Pulmonary expression of the human haptoglobin gene.

Haptoglobin (Hp), a member of the acute-phase reactants, has long been known as a major hemoglobin-binding protein associated with hemoglobin catabolism. Recent studies indicate that another important biologic function of Hp is the modulation of the immune response. We found that Hp is expressed at high levels in specific cells, including alveolar macrophages and eosinophils in diseased or inflamed human lung tissues, but not in the normal lung. Expression of the human Hp gene was studied in two transgenic mouse lines carrying a 9-kb human Hp 2 gene. In both lines, the human Hp transgene was expressed constitutively in alveolar macrophages at a high level, whereas the endogenous mouse Hp was synthesized in airway epithelial cells. Expression of the human Hp transgene in lung cells was upregulated when the transgenic mice were treated with endotoxin. In humans and in Hp transgenic mice, human Hp messenger RNA was also detected in circulating eosinophils, but not in other blood cells. Our findings suggest that Hp is involved in a variety of lung inflammatory diseases, including respiratory allergy and asthma. The transgenic mouse line that overexpresses the human Hp gene in alveolar macrophages and eosinophils is a promising system for investigating the function of Hp in vivo during lung inflammation.

A comparison of the suppression of human transferrin synthesis by lead and lipopolysaccharide.

Transferrin, as the major iron-transport protein in serum and other body fluids, has a central role in managing iron the body receives. Liver is a major site of transferrin synthesis, and in this study we present evidence that liver synthesis of human transferrin is suppressed by both the toxic metal lead and bacterial lipopolysaccharide, an inducer of the hepatic acute phase response. The responses of intact endogenous transferrin in the human hepatoma cell line HepG2 and chimeric human transferrin-chloramphenicol acetyltransferase genes in transgenic mice were examined. In HepG2 cells, 35S-transferrin protein synthesis and mRNA levels were suppressed by 100 microM and 10 microM lead acetate as early as 24 h after the initial treatment. Yet, synthesis of two proteins known to respond in the hepatic acute phase reaction, complement C3 and albumin, was not altered by the lead treatment. In transgenic mouse liver, lead suppressed expression of chimeric human transferrin genes at both the protein and mRNA levels, but LPS only suppressed at the protein level. The study indicates that lead suppresses human transferrin synthesis by a mechanism that differs from the hepatic acute phase response and that lead may also affect iron metabolism in humans by interfering with transferrin levels.

Mg(2+)-dependent and Ca2+, Mg(2+)-dependent ATPase activities in the harderian gland of rodents: age and sex influences.

Three experiments employing male and female Syrian hamsters (aged 1, 2, and 8-10 months), male Sprague-Dawley rats (aged 1, 2, and 10 months) and male C57B1 mice (aged 2, 7, 13, and 29 months) examined the effects of age and sex on Mg(2+)-dependent and Ca2+, Mg(2+)-dependent ATPase activity in the Harderian gland. Significant differences due to age and sex were observed in the hamsters and rats but not with age in mice. Generally, male hamsters had significantly higher Mg(2+)-dependent and Ca2+, Mg(2+)-dependent (exception at one timepoint) ATPase activity than did females. Age-matched male and female rats had similar values of Mg(2+)-dependent ATPase activity, but males had significantly higher Ca2+, Mg(2+)-dependent ATPase activity than females at 2 months of age.

Cellular expression of ceruloplasmin in baboon and mouse lung during development and inflammation.

Ceruloplasmin (CP) is an important extracellular antioxidant and free radical scavenger. Although CP is expressed mainly in the liver, recent studies have identified the lung as another major site of CP synthesis. The sites and cell types that are responsible for CP expression in baboon and mouse lung are described. CP mRNA is detected in primordial bronchial epithelium in baboon fetuses by 60 days of gestation. At 140 days of gestation and thereafter, CP mRNA is found in airway epithelium and in the ductal cells of the submucosal glands. In developing and mature mice, CP mRNA is present in epithelial cells throughout the airway. In endotoxin-treated mice, the amount of CP mRNA increases several-fold in large airways but increases only moderately in small airways. This suggests that the high concentration of CP in the mucus lining of the upper airway, which serves to filter harmful substances, is particularly important during stressful conditions. Endotoxin treatment in mice also results in the induction of high levels of CP mRNA in a subset of alveolar wall cells. The data suggest that the airway epithelial cells are the major source of CP in the lung fluid and support ceruloplasmin's critical role in host defense against oxidative damage and infection in the lung.

Targeted overexpression of androgen receptor with a liver-specific promoter in transgenic mice.

