Meningoradiculitis due to varicella zoster virus reactivation in a patient treated with ixekizumab.

INTRODUCTION: Ixekizumab is a recently developed biopharmaceutical used since 2016 in the US and Europe for the treatment of plaque psoriasis. Few adverse effects have been reported in the literature and ixekizumab is not known to increase the risk of viral reactivation. Herein we report the case of an immunocompetent female patient treated with ixekizumab who presented meningoradiculitis due to varicella zoster virus (VZV) reactivation. PATIENTS AND METHODS: A 51-year-old woman, treated with ixekizumab for psoriasis, consulted in March 2018 for left hemicrania headache associated with left facial paralysis, but without fever. Brain MRI scans revealed cerebral venous thrombosis of the superior sagittal sinus. Analysis of cerebrospinal fluid (CSF) revealed lymphocytic meningitis and positive VZV-PCR. PCR blood assay for VZV was negative. Blood concentrations of anti-VZV IgG antibody increased while Anti-VZV IgM antibodies remained negative. The final diagnosis was VZV meningoradiculitis complicated by cerebral thrombophlebitis. The absence of skin rash, the rapid increase in anti-VZV IgG antibodies, the absence of anti-VZV IgM antibodies, and the negative blood PCR assay suggested viral reactivation rather than primary infection. The patient received acyclovir and coumadin and her condition improved rapidly. After multidisciplinary discussion, ixekizumab was reintroduced together with valacyclovir prophylaxis. CONCLUSION: This case raises the question of the risk of viral reactivation during treatment with an IL-17 inhibitor and with biologics in general. Neurological and vascular complications of VZV may occur without skin lesions and their occurrence during ixekizumab therapy must be investigated by PCR testing of CSF for VZV DNA.

Impacts on human mortality due to reductions in PM10 concentrations through different traffic scenarios in Paris, France.

OBJECTIVES: Air pollution is a well-known burden for population health and health systems worldwide. Reduction in air pollution is associated with improvements in mortality and rates of respiratory, cardiovascular and other diseases. Though air quality is a problem globally, efforts to lower air pollutant concentrations are usually regional or local. In industrialized countries, most urban air pollution is caused by vehicles, suggesting reductions in traffic would result in reductions of pollution. However, detailed data on how such reductions can be achieved and impact public health is just beginning to emerge, and other influencing factors, including vehicle flow or urban landscape are largely unaccounted for. METHODS: We utilized a unique combination of vehicle emission measurements combined with simulations of traffic and vehicle variations, as well as urban topographies, to quantify health impacts of PM10 reduction in a single district of Paris, France, for various methods of traffic improvement. Here we rank and evaluate improvements in non-accidental mortality for thirteen possible scenarios to reduce traffic related PM10 emissions. RESULTS: The maximum impact scenario requires all passenger vehicles to meet Euro 5 standards and excludes diesel vehicles, resulting in long-term decreases in non-accidental mortality of 148.79 people per year, or 104.40 per 100,000 people. Similar reductions hold for the scenario requiring a completely electric passenger fleet, with long-term annual reductions of 137.14 premature mortalities. Removing all diesel vehicles is the third most impactful scenario, preventing 135.55 deaths yearly. DISCUSSION: PARTLESS provides comparisons between thirteen different traffic-related air quality reduction mechanisms in terms of improvements in mortality rates. Improving emissions standards, increasing electric vehicle use and removing diesel vehicles can prevent more than 148 deaths per year in this district alone. Further improvements in mortality reduction may require changes to the composition of vehicle components, asphalt or to the management of resuspended particulate matter.

Multi-luminescent hybrid gadolinium oxide nanoparticles as potential cell labeling.

