The zebrafish Pax3 and Pax7 homologues are highly conserved, encode multiple isoforms and show dynamic segment-like expression in the developing brain

This study describes the isolation and characterization of zebrafish homologues of the mammalian Pax3 and Pax7 genes. The proteins encoded by both zebrafish genes are highly conserved (>83%) relative to the known mammalian sequences. Also the neural expression patterns during embryogenesis are very similar to the murine homologues. However, observed differences in neural crest and mesodermal expression relative to mammals could reflect some functional divergence in the development of these tissues. For the zebrafish Pax7 protein we report the first full-length amino acid sequences in vertebrates and show the existence of three additional isoforms which have truncations in the homeodomain and/or the C-terminal region. These novel variants provide evidence for additional isoform diversity of vertebrate Pax proteins.     

Genetic background effects on dental and other craniofacial abnormalities in homozygous small eye (Pax6Sey/Pax6Sey) mice

Blockade of the N-methyl D-aspartate (NMDA) receptor by the ion-channel-blocking drug aptiganel hydrochloride (CNS 1102, Cerestat) is neuroprotective in focal cerebral ischemia. Short intravenous infusions of up to 30 microg/kg have been well tolerated by healthy male volunteers. We undertook a randomized, double-blind, placebo-controlled study in 20 male volunteers to examine the safety, tolerability, and cardiovascular and psychomotor effects of a dosing paradigm similar to that envisaged for therapeutic use. Aptiganel HCl was infused over 4 h in total doses of 15, 32, 50, or 73 microg kg. Mean arterial pressure increased significantly with dose group (p < 0.01, analysis of covariance). Motor reaction time was related to maximal plasma concentration (r2 = 0.21, p < 0.001). Transient symptoms and signs of peripheral paresthesiae, light-headedness, and euphoria were seen at total doses of 32 microg/ kg. Higher doses were associated with motor retardation, perceptual disturbances, and hallucinations (one case). Clearance was 125 +/- 55 L/h, and volume of distribution was 537 +/- 1,261. Total doses of up to 32 microg/kg of aptiganel HCl infused over 4 h are well tolerated by healthy males. Aptiganel HCl causes elevation of blood pressure and is associated with central nervous system symptoms and signs similar to other noncompetitive NMDA antagonists.     

Regulation of plasminogen gene expression by interleukin-6

Plasmin, the primary fibrinolytic enzyme, has a broad substrate spectrum and participates in other biological processes dependent upon proteolytic activity. Consequently, plasmin activity is tightly regulated by plasminogen activators and protease inhibitors. In this study, we examined whether regulation of plasminogen gene expression also might provide a new mechanism for controlling this system. We examined the effects of recombinant human interleukin-6 (rhIL-6), a pleiotropic cytokine, on plasminogen mRNA expression in primary murine hepatocytes and Hep3B human hepatoma cells. In primary hepatocytes, rhIL-6 and hydrocortisone separately increased plasminogen mRNA expression, but hydrocortisone did not markedly enhance the response to rhIL-6. Hep3B hepatoma cells exhibited more modest responses to rhIL-6. We used the polymerase chain reaction to amplify a 1,067-bp fragment of the human plasminogen promoter/5' flanking region. This fragment was cloned upstream of a luciferase reporter gene. Hep3B cells transiently transfected with this construct provided approximately 100-fold higher luciferase activity compared to cells transfected with control plasmids, and luciferase activity was increased approximately 4.5-fold when these cells were treated with rhIL-6. Furthermore, mice injected with rhIL-6 exhibited increases in hepatic plasminogen mRNA. Circulating plasminogen levels were significantly higher in the mice injected with rhIL-6 compared to mice injected with saline. Mice injected with lipopolysaccharide (an inducer of IL-6 in vivo) also showed increased hepatic plasminogen mRNA. Thus, plasminogen gene expression can be modulated by rhIL-6, suggesting a new mechanism for regulating biological systems that use plasmin.     