The rodent liver displays marked age- and sex-dependent changes in androgen sensitivity due to the sexually dimorphic and temporally programmed expression of the androgen receptor (AR) gene. We have altered this normal phenotype by constitutive overexpression of the rat AR transgene in the mouse liver by targeting it via the human phenylalanine hydroxylase (hPAH) gene promoter. These transgenic animals in their heterozygous state produce an approximately 30-fold higher level of the AR in the liver as compared with the nontransgenic control. Androgen inactivation via sulfonation of the hormone by dehydroepiandrosterone sulfotransferase (DST), an androgen-repressible enzyme, also contributes to the age- and sex-dependent regulation of hepatic androgen sensitivity. DST has a broad range of substrate specificity and is responsible for the age- and sex-specific activation of certain polycyclic aromatic hepatocarcinogens as well, by converting them to electrophilic sulfonated derivatives. In the transgenic female, the hepatic expression of DST was approximately 4-fold lower than in normal females, a level comparable to that in normal males. The hPAH-AR mice will serve as a valuable model for studying the sex- and age-invariant expression of liver-specific genes, particularly those involved in the activation of environmental hepatocarcinogens such as the aromatic hydrocarbons.

YY1 and Sp1 transcription factors bind the human transferrin gene in an age-related manner.

The iron-binding protein transferrin has major roles in transporting, delivering, and sequestering ferric ions acquired by body tissues. Yet, during aging, serum transferrin levels decrease in humans. Likewise, in transgenic mice carrying chimeric human transferrin transgenes, liver expression of transferrin transgenes decreases with age. The aging regulation is due to decreased gene transcription. Electrophoretic mobility shift assays and antibody-recognition have revealed the binding of 5' regulatory elements of the human transferrin gene by three YY1 proteins, called YY1, YY1-a, and YY1-b, and an Sp1-a transcription factor. An age-related increase in YY1-a and YY1-b binding activities and a decrease in Sp1-like binding activity were shown. Since Sp1 is a positive transcription factor and YY1 can be a negative transcription factor, the alterations in their binding with age could cause the decreased transcription of the human transferrin transgene, and also the age-related decreased serum transferrin levels in humans.

Discovery of a brain promoter from the human transferrin gene and its utilization for development of transgenic mice that express human apolipoprotein E alleles.

Transgenic mice carrying heterologous genes directed by a 670-bp segment of the regulatory sequence from the human transferrin (TF) gene demonstrated high expression in brain. Mice carrying the chimeric 0.67kbTF-CAT gene expressed TF-CAT in neurons and glial cells of the nucleus basalis, the cerebrum, corpus callosum, cerebellum, and hippocampus. In brains from two independent TF-CAT transgenic founder lines, copy number of TF-CAT mRNA exceeded the number of mRNA transcripts encoding either mouse endogenous transferrin or mouse endogenous amyloid precursor protein. In two transgenic founder lines, the chloramphenicol acetyltransferase (CAT) protein synthesized from the TF-CAT mRNA was estimated to be 0.10-0.15% of the total soluble proteins of the brain. High expression observed in brain indicates that the 0.67kbTF promoter is a promising director of brain expression of heterologous genes. Therefore, the promoter has been used to express the three common human apolipoprotein E (apoE) alleles in transgenic mouse brains. The apoE alleles have been implicated in the expression of Alzheimer disease, and the human apoE isoforms are reported to interact with different affinities to the brain beta-amyloid and tau protein in vitro. Results of this study demonstrate high expression and production of human apoE proteins in transgenic mouse brains. The model may be used to characterize the interaction of human apoE isoforms with other brain proteins and provide information helpful in designing therapeutic strategies for Alzheimer disease.

Age-specific regulation of clotting factor IX gene expression in normal and transgenic mice.

Factor IX (FIX), a circulating serine protease that serves as an essential component of the blood coagulation pathway, has been shown to increase with age in humans. We show here that murine FIX mRNA and activity levels also increase with age. Furthermore, one form of hemophilia B, hemophilia B Leyden, which is caused by mutations within the promoter region of the FIX gene, has a distinct age-dependent phenotype. To determine the source of the age-related increases in FIX gene expression, we have analyzed the regulation of the normal FIX gene promoter and FIX Leyden gene promoter with the +13 mutation during aging by generating transgenic mice that contain the -189 to +21 bp promoter segment ligated to a chloramphenicol acetyltransferase reporter gene. We have established that the normal FIX promoter and the Leyden promoter transgenes are expressed in a tissue-specific manner in vivo. The normal FIX promoter transgene does not show any differences in the pattern of expression with age or sex of the organism, whereas the Leyden promoter transgene showed age-dependent male-specific expression. This is the first demonstration of the FIX Leyden phenotype in a transgenic mouse model.