This manuscript analyses the use of newly developed hybrid gadolinium oxide nanoparticles as cell-labeling tracers. The nanoparticles are core-shell particles composed of a core of gadolinium oxide of [2-4] nm and a protecting shell of polysiloxane [1-3 nm] where different organic dyes (fluoresceine isothiocyanate (FITC) or rhodamine B isothiocyanate (RBITC)) are embedded. They are functionalized with poly(ethylene glycol)bis(carboxymethyl) to ensure their colloidal stability in biological buffers. These particles are potential multi-labeling tracers (magnetic and optical). In this paper, we show by optical imaging that they can be efficiently internalized in cells without cell alteration. The in-vitro uptake of the nanoparticles was followed in two cell lines (human fibroblasts and a human adenocarnima cell lines MCF7 cells). Nanoparticles distribution within cells was analysed by confocal analysis, and gadolinium concentration within cells was quantified by mass spectrometry (ICP-MS analysis). Nanoparticles uptake is found to be fast and efficient for both cell lines, with fluorescent labeling visible after 10 min of incubation whatever the nature of the fluorophore. The fluorescent intensity is mainly found as concentrated dots in the perinuclear region of the cells and decreases with the number of days in culture, but is still easily detectable after 3 days in culture. No significant effect on cell growth was detected. Finally, we show in this study the protective effect of the polysiloxane layer: encapsulation of RBITC within the polysiloxane shell, leads to a better photostability of this low cost dye than Cy3 and even reach a level comparable to Alexa 595. With their high photostability and long-lasting contrast properties, these hybrid luminescent nanoparticles appears thus as a versatile solution to assess multiple cell fate both in in-vitro cell model as well as in-vivo.

Characterization of an acquired dps-containing gene island in the lactic acid bacterium Oenococcus oeni.

AIMS: To identify novel actors responsible for the marked adaptation of the Oenococcus oeni species to its environment. METHODS AND RESULTS: Genomic surveillance of the available genome sequences from O. oeni indicated the presence of a small ORF, encoding a protein named Dps(A). The cloned gene complemented the dps(-) mutant of Escherichia coli and conferred resistance to hydrogen peroxide, wine, and metals. The dps(A) gene was flanked by IS-related elements. The entire region was characterized by an anomalously high GC content compared to those reported for oenococcal genomes. The dps(A) gene was present in 15 of the 38 tested isolates. Positive strains originated from different geographical areas and sources. No change in tolerance to wine or to oxidative stress was observed between O. oeni strains harbouring dps(A) and those not harbouring this gene. CONCLUSIONS: Some O. oeni have acquired a functional homologue to the dps gene from E. coli as part of a mobile element. SIGNIFICANCE AND IMPACT OF THE STUDY: Dps(A) probably increases the bacterial fitness in response to environmental challenges. However, the physiological condition under which it adds a selective advantage to O. oeni during winemaking remains to be found.

Gene therapy in the neuroendocrine system.

The implementation of experimental gene therapy in animal models of neuroendocrine diseases is an area of growing interest. In the hypothalamus, restorative gene therapy has been successfully implemented in Brattleboro rats, an arginine vasopressin (AVP) mutant which suffers from diabetes insipidus, and in Koletsky (fa(k)/fa(k)) and in Zucker (fa/fa) rats which have leptin receptor mutations that render them obese, hyperphagic and hyperinsulinemic. In the above models, viral vectors expressing AVP, leptin receptor b and proopiomelanocortin, respectively, were stereotaxically injected in the relevant hypothalamic regions. In rats, aging brings about a progressive degeneration and loss of hypothalamic tuberoinfundibular dopaminergic (TIDA) neurons, which are involved in the tonic inhibitory control of prolactin secretion and lactotropic cell proliferation. Stereotaxic injection of an adenoviral vector expressing insulin-like growth factor I corrected their chronic hyperprolactinemia and restored TIDA neuron numbers. Spontaneous intermediate lobe pituitary tumors in a retinoblastoma (Rb) gene mutant mouse were corrected by injection of an adenoviral vector expressing the human Rb cDNA and experimental prolactinomas in rats were partially reduced by intrapituitary injection of an adenoviral vector expressing the HSV1-thymidine kinase suicide gene. These results suggest that further implementation of gene therapy strategies in neuroendocrine models may be highly rewarding.

Developmental toxicity of combined ethylbenzene and methylethylketone administered by inhalation to rats.