Regulation of intestinal nutrient transport is impaired in aged mice

To determine the effect of age on the regulation of intestinal nutrient absorption, we fed young (7.6-mo-old) and aged (24.8-mo-old) C57BL mice diets designed to stimulate in vitro sugar or amino acid uptake in the isolated small intestine. In each age group, diet had no effect on feeding rates and body weights. D-Glucose and D-fructose uptakes by the small intestine each increased by about two times in young and 1.5 times in aged mice fed high carbohydrate diets as compared with those fed no carbohydrate. Adaptive increases in uptake by the aged group were not only reduced but also restricted to more proximal regions of the small intestine. In both age groups, diet-stimulated increases in D-glucose transport were accompanied by parallel increases in number of Na(+)-D-glucose cotransporters as estimated by specific phlorizin binding. Diet had no effect on transporter Kd for phlorizin, turnover rate of each transporter, mucosal mass or mucosal permeability. A high protein diet stimulated the uptake of L-aspartate and L-proline in young mice and of only L-aspartate in aged mice. Uptake of essential amino acids and of nonessential amino acids sharing transporters with essential ones were not regulated. Although aged mice possess adaptive mechanisms to diet that are similar to those in young mice, the effectiveness of these mechanisms may be impaired with age and may result in malabsorption symptoms so prevalent in the elderly.     

Regulation of muscarinic receptor expression and function in cultured cells and in knock-out mice

We have investigated the molecular and cellular basis for the regulation of expression and function of the muscarinic acetylcholine receptors. Treatment of cultured chick cardiac cells with the agonist carbachol results in decreased levels of mRNA encoding the m2 and m4 receptors. Treatment of chick embryos in ovo with carbachol results in decreased levels of mRNA encoding the potassium channel subunits GIRK1 and GIRK4 as well as the m2 receptor. There are thus multiple pathways for the regulation of mAChR responsiveness by long-term agonist exposure. Immunoblot, immunoprecipitation, and solution hybridization analyses have been used to quantitate the regulation of mAChR expression in chick retina during embryonic development. The m4 receptor is the predominant subtype expressed early in development, while the expression of the m3 and m2 receptors increases later in development. A cAMP-regulated luciferase reporter gene has been used to demonstrate that the m2 and m4 receptors have distinct specificities for coupling to G-protein subtypes to mediate inhibition of adenylyl cyclase. This system has also been used to demonstrate that beta-arrestin1 and beta-adrenergic receptor kinase-1 act synergistically to promote receptor desensitization. We have isolated the promoter region for the chick m2 receptor gene, identified regions of the promoter required to drive high level expression in cardiac and neural cells, and have identified a region which confers sensitivity of gene expression to neurally active cytokines. Finally, in order to determine the role of individual receptor subtypes in muscarinic-mediated responses in vivo, we have used the method of targeted gene disruption by homologous recombination to generate mice deficient in the m1 receptor.     

The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression

We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.     

Immobilization stress increases hepatic IL-6 expression in mice

Male Djungarian hamsters (Phodopus sungorus; 45 animals per group) were either sham-exposed or exposed to a sinusoidal magnetic field for 56 days (Experiment 1: 50 Hz, 450 microTesla peak; max. dB/dt = 140 mTesla s(-1); 24 hrs day(-1)). Except for day 7, no effects were observed with respect to body weights during exposure. However, testicular cell numbers were significantly increased by exposure (tetraploid (4C): p=0.022; diploid (2C): p=0.039). Rectangular magnetic fields (Experiment 2: 360 microTesla; max. dB/dt = 2.5 Tesla s(-1)) caused a significant (p<0.001) but transient suppressing effect on body weights. Significant increases were also observed in testicular cell numbers (4C: p=0.034; haploid (1C): p=0.014) and in serum melatonin (p=0.001). It is concluded that weak magnetic fields may affect reproductive and physiological functions in the mammalian species tested and that the degree of these effects depends upon the fields' gradients.     