A human (3.3 kb) haptoglobin-CAT transgene is modulated in lungs of transgenic mice by inflammation.

Four independent lines of transgenic mice were produced carrying integrated copies of a chimeric gene composed of 3.3 kb of the human haptoglobin 5' regulatory region fused to the CAT (chloramphenicol acetyl transferase) reporter gene. Although the endogenous mouse haptoglobin (Hp) and human haptoglobin (HP) genes express mainly in liver and lung, expression of the human 3.3-kb HP-CAT transgene was not detected until after induction of inflammation and then only in lungs. The results indicated that the transgene maintained the regulatory DNA elements required for lung specific responsiveness to inflammation in vivo but lacked the DNA sequence required for robust expression in liver. The DNA sequence(s) responsible for the normally high level of HP expression in liver either reside outside the 3.3-kb regulatory region of the HP chimeric gene or this region contains a suppressor sequence affecting tissue specific expression in the liver.

Brain and liver targeted overexpression of O6-methylguanine DNA methyltransferase in transgenic mice.

O6-Methylguanine DNA methyltransferase (MGMT; EC 2.1.1.63) is an unusual DNA repair protein in that it directly and specifically repairs a premutagenic DNA lesion without involving other proteins. MGMT removes the alkyl group from O6-alkylguanine in DNA in a unique stoichiometric reaction by accepting the alkyl group on a cysteine residue. The intracellular level of MGMT varies among tissues and appears to be inversely correlated to tissue-specific tumorigenesis induced by monofunctional alkylating agents. Because MGMT acts in solo, genetic manipulation of its expression may provide valuable insight into its contribution to cellular resistance to alkylation toxicity and to tumor induction. The human MGMT full length cDNA has been fused with a portion of the human transferrin (TF) 5'-flanking region (TF/MGMT). Transgenic founder mice were produced carrying the TF/MGMT transgene and then bred to establish stable transgenic lines. Human MGMT transcripts were specifically expressed in abundance in transgenic brain and liver tissues. In vitro MGMT assays revealed approximately 150-fold and approximately 25-fold increases in MGMT activity in transgenic brain and liver extracts respectively. Western blot analysis confirmed that human MGMT protein is specifically synthesized in transgenic brain and liver tissues.

Expression of chimeric human transferrin-chloramphenicol acetyltransferase genes in liver and brain of transgenic mice during development.

Transferrin (TF) gene expression is tissue specific and is regulated during development. Transgenic mice have been developed which carry 1.2 or 0.67 kb of the TF 5' flanking region of the human TF gene fused to the bacterial chloramphenicol acetyltransferase (CAT) gene. The onset of expression of the chimeric human TF-CAT transgenes in liver and brain during development has been studied in these transgenic mice. In brain, the TF(0.67)CAT transgene began to express between 5 and 10 days after birth; in liver, the TF(0.67)CAT transgene was turned on between 10 and 20 days after birth. Endogenous mouse TF mRNA levels in liver and brain have also been measured during development by Northern analysis. In brain, the developmental expression pattern of the TF(0.67)CAT transgene is the same as the mouse endogenous TF gene; in liver, the transgene is turned on later than the endogenous mouse TF gene. DNA-protein mobility shift assays and DNase I footprinting analyses were conducted in the region of -621 to -409 bp of the human TF gene by using TF-CAT expressing liver nuclear extract from 27-day-old mice and nonexpressing liver nuclear extract from 7-day-old mice. The level of protein-DNA complex formation is several times higher in the expressing extracts, and the region from -481 to -463 bp of human TF gene is protected by the expressing extract but not the nonexpressing extracts. As demonstrated by this and other studies, the transgenic mouse model furnishes a unique opportunity to analyze developmental regulation of human transgenes.

Lead suppresses chimeric human transferrin gene expression in transgenic mouse liver.

