Sequencing of the rat light neurofilament promoter reveals differences in methylation between expressing and non-expressing cell lines, but not tissues.

The DNA methylation pattern of the promoter (pNF-L) region of the rat light-neurofilament-encoding gene (NF-L), a neuron-specific gene, was assessed in NF-L expressing and non-expressing cell lines and tissues by genomic sequencing using PCR amplification of bisulfite-modified DNA. We analysed twenty-five potential CpG methylation sites between nucleotide (nt) positions -311 and +103 of pNF-L, containing Sp1- and AP-2-binding sites, a CGCCCCCGC box and a cAMP-responsive element. Six out of 25 possible CpG methylation sites are within these elements. The pNF-L promoter was unmethylated in NF-L-expressing rat brain, as well as in liver not expressing NF-L. In NF-L-expressing PC12 cells, the promoter was unmethylated, whereas in non-expressing glioma C6 cells intensive methylation occurred. A cluster of methylated CpG dinucleotides spanned the region from nt -176 to -67 bp. Thus, methylation of this promoter region could play a role in silencing NF-L in the glioma cell line in vitro, but not in liver tissue in vivo. In a non-CpG sequence, in the CpApG trinucleotide at nt position -114, cytosine was found to be partially methylated. It is thus possible to describe the methylation state of each cytosine present in the area of genomic DNA of interest.

Different sensitivities of human and rat rho(1) GABA receptors to extracellular pH.

We have examined the sensitivity of human and rat homo-oligomeric rho(1) GABA receptors to variations in extracellular pH (pH(o)) using the whole-cell patch clamp technique. The GABA-induced conductance mediated by the rat rho(1) receptor (rho(1)-R) decreased with a decrease in pH(o) between 9.0 to 5.4. Below pH(o) 7.4 the effect of protons on the GABA-induced conductance was apparently competitive, but above pH(o) 7.4 the inhibitory effect of extracellular protons was almost independent on the GABA concentration. Titration of the GABA-induced conductance at 3 microM GABA revealed two protonation sites on rat rho(1)-R with pKa 6.4 and pKa 8.2. At 10 microM GABA the low pKa (6.4) was shifted to a clearly lower value (5.6), but the high pKa was only slightly decreased (from 8.2 to 7.9). Zn(2+) ions were capable of relieving the proton inhibition at low pH(o) indicating that Zn(2+) interacts with the low pKa site. Unlike the rat rho(1)-R, the human rho(1)-R was sensitive only to changes in pH(o) at acidic levels. Proton inhibition of human rho(1)-R was apparently competitive, as observed on rat-rho(1) at acidic pH(o). Titration of the human rho(1)-R gave a single H(+) binding site with a pKa of 6.3, similar to the value for the low pKa on rat rho(1)-R. The pKa value of human rho(1)-R was not dependent on the GABA concentration. A chimeric receptor, consisting of the N-terminal part of the rat rho(1)-R and C-terminal part of the human rho(1)-R, displayed pH(o) sensitivity similar to that observed for rat rho(1)-R. This indicates that the high pKa of rat rho(1)-R is attributable to the 11 amino acid differences between the rat and human rho(1)-R extracellular domains.

The messenger RNAs for both glial cell line-derived neurotrophic factor receptors, c-ret and GDNFRalpha, are induced in the rat brain in response to kainate-induced excitation.

