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.

The primate spinal cord is a target for gonadal steroids.

The nuclear uptake and retention of 3H-dihydrotestosterone (3H-DHT) or one of its metabolites was studied in the spinal cord of the rhesus monkey. Normally-cycling adult female rhesus monkeys which were castrated and adrenalectomized prior to the experiment were injected with 1 microgram of 3H-DHT (107 Ci/mmole)/kg body weight and killed 90 minutes later. The spinal cords were removed and segments processed for autoradiography. Nuclear uptake and retention were found in both the visceral and somatic motor systems and, in addition, in the nociceptive system. The data suggest a role for androgen in sexual reflexes and possibly pain perception at the level of the spinal cord in the primate and provide further support for a role of androgen in amyotropic lateral sclerosis.

Androgen-concentrating cells in the periventricular brain of the female rhesus monkey.

Although androgens act on the primate central nervous system to modulate both endocrine functions and a number of limbic-related behaviors, little is known about the anatomical location of the neurons which sequester these steroids in primates. To determine the prime location of these androgen-concentrating neurons in the forebrain of the primate, we injected three castrated female rhesus monkeys in the femoral vein with 1 microgram of 5 alpha-dihydro (1,2,4,5,6,7-3H) testosterone (3H-DHT, 107 Ci/mmole) per kg of body weight. One of these animals also received an IV injection of 100 micrograms/kg body weight of unlabeled dihydrotestosterone (DHT) to serve as a control. One hour after the injection of 3H-DHT we rapidly exsanguinated each animal. The forebrain was sliced and blocks containing the amygdala, diencephalon, frontal pole, and hippocampus were frozen and stored in liquid nitrogen until processing. The tissue was then processed for autoradiography. A specific topographic pattern of nuclear concentration of DHT or one of its metabolites was obtained in neurons of the basal hypothalamus, preoptic region, amygdala, and hippocampus. This pattern was similar to that found in rodent species.

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.

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.

Autoradiographic localization of estrogen target cells in the spinal cord of the armadillo and baboon: a comparative study.

The uptake and retention of radiolabeled estradiol by the spinal cord were examined in the baboon and the armadillo and compared to previous observations in the rat. Four females of each species were injected intracardially with 1.0-1.4 micrograms/kg body weight of 3H-estradiol and two females, one baboon and one armadillo, were injected with both labeled and 100-140 micrograms/kg body weight of unlabeled estradiol. One hour after the injections, the animals were killed and segments from the cervical, thoracic, lumbar and sacral cord were removed and processed for autoradiography. In the armadillo, labeling of neuronal nuclei were noted in laminae I & II and in alpha motor neurons. In addition, nuclei of the ependymal cells of the ventral portion of the central canal in the cervical cord concentrated radioactivity. In contrast, the baboon demonstrated only sporadic labeling of neurons in lamina II in all levels of the spinal cord. The comparison of our observations with that of the rat suggest that estrogen mediated sensations are probably coordinated at higher brain centers in the primate as opposed to the more primitive mammals.

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.

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.

Androgen receptor systems in the brain stem of the primate.

The nuclear uptake and retention of [3H]dihydrotestosterone ([3H]DHT) or one of its metabolites was studied in the cerebellum and brain stem of the rhesus monkey. Three days before the injection of the tritiated hormone, we removed both ovaries and the right adrenal gland of 3 female rhesus monkeys under ketamine and halothane or fluothane anesthesia with aseptic surgical procedures. Two days later, we removed the left adrenal gland under similar conditions. Shortly after the second operation, each animal received 100 mg prednisolone sodium succinate. On the day of autopsy, we injected into a femoral vein of each animal 1 microgram of 5 alpha-dihydro[1,2,4,5,6,7-3H]-testosterone (107 Ci/mmol). One of these animals also received an i.v. injection of 100 micrograms/kg body weight of unlabeled DHT to serve as a control. One hour after injection, we rapidly exsanguinated each animal through a femoral venous catheter and perfused the vascular system with approximately 3 1 of Ringer's solution through a femoral arterial catheter. The cerebella and brain stems were removed and processed for autoradiography. Unlike in the rat, nuclear uptake and retention of [3H]DHT was found in both motor and sensory systems of these monkeys and in other areas less well defined. These data indicate that there are major species differences in the nuclear uptake and retention of androgen by the cerebellum and brain stem of rats and primates.

