Three-dimensional analysis of the islet vasculature.

The pancreatic islets of Langerhans are highly vascularized structures scattered throughout the pancreas that contain a capillary network 5-10 times denser than that of the exocrine pancreas. A simple method for three-dimensional (3D) analysis of this intricate intraislet vasculature has been difficult because of the intrinsic opacity of the pancreas. We developed a whole-mount imaging technique that allows relatively easy visualization of the islet vasculature. In combination with confocal microscopy and the use of 3D imaging software, we were able to readily reconstruct the 3D architecture of an islet, allowing delineation of the islet volume, length of the intraislet vessels, and the number of vessel branch-points. This technique allows for straightforward 3D image analysis that may help toward understanding islet function.

Device based left ventricular shape change: validation of conductance technology in shape changed hearts.

We have reported that device based left ventricular (LV) shape change, accomplished by Myosplint, improved LV systolic function by three-dimensional echocardiography (3-D echo). However, evaluation of this device using the pressure-volume relationship is still important. This study was conducted to validate the use of conductance technology for this evaluation in shape-changed hearts. An ex vivo study using excised ovine hearts (n = 11) and an in vivo study using a canine pacing-induced heart failure model (n = 11) were performed. Three Myosplints were implanted. Before and after the shape changes, volumes measured by a conductance catheter were compared with volumes measured by the amount of saline in the ex vivo study or by 3-D echo in the in vivo study. The conductance volumes were linearly correlated with the saline volumes (r2 = 0.961+/-0.046; p < 0.0001) in the ex vivo study and with 3-D echo volumes (r2 = 0.757+/-0.220; p < 0.0001) in the in vivo study. The conductance volumes were linearly correlated with LV volumes even in the shape-changed hearts. This technology can be used to evaluate pressure-volume loops in the shape-changed hearts as long as the conductance volume is calibrated by a reliable method.

Localization and characterization of a short isoform of the corticotropin-releasing factor receptor type 2alpha (CRF(2)alpha-tr) in the rat brain.

We have recently isolated a cDNA encoding a short isoform of the corticotropin-releasing factor (CRF) receptor subtype, referred to as CRF(2)alpha-tr, from the rat amygdala. The present study determined the localization of the truncated receptor mRNA in the rat brain by in situ hybridization histochemistry. The results showed significant levels of hybridization in the lateral septum, central nucleus of the amygdala, cortico-amygdaloid nucleus, ventromedial nucleus of the hypothalamus (VMH), and frontal cortex. In the physiological study, antidepressive drugs increased the expression of CRF(2)alpha-tr mRNA and the total binding activity to CRF in the rat amygdala. These findings suggest that CRF(2)alpha-tr may regulate endogeneous CRF release in the amygdala.

Characterization of glucokinase regulatory protein-deficient mice.

The glucokinase regulatory protein (GKRP) inhibits glucokinase competitively with respect to glucose by forming a protein-protein complex with this enzyme. The physiological role of GKRP in controlling hepatic glucokinase activity was addressed using gene targeting to disrupt GKRP gene expression. Heterozygote and homozygote knockout mice have a substantial decrease in hepatic glucokinase expression and enzymatic activity as measured at saturating glucose concentrations when compared with wild-type mice, with no change in basal blood glucose levels. Interestingly, when assayed under conditions to promote the association between glucokinase and GKRP, liver glucokinase activity in wild-type and null mice displayed comparable glucose phosphorylation capacities at physiological glucose concentrations (5 mM). Thus, despite reduced hepatic glucokinase expression levels in the null mice, glucokinase activity in the liver homogenates was maintained at nearly normal levels due to the absence of the inhibitory effects of GKRP. However, following a glucose tolerance test, the homozygote knockout mice show impaired glucose clearance, indicating that they cannot recruit sufficient glucokinase due to the absence of a nuclear reserve. These data suggest both a regulatory and a stabilizing role for GKRP in maintaining adequate glucokinase in the liver. Furthermore, this study provides evidence for the important role GKRP plays in acutely regulating of hepatic glucose metabolism.

Nuclear import of hepatic glucokinase depends upon glucokinase regulatory protein, whereas export is due to a nuclear export signal sequence in glucokinase.