Pregnant Sprague-Dawley rats were exposed to ethylbenzene (EB; 0, 250, or 1000 ppm) and methylethylketone (MEK; 0, 1000, or 3000 ppm), alone and in combination, by inhalation, for 6h/day, during days 6-20 of gestation. Maternal toxicity, evidenced by decreased in body weight gain and food consumption, tended to be greater after simultaneous exposures to the high concentrations of 1000 ppm EB and 3000 ppm MEK, when compared to the treatments with individual compounds. No significant increase in embryo/fetal lethality or incidence of malformations and variations was observed in any of the treatment groups. Fetal body weight was significantly reduced after individual treatment with 1000 ppm EB or 3000 ppm MEK, and in the combined groups. There was no evidence of interaction between EB and MEK in causing developmental toxicity.

Leukemia inhibitory factor is a key signal for injury-induced neurogenesis in the adult mouse olfactory epithelium.

The mammalian olfactory epithelium (OE) is composed of primary olfactory sensory neurons (OSNs) that are renewed throughout adulthood by local, restricted neuronal progenitor cells. The molecular signals that control this neurogenesis in vivo are unknown. Using olfactory bulb ablation (OBX) in adult mice to trigger synchronous mitotic stimulation of neuronal progenitors in the OE, we show the in vivo involvement of a cytokine in the cellular events leading to the regeneration of the OE. We find that, of many potential mitogenic signals, only leukemia inhibitory factor (LIF) is induced before the onset of neuronal progenitor proliferation. The rise in LIF mRNA expression peaks at 8 hr after OBX, and in situ RT-PCR and immunocytochemistry indicate that LIF is upregulated, in part, in the injured neurons themselves. This rise in LIF is necessary for injury-induced neurogenesis, as OBX in the LIF knock-out mouse fails to stimulate cell proliferation in the OE. Moreover, delivery of exogenous LIF to the intact adult OE using an adenoviral vector stimulates BrdU labeling in the apical OE. Taken together, these results suggest that injured OSNs release LIF as a stimulus to initiate their own replacement.

Cellular distribution of growth hormone-releasing hormone receptor in human reproductive system and breast and prostate cancers.

Growth hormone releasing hormone receptor (GHRH-R) mRNA and protein was first localized to the anterior pituitary gland, consequent with the action of its ligand on GH synthesis and release. Subsequent studies found GHRH-R also expressed in the hypothalamus and in systemic tissues including those of the reproductive system. In the present work, we studied the distribution of GHRH-R in human reproductive system of males and females by immunohistochemical method. GHRH-R immunostaining was localized in male reproductive system: Leydig cells, Sertoli and basal germ cells of the seminiferous tubules and prostate secretory cells. GHRH-R immunostaining was also demonstrated in the ovary: oocytes, follicular cells, granulosa, thecal and corpus luteum cells. Endometrial glands, placenta and normal mammary glands also showed GHRH-R immunostaining. Our results demonstrate the localization of GHRH-R in the reproductive system, which may mediate the direct action of GHRH in these tissues. Moreover, GHRH-R was demonstrated in prostate and breast carcinomas, opening a variety of possibilities for the use of GHRH antagonists in the treatment of prostatic and mammary tumors.

Developmental toxicity of two trimethylbenzene isomers, mesitylene and pseudocumene, in rats following inhalation exposure.

The developmental toxicity of two trimethylbenzene isomers, mesitylene (1,3,5-trimethylbenzene) and pseudocumene (1,2,4-trimethylbenzene) was studied in Sprague-Dawley rats following inhalation exposure. Pregnant rats were exposed whole body to vapours of mesitylene (0, 100, 300, 600, and 1200 ppm) or pseudocumene (0, 100, 300, 600, and 900 ppm), 6h/day, on gestational days (GD) 6 through 20. Significant decrease in maternal body weight gain and food consumption was observed at concentrations of 300 ppm mesitylene, 600 ppm pseudocumene, or greater. Fetal toxicity, expressed as significant reduction in fetal body weight, occurred at 600 and 1200 ppm mesitylene, and at 600 and 900 ppm pseudocumene. There was no evidence of embryolethal or teratogenic effects following inhalation exposure to either of these chemicals. In summary, the no-observed-adverse-effect-level (NOAEL) for maternal toxicity was 100 ppm for mesitylene and 300 ppm for pseudocumene, and the NOAEL for developmental toxicity was 300 ppm for mesitylene and pseudocumene.

Combined effects of noise and styrene on hearing: comparison between active and sedentary rats.

