Expression and regulation of the LIM-class homeobox gene rlim-1 in neuronal progenitors of the rat cerebellum.

To investigate LIM gene function in the rat cerebellar system, we analyzed expression and regulation of the rat homologue of frog Xlim-1 (rlim-1) in vivo and in cultured cells. In developing cerebellum, peak levels of rlim-1 mRNA at postnatal day 8 (p8) are coincident with the peak period of granule cell proliferation. Analysis of rlim-1 protein with a specific antibody showed that expression was also maximal at p8. In situ hybridization showed that at p8 rlim-1 mRNA was expressed in Purkinje and granule cells. Both the proliferative and the premigratory granule cells in the external germinal zone displayed high levels of rlim-1 mRNA expression. Immunocytochemical staining demonstrated that at p8 rlim-1 protein was also present in proliferative and premigratory granule cells. In adult cerebellum (p30), rlim-1 mRNA and protein expression in granule cells was strongly attenuated. The down-regulation of rlim-1 mRNA occurred in granule cells just after the time of final division, coinciding with the onset of their migration. rlim-1 protein was detected in migratory granule neurons. The developmental decrease in rlim-1 mRNA and protein found in vivo was reproduced in pure cerebellar granule cell cultures. In these cultures, granule neurons were postmitotic 1 day after plating but still displayed high levels of rlim-1 protein expression up to 3 days in vitro. Our findings indicate that 1) rlim-1 is likely to act in concert with other genes to specify granule cell fate, 2) rlim-1 expression in granule neurons is regulated autonomously, and 3) rlim-1 protein may also play an important role in granule neuron differentiation and survival. Published 2001 Wiley-Liss, Inc.

Immunological identification and localization of yeast aspartic protease 3-like prohormone-processing enzymes in mammalian brain and pituitary.

The novel aspartic proteases, yeast aspartic protease 3 and the mammalian POMC-converting enzyme (PCE), can process prohormones at specific basic residue cleavage sites. We show that an antibody against yeast aspartic protease 3 (YAP3p) cross-reacted with purified bovine PCE on Western blot, indicating structural homology between these two enzymes, but not with other aspartic proteases, such as renin or cathepsin D. A PCE-sized anti-YAP3p-immunoreactive band was detected on Western blots of bovine intermediate lobe where PCE activity has been found. YAP3p antiserum also cross-reacted with a protein of approximately 90 kDa from mouse hypothalamus and anterior pituitary, and bovine anterior pituitary secretory granules. Distribution studies showed the presence of anti-YAP3p-immunopositive cells in bovine pituitary and peptide-rich brain regions, including the mouse arcuate nucleus and hippocampus and the rat supraoptic nucleus, paraventricular nucleus, cortex, striatum, and reticular nucleus. In the bovine intermediate pituitary, a subpopulation of cells was intensely stained with the YAP3p antiserum, and in combination with in situ hybridization, these cells were shown to contain POMC messenger RNA (mRNA). Only a subpopulation of cells was immunopositive for anti-YAP3p in bovine anterior pituitary, and most of these cells were identified by double immunostaining with ACTH antiserum as corticotrophs. In situ hybridization in combination with immunocytochemistry provided evidence for the localization of arginine vasopressin mRNA in YAP3p-immunopositive neurons in the rat supraoptic nucleus, whereas cholecystokinin mRNA was detected in YAP3p-immunopositive cells in the rat cortex and hippocampus. These results support the hypothesis that YAP3p-like aspartic proteases, including PCE, play a role in prohormone processing in endocrine/neuroendocrine cells in vivo.

Differential coexpression of genes encoding prothyrotropin-releasing hormone (pro-TRH) and prohormone convertases (PC1 and PC2) in rat brain neurons: implications for differential processing of pro-TRH.