Glial cell line-derived neurotrophic factor (GDNF) has two receptors, receptor-tyrosine kinase c-ret and glycosylphosphatidylinositol-linked cell surface receptor GDNFRalpha. Kainate-induced seizures, a widely studied model of neuronal plasticity and human epilepsy, have been shown to increase gene expression of several trophic factors, including GDNF, in the rat hippocampus. Here we show that systemic kainate-induced excitation leads to a transient increase of both c-ret and GDNFRalpha messenger RNAs in the rat brain. Northern analysis demonstrated that, in the hippocampus, the maximal 2.5-fold increase of c-ret and four-fold increase of GDNFRalpha messenger RNAs was observed after 12 h of kainate injection, in contrast to GDNF messenger RNA, which reaches its maximum in 4-6 h. The blocking of de novo protein synthesis by cycloheximide inhibited the induction of GDNF receptors by kainate, whereas blocking of the N-methyl-D-aspartate-type glutamate receptors by the antagonist dizocilpine maleate did not significantly alter the response. Thus, GDNF receptor messenger RNA increase by kainate depends on protein synthesis, but is not mediated by the N-methyl-D-aspartate receptor. GDNFRalpha and c-ret show distinct, but partially overlapping, patterns of expression in the brain after kainate treatment. GDNFRalpha messenger RNA was prominently induced in the dentate gyrus of the rat hippocampus, less in the habenular and reticular thalamic nuclei and cerebral cortex as revealed by in situ hybridization. C-ret transcripts were induced in the hilus of the hippocampus, several thalamic and amygdala nuclei and in superficial layers of the piriform cortex. These data suggest that GDNF and its receptors may play a local role in neuronal plasticity and in neuronal protection following epileptic insults.

The rho 1 GABA receptor cloned from rat retina is down-modulated by protons.

We have cloned and sequenced the full-length cDNA of the rat GABA (gamma-aminobutyric acid) rho 1 receptor subunit. The deduced amino acid sequence (474 amino acids) of the rat rho 1 receptor is 95% homologous to the previously cloned human rho 1 receptor. The rho 1 cDNA includes a 5' 129 bp and a 3' 2.6 kb untranslated region, which contains a sequence homologous to the human medium reiteration frequency repetitive sequence, MER18. The rat rho 1 receptor shows 45-50% similarity to the GABAA receptor beta subunits. This similarity is among the highest between all GABAA receptor subunit classes, giving no support at the molecular level to the classification of the rho subunits to a novel GABAC receptor class. The rat rho 1 cDNA formed functional GABA receptors insensitive to bicuculline, but sensitive to blockade by picrotoxin, when transiently expressed in the human embryonic kidney cell line HEK 293. We studied the sensitivity of the rho 1-mediated GABA current to variations in extracellular pH (pH0), and found that these receptors are strongly down-modulated by H+ ions. A decrease in pH0 from 7.4 to 6.4 decreased the GABA current by 51 +/- 8%, whereas an increase in pH0 from 7.4 to 8.4 increased the current by 77 +/- 8%. In view of the up-regulatory effect of protons on the GABA current observed in a number of preparations, the rho 1 receptors may contain a novel down-regulatory binding site for protons characteristic to these receptors.

Hepatic expression of the human insulin gene reduces glucose levels in vivo in diabetic mice model.

OBJECTIVES: The objective of these studies was to evaluate human insulin gene expression following intraliver plasmid injection in diabetic mice as a potential approach to gene therapy for insulin-dependent diabetes mellitus. METHODS: The fragment containing human proinsulin gene lacking its own promoter, was cloned into plasmids containing promoter and enhancer of cytomegalovirus or human hepatitis B virus. The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo applying streptozotocin-induced diabetic mice. RESULTS: We found significant reduction in glucose levels in both experimental systems, giving evidence that prolonged constitutive systemic secretion of bioactive human (pro)insulin has been attained in non-neuroendocrine cell line in vitro and in mice following intra-liver plasmid injection. CONCLUSION: Our data demonstrate the reduction of glucose levels in vitro in 3T3 fibroblast cells and in vivo in diabetic mice after treatment with plasmids expressing proinsulin, giving evidence that those constructs may have certain usage also in human gene therapy of diabetes mellitus type 1.

Sequence variations and two levels of MCT1 and CD147 expression in red blood cells and gluteus muscle of horses.

