyanogen bromide fragments to design oligonucleotide probes for the screening of a human placental cDNA library. In addition to the PP4 cDNA we isolated a cDNA coding for a protein with considerable homology which we subsequently termed PP4-X. PP4 and PP4-X belong to the phospholipase A2 inhibitor family, as judged by their homology to lipocortin I and calpactin I3. The full-length PP4-X cDNA encodes a protein of 321 amino acid residues including a fourfold repeat structure. Northern blot analysis using the PP4-X cDNA reveals two hybridizing RNA species of approximately 1400 nucleotides and 2500 nucleotides, respectively. The shorter one could well represent the PP4-X transcript which is in good agreement with the isolated cDNA insert of 1326 nucleotides. Expression of the PP4-X coding sequence in E. coli resulted in the appearance of a protein which crossreacts with antibodies raised against PP4.

Denaturation-renaturation of the fibrin-stabilizing factor XIII a-chain isolated from human placenta. Properties of the native and reconstituted protein.

The denaturation-renaturation transition between the native and unfolded states of the dimeric blood coagulation factor XIIIa has been examined by far-UV circular dichroism, fluorescence spectroscopy, activity measurements, sedimentation equilibrium analysis, and size exclusion high performance liquid chromatography. Guanidine hydrochloride and urea-dependent denaturation in the absence and in the presence of 5mM dithioerythritol or glutathione (5mM GSH) exhibit biphasic transitions. The first stage represents a sharp transition characterized by a change in secondary structure without subunit dissociation. This step is accompanied by the irreversible loss of biological activity. The second transition reflects the dissociation and complete unfolding of the protein to a random coil. After loss of biological activity no reactivation can be accomplished under any of the following conditions: (i) denaturation and renaturation under reducing or non-reducing conditions, (ii) variation of the protein concentration and temperature, (iii) addition of specific ligands (Ca2+, substrate), (iv) presence of stabilizing and/or destabilizing agents. Attempts to renature the protein under standard conditions (0.1 M Tris/HCl pH 7.5-9.0, 5mM DTE, 5mM EDTA) lead to refolding intermediates which exhibit a strong tendency to aggregate. A soluble product of reconstitution can be obtained by refolding at low protein concentration, low temperature, and in the presence of small amounts of destabilizing agents such as arginine or urea in the renaturation buffer at pH 7.5 to 9. The spectroscopic and hydrodynamic characterization of the partially reconstituted (non-native inactive) protein shows that partially reconstituted factor XIIIa exhibits the fluorescence properties and the dimeric structure of the native protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Localization of the gene for ATP citrate lyase (ACLY) distal to gastrin(GAS) and proximal to D17S856 on chromosome 17q12-q21.

The gene encoding ATP-citrate lyase, designated ACLY, was mapped to human chromosome 17q12-q21 by PCR on a panel of human/rodent somatic cell hybrids and localized to 17q21.1 by PCR on a panel of radiation hybrids. The radiation hybrid panel indicates that the most likely position of ACLY on 17q21.1 is between gastrin (GAS) and D17S856 at a distance of 170-290 kb from the GAS locus.

Structural organization and mapping of the human TCF11 gene.

We recently cloned human cDNA representing several mRNA isoforms of human TCF11, a transcription factor of the basic-region, leucine-zipper (bZIP) family located on chromosome 17q22 as well as a genomic clone of this gene. We have now determined the complete genomic organization of the TCF11 gene, which consists of 9 exons distributed over 15 kb of genomic DNA. Pulsed-field gel electrophoresis was used to construct a physical map around TCF11, to characterize a 450-kb YAC clone that contains the gene, and to link TCF11 physically to BTR, a marker on chromosome 17.

Mouse Brca1: localization sequence analysis and identification of evolutionarily conserved domains.

The human genes BRCA1, conferring susceptibility to early-onset breast and ovarian cancer, has recently been isolated. Here we describe isolation of cDNAs, sequence analysis, and genomic localization of the murine homolog, Brac1. The mouse cDNA sequence predicts a protein of 1812 amino acids; a number of small gaps account for the 51 fewer residues in the mouse protein relative to human BRCA1. While the predicted mouse and human proteins display on the whole a high level of homology (58% identity, 73% similarity), the regions of greatest homology are at the respective amino and carboxyl termini. Most reported disease-associated missense mutations in human BCRA1 occurred within these more highly conserved terminal regions. A predicted zinc-building RING finger domain near the amino terminus lies within a 50 amino acid stretch that is perfectly conserved in both species. The strong conservation during mammalian evolution argues for the importance of this domain, perhaps mediating a role for BRCA1 in DNA and/or protein binding. We have also identified a conserved highly acidic domain in the carboxyl terminal half of the BCRA1 protein resembling acidic transactivation domains of certain transcription factors. Using an interspecific backcross panel, Brca1 was mapped to a region of mouse chromosome 11 that exhibits conserved linkage with 17q21. The sequence and isolated cDNAs will provide useful reagents for studying the expression of Brca1 in the mouse, and for testing the importance of the evolutionarily conserved domains.

A chromosome 11 YAC library.

A targeted yeast artificial chromosome (YAC) library for chromosome 11 has been constructed from the J1 cell line that carries a single human chromosome 11 within a hamster DNA background. Interspecies chimeric clones generated during construction of the library were detected during the screening process and eliminated from the library. Contig assembly becomes much less difficult using such a library as the complexity is decreased and the ends of the clone inserts can be rescued for walking to neighboring clones. The library contains > 1824 clones with an average insert length of 337 kb. This represents a fourfold coverage of chromosome 11 or a > 95% chance of recovering a unique single-copy sequence from the library. Two hundred YAC clones were localized by fluorescence in situ hybridization and found to be randomly distributed along the chromosome. The library has been screened with probes for the chromosome 11 markers HBB, GLUR4, H19, and D11S193. Corresponding YAC clones have been isolated for each locus. This analysis has indicated that the library is unbiased, that cognate YAC clones can be recovered with chromosome 11 markers, and that extensive contig assembly should be feasible.

Linkage of Agt and Actsk-1 to distal mouse chromosome 8 loci: a new conserved linkage.

Angiotensinogen is an alpha 2-globulin involved in the maintenance of blood pressure and electrolyte balance. We have refined the position of the mouse angiotensinogen locus (Agt) on Chromosome (Chr) 8 and have also confirmed the assignment of the human angiotensinogen locus (AGT) to Chr 1. The segregation of several restriction fragment length variants (RFLVs) was followed in two interspecific backcross sets and in four recombinant inbred (RI) mouse sets. Analysis of the segregation patterns closely linked Agt to Aprt and Emv-2, which places the angiotensinogen locus on the distal end of mouse Chr 8. Additionally, a literature search has revealed that the strain distribution pattern (SDP) for the mouse skeletal alpha-actin locus 1 (Actsk-1, previously Acta1, Acta, or Acts) is nearly identical to the SDP for Agt in two RI sets. On the basis of this information we were able to reassign Actsk-1 to mouse Chr 8. By screening a panel of human-mouse somatic cell hybrids, we confirmed that the human angiotensinogen locus lies on Chr 1. This information describes a new region of conserved linkage homology between mouse Chr 8 and human Chr 1. It also defines the end of a large region of conserved linkage homology between mouse Chr 8 and human Chr 16.