In this study, two investigations were carried out with adult Long-Evans rats exposed to increasing concentrations of styrene. In the first experiment, the hearing of rats, which were forced to walk in a special wheel during the exposure, was compared to that of rats which were sleepy in their cage. The active rats were exposed to styrene concentrations ranging from 300 to 600 ppm, whereas the sedentary rats were exposed from 500 to 1000 ppm for 4 weeks, 5 days per week, 6 hours per day. In the second experiment, designed to evaluate the hearing risks at threshold limit values, active rats were exposed either to a noise having a Leq8h of 85 dB (equivalent level of a continuous noise for a typical 8-h workday), or to 400-ppm styrene or to a simultaneous exposure to noise and styrene. In both experiments, auditory function was tested by auditory-evoked potentials from the inferior colliculus and completed by morphological analyses of the organ of Corti. The results of the first experiment showed that the same amount of styrene-induced hearing loss can be obtained by using concentrations approximately 200 ppm lower in active rats than in sedentary rats. The second investigation showed that, in spite of the low-intensity noise and the low-concentration of styrene, there is a clear risk of potentiation of styrene-induced hearing loss by noise. These findings and exposure conditions were discussed and extrapolated with regard to the risk assessment for human beings. The authors propose to decrease the French threshold limit value of styrene for ensuring a high level of protection for human hearing.

Monoclonal antibodies to bovine serum albumin: affinity and specificity determinations.

A panel of 12 monoclonal antibodies (MAb) to bovine serum albumin (BSA) was developed and characterized as to their physiochemical and immunological properties. Affinity constants of the MAb varied over a wide range from 10(5) to 10(8) M-1. MAb were assembled into several groups of non- or minimally interacting antibodies by analysis of competitive binding experiments, and BSA domain and subdomain specificities of the MAb were assigned by analysis of results of MAb binding to purified BSA fragments. Further fine specificity delineation was accomplished by examination of cross-reactivity patterns to several mammalian albumins. The data suggest that some of the low affinity MAb recognize sites on different portions of the BSA molecule, indicating that similar epitopes exist on different domains of the BSA molecule.

Cognitive impairment and morphological changes in the dorsal hippocampus of very old female rats.

The hippocampus, a medial temporal lobe structure necessary for the formation of spatial memory, is particularly affected by both normal and pathologic aging. In previous studies, we observed a significant age-related increase in dopaminergic neuron loss in the hypothalamus and the substantia nigra of female rats, which becomes more conspicuous at extreme ages. Here, we extend our studies by assessing spatial memory in 4-6 month-old (young), 26-month-old (old) and 29-32-month-old (senile) Sprague-Dawley female rats as well as the age-related histopathological changes in their dorsal hippocampus. Age changes in spatial memory performance were assessed with a modified version of the Barnes maze test. We employed two probe trials (PTs), one and five days after training, respectively, in order to evaluate learning ability as well as short-term and longer-term spatial memory retention. A set of relevant hippocampal cell markers was also quantitated in the animals by means of an unbiased stereological approach. The results revealed that old rats perform better than senile rats in acquisition trials and young rats perform better than both aging groups. However, during short-term PT both aging groups showed a preserved spatial memory while in longer-term PT, spatial memory showed deterioration in both aged groups. Morphological analysis showed a marked decrease (94-97%) in doublecortin neuron number in the dentate gyrus in both aged groups and a reduction in glial fibrillary acidic protein-positive cell number in the stratum radiatum of aging rats. Astroglial process length and branching complexity decreased in aged rats. We conclude that while target-seeking activity and learning ability decrease in aged females, spatial memory only declines in the longer-term tests. The reduction in neuroblast number and astroglial arborescence complexity in the dorsal hippocampus are likely to play a role in the cognitive deficits of aging rats.

Evidence for the presence of alpha and beta-related T3 receptors in rat liver mitochondria.

Although the dependence of mitochondrial structure and function on thyroid hormone status is well established, several attempts to demonstrate a direct pathway of T3 action on mitochondria have been made during the last decade without being firmly conclusive. In this study, we present evidence firstly for the presence of specific binding sites for [125I]-T3 in rat liver mitochondria 5 min after injection, as assessed by ultrastructural autoradiography. In the same way, using immunocytological techniques and protein immunoblotting, T3 receptor-like immunoreactivity was revealed mainly in the nucleus and mitochondria of hepatocytes. Whereas the colloidal gold labeling over mitochondria was found to be specific at the ultrastructural level, these results were confirmed biochemically by Western blotting experiments which revealed the presence of two protein bands in mitochondria: a stronger one of 55 kDa and a weaker one of 48 kDa. At the opposite, receptor T3 mRNAs were not detected in mitochondria by ultrastructural in situ hybridization thus confirming that the synthesis of receptor T3 occurs in the cytoplasm and that nuclear-encoded T3 receptors may belong to the bulk of cytosolic precursor polypeptides which are targeted to and imported into mitochondria. These results confirm that a direct pathway of T3 action on mitochondria occurs in situ which could now explain how the rapid activation of several mitochondrial functions can take place within minutes after thyroid hormone injection.

Uptake and ultrastructural localization of a [125I] growth hormone releasing factor agonist in male rat pituitary gland: evidence for internalization.

GRF was isolated from a human tumor of the pancreas and characterized. GRF stimulates the in vivo and in vitro secretion of GH. The present study was designed to find out whether human (h) GRF agonist could be internalized and to determine the subcellular localization of internalized peptide in somatotrophs. Autoradiography was performed on rat anterior pituitary glands removed at specific time intervals (2-60 min) after iv injection of monoradioiodinated [125I] (His1,Nle27) hGRF (1-32) NH2. Administration of an excess of unlabeled hGRF agonist along with the radioiodinated hormone prevented the uptake, indicating the specificity of the reaction. At the ultrastructural level only the somatotrophs appeared to contain silver grains. The main effect of hGRF agonist injection on the cytological aspect of the somatotrophs was a decrease in the area occupied by secretory granules, accompanied inversely, by an increase in that of the Golgi complex. The time course study in somatotrophs showed that five compartments (plasma membrane, secretory granules, cytoplasmic matrix, nuclear membrane, and lysosomes) have distinct marked labeling patterns. Plasma membrane, secretory granules, and nuclear membrane were labeled throughout the time course studied (2-60 min after injection). Cytoplasmic matrix was labeled 5 min post injection and lysosomes 15 and 30 min after injection. The Golgi complex, mitochondria, rough endoplasmic reticulum, and nucleus matrix were not labeled. The findings show the cellular specificity of GRF uptake by somatotrophs and the internalization process from the plasma membrane to the intracellular organelles (secretory granules, lysosomes, and nuclear membrane). Labeling of the secretory granule compartment suggests that granules may bind and protect internalized peptide from lysosomal degradation. The appearance of label on the nuclear membrane suggests that GRF may have effects on the translocation of messenger RNA from nucleus to cytoplasm in somatotrophs.

Internalization and subcellular distribution of radiolabeled somatostatin-28 in mouse anterior pituitary tumor cells.

The ACTH-secreting mouse AtT-20/D16-16 (AtT-20) tumor corticotroph possesses receptors for the tetradecapeptide somatostatin (S-14) to which the 14-amino acid N-terminal extension somatostatin-28 (S-28) also binds. When AtT-20 cells are exposed to either S-14 or S-28 for extended periods of time, a marked decrease in S-14 receptor density is observed. Since receptor down-regulation is frequently associated with internalization of ligand and/or receptor, the present study was designed to establish whether AtT-20 cells could in fact internalize S-28 and to determine the subcellular localization of internalized peptide. Cells were incubated in the presence of [Leu8, D-Trp22,125I-Tyr25] S-28 for 1, 4, and 18 h; washed with PBS; and harvested. Cell pellets were fixed, sectioned, and analyzed by light and electron microscopic autoradiography. Uptake of radiolabeled S-28 (90% of all cells) was inhibited by 80% when unlabeled S-14 was coincubated with [125I]S-28. Of the cell compartments examined, plasma membrane, secretory granules, lysosomes, Golgi apparatus, and nuclear membrane all had distinct time-dependent labeling patterns. Plasma membranes were maximally labeled 1 h after exposure to [125 I] S-28. Secretory granule and lysosomal labeling was observed within 1 h, but was maximal after 18 h of exposure. Labeling of the granule compartment preceded that of the Golgi apparatus. The nuclear compartment (membranes plus nuclei) was also labeled significantly after 18 h of incubation. However, the nuclear membrane itself was labeled after only 1 h of exposure to the ligand. The data suggest that radiolabel is transferred from the plasma membrane to the intracellular organelles as a function of exposure time. Labeling of the secretory compartment suggests that granules may bind and protect internalized peptide from lysosomal degradation. Appearance of label in the nuclear compartment suggests that S-28 (and S-14) may have effects on transcriptional activity in AtT-20 cells.

Ultrastructural autoradiographic localization of somatostatin-28 in the rat pituitary gland.

To identify the anterior pituitary cell type(s) containing somatostatin-28 (SS-28)-binding sites and to study the internalization processes of this peptide by the target cells, autoradiography was performed on rat anterior pituitaries removed at specific intervals (2-60 min) after iv injection of the [125I]iodo-SS-28 agonist [Leu8,D-Trp22,Tyr25]SS-28 into intact, adrenalectomized, or castrated male rats. At the light microscopic level, the silver grains were found in 75% of cells. Concomitant injection of an excess of unlabeled peptide prevented the binding of label, verifying the specificity of binding. No specific labeling could be detected in the adrenocorticotrophs of adrenalectomized rats or gonadotrophs (castration cells) of castrated rats. At the electron microscopic level, three cell types (somatotrophs, thyrotrophs, and mammotrophs) appear to contain radiolabeled SS-28. The time-course study in somatotrophs of intact animals showed that 2 min after injection, most silver grains were associated with the plasma membrane. Five to 15 min after injection, label was found over both the plasma membrane and cytoplasmic organelles, especially the Golgi apparatus, lysosomes, and secretory granules. At the longest time interval (60 min), labeling was mostly associated with the cytoplasmic organelles. These results indicate that SS-28-binding sites are present only in those cell types in which somatostatin is known to regulate secretory functions. The present data also show that a rapid internalization of the radiolabeled peptide occurs.

Visualization of gene expression of short and long forms of prolactin receptor in the rat.

PRL receptor gene expression was visualized in various tissues by in situ hybridization, using 35S-labeled probes unique to each form of receptor. Tissues were removed rapidly from adult male and female rats and placed in liquid nitrogen. Cryostat sections (10 microns) were prepared, fixed, pretreated, and dehydrated before incubation with the various probes. Hybridization was performed overnight, after which the slides were first exposed to autoradiographic film and then dipped in nuclear emulsion and exposed for 1-2 weeks. The specificity of the signal was studied by competition and using radiolabeled heterologous probes. Some tissues show no expression of either form of receptor mRNA, such as olfactory bulb and penis. Macroautoradiogram signals (optical density) were compared to a standard curve to observe the variation in mRNA expression, which was expressed in arbitrary units. Sex differences in the expression of PRL receptors were seen in a number of tissues, such as adrenal gland and pituitary. Expression of mRNAs specific to the long form of PRL receptor was predominant in adrenal gland, pituitary, thymus, spleen, skin, heart, and skeletal muscle, whereas the short form was expressed to a greater extent in kidney and lung. At the light microscopic level, the silver grains observed by epipolarization or light field were seen in the specific regions or cells that express PRL receptor mRNAs. In conclusion, the long form transcript predominates in most tissues, except kidney and lung. The advantage of in situ hybridization is that it allows the identification of specific regions or cells expressing mRNAs to be identified. The actions of PRL have not been identified in all tissues expressing PRL receptor transcripts, nor has any definitive correlation been made with the expression of short and long forms of PRL receptor and function.

Is atrial natriuretic peptide synthesized and internalized by gonadotrophs?