Hepatic glucokinase (GK) moves between the nucleus and cytoplasm in response to metabolic alterations. Here, using heterologous cell systems, we have found that at least two different mechanisms are involved in the intracellular movement of GK. In the absence of the GK regulatory protein (GKRP) GK resides only in the cytoplasm. However, in the presence of GKRP, GK moves to the nucleus and resides there in association with this protein until changes in the metabolic milieu prompt its release. GK does not contain a nuclear localization signal sequence and does not enter the nucleus in a GKRP-independent manner because cells treated with leptomycin B, a specific inhibitor of leucine-rich NES-dependent nuclear export, do not accumulate GK in the nucleus. Instead, entry of GK into the nucleus appears to occur via a piggy-back mechanism that involves binding to GKRP. Nuclear export of GK, which occurs after its release from GKRP, is due to a leucine-rich nuclear export signal within the protein ((300)ELVRLVLLKLV(310)). Thus, GKRP appears to function as both a nuclear chaperone and metabolic sensor and is a critical component of a hepatic GK translocation cycle for regulating the activity of this enzyme in response to metabolic alterations.

Cloning and characterization of a short variant of the corticotropin-releasing factor receptor subtype from rat amygdala.

We have identified and characterized a cDNA encoding a novel isoform of the corticotropin-releasing factor (CRF) receptor, referred to as CRF2alpha-tr, from the rat amygdala cDNA library. The nucleotide sequence of the cloned cDNA has a structure of an alternatively spliced form of the CRF2alpha receptor, which contains unspliced introns 6 and 7 in the message, and encodes a 236-amino-acid truncated protein that comprises three unique transmembrane domains. Northern blot analysis shows that the CRF2alpha-tr receptor is more strongly expressed in the rat amygdala, thalamus, and hypothalamus than the intact CRF2alpha receptor. Western blot analysis also reveals that the CRF2alpha-tr protein can be expressed in transfected COS-7 cells as well as CRF2alpha. Furthermore, this receptor binds rat/human CRF with almost the same low affinity (Kd = 12.7 nM) as the CRF2alpha and without accumulation of intracellular cAMP. Interestingly, it does not bind sauvagine or rat urocortin. These findings suggest that this truncated CRF receptor is the major isoform of CRF2alpha receptor mRNA transcripts in the amygdala and would mediate some functions of CRF pathways in the central nervous system.

Effects on blood pressure and exploratory behaviour of mice lacking angiotensin II type-2 receptor.

There are two major angiotensin II receptor isoforms, AT1 and AT2. AT1 mediates the well-known pressor and mitogenic effects of angiotensin II, but the signalling mechanism and physiological role of AT2 has not been established. Its abundant expression in fetal tissues and certain brain nuclei suggest possible roles in growth, development and neuronal functions. Here we report the unexpected finding that the targeted disruption of the mouse AT2 gene resulted in a significant increase in blood pressure and increased sensitivity to the pressor action of angiotensin II. Thus AT2 mediates a depressor effect and antagonizes the AT1-mediated pressor action of angiotensin II. In addition, disruption of the AT2 gene attenuated exploratory behaviour and lowered body temperature. Our results show that angiotensin II activates AT1 and AT2, which have mutually counteracting haemodynamic effects, and that AT2 regulates central nervous system functions, including behaviour.

Novel isoforms of mouse myelin basic protein predominantly expressed in embryonic stage.

Myelin basic protein (MBP), a major protein of myelin, is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages. These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of 18 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here.

Concentration of serum laminin and type IV collagen in liver diseases assayed by a sandwich enzyme-immunoassay using monoclonal antibodies.

Serum laminin (P1 fragment) and type IV collagen levels were determined in patients with hepatic disorders. The method was based on a sandwich enzyme-immunoassay using two monoclonal antibodies that recognize different epitopes of either laminin or type IV collagen molecule. Laminin and type IV collagen levels in the serum of patients with chronic hepatic disorders were higher as compared with those in healthy control subjects, with the increment of serum type IV collagen being far greater than that of laminin. Since type IV collagen and laminin are major basement membrane components, it is suggested that the higher levels of these peptides may reflect a so-called capillarization of the perisinusoidal wall encountered in hepatic fibrogenesis. The assay system used in this experiment is simple and sensitive and can be applied to clinical evaluation of hepatic fibrosis.

alpha 1(VIII)-collagen gene transcripts encode a short-chain collagen polypeptide and are expressed by various epithelial, endothelial and mesenchymal cells in newborn mouse tissues.

