Biomedical subjects

D C Herbert

Publications and source records attributed to D C Herbert.

At least 37 records · Page 2Linked to original sources

PubMed1995

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.

Alleles↗

PubMed1995

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.

Age Factors↗

PubMed1995

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.

Animals↗

PubMed1995

Melatonin inhibits luteinizing hormone releasing hormone (LHRH) induction of LH release from fetal rat pituitary cells.

The in vitro effect of melatonin on the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from fetal rat pituitary cells was investigated. A significant inhibition of LH release induced by 10(-9) M luteinizing hormone releasing hormone (LHRH) was seen when cells were incubated with 10(-9) M melatonin. FSH release was unaffected by either LHRH alone or LHRH in combination with melatonin. In addition, the significant inhibitory effect of melatonin was reduced by pretreatment of the pituitary cells with 10(-10) M melatonin. These findings indicate that melatonin can act directly on the fetal pituitary gland to suppress LHRH-induced release of LH perhaps by a mechanism which eventually involves down-regulation of the melatonin receptors.

Animals↗

PubMed1994

Septate-like junctions in the normal male rat pituitary gland.

Septate-like junctions were observed in the rat anterior pituitary gland of the adult male solely between adjacent folliculo-stellate cells. Considering their location, it is presumed that their function is cellular adhesion and mechanical support for the hypophyseal follicles.

Animals↗

PubMed1994

Immunohistochemical study of the post-natal development of the folliculo-stellate cells in the rat anterior pituitary gland.

We investigated the post-natal development of cell-to-cell communication within the rat anterior pituitary gland cells using immunohistochemistry of the S-100 protein. Tissues of animals from 10 to 60 days of age were analyzed. At 10 days of age, S-100 protein-containing cells were rarely observed. With age, the population of S-100 immunostained cells increased until day 40 when they were found to be quite numerous. No further changes were noted from day 40 through day 60. From our previous studies, we conclude that the cells which reacted with the S-100 antiserum were folliculo-stellate cells and their developmental pattern parallels that of the hypophyseal-gonadal axis.

Animals↗

PubMed1994

The pineal melatonin rhythm and its regulation by light in a subterranean rodent, the valley pocket gopher (Thomomys bottae).

The daytime and nightime levels of pineal N-acetyltransferase (NAT) activity, hydroxyindole-O-methyltransferase (HIOMT) activity, and melatonin were measured in adult male and female valley pocket gophers, Thomomys bottae. This species was chosen for study because it is a subterranean rodent that inhabits burrows whose openings to the surface are closed. Therefore, under field conditions it is estimated that the pocket gopher spends roughly 99% of its time in absolute darkness in underground burrows. When wild captured pocket gophers were maintained under a light:dark cycle (light intensity during the day of roughly 140 microW/cm2), nightime levels of pineal NAt activity and melatonin content were higher than values measured during the day; on the other hand, HIOMT activity in the pineal gland was similar in the day and at night. When pocket gophers were exposed to an extended light period (220 microW/cm2) 4 hr into the night, the rise in melatonin synthesis normally associated with darkness onset was not inhibited. Also, when gophers were acutely exposed to a light intensity of 400 microW/cm2 for 1 hr beginning 4 hr after darkness onset, neither high nocturnal levels of pineal NAT nor pineal melatonin contents were reduced. Finally, when pocket gophers were exposed to a 600 microW/cm2 light intensity at either 4 hr or 8 hr into the dark period, pineal melatonin synthesis remained elevated at a level comparable to that measured in dark-exposed controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylserotonin O-Methyltransferase↗

PubMed1994

Heterozygous Rb-1 delta 20/+mice are predisposed to tumors of the pituitary gland with a nearly complete penetrance.

Humans with a germline mutation of the RB gene are predisposed to retinoblastoma with a 90% penetrance and are at higher risk for other rare tumors. We have established a mouse strain carrying a germ-line mutation of the Rb-1 gene. Here we describe a nearly 100% incidence of spontaneous pituitary tumors which arise in Rb-1 heterozygous mice from ages 2 to 11 months. Histological and immunological analysis indicate that these tumors originate from the intermediate lobe of the pituitary gland, which is a vestigial structure in adult humans. Progression of the tumors can be followed by the elevated level of a specific proteolytic product of pro-opiomelanocortin prohormone, alpha-melanocyte stimulating hormone, in the circulating blood of the tumor-bearing animals. Multiple foci are frequently seen in the tiny intermediate lobe of the pituitary gland which contains approximately 1.5 x 10(5) cells. The tumor tissues invariably lose expression of full-length Rb protein due to loss of the single wild-type Rb-1 allele. The Rb knock out mouse strain provides a unique model for addressing tissue-specific tumor predisposition by inactivation of a ubiquitously expressed tumor suppressor gene.

Age Factors↗

PubMed1993

Evaluation of the molecular and cellular basis for the modulation of thyroid C-cell hormones by aging and food restriction.

Male F344 rats fed ad libitum or maintained on 60% of the ad libitum food intake were sacrificed at 6, 12, 18 and 24 months of age. The thyroids were removed for the analysis of the C-cell hormones, calcitonin, calcitonin gene-related peptide (CGRP) and somatostatin. In the animals fed ad libitum, the peptide content of all three hormones and their mRNA pools increased significantly with age. The increases were markedly suppressed by food restriction. Similarly, the rate of mRNA synthesis of the hormones increased with age and was attenuated by food restriction. Calcitonin and CGRP containing cells increased in number with age in the ad libitum fed animals. In the food restricted animals the numbers of calcitonin positive cells were consistently but not significantly lower than those of ad libitum fed animals at similar ages. In the case of CGRP containing cells, their numbers were significantly lower in the food restricted than in the ad libitum fed animals from 18 months of age. Our findings indicate that aging and food restriction modulate the levels of the thyroidal C-cell hormones at the levels of cell proliferation and possibly gene transcription.

Aging↗

PubMed1993

Quantitative morphological analysis of the pituitary gland in protein-calorie malnourished rats.

A quantitative analysis of the pituitary gland was conducted to ascertain the effects of protein-calorie malnutrition on the morphology of the somatotrophs, gonadotrophs, thyrotrophs, and corticotrophs. Male rats were fed a low, 8% protein diet from 20 to 50 days of age, while their age-matched controls were given a diet containing 27% protein. The hypophyses were then processed for light microscopic immunocytochemical staining using antibodies to growth hormone, the beta subunits of luteinizing hormone and thyroid stimulating hormone, and adrenal corticotrophic hormone. The number of each cell type along with an evaluation of the cell, cytoplasmic, and nuclear areas was conducted using a computerized image analyzer. All of these parameters were reduced significantly in the somatotrophs as a result of the low protein diet, while in the gonadotrophs, the cell, cytoplasmic, and nuclear areas were similarly affected. Smaller cell number, cell area, and nuclear area were noted in the corticotrophs of the malnourished animals, while in the thyrotrophs, only the cell and nuclear areas were reduced. The data demonstrate that each pituitary cell type responds in a unique manner to undernutrition.

Adrenal Glands↗

PubMed1993

Intercellular communication within the rat anterior pituitary gland: V. Changes in cell-to-cell communications as a function of the timing of castration in male rats.

Cell-to-cell communication by gap junctions was investigated in the male rat anterior pituitary gland following several experimental regimens involving castration. The regimens included the following animals: (1) Group 1, castrated at 10-day intervals from day 10 to 50 and sacrificed at 60 days of age; (2) Group 2, castrated every 10 days from days 10 to 50 and sacrificed 50 days after castration; (3) Group 3, castrated at 5 days of age and sacrificed every 10 days from day 10 to 60; or (4) Group 4, remained intact and sacrificed every 10 days from days 10 to 60. In all of the castrated animals, numerous so-called castration cells were scattered throughout the pars distalis of the pituitary gland, with occasional "signet ring cells" being observed. In Groups 1 and 2, the pattern of gap junction development and their number was no different from the intact control (Group 4). In contrast, the number of gap junctions in the animals castrated on day 5 remained very small even into adulthood. These data demonstrate that gonadal steroids are important in the initial development of gap junctions within the pituitary gland but are not necessary to sustain their presence once an animal becomes an adult.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

Evidence that Müller cells can phagocytize egg-lecithin-coated silicone particles.

This study is designed to better understand the function of the Müller cells of the retina through the use of silicon particles that had been injected into the vitreous body of the eye and via an analysis of the number of gap junctions associated with these cells. Following intraocular injection of a silicone oil mixture into the rabbit retina, numerous small silicone particles less than 1 micron in diameter were found attached to the basement membrane of the inner limiting membrane, subjacent to the Müller cells. On the sites to which the silicone particles attached, the basement membrane was reduced in thickness or completely disappeared. The 'end-feet' of the cell membrane facing the silicone particles became concave appearing as if they were in the process of incorporating the particles into the cell. Similarly, the Müller cells occasionally extended cytoplasmic processes through the basement membrane and towards the silicone particles, as if to engulf them. Gap junctions were observed but only in association with the Müller cells. They were often found between the 'end-feet' of adjacent Müller cells or the 'end-feet' of one cell and very thin cytoplasmic process of a neighboring cell. On rare occasions, they were located between 'end-feet' of the same Müller cell. The percentage of 'end-feet' displaying gap junctions was 38 +/- 8% (mean +/- SE). These data suggest that the Müller cells are important in the maintenance of normal retinal function by their ability to phagocytize foreign substances and by their ability to transmit information to various parts of the retina through cell-to-cell connections established by gap junctions.

Animals↗

PubMed1993

Evidence for estrogen receptor-linked calcium transport in the intestine.

Intestinal calcium malabsorption in postmenopausal osteoporotic women is often linked indirectly to decreased serum 1,25(OH)2 vitamin D or to intestinal resistance to its action, rather than directly to the low circulating estrogen that results following menopause. The studies presented indicate that the intestinal mucosal cells of rats contain estrogen receptor immunoreactivity, express the mRNA for estrogen receptors, and respond directly to 17 beta-estradiol with enhanced calcium transport that is suppressed by gene transcription and protein synthesis inhibitors. These findings suggest that estrogen has a physiological role in the regulation of intestinal calcium absorption and that its deficiency in postmenopausal osteoporosis, and following therapeutic oophorectomy, may result directly in calcium malabsorption that is believed to be an important factor in the bone loss that occurs in these conditions.

Animals↗

PubMed1993

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.

Animals↗

PubMed1993

Phototransduction-related circadian changes in indoleamine metabolism in the chick pineal gland in vivo.

The purpose of this study was to examine the day/night levels of pineal melatonin and its rate limiting enzyme N-acetyltransferase (NAT) in relationship to the ratio of 11-cis- to all-trans-retinal. Three-week-old chicks were placed in 12:12 light:dark (LD 12:12) cycle for one week, pineals were collected during the light phase at 1500 (i.e., after 10 hr light), during the dark phase at 1900 (i.e., 2 hr after dark), at 2100 (i.e., 4 hr after dark), and at 2300 (i.e., 6 hr after dark) and after light extension to 1900. The results show that light-sensitive 11-cis-retinal in the chick pineal has the same diurnal rhythm as NAT and melatonin; all constituents increased within 2 hr of darkness onset (at 1900) and reached their peak after 4 hr of dark. All values were lowest during the light phase at 1500. Low values for 11-cis-retinal, NAT, and melatonin were also seen in the group of chicks which experienced light extension to 1900. The data indicate that in vivo light plays a major role in triggering rhodopsin-bound 11-cis-retinal production within 2-4 hr after darkness onset; this change likely serves as the signal for the subsequent formation of the hormonal product of the pineal gland, melatonin.

Acetylserotonin O-Methyltransferase↗

PubMed1993

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.

Animals↗

PubMed1992

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.

Adolescent↗