Regulation of renal EGF receptor expression is normal in Denys-Drash syndrome

We evaluated 819 isolates referred to us as "Burkholderia cepacia" from cystic fibrosis (CF) clinics and research laboratories from five countries; 28 (3.4%) were not B. cepacia. A further 12 (1.5%) organisms appeared to be other Burkholderia species, but identification could not be confirmed by conventional means. The most prevalently misidentified organisms were Stenotrophomonas maltophilia, Alcaligenes xylosoxidans, and Comamonas acidovorans. Many of these organisms grew on oxidation-fermentation polymyxin-bacitracin-lactose (OFPBL) and Pseudomonas cepacia agars, selective media currently used for B. cepacia isolation. We developed a new medium, B. cepacia selective agar (BCSA), which is more enriched for the growth of B. cepacia yet which is more selective against other organisms than currently available selective agars. A total of 190 of 191 (99.5%) isolates of B. cepacia from patients with CF grew on BCSA without vancomycin, whereas 100% grew on OFPBL agar and 179 (94.2%) grew on P. cepacia agar. Of 189 other gram-negative and gram-positive organisms tested, 10 (5.3%) grew on BCSA without vancomycin. The addition of vancomycin to BCSA lowered the false positivity rate to 3.7% without further inhibition of B. cepacia. The false positivity rates for OFPBL and P. cepacia agars were 19.6 and 13.8%, respectively. Isolates of B. cepacia from CF patients grew most quickly on BCSA, with 201 of 205 (98.0%) being readily visible within 24 h, whereas 182 (88.8%) grew on OFPBL agar and 162 (79.0%) grew on P. cepacia agar within 24 h. We propose that the use of BCSA will allow investigators to overcome many of the difficulties associated with the identification of B. cepacia and should be considered for use as a primary isolation agar for specimens from patients with CF.     

The COP9 complex is conserved between plants and mammals and is related to the 26S proteasome regulatory complex

The COP9 complex, genetically identified in Arabidopsis as a repressor of photomorphogenesis, is composed of multiple subunits including COP9, FUS6 (also known as COP11) and the Arabidopsis JAB1 homolog 1 (AJH1) ([1-3]; unpublished observations). We have previously demonstrated the existence of the mammalian counterpart of the COP9 complex and purified the complex by conventional biochemical and immunoaffinity procedures [4]. Here, we report the molecular identities of all eight subunits of the mammalian COP9 complex. We show that the COP9 complex is highly conserved between mammals and higher plants, and probably among most multicellular eukaryotes. It is not present in the single-cell eukaryote Saccharomyces cerevisiae, however. All of the subunits of the COP9 complex contain structural features that are also present in the components of the proteasome regulatory complex and the translation initiation factor eIF3 complex. Six subunits of the COP9 complex have overall similarity with six distinct non-ATPase regulatory subunits of the 26S proteasome, suggesting that the COP9 complex and the proteasome regulatory complex are closely related in their evolutionary origin. Subunits of the COP9 complex include regulators of the Jun N-terminal kinase (JNK) and c-Jun, a nuclear hormone receptor binding protein and a cell-cycle regulator. This suggests that the COP9 complex is an important cellular regulator modulating multiple signaling pathways.     

Definition of family of coronin-related proteins conserved between humans and mice: close genetic linkage between coronin-2 and CD45-associated protein

Cell adhesion and signal transduction are coordinated processes that may be linked through regulatory elements such as actin-binding proteins. One such protein that may fulfill this role is coronin. In Dictyostelium discoideum, coronin is involved in cellular processes such as mitosis, cell motility, and phagocytosis. In addition, a human coronin, p57, has been described which interacts with the p47 component of phox proteins and may be involved in the formation of phagocytic vacuoles. Here, we describe a family of four mouse proteins which share 38% identity with Dictyostelium coronin and thus are designated coronin-1, -2, -3, and -4. The gene for coronin-2 is localized to mouse chromosome 19, 5' of the gene for CD45-associated protein. All the coronin proteins contain five highly conserved WD domains. However, their carboxyl regions are quite distinct. Three of the four proteins are ubiquitously expressed, whereas coronin-1, the mouse ortholog of p57, demonstrates expression restricted to hematopoietic cells. Comparison of expressed sequence tag cDNAs indicates that coronin-1, -2, -3, and -4 are highly conserved between mice and humans.     