Type-VIII collagen is a major constituent of Descemet's membrane and contains two genetically distinct alpha chains, alpha 1(VIII) and alpha 2(VIII). We have previously cloned the human alpha 1(VIII) and alpha 2(VIII) genes and determined that they are located on chromosomes 3 and 1, respectively. Comparison of the alpha 1(VIII) and alpha 2(VIII) genes with the alpha 1(X)-collagen gene showed that the structure of the three genes and the sequence of their collagen polypeptides were strikingly similar. Therefore, we have grouped the three genes in a common subclass of collagens which we have named the short-chain collagens because of the relatively small size of their triple-helical domains. In the present study, we have isolated and characterized a mouse gene fragment encoding the entire mouse alpha 1(VIII)-collagen chain and determined the complete primary structure of the polypeptide chain. The size of mouse alpha 1(VIII)-collagen mRNA, as estimated by Northern-blot analysis, was 4.2 kb, compared with 2.8 kb previously reported for the corresponding rabbit mRNA. By cloning and sequencing of four overlapping cDNA, we demonstrate that this larger size of mouse alpha 1(VIII) mRNA is due to a larger 3' untranslated region in the mouse transcript. Using the gene fragment as a probe, we performed Northern-blot hybridization analysis of RNA prepared from newborn mice and demonstrated that alpha 1(VIII) collagen mRNA is expressed at high levels in the calvarium, eye and skin. In situ hybridization revealed that alpha 1(VIII) RNA is present in skin keratinocytes, corneal epithelial and endothelial cells, lens epithelial cells, as well as mesenchymal cells surrounding cartilage and calvarial bone and in the meninges surrounding the brain.

Differential localization of alternative spliced transcripts encoding inositol 1,4,5-trisphosphate receptors in mouse cerebellum and hippocampus: in situ hybridization study.

Expression of inositol 1,4,5-trisphosphate (InsP3) receptor subtypes was determined in mouse brain using oligonucleotide-specific in situ hybridization. All subtypes, except one that deletes 120 bp from the full-length InsP3 receptor cDNA, were expressed in cerebellar Purkinje cells. In hippocampus, various subtypes showed distinct expression patterns. These results suggest that regionally selective expression of InsP3 receptor subtypes may result in the generation of functionally distinct channels.

Developmental expression of myelin protein genes in dysmyelinating mutant mice: analysis by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry.

Gene expression for myelin proteolipid protein (PLP) and myelin basic protein (MBP) in the dysmyelinating mutant mice shiverer and jimpy was analyzed by nuclear run-off transcription assay, in situ hybridization, and immunohistochemistry. The level of PLP transcription in shiverer brains was lower than that in controls at postnatal day 18 but relatively higher at later stages. In spite of the considerable amount of hybridization with PLP cDNA, immunoreaction for PLP was greatly reduced in shiverer mice throughout their lives, probably owing to a defect in the assembly of PLP into myelin. Abnormal deposition of PLP in oligodendroglial cell bodies suggested that transport of PLP to myelin is delayed in shiverer brains. The number of oligodendrocytes expressing PLP mRNA was drastically reduced in jimpy mice. MBP mRNA in jimpy mice is localized preferentially in oligodendroglial cell bodies, a result suggesting that oligodendrocytes in jimpy are mostly the immature type. Although transcriptional activity of the MBP gene in jimpy was greatly reduced, a finding reflecting the decrease in the number of mature oligodendrocytes, that of the PLP gene remained high at early stages. The discrepancy of the two gene expressions is discussed relative to the role of PLP transcripts at early stages of myelination.

Myelin basic protein gene and the function of antisense RNA in its repression in myelin-deficient mutant mouse.

The myelin-deficient (mld) mouse is an autosomal recessive mutant characterized by hypomyelination of the CNS due to reduced expression of the myelin basic protein (MBP) gene. In the mld mutant, the MBP gene is duplicated in tandem. One gene is intact, but a large portion is inverted upstream of the other copy, and its transcription yields the antisense RNA. This antisense RNA was shown to be localized in the nucleus and to form an RNA:RNA duplex with sense RNA. These findings suggested that inhibition of transport from the nucleus or selective degradation of the duplex is responsible for the reduced expression of the MBP gene in the mld mutant. The mechanism of gene rearrangement at the MBP locus was also characterized. Cosmid clones encompassing whole MBP gene loci from control and mld genomic DNA libraries were isolated. The recombination points indicated that the duplication and inversion observed in mld occurred due to nonhomologous recombination.