Pro-TRH is cleaved at paired basic residues to yield five copies of TRH and cryptic peptides. Recent studies have shown that the prohormone convertases, PC1 and PC2, can process pro-TRH correctly. To determine whether these two enzymes could play a role in pro-TRH processing in vivo, the regional and cellular colocalization of pro-TRH messenger RNA (mRNA) with the mRNAs encoding the prohormone convertases PC1 and PC2 was examined in rat brain, using in situ hybridization histochemistry. Differential regional distribution of pro-TRH mRNA with PC1 and/or PC2 mRNA was found in several brain regions. For example, in the olfactory regions, there was coexpression of pro-TRH mRNA in the glomerular layer with PC2 mRNA, but not PC1 mRNA, whereas in the tenia tecta, coexpression of pro-TRH and PC1 mRNAs was evident, but PC2 mRNA was absent. Pro-TRH mRNA in the paraventricular nucleus was coexpressed with both PC1 and PC2 mRNAs, whereas the basal lateral hypothalamus showed coexistence of pro-TRH mRNA with PC2 mRNA, but not PC1 mRNA. Interestingly, pro-TRH was expressed in the thalamic reticular nucleus, but neither PC1 nor PC2 was detectable in this region. Cellular colocalization studies using double in situ hybridization histochemistry showed the presence of PC2 mRNA in the pro-TRH neurons of the olfactory glomerular layer and basal lateral hypothalamus, and PC1 mRNA in the pro-TRH neurons in the paraventricular nucleus. These results suggest that PC1 and PC2 are enzyme candidates for the processing of pro-TRH in vivo. Moreover, the differential distribution of PC1 and PC2 mRNAs with pro-TRH mRNA may be responsible for the differential processing of this prohormone in the central nervous system. The absence of PC1 and PC2 mRNAs in certain TRH neurons raises the possibility that prohormone convertases other than PC1 and PC2 may be involved in the processing of brain pro-TRH.

Localization of vasopressin mRNA and immunoreactivity in pituicytes of pituitary stalk-transected rats after osmotic stimulation.

The presence of [arginine] vasopressin (AVP) mRNA and AVP immunoreactivity in pituicytes of the neural lobe (NL) of intact and pituitary stalk-transected rats, with and without osmotic stimulation, was examined. AVP mRNA was analyzed by Northern blotting, as well as by in situ hybridization in combination with immunocytochemistry using anti-glial fibrillary acidic protein (GFAP) as a marker for pituicytes. In intact rats, a poly(A) tail-truncated 0.62-kb AVP mRNA was detected in the NL and was found to increase 10-fold with 7 days of continuous salt loading. Morphological analysis of the NL of 7-day salt-loaded rats revealed the presence of AVP mRNA in a significant number of GFAP-positive pituicytes in the NL and in areas most probably containing nerve fibers. Eight days after pituitary stalk transection the NL AVP mRNA diminished in animals given water to drink, whereas in those given 2% saline for 18 h followed by 6 h of water, a treatment repeated on 6 successive days beginning 2 days after surgery, the 0.62-kb AVP mRNA was present. The AVP mRNA in the pituitary stalk-transected, salt-loaded rats showed an exclusive cellular distribution in the NL, indicative of localization in pituicytes. Immunoelectron microscopy showed the presence of AVP immunoreactivity in a subpopulation of pituicytes 7 and 10 days after pituitary stalk transection in salt-loaded animals, when almost all AVP fibers had disappeared from the NL. These data show that a subset of pituicytes in the NL is activated to synthesize AVP mRNA and AVP in response to osmotic stimulation.

Comparison of the molecular forms of the Kex2/subtilisin-like serine proteases SPC2, SPC3, and furin in neuroendocrine secretory vesicles reveals differences in carboxyl-terminus truncation and membrane association.

The molecular forms and membrane association of SPC2, SPC3, and furin were investigated in neuroendocrine secretory vesicles from the anterior, intermediate, and neural lobes of bovine pituitary and bovine adrenal medulla. The major immunoreactive form of SPC2 was the full-length enzyme with a molecular mass of 64 kDa. The major immunoreactive form of SPC3 was truncated at the carboxyl terminus and had a molecular mass of 64 kDa. Full-length 86-kDa SPC3 with an intact carboxyl terminus was found only in bovine chromaffin granules. Immunoreactive furin was also detected in secretory vesicles. The molecular masses of 80 and 76 kDa were consistent with carboxyl-terminal truncation of furin to remove the transmembrane domain. All three enzymes were distributed between the soluble and membrane fractions of secretory vesicles although the degree of membrane association was tissue specific and, in the case of SPC3, dependent on the molecular form of the enzyme. Significant amounts of membrane-associated and soluble forms of SPC2, SPC3, and furin were found in pituitary secretory vesicles, whereas the majority of the immunoreactivity in chromaffin granules was membrane associated. More detailed analyses of chromaffin granule membranes revealed that 86-kDa SPC3 was more tightly associated with the membrane fraction than the carboxyl terminus-truncated 64-kDa form.