Alternative splicing of Pax6 in bovine eye and evolutionary conservation of intron sequences

There are now six recognized neuropeptide Y (NPY) receptor subtypes (Y1-Y4 and two recently cloned distinct receptors labeled Y5), of which Y1 and one of the Y5's have been suggested could mediate the effect of NPY on feeding. The fragments NPY(2-36) and NPY(3-36), which bind Y1 only poorly, were injected intracerebroventricularly (icv) and found to have similar dose-response relationships to NPY in the stimulation of feeding. However NPY (13-36), which stimulates both Y2 and Y5, caused no increase in food intake, even at high doses. Maximal stimulation with the classical Y1 agonist [Pro34]-NPY produced only 50% of the maximum effect of NPY itself despite fully inhibiting adenylyl cyclase activity in vitro in a Y1 system. The novel fragment [Pro34]-NPY(3-36) is as effective at stimulating food intake as the classical Y1 analogue [Pro34]-NPY but bound to the Y1 receptor with only 1/20th of the affinity of NPY and failed to inhibit adenylyl cyclase through this receptor. [Pro34]-NPY(3-36) is therefore a relatively appetite-selective ligand. Coadministration of high dose NPY(13-36) and [Pro34]NPY did not enhance feeding compared with [Pro34]-NPY alone. In addition, the NPY Y1 receptor antagonist BIBP-3226, which does not bind Y2, Y4, or Y5 receptors, significantly reduced NPY induced feeding. These results indicate that the feeding effect of icv NPY involves a novel receptor and that it is functionally distinct from the recognized receptor subtypes.     

Localization of metallothionein-I and -III expression in the CNS of transgenic mice with astrocyte-targeted expression of interleukin 6

The effect of interleukin-6 (IL-6) on metallothionein-I (MT-I) and MT-III expression in the brain has been studied in transgenic mice expressing IL-6 under the regulatory control of the glial fibrillary acidic protein gene promoter (GFAP-IL6 mice), which develop chronic progressive neurodegenerative disease. In situ hybridization analysis revealed that GFAP-IL6 (G16-low expressor line, and G36-high expressor line) mice had strongly increased MT-I mRNA levels in the cerebellum (Purkinje and granular layers of the cerebellar cortex and basal nuclei) and, to a lesser degree, in thalamus (only G36 line) and hypothalamus, whereas no significant alterations were observed in other brain areas studied. Microautoradiography and immunocytochemistry studies suggest that the MT-I expression is predominantly localized to astrocytes throughout the cerebrum and especially in Bergman glia in the cerebellum. However, a significant expression was also observed in microglia of the GFAP-IL6 mice. MT-III expression was significantly increased in the Purkinje cell layer and basal nuclei of the cerebellum, which was confirmed by Northern blot analysis of poly(A)+ mRNA and by ELISA of the MT-III protein. In contrast, in the G36 but not G16 mice, transgene expression of IL-6 was associated with significantly decreased MT-III RNA levels in the dentate gyrus and CA3 pyramidal neuron layer of the hippocampus and, in both G36 and G16 mice, in the occipital but not frontal cortex and in ependymal cells. Thus, both the widely expressed MT-I isoform and the CNS specific MT-III isoform are significantly affected in a MT isoform- and CNS area-specific manner in the GFAP-IL6 mice, a chronic model of brain damage.     

IL-6 expression by oral fibroblasts is regulated by androgen

The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'-phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme.