Nonsense mutations at Arg-1947 in two cases of familial neurofibromatosis type 1 in Japanese

We report two familial cases of NF1 presenting as C to T transitions changing an Arg-1947 codon to a stop codon. In one of the two families, cosegregation of the mutation with NF1 was demonstrated, indicating this mutation causes the disease in this family. As the same mutation at Arg-1947 has been reported previously in three cases of unrelated Caucasians (two are sporadic; the origin of the other is not reported), the codon at Arg-1947 (CGA) in the NF1 gene is considered to be a hotspot common among different ethnic groups and also among familial and sporadic cases.     

Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.     

Insulin resistance induces earlier initiation of cognitive dysfunction mediated by cholinergic deregulation in a mouse model of Alzheimer's disease

Although insulin resistance increases the risk of Alzheimer's disease (AD), the mechanisms remain unclear, partly because no animal model exhibits the insulin-resistant phenotype without persistent hyperglycemia. Here we established an AD model with whole-body insulin resistance without persistent hyperglycemia (APP/IR-dKI mice) by crossbreeding constitutive knock-in mice with P1195L-mutated insulin receptor (IR-KI mice) and those with mutated amyloid precursor protein (App^NL-G-F mice: APP-KI mice). APP/IR-dKI mice exhibited cognitive impairment at an earlier age than APP-KI mice. Since cholinergic dysfunction is a major characteristic of AD, pharmacological interventions on the cholinergic system were performed to investigate the mechanism. Antagonism to a nicotinic acetylcholine receptor α7 (nAChRα7) suppressed cognitive function and cortical blood flow (CBF) response to cholinergic-regulated peripheral stimulation in APP-KI mice but not APP/IR-dKI mice. Cortical expression of Chrna7, encoding nAChRα7, was downregulated in APP/IR-dKI mice compared with APP-KI. Amyloid β burden did not differ between APP-KI and APP/IR-dKI mice. Therefore, insulin resistance, not persistent hyperglycemia, induces the earlier onset of cognitive dysfunction and CBF deregulation mediated by nAChRα7 downregulation. Our mouse model will help clarify the association between type 2 diabetes mellitus and AD.     

ACETYL-CoA SYNTHESIZING ENZYME ACTIVITIES IN SINGLE NERVE CELL BODIES OF RABBIT

Activities of five enzymes (pyruvate dehydrogenase complex; citrate synthase, EC 4.1.3.7; carnitine acetyltransferase, EC 2.3.1.7; acetyl-CoA synthetase, EC 6.2.1.1; and ATP citrate lyase, EC 4.1.3.8) were determined in cell bodies of anterior horn cells and dorsal root ganglion cells from the rabbit. For comparison, molecular layer, granular layer and white matter from rabbit and mouse cerebella and cerebral cortex and striatum from the mouse were analyzed. Samples (3–85 ng dry weight) were assayed in 180 to 370 ml of assay reagents containing CoASH and other substrates in excess. By using ‘CoA cycling’, the assay systems were devised to amplify and measure small amounts of acetyl-CoA formed during the enzyme reactions. Carnitine acetyltransferase was the most active enzyme in single nerve cell bodies and all layer samples, except for rabbit and mouse cerebellar white matter. Citrate synthetase was the lowest in single cell bodies. The activities of carnitine acetyltransferase and acetyl-CoA synthetase (656 and 89.8 mmoles of acetyl-CoA formed/kg of dry weight/h at 38°C) from dorsal root ganglion cells were about 2-fold higher than those from anterior horn cells. The activity of ATP citrate lyase (134mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) from anterior horn cells was approximately twice that from dorsal root ganglion cells. The activity of this enzyme was distributed in a wider range in anterior horn cells than dorsal root ganglion cells. The second highest activity (80.0 mmol of acetyl-CoA formed/kg of dry weight/h at 38°C) of ATP citrate lyase was found in striatum where cholinergic interneurones are abundant. Relatively higher activities of this enzyme were found in cerebellar granular layer and white matter which are known to contain the cholinergic mossy fibers. These results suggested that cholinergic neurones contain higher activity of ATP citrate lyase which is thought to supply acetyl-CoA to choline acetyltransferase (EC 2.3.1.6) as a substrate to form acetylcholine.     

Ligation and synthesis of chromatin deoxyribonucleic acid in vitro in neuronal, glial and liver nuclei isolated from adult guinea pig

Neuron-rich and glial nuclear preparations and liver nuclei were isolated from adult guinea pigs. These nuclei were incubated to carry out DNA-ligation and -synthesis reactions. Before and after incubation, the sizes of single-standed DNA and DNA-synthesis patterns in single strands were analysed by using alkaline sucrose-density-gradient centrifugation. Isolation of nuclei by cell-fractionation technique shortened chromatin DNA and decreased markedly the number-average molecular weight of DNA strands. Chromatin DNA in neuronal and glial nuclei was ligated at the nicks during incubation in a reaction mixture containing ATP, Mg(2+), dithiothreitol and four deoxyribonucleotides. The number-average molecular weights were estimated to increase 1.1-and 2.1-fold in neuronal and glial nuclei respectively. DNA strands in liver nuclei were shortened during incubation, but elongated under conditions that inhibit deoxyribonuclease. Since the endogenous deoxyribounuclease activity was conspicuously higher in liver nuclei than in neuronal and glial nuclei, the shortening and elongation were thought to depend on the balance between DNA ligase and deoxyribonuclease reactions. DNA synthesis