The Neuro-2a neuroblastoma cell line expresses [Met]-enkephalin and vasopressin mRNA and peptide.

Mouse neuroblastoma Neuro-2a cells were examined for the expression of pro-enkephalin mRNA, protein, and Met-enkephalin ([Met]-Enk) peptide. Reverse transcriptase/polymerase chain reaction (RT/PCR) and in situ hybridization demonstrated the presence of pro-enkephalin mRNA in these cells. Immunocytochemistry using an antibody which recognizes pro-enkephalin and high pressure liquid chromatography (HPLC) followed by radioimmunoassay indicated that pro-enkephalin was synthesized in these cells and processed to yield the bioactive pentapeptide, [Met]-Enk. Furthermore, release studies showed that the [Met]-Enk was secreted from these cells with high K+ stimulation. Using double labeling, in situ hybridization combined with immunocytochemistry, we demonstrated that prohormone convertase 2 (PC2) mRNA is colocalized with pro-enkephalin in the same Neuro-2a cells, suggesting that this enzyme may be responsible for processing this precursor. we also showed the presence of vasopressin mRNA and arginine-vasopressin peptide in these cells using in situ hybridization and immunocytochemistry, respectively. Thus, the Neuro-2a cells are a multiple neuropeptide-producing cell line and an excellent model for studying the mechanisms involved in the synthesis, intracellular targeting and processing of endogenous pro-enkephalin and pro-vasopressin, as well as other transfected neuropeptide precursors.

Expression of pit-1 messenger ribonucleic acid and protein in the human placenta.

It is well established that the human placenta produces a wide range of hormones similar to those secreted by the pituitary and hypothalamus. However, the physiological role and regulation of placental hormone synthesis and release are still largely unknown. GH (GH-N) is expressed in the pituitary, where it requires the tissue-specific transcription factor Pit-1. Chorionic somatomammotropin A (CS-A) and CS-B as well as the placental GH variant (GH-V), which also belong to the GH gene family and are located in the same chromosomal cluster, are expressed in the placental syncytiotrophoblast. The presence of Pit-1-binding sites in the CS-A and GH-V promoter regions predicts that Pit-1 may be expressed in the placenta. However, this has not yet been demonstrated. To examine possible similarities in the regulation of these genes in the pituitary and placenta, we studied the expression of pit-1 messenger ribonucleic acid (mRNA) in the human placenta, transformed human placental cells, and the JEG-3 choriocarcinoma cell line. Polymerase chain reaction (PCR) products of the expected size were amplified from first and third trimester placentas, transformed placental cells, and JEG-3 complementary DNA by reverse transcription-PCR. The pit-1-specific sequence was confirmed by restriction endonuclease digestion, Southern hybridization, and DNA sequencing. Human pituitary tissue was used as a positive control; no PCR product was obtained from hippocampus (negative control). In situ hybridization of placental tissue sections revealed the presence of pit-1 mRNA in first and third trimester syncytiotrophoblast. Pit-1 protein was localized by immunohistochemistry with the same tissue distribution and a nuclear localization pattern. These data demonstrate expression of pit-1 mRNA and Pit-1 protein in the human placenta, thus questioning its role as a pituitary-specific regulator of GH-N gene transcription. The expression of Pit-1 in the placenta, together with its previously demonstrated capability to bind to and activate the CS-A and the GH-V promoters, suggests that it may play a role in the regulation of hormones belonging to the GH gene family in both pituitary and placenta.

Secretion of yeast aspartic protease 3 is regulated by its carboxy-terminal tail: characterization of secreted YAP3p.

Yeast aspartic protease 3 (YAP3p), a basic-residue specific proprotein processing enzyme, was shown to be a membrane-associated protease. The membrane association of YAP3p was demonstrated to be through a glycophosphatidylinositol anchor situated in the carboxy terminus of the enzyme. Carboxy-terminal truncation of YAP3p by 37 amino acids resulted in secretion of YAP3p into the growth medium. Western blot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed two secreted forms of YAP3p with apparent molecular masses of approximately 180 and approximately 90 kDa. YAP3p has an isoelectric point of approximately 4.5 as determined by isoelectric focusing gel electrophoresis. Treatment of YAP3p with endoglycosidase H reduced the size of both forms of the protein to approximately 65 kDa, consistent with the presence of 10 potential N-linked glycosylation sites in the deduced amino acid sequence of this protein. Removal of the N-linked sugars did not affect the enzymatic activity of YAP3p. Analysis of the effect of temperature on the stability and the rate of enzymatic activity of YAP3p showed that the enzyme retained 100% of its activity when incubated for 1 h at 37 degrees C, while incubation at 50 degrees C for 1 h resulted in approximately 80% loss of activity. The dependence of activity on temperature demonstrated a calculated Q10 of 1.95.

Frog prohormone convertase PC2 mRNA has a mammalian-like expression pattern in the central nervous system and is colocalized with a subset of thyrotropin-releasing hormone-expressing neurons.

The prohormone convertase (PC2) is expressed in the mammalian central nervous system (CNS) and has been shown to play an important role in the processing of certain neuropeptide precursors and prohormones at paired basic residues. Amphibian PC2 cDNA was recently cloned for the frog Xenopus laevis, and both its sequence and its pituitary expression pattern were shown to be very similar to those of mammalian PC2. To investigate further the function of PC2 in the vertebrate CNS, we used in situ hybridization histochemistry to localize the distribution of cells expressing PC2 mRNA in the frog brain and the spinal cord. The distribution of PC2-expressing cells was also compared with that of cells expressing thyrotropin-releasing hormone (TRH) mRNA or peptide. PC2-expressing cells were detected in specific nuclei that were widely distributed in the frog CNS. In forebrain, telencephalic PC2 mRNA was found in the olfactory bulb, pallium, striatum, amygdala, and septum, and diencephalic PC2 mRNA was seen in the preoptic area, thalamus, and hypothalamus. More posteriorly, PC2 cells were localized to midbrain tegmentum, the torus semicircularis, and the optic tectum, as well as the cerebellum, brainstem, and spinal cord. Despite this wide distribution steady-state levels of PC2 mRNA were clearly different in various brain nuclei. Regions with higher levels showed good correspondence to areas shown by others in frog to contain large numbers of neuropeptide-expressing cells, including TRH cells. On the other hand, not all brain areas with high levels of TRH mRNA had high levels of PC2 mRNA. Localization studies combining in situ hybridization and immunocytochemistry showed that, at least in optic tectum and brainstem, PC2 mRNA and pro-TRH peptide coexist. These findings suggest that pro-TRH is processed by PC2 in some, but possibly not all, brain regions. Thus, different converting enzymes may be involved in pro-TRH processing in different brain regions.

Fetal development of delta-sleep-inducing-peptide-like immunoreactivity in hypothalamus of guinea pig with special regard to the prenatal colocalization with gonadotropin-releasing-hormone-like immunoreactivity.

Delta-sleep-inducing peptide (DSIP) colocalizes within gonadotropin-releasing-hormone (GnRH)-containing neurons in adult hypothalamus and could play a role in the regulation of hypothalamic-pituitary axis in adults. To support the possibility that DSIP also participates in fetal neuroendocrine events and to demonstrate the ontogenic evidence of coexisting neuropeptides, we have performed a detailed immunocytochemical study of DSIP- and GnRH-immunoreactivity in fetal hypothalamus of guinea pig. Using indirect immunofluorescent and sequential double-immunolabeling (elution-restaining) techniques, the results indicated that DSIP immunoreactivity was initially detected at the 38th day of gestation. In contrast to the first appearance of GnRH immunoreactivity at day 28, therefore, a 10-day delay was found. Such a delay remains as yet unexplained. From its first occurrence, DSIP immunoreactivity was always labeled with GnRH, whereas some of GnRH-immunoreactive structures did not display a DSIP immunoreactivity. But with the growth of fetus, especially before and after birth, a complete overlap between DSIP and GnRH immunoreactivity was observed throughout various regions of hypothalamus. Attention was also focused on prenatal morphological development of DSIP/GnRH- and GnRH-immunolabeled neurons. Initially, labeled neurons were visualized as uni- or bipolar types. Thereafter, their smooth and irregular subtypes could be distinguished. During later fetal age, relatively mature features were evident such as the increase of multipolar and irregularly labeled neurons. Taken together, these data provide, for the first time, anatomical evidence that DSIP exists in fetal hypothalamus and that, like GnRH, it could regulate the hypothalamic-pituitary axis during ontogenesis.

The distribution and development of delta sleep-inducing Peptide-like immunoreactivity in postnatal and prepubertal Guinea-pig brain.

The distribution and development of delta sleep-inducing peptide (DSIP) in the guinea-pig brain were studied in 2- to 60-day-old animals by using the indirect immunofluorescence method. DSIP-immunoreactive perikarya were observed in the olfactory bulb and tubercle, diagonal band of Broca, septum, preoptic area, anterior and lateral hypothalamus, arcuate nucleus and hippocampus. In addition to the densest innervation of the median eminence, DSIP-immunoreactive fibres were widely localized from forebrain to mesencephalon. The field of immunoreactive fibre endings appeared to be in close association with either the blood vessels of brain, ventricles, subarachnoid space or immunolabelled perikarya. Furthermore, throughout development the topographic distribution pattern of immunolabelled neuronal elements seemed to be similar. However, a generalized increase in number, immunofluorescence intensity and varicosities of DSIP fibres was displayed with the growth. The present results provide an anatomical basis for understanding multiple actions of DSIP in the central nervous system and future research for DSIP on development.

Light and electron microscopic immunocytochemical evidence that delta sleep-inducing peptide and gonadotropin-releasing hormone are coexpressed in the same nerve structures in the guinea pig median eminence.

The relationships between delta sleep-inducing peptide (DSIP) and GnRH immunoreactivity within the guinea pig median eminence are investigated by light and electron microscopic immunocytochemistry. Indirect immunofluorescence and elution-restaining experiments show that at the light microscopic level the distribution patterns of DSIP and GnRH immunoreactivity are indistinguishable. This suggested the possible coexistence of both immunoreactivities within the same fibers and neurosecretory endings. At the electron microscopic level, a preembedding double-immunolabeling technique using both indirect immunoperoxidase and immunogold methods, clearly indicate that DSIP and GnRH immunoreactivity are frequently colocalized within single secretory granules. In addition DSIP/GnRH immunoreactive nerve endings were also observed often in close proximity to tanycyte elements. Taken together, the present results provide for the first time ultrastructural evidence for the presence of DSIP immunoreactivity and demonstrate that DSIP and GnRH immunoreactivities may be coexpressed within the same neuronal elements in the median eminence.

Doppler ultrasonic diagnosis for deep venous thrombosis of lower limbs.

Seventy-one patients with 80 lower limbs clinically suspected of deep venous thrombosis (DVT) were investigated by both Doppler ultrasound and venography. Sixty-seven lower limbs demonstrated by venography to have DVT, in 5 the thrombus was confined to the calf, and all were detected by the Doppler ultrasound. In 62 limbs thrombus extended into the popliteal and more proximal veins, the Doppler ultrasound detected 61 of them, a sensitivity of 98%. Venography showed a deep venous system without any evidence of thrombosis in 13, the Doppler findings were normal in all of the 13 lower limbs, a specificity of 100%. We found that the examination of the popliteal vein with patient in a semi-lateral prone position was important in detecting the presence of thrombosis in popliteal and more proximal vein.

Recognition and management of Budd-Chiari syndrome: report of one hundred cases.

Budd-Chiari syndrome is an unusual form of portal hypertension caused by occlusion of the hepatic venous outflow, and it is often confused with portal hypertension caused by liver cirrhosis. Its prognosis is poor, and optimal therapy remains to be established. This is a report of 100 confirmed cases of this syndrome treated from December 1982 to March 1988 at two vascular centers in China. Sixty-two male and 38 female patients, 15 to 62 years of age (mean age, 32.6 years) were treated. Seventy-six patients had intractable ascites, 56 had esophageal varices, and 22 had upper gastrointestinal bleeding. There were 37 cases of membranous obstruction, 57 cases of occlusion of the inferior vena cava above the confluence of the hepatic veins, and 6 cases of occlusion of the hepatic veins. Eighty-one patients were operated on. Operative mortality rate was 8.6% (7/81). Follow-up from 2 to 66 months revealed that 58 of the patients operated on (72%) had good results, whereas 11 of 19 (58%) patients treated nonoperatively died within 2 months after admission. On the basis of these data we conclude that the operative procedure must be tailored to the cause and underlying pathologic characteristics. Mesoatrial shunting is the operation of choice for patients with occlusion of the retrohepatic inferior vena cava and hepatic veins, and inferior vena cava--atrial shunting is the operation of choice for patients with occlusion of the inferior vena cava and patency of the hepatic veins. Membranotomy is used for patients with inferior vena cava webbing, and mesocaval shunting is used for patients with intrahepatic venous occlusion only.(ABSTRACT TRUNCATED AT 250 WORDS)