MCT1-CD147 complex is the prime lactate transporter in mammalian plasma membranes. In equine red blood cells (RBCs), activity of the complex and expression of MCT1 and CD147 is bimodal; high in 70% and low in 30%. We studied whether sequence variations contribute to the bimodal expression of MCT1 and CD147. Samples of blood and cremaster muscle were collected in connection of castration from 24 horses. Additional gluteus muscle samples were collected from 15 Standardbreds of which seven were known to express low amounts of CD147 in RBCs. The cDNA of MCT1 and CD147 together with a promoter region of CD147 was sequenced. The amounts of MCT1 and CD147 expressed in RBC and muscle membranes were measured by Western blot and mRNA levels in muscles by qPCR. MCT1 and CD147 were expressed in 20 castrates, and in four only were traces found. Sequence variations found in MCT1 were not linked to MCT1 expression. In CD147 linked heterozygous single nucleotide polymorphisms (SNPs) 389A>G (Met(125)Val) and 990C>T (3'-UTR) were associated to low expression of CD147. Also a mutation 168A>G (Ile(51)Val) in CD147 was associated to low MCT1 and CD147 expression. Low MCT1 and CD147 mRNA levels in gluteus were found in Standardbreds with low CD147 expression in RBCs. The results suggest that sequence variations affect the expression level of CD147, but do not explain its bimodality. The levels of MCT1 and CD147 mRNA correlated with the expression of CD147 and suggest that bimodality of their expression is regulated at transcriptional level.

MCT1, MCT4 and CD147 gene polymorphisms in healthy horses and horses with myopathy.

Polymorphisms in human lactate transporter proteins (monocarboxylate transporters; MCTs), especially the MCT1 isoform, can affect lactate transport activity and cause signs of exercise-induced myopathy. Muscles express MCT1, MCT4 and CD147, an ancillary protein, indispensable for the activity of MCT1 and MCT4. We sequenced the coding sequence (cDNA) of horse MCT4 for the first time and examined polymorphisms in the cDNA of MCT1, MCT4 and CD147 of 16 healthy horses. To study whether signs of myopathy are linked to the polymorphisms, biopsy samples were taken from 26 horses with exercise-induced recurrent myopathy. Two polymorphisms that cause a change in amino acid sequence were found in MCT1 (Val(432)Ile and Lys(457)Gln) and one in CD147 (Met(125)Val). All polymorphisms in MCT4 were silent. Mutations in MCT1 or CD147 in equine muscle were not associated with myopathy. In the future, a functional study design is needed to evaluate the physiological role of the polymorphisms found.

MCT1 and CD147 gene polymorphisms in standardbred horses.

REASONS FOR PERFORMING STUDY: Transport of lactate across membranes is facilitated by proton-monocarboxylate transporters (MCT). The most widely distributed isoform is MCT1, which needs an ancillary protein CD147. Studies on erythrocytes have shown that high activity of MCT1 is inherited as the dominant allele and that activity is regulated through CD147. Mutations of human MCT1 have been described that appear to impair lactate transport in muscles and cause exertional rhabdomyolysis. There are no reports of this potential relationship in the horse. OBJECTIVES: To obtain sequences of equine MCT1 and CD147 to examine differences between horses with high and low lactate transport activity in their erythrocytes. METHODS: Muscle biopsy samples were taken from 3 healthy Standardbred horses and from 7 horses which according to the owners had signs of myopathy after intense exercise. DNA and RNA were isolated and PCR analysis and sequencing performed. RESULTS: Currently, PCR fragments covering 100% of MCT1 and 70% of CD147 coding region are retained and sequence analysis has demonstrated one single nucleotide polymorphism (SNP) in the C-terminal area of MCT1 and one SNP in the extracellular domain of CD147. Both cause an amino acid change. The SNPs found are not related to lactate transport activity in erythrocytes or signs of myopathy. CONCLUSIONS: More samples need to be analysed to make conclusions on the significance of the polymorphisms found. Furthermore, full sequence coverage of the coding region of CD147 is needed. POTENTIAL RELEVANCE: The molecular probes produced could be used as tools to study gene regulation of lactate transport.

[Monoclonal antibodies to cyclodextrin-glycosyltransferase from Bacillus macerans]. / Monoklonal'nye antitela k tsiklodekstrin-gliukanotransferaze Bacillus macerans.

Cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) was isolated from the cell-free culture medium of Bacillus macerans strain BKMB-506. The purified enzyme was used to immunize BALB/c mice. Polyclonal antibodies (PAbs) and 15 hybridoma cells producing monoclonal antibodies (MAbs) against CGTase were obtained. The properties of MAbs were studied using ELISA and immunoblotting. A method for detecting small amounts of CGTase was developed. The interactions of PAbs and MAbs with CGTase of Bac. circulans var. alkalophilis were investigated.