Distribution of inositol 1,4,5-trisphosphate receptor mRNA in mouse tissues.

Northern blot analysis demonstrated that the concentration of inositol 1,4,5-triphosphate (IP3) receptor mRNA was greatest in cerebellar tissue. Moderate amounts of IP3 mRNA were present in brain tissue without cerebellum and tissue of the thymus, heart, lung, liver, spleen, kidney, and uterus. Small amounts of IP3 receptor mRNA were observed in skeletal muscle and testicular tissue. Regional distribution of IP3 mRNA in various tissues was also examined by in situ hybridization. A considerable amount of IP3 receptor mRNA was located in smooth muscle cells, such as those of the arteries, bronchioles, oviduct and uterus. In addition, secondary oocytes surrounded by Graafian follicles in the ovary were found to have large amounts of IP3 receptor mRNA. The present studies suggest a functional importance of the IP3 second-messenger system in these cell types.

Developmental profile and differential localization of mRNAs of myelin proteins (MBP and PLP) in oligodendrocytes in the brain and in culture.

We applied the in situ hybridization technique to localize the mRNAs for two myelin proteins: proteolipid protein (PLP) and myelin basic protein (MBP). In the oligodendrocyte in primary culture, PLP mRNA was located exclusively in the cell body throughout development. However, MBP mRNA was first located in the cell body and was rapidly distributed to the processes but not to the membranous sheets formed from the tips and lengths of the processes. Expression of PLP and MBP genes progressed in the caudo-cranial direction in the brain as far as we examined it in the tissue sections up to the 30th postnatal day: mRNAs of both genes were first detected in the pons and the medulla oblongata on the 3rd postnatal day and then in the cerebellum and the anterior part of the brain. PLP mRNA was located exclusively in the cell body throughout development. The number of PLP mRNA-positive cells reached a plateau in the posterior part of the brain on the 18th postnatal day, whereas it continued to increase in the anterior part of the brain by the 30th day. MBP mRNA was first expressed in the cell body, but later, it was found along the myelin sheath.

Subcortical crossed axonal projections to the caudate nucleus of the cat: a double-labelling study.

The anatomical organization of the interhemispheric projections of subcortical caudate nucleus (Cd) input neurons in the cat was assessed by the retrograde axonal transport of multiple marker substances. These double-labelling methods indicated the existence of two types of subcortical afferents to the Cd. (1) Uncrossed projections terminating in the ipsilateral Cd (but not the contralateral Cd) originated from the globus pallidus, thalamus, substantia nigra and midbrain raphe nuclei. The uncrossed axons provided the vast bulk of the subcortical Cd inputs. (2) Crossed projections terminating in the contralateral Cd (but not the ipsilateral Cd) originated from the substantia nigra and raphe nuclei. The crossed projections from the midbrain provided a very small Cd input compared to the crossed and divergent corticocaudate projections. Therefore, interhemispheric connections of the Cds may be subserved primarily by arrangement of corticocaudate projections. Monosynaptic interhemispheric subcortical inputs to the Cds are minor. Multisynaptic pathways could provide alternative, but less tightly coupled, interhemispheric linkages of the Cds.

Interhemispheric organization of corticocaudate projections in the cat: a retrograde double-labelling study.

The organization of interhemispheric corticocaudate projections in the cat was assessed by the retrograde axonal transport of different marker substances. These double-labelling methods indicated the existence of three types of neocortical efferents terminating in the caudate nucleus (Cd): (1) uncrossed axons projecting only to the ipsilateral Cd; (2) crossed axons projecting strictly to the contralateral Cd; and, (3) divergent axons projecting to both Cds. In all cases, the corticocaudate projections originated from small-to-medium sized pyramidal neurons situated in layers III, IV and V of the rostral neocortex. Interhemispheric inputs to the Cd may account, to some extent, for the functional coupling of the right and left basal ganglia.