The present study was designed to determine whether atrial natriuretic peptide (ANP) could be both synthesized and internalized by rat anterior pituitary gonadotrophs. ANP synthesis was assessed by in situ hybridization of ultrathin frozen sections of anterior pituitary to a biotinylated 30-base oligonucleotide to rat ANP mRNA. As revealed by the immunogold technique, only gonadotrophs were labeled by the probe. At the subcellular level, ANP mRNA was observed at both the nuclear and cytoplasmic levels in gonadotrophs, and labeling of the latter compartment was quantitatively more intense. Internalization of ANP was investigated by an in vivo ultrastructural autoradiographic approach. Intravenous injection of [125I]ANP resulted in rapid labeling within 1 min of the plasma membrane, cytoplasmic matrix, secretory vesicle, and mitochondrial compartments and the Golgi apparatus; these compartments were labeled throughout the remainder of the time course studied (1-30 min). Peak labeling of the plasma membrane compartment was at 1 min and diminished from that point; labeling in the Golgi apparatus peaked 5 min postinjection, while in the other compartments labeling was fairly uniform over the time course. The lysosomal compartment was also radiolabeled; however, only 2 and 5 min after injection of [125I]ANP. The findings demonstrate that gonadotrophs can both synthesize and internalize extracellular ANP. These observations can be extended to suggest that ANP has both autocrine and paracrine actions in the anterior pituitary gland. Since the peptide neither stimulates nor antagonizes the release of any anterior pituitary hormone, these actions are probably unrelated to the adenohypophyseal secretory function.

Oxytocin receptor messenger ribonucleic acid: characterization, regulation, and cellular localization in the rat pituitary gland.

The hypothalamic neuropeptide oxytocin (OT) stimulates the release of several pituitary hormones, including ACTH, LH, and PRL. Although specific OT receptors have been identified in anterior pituitary membranes, the structure and cellular localization of these binding sites have not been elucidated. We previously cloned a rat OT receptor (OTR) gene and showed that its expression in rat uterus results in several transcripts ranging in size from 2.9-6.7 kilobases. In this study we show, by using Northern blot analysis, reverse transcriptase-polymerase chain reaction, and ultrastructural in situ hybridization that the same OTR gene is also expressed in the pituitary, where it gives rise to a 6.7- and a 4.8-kilobase messenger RNA. Ultrastructural in situ hybridization combined with immunogold labeling indicated that pituitary OTR gene expression is highly cell-specific and restricted to lactotrophs. In accordance with this finding, only the lactotroph-derived cell line MMQ expressed the OTR gene among several pituitary cell lines tested. Northern blot analysis, reverse transcriptase-polymerase chain reaction, and in situ hybridization analysis indicated a dramatic increase in pituitary OTR gene expression at the end of gestation and after estrogen treatment. Our results suggest that the OT effect on lactotrophs is direct, whereas OT actions on gonadotrophs and corticotrophs are either indirect or mediated via different receptors. Moreover, our findings imply that OT exerts its full potential as a physiological PRL-releasing factor only towards the end of gestation, and that therefore the role of OT as a hypothalamic PRL-releasing factor may so far have been underestimated.

Constitutive nuclear localization of Janus kinases 1 and 2.

Both GH and the GH receptor have been reported to undergo rapid nuclear translocation. Janus kinases (JAK) 1 and 2 have been implicated in GH receptor signaling, and both of these kinases are phosphorylated by GH stimulation. In this report, we have investigated the subcellular distribution of JAK1 and JAK2. Both JAK1 and JAK2 exhibit a nucleocytoplasmic distribution by immunocytochemistry in unstimulated serum deprived CHO cells stably transfected with rat GH receptor complementary DNA (cDNA). The nucleocytoplasmic localization of JAK2 was verified by immunogold electron microscopy in both rat liver hepatocytes and CHO cells stably transfected with rat GH receptor cDNA. Nucleocytoplasmic localization of JAK2 was also verified by transient tranfection of CHO cells with a Haemophilus influenzae haemagglutinin (HA) epitope tagged JAK2 expression plasmid and subsequent localization of HA immunoreactivity. Western blot analysis of purified nuclear extracts revealed the presence of immunoreactive JAK1 at 130 kDa and immunoreactive JAK2 at 128 kDa. No change in the nuclear content of JAK1 or JAK2 was observed upon ligand stimulation of GH receptor cDNA transfected cells with 100 nM human GH for 5, 10, 15, 30, or 60 min. GH stimulation caused, however, the appearance of tyrosine phosphorylated 42- and 44-kDa proteins as well as tyrosine phosphorylated JAK2 in the nucleus. The constitutive nuclear localization of the Janus Kinases is suggestive of a novel nuclear role for JAK family members, in addition to their described cytosolic function and presents an interesting challenge to the subcellular site of hormone action.