The major iron-transport protein in serum is transferrin (TF) which also has the capacity to transport other metals. This report presents evidence that synthesis of human TF can be regulated by the metal lead. Transgenic mice carrying chimeric human TF-chloramphenicol acetyl transferase (CAT) genes received lead or sodium salts by intraperitoneal injections or in drinking water. Transgene expression in liver was suppressed 31 to 50% by the lead treatment. Lead regulates human TF transgenes at the mRNA level since liver CAT enzyme activity, CAT protein, and TF-CAT mRNA levels were all suppressed. The dosages of lead did not alter synthesis of the other liver proteins, mouse TF and albumin, as measured by Northern blot analysis of total liver RNA and rocket immunoelectrophoresis of mouse sera. Moderate levels of lead exposure were sufficient to evoke the human TF transgene response; blood lead levels in mice that received lead acetate in drinking water ranged from 30 micrograms/dl to 56 micrograms/dl. In addition to suppressing expression of TF-CAT genes in transgenic mice, lead also suppressed synthesis of TF protein in cultured human hepatoma HepG2 cells. The regulation of human TF apparently differs from the regulation of mouse TF which is unresponsive to lead exposure.

The expression of transferrin mRNA in the salivary glands and other tissues of baboons.

In order to determine whether all the extrinsic salivary glands synthesize transferrin mRNA, the polyadenylated ribonucleic acids [poly(A)+ RNAs] from parotid, submandibular, and sublingual glands, liver, midbrain, testis, spleen, heart, kidney, and the mucosae of oesophagus and stomach from adult male baboons were analysed, using oligo(dT)-cellulose chromatography, agarose gel electrophoresis, followed by transfer of the mRNAs to nitrocellulose filters and identification with transferrin and tubulin cDNA probes. Transferrin and tubulin mRNAs were visualized by autoradiography and analysed by measuring specific activity from beta emitting nuclides following transfer to nitrocellulose filters and hybridizing with [alpha-32P]-labelled human transferrin and tubulin cDNA probes. The results indicate that transferrin mRNA is present in all the extrinsic salivary glands (submandibular, sublingual, parotid) of baboons.

Expression of a human chimeric transferrin gene in senescent transgenic mice reflects the decrease of transferrin levels in aging humans.

Transgenic mice provide a means to study human gene expression in vivo throughout the aging process. A DNA sequence containing 668 bp of the 5' regulatory region of the human transferrin gene was fused to the bacterial reporter gene chloramphenicol acetyl transferase (TF-CAT) and introduced into the mouse genome. Expression of the human chimeric transferrin gene was similar to the tissue patterns of mouse and human transferrin. In aging transgenic mice, expression of the human chimeric transferrin gene was found to diminish 40% in livers between 18 and 26 months of age. Transferrin levels and serum iron levels in aging humans also diminish, as observed from measurements of total iron binding capacity and percent iron saturation in sera from 701 individuals ranging from 0 to 99 years of age. In contrast, in transgenic mice and nontransgenic mice, the mouse endogenous plasma transferrin and endogenous Tf mRNA increase significantly during aging. Neither the decrease of human TF-CAT nor the increase of mouse transferrin during aging appears to be part of a typical inflammatory reaction. Although the 5' regions of the human transferrin and mouse transferrin genes are homologous, sequence diversities exist which could account for the different responses to inflammation and aging observed.

Tissue specific expression of mouse transferrin during development and aging.

Transferrin (TF) is a major plasma protein that binds ferric iron and transports it to all target tissues of the body. This study is the first step to identify the tissue specific expression of the transferrin gene in mice during development, into maturity and throughout the aging process. The transferrin gene expresses mainly in mouse liver, the cerebral hemispheres and cerebellum. In mouse, transferrin is expressed in peritoneal macrophages and in mouse macrophage cell line MO59. At 19 days of gestation, transferrin mRNA is detected in the fetal lung, heart, stomach and kidney. TF mRNA levels increase in liver throughout gestation with maximum expression occurring at 19 days. Transferrin mRNA was detected in placentas of pregnant mice, with levels progressively increasing throughout the term of pregnancy. The levels of liver TF mRNA in mouse vary in a cyclic manner during the development increasing with the aging processes. Because of the dynamic nature of tissue requirements for transferrin during homeostasis the TF gene serves as a promising system for analyzing tissue-specific regulation in vivo during development and aging. Results from this study designate periods in the life-span of the mouse where regulatory mechanisms interacting with the TF gene appear to dynamically alter its expression.

Human transferrin. Expression and iron modulation of chimeric genes in transgenic mice.

Transferrin (TF) is a plasma protein that transports and is regulated by iron. The aim of this study was to characterize human TF gene sequences that respond in vivo to cellular signals affecting expression in various tissues and to iron administration. Chimeric genes were constructed containing 152, 622, and 1152 base pairs (bp) of the human TF5'-flanking region with the coding region of a reporter gene, CAT (chloramphenicol acetyltransferase), and introduced into the germ line of mice. Transgenes containing TF 5'-flanking sequences to -152 bp were expressed poorly in all tissues examined. In contrast, transgenes containing TF sequences to -622 or -1152 bp were expressed at high levels in brain and liver, greater than or equal to 1000-fold higher than tissues such as heart and testes. Liver and brain are major sites of endogenous TF mRNA synthesis, but liver mRNA levels are 10-fold higher than brain. A significant diminution of CAT enzymatic activity in liver accompanied iron administration in both TF(0.67) and TF(1.2)CAT transgenic mice, mimicking the decrease of transferrin in humans following iron overload. Levels of endogenous plasma transferrin also decreased in iron-treated transgenic mice. Transgenic mouse lines carrying human TF chimeric genes will be useful models for analyzing the regulation of human transferrin by iron and for determining the molecular basis of transferrin regulation throughout mammalian development into the aging process.

Proto-oncogene analyses in brain tumors.

The present study determined which oncogenes (N-myc, c-myc, v-sis, or v-fos) were amplified and which messenger ribonucleic acids (mRNA's) accumulated in 10 primary human brain tumors of neuroectodermal origin. The tumors included four glioblastomas multiforme, one mixed glioma (astrocytoma grade I and ependymoma), one astrocytoma grade II, one cystic cerebellar astrocytoma, one ependymoma, one ganglioglioma, and one medulloblastoma. The relative amounts of polyadenylated (poly(A)+) RNA's homologous to these genes and their copy number were determined using the RNA and deoxyribonucleic acid blot hybridization techniques. The N-myc and v-sis probes hybridized strongly to the poly(A)+ RNA from the same recurrent glioblastoma with gene amplifications (N-myc 80 copies; v-sis three to four copies). The c-myc probe hybridized strongly to the recurrent medulloblastoma without gene amplification. The amplification or abundant accumulation of mRNA's homologous to their oncogenes may be involved in tumorigenesis or the aggressiveness of these malignant brain tumors of neuroectodermal origin and may be good molecular indicators of an extremely malignant state in these tumors.

Expression of three viral oncogenes (v-sis, v-myc, v-fos) in primary human brain tumors of neuroectodermal origin.

We determined which viral oncogenes (v-sis, v-myc, and v-fos) were expressed in five primary human brain tumors of neuroectodermal origin (two glioblastomas multiforme, one medulloblastoma, one cystic cerebellar astrocytoma, and one ganglioglioma) and which of these oncogenes is correlated with malignancy. Using the dot hybridization technique, we determined the relative amounts of mRNA coded by these genes using the same nitrocellulose filter. The v-myc probe showed a 4- to 12-fold greater hybridization to the mRNA from two glioblastomas and the medulloblastoma (malignant group) than the mRNA from the cystic cerebellar astrocytoma or the ganglioglioma (benign group). In contrast, RNA hybridizing to v-sis and v-fos were accumulated to a greater extent in the benign tumors. These data suggest that the amount of myc expression may be correlated with the degree of malignancy of brain tumors of neuroectodermal origin.

Nyctohemeral rhythms of gonadal, thyroid and pineal function in the hyperprolactinemic male rat.

In young adult male rats bearing a donor anterior pituitary gland grafted for 3 weeks under a kidney capsule, serum prolactin (PRL) concentrations were elevated and exhibited a rhythm with the highest values in the light phase. Serum PRL in control animals did not exhibit a significant rhythm. Eutopic pituitary PRL content, manifesting a biphasic (12-hr) rhythm with crests during the day and night in controls, exhibited a similar pattern in grafted rats though an overall reduction in pituitary PRL content was seen in the grafted animals. Neither the normal biphasic serum testosterone rhythm nor the normal 24-hr rhythm (nocturnal surge) of pineal N-acetyltransferase activity and melatonin content were altered in the hyperprolactinemic rats. Serum thyroxine (T4) and triiodothyronine (T3) and their free indices (FT4I, FT3I) and serum thyrotropin (TSH) were highest during the day in controls and grafted rats and a 12-hr rhythmic component was detected in data for these variables. In the grafted animals, the 12-hr component was reflected in an additional peak at night detectable by testing of means. The overall serum T4, FT4I, and TSH levels were lower in grafted rats though overall T3 and FT3I levels did not differ between grafted and controls. T3 uptake (T3U) values were similar between controls and grafted rats, in both cases exhibiting a fall during the night. Changes in serum thyronines could not be explained by changes in serum binding as assessed by the T3U, and thus may represent changes in thyroidal secretion of T4.(ABSTRACT TRUNCATED AT 250 WORDS)