Tissue-specific expression of rat light neurofilament promoter-driven reporter gene in transgenic mice.

We have produced nine transgenic mice lines carrying either 5 kbp or 407 bp of the 5' flanking sequence of the rat light neurofilament gene linked to the chloramphenicol acetyltransferase (CAT) structural gene. With the 5kb light neurofilament 5' flanking region governing the expression of CAT, reporter gene activity was detected not only in brain but also in the eye lens and skeletal muscle, yet not in other tissues. With the 407 bp construct, reporter gene activity was detected only in the brain, although expression was approximately one tenth of that found with the 5 kb 5' region. These results, together with earlier observations, indicate that the sequence -407 to -292 of the proximal promoter region for the light neurofilament gene or sequence +15 to +75 bp after the transcription initiation site is crucial for brain-specific expression of a fusion gene in transgenic mice.

Characterization of the rat light neurofilament (NF-L) gene promoter and identification of NGF and cAMP responsive regions.

We have isolated a genomic DNA clone covering the coding and 14 kb upstream region of the rat light neurofilament (NF-L) gene and sequenced 2.3 kb of its promoter. DNase I hypersensitive sites have been mapped in PC12 cells. For functional analysis of the NF-L promoter, constructs carrying 38, 97, 407, 564, 650, 1,099, 1,660, 2,003 base pairs (bp) upstream region in front of the chloramphenicol acetyltransferase (CAT) reporter gene were tested for their capability to direct CAT expression after transient transfection into various cell lines. Similar CAT activities were recorded both in rat pheochromocytoma (PC12) and mouse neuroblastoma N115 cells and also in several nonneural cell lines (HeLa, C127, NIH 3T3). Regions responsible for the basic promoter activity were located between -407 and +75 bp from the transcription initiation site. The NGF-responsive element was located between -38 and +75 bp, and sequence -97 to -38 was found to contain a functional cAMP-responsive element. In PC12 cells in which nerve growth factor (NGF) induces neurite outgrowth and NF-L transcription, NF-L promoter-driven CAT expression was stimulated up to 12-fold within three days of NGF treatment, whereas epidermal growth factor (EGF) had no effect. Rat NF-L promoter contained Sp1, AP-2 and CGCCCCCGC elements. In PC12 cells, NGF transiently induced the binding of transcription factors to the deoxyoligonucleotide probes containing the binding sites of these elements. The role of these factors in NF-L gene transcriptional induction by NGF in PC12 cells is discussed.

Transcriptional regulation of neurofilament expression by protein kinase A.

RN46A cells, a conditionally immortalized neuronal cell line derived from E12 rat medullary raphe nucleus, upregulate low M(r) (68 kDa, neurofilament [NF]-L) and medium M(r) (160 kDa, NF-M) neurofilament protein expression upon activation of protein kinase A (PKA). To examine possible transcriptional regulation of neurofilament protein expression by PKA, two cell lines were used; RN46A cells and C alpha EV6 cells, a cell line derived from RN46A cells that stably expresses the catalytic subunit of PKA under the control of the metallothionein promoter. Treatment of RN46A cells with dbcAMP resulted in an increase in the steady-state levels of both NF-L and NF-M, but not high M(r) (200 kDa, NF-H) neurofilament mRNA. These increases were both time and dose dependent and were sensitive to treatment with the protein synthesis inhibitor cycloheximide. In C alpha EV6 cells, activation of PKA by 80 microM ZnSO4 upregulated the expression of C alpha mRNA with maximal levels reached 8 hr post-treatment and maintained at 24 hr. Reporter gene assays in C alpha EV6 cells following transfection with increasing lengths of the NF-L promoter demonstrated that both a putative Sp1-like and a cAMP response (CRE), but not a NGFI-A, element were likely involved in PKA-dependent activation of the NF-L promoter. Electrophoretic mobility shift assays confirmed these results but showed that the nuclear proteins induced by PKA which bound to the NF-L promoter Sp1-like sequence were not Sp1. Collectively, these data suggest that constitutively expressed Sp1 may be involved in basal NF-L promoter activity, and newly synthesized, PKA-dependent nuclear proteins may synergistically activate the rat NF-L promoter.

Induced expression of neurotrophins in transgenic mice overexpressing ornithine decarboxylase and overproducing putrescine.

Transgenic mice overexpressing ornithine decarboxylase (ODC) were recently generated (Halmekytö et al.: Biochem Biophys Res Commun 180:262-267, 1991). The dramatic ODC overexpression resulted in a very high accumulation of the polyamine putrescine in the brain. As elevated polyamine levels in the brain are believed to be associated with neuronal damage, we studied whether enhanced putrescine accumulation in the brain of these mice affects the expression of neurotrophins and their high affinity receptors. Northern blot analysis indicated that mRNA levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF) and neurotrophin-3 (NT-3) were significantly elevated about 1.5-fold in the hippocampus of ODC transgenic mice as compared with control animals. The levels of BDNF, NGF and NT-3 mRNA were also elevated in the kidneys of the transgenic mice. In eight other tissues examined there were no significant differences. The expression pattern of BDNF mRNA and of trkB and trkC (high affinity receptors for BDNF and NT-3 respectively) immunoreactivity in the hippocampal formation did not reveal significant differences. The induction of the expression of neurotrophins could belong to neuroprotective measures triggered by ODC overexpression.

Seizures induce widespread upregulation of cystatin B, the gene mutated in progressive myoclonus epilepsy, in rat forebrain neurons.

Loss of function mutations in the gene encoding the cysteine protease inhibitor, cystatin B (CSTB), are responsible for the primary defect in human progressive myoclonus epilepsy (EPM1). CSTB inhibits the cathepsins B, H, L and S by tight reversible binding, but little is known regarding its localization and physiological function in the brain and the relation between the depletion of the CSTB protein and the clinical symptoms in EPM1. We have analysed the expression of mRNA and protein for CSTB in the adult rat brain using in situ hybridization and immunocytochemistry. In the control brains, the CSTB gene was differentially expressed with the highest levels in the hippocampal formation and reticular thalamic nucleus, and moderate levels in amygdala, thalamus, hypothalamus and cortical areas. Detectable levels of CSTB were found in virtually all forebrain neurons but not in glial cells. Following 40 rapidly recurring seizures evoked by hippocampal kindling stimulations, CSTB mRNA expression showed marked bilateral increases in the dentate granule cell layer, CA1 and CA4 pyramidal layers, amygdala, and piriform and parietal cortices. Maximum levels were detected at 6 or 24 h, and expression had reached control values at 1 week post-seizures. The changes of mRNA expression were accompanied by transient elevations (at 6-24 h) of CSTB protein in the same brain areas. These findings demonstrate that seizure activity leads to rapid and widespread increases of the synthesis of CSTB in forebrain neurons. We propose that the upregulation of CSTB following seizures may counteract apoptosis by binding cysteine proteases.

Distribution of GABA receptor rho subunit transcripts in the rat brain.

The gamma-aminobutyric acid (GABA) receptor rho subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABA(C). In this work we have examined the distribution of rho1, rho2 and rho3 subunits, and found expression of all three transcripts in several regions of the rat nervous system. In situ hybridization revealed expression of rho2 in the adult rat retina and some other parts of the visual pathways. A high local rho2 expression was seen in the superficial grey layer of the superior colliculus, and in the dorsal lateral geniculate nucleus. Expression was also detected in the 6th layer of visual cortex and in the CA1 pyramidal cell layer of hippocampus. With reverse transcriptase-polymerase chain reaction, expression of rho1 was mainly seen in the adult rat retina and dorsal root ganglia, as well as, at a significantly lower level, in the superior colliculus, hippocampus, brain stem, thalamus, postnatal day 8 (P8) superior colliculus and P8 hippocampus. Expression pattern of rho3 mRNA was clearly different from that of rho1 and rho2, being strongest in the hippocampus, and significantly lower in the retina, dorsal root ganglia and cortex. No rho3 expression was observed in adult or P8 superior colliculus or in P8 hippocampus. The present results clearly demonstrate that expression of GABA receptor rho subunits is not restricted to the retina, but significant expression can also be detected in many other brain regions, especially in those belonging to the visual pathways. The expression pattern of the rho subunits may be helpful in solving the functional significance of the receptors formed from these subunits.