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.

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.

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.

Localization of 3H-estradiol in the reproductive organs of male and female baboons.

The uptake and retention of radiolabeled estradiol by both the male and female reproductive organs were examined in the baboon. Two male and two female baboons were injected intracardially with 1 microgram/kg body weight of 3H-estradiol and two animals, one male and one female, were injected with both labeled and 100 micrograms/kg body weight of unlabeled estradiol. One and a half hours after the injections, the animals were sacrificed and the uterus, cervix, vagina, oviduct, seminal vesicles, and prostate gland were removed and processed for autoradiography. The stratified squamous epithelia of the cervix and vagina demonstrated a light uptake of the label in the germinative, but not in the superficial cell layers. The columnar cells lining the oviduct and uterine glands were labeled, whereas the luminal epithelium of the uterus and the glandular epithelia of the seminal vesicles and prostate gland did not sequester the tritiated steroid. The interstitial cells of all the organs studied demonstrated a moderate to heavy uptake of the radioactivity, whereas the smooth muscle cells were lightly labeled except in the vagina, in which these cells displayed a moderate number of silver grains.

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)

Spermatogonia and the cycle of the seminiferous epithelium in the nine-banded armadillo.

The cycle of the seminiferous epithelium of the nine-banded armadillo can be divided into ten stages. As in most mammals, only one stage is observed per tubular cross-section. The process of spermiogenesis can be divided into thirteen steps according to the development of the acrosomal system and the flagellum. Four generations of spermatogonia are abserved in the germinal epithelium: 1) stem cells, 2)type "A", 3) intermediate, and 4) type "B" spermatogonia. The stem cell is characterized by a highly irregular nucleus and the presence of glycogen in its cytoplasm. The type "A" spermatogonium contains an oblong nucleus with one or two shallow infoldings of the nuclear membrane. The intermediate spermatogonium contins an ovoid nucleus characterized by one or two nuclei and heterochromatin scattered in the nuceoplasm. The nucleus of the type "B" spermatogonium is more spherically shaped with a centrally placed nucleolus and heterochromatin associated with the nuclear envelope.

Light microscopic and ultrastructural features of the Harderian gland of the nine-banded armadillo.

The Harderian gland of the nine-banded armadillo is a mixed, compound tubuloacinar gland located in the medioposterior aspect of the orbit. The gland is lobulated and appears homogeneous in colour and texture. Harderian glands of both male and female fluoresce a pale yellow-green, indicating the lack of porphyrins. At the light microscopic level, the gland contains lobules which synthesize different secretory products. The lobules of the gland adjacent to the eyeball (the proximal point) contain predominantly mucus-secreting acini which empty into a well developed system of intralobular ducts. The lobules of the major portion of the gland (the distal part) contain lipid-secreting acini. The cells contain numerous lipid vacuoles which are colourless when fixed with osmium tetroxide and stain blue with the perchloric acid-naphthoquinone technique for cholesterol. The duct system of this portion of the gland is less developed than the mucous portion; however, these lobules contain a well developed network of fenestrated capillaries. At the ultrastructural level, the secretory cells of the lipid acini are characterized by an expansive network of smooth endoplasmic reticulum and numerous lipid vacuoles. Additionally, these cells contain a well developed Golgi apparatus with associated vesicles, condensing vacuoles, and secretory granules. The free surface of the cells possess microvilli; however, the microvilli appear longer and more concentrated on the surface of the cells adjacent to the fenestrated capillaries. The possible functions of this gland in the armadillo, which possesses a rudimentary pineal gland, are discussed.

The fine structure of the interstitial tissue of the testis of the nine-banded armadillo.

The interstitial tissue of the testis of the nine-banded armadillo is composed of blood vessels, clusters of Leydig cells, the usual connective tissue elements, and a network of lymphatic sinusoids. The endothelial walls of the sinusoids are separated from the peritubular contractile cell layer surrounding the seminiferous tubules by a thin layer of collagen. The pertibular contractile cell is characterized by filaments and dense bodies within the cytoplasm, whereas the endothelial cells lack these structures. Within each cluster, several Leydig cells surround one or more blood vessels. Adjacent Leydig cells are jointed by 2- to 3-nm wide gap junctions and desmosome-like specializations. The Leydig cell is polygonal in shape with an ovoid nucleus. The cell is characterized by an abundance of smooth endoplasmic reticulum which appears as sheets of membranes, concentric whorls around vacuoles, and a random tubular network. Only a few short cisternae of rough endoplasmic reticulum are observed. Centrioles are closely associated with the Golgi apparatus. Rod-like mitochondria with tubular cristae are scattered throughout the cytoplasm. In addition, the cells contain vacuoles resulting from lipid extraction, filaments, microtubules, and glycogen. The surface of the cell exposed to the intercellular spaces exhibit numberous pinocytotic vesicles and cell processes which indicate active movement of material across the plasma membrane. In comparison to other mammalian species, the ultrastructural organization of the interstitium and the fine structure of Leydig cell of the armadillo resemble those of the guinea pig.

Pineal gland morphology in rats with experimentally induced protein-calorie malnutrition.

The morphology of the pineal gland was studied in protein-calorie-malnourished (PCM) rats. Twenty-day-old male Sprague-Dawley rats were placed in a 14:10 photoperiod and fed either an 8% low protein diet (LPD) or a standard laboratory diet (SLD) containing 27% protein for 30 d. At 50 d of age, rats from both animal groups were sacrificed at 0900 h and at 2400 h, and the pineal glands were immersion-fixed for either light or electron microscopic analysis. The cytoplasm and nuclei of the pinealocytes from the SLD-fed rats were consistently larger than those of the animals maintained on the LPD. Additionally, the lipid droplets were larger and more prominent in the controls at both 0900 h and 2400 h. Even though the size of these inclusions did not vary among animals given the same diet as a function of the time of sacrifice, they were more numerous in both the well-fed and malnourished rats during the dark phase of the photoperiod. In contrast neither diet nor sampling time affected the size or number of pinealocyte mitochondria. These morphological observations lend further support to the premise than PCM impairs the cellular activity of the pinealocytes.

Chromatin ultrastructure changes during spermatogenesis in armadillos.

The chromatin fibres of the male gamete nucleus of Dasypus novemcinctus contain fibrils. Measurements indicate that the chromatin fibre diameter and the number of fibrils within the fibre increase, whereas the diameter of the fibrils decreases during spermiogenesis. It is suggested that the fibrils represent a DNA-protein complex. It is further suggested that the increased fibre diameter, and the increased number of fibrils correlates with the formation of larger fibres during the condensation of the spermatid nucleus, whereas the decrease in fibril diameter is correlated with the replacement of histones with new low molecular weight acid-soluble proteins.

Postnatal development of the ventral prostate gland in normal versus protein-calorie malnourished rats.

The ultrastructure of the postnatal development of the secretory cells within the ventral prostate gland was studied in the protein-calorie malnourished rat. Twenty-day-old Sprague-Dawley rats were fed either a low protein diet (LPD) containing 8% protein in the form of vitamin free casein or a standard laboratory diet (SLD) containing 27% protein. Animals maintained on the LPD and SLD were sacrificed every 7 days, beginning on Day 27 and ending on Day 55. The ventral prostate glands were perfused by whole body vascular perfusion via the left ventricle and processed for electron microscopy. The secretory cells of the prostates of the SLD-fed rats demonstrated progressive development of the organelles involved in protein synthesis which included an increase in cisternal profiles of the rough endoplasmic reticulum (RER), an enlargement of the Golgi apparatus, and an increase in the number of mature secretory granules. On the other hand, the growth of the secretory cells of the animals fed the LPD was retarded. The cells had an overall reduction in RER, the elements of the Golgi complex and the size of the nuclei as compared to age-matched controls. Additionally, they were characterized by an increase in various vacuole-like structures along with a concomitant decrease in mature secretory granules. These morphological observations suggest that protein-calorie malnutrition impedes both the development of the ventral prostate gland and the secretory activity of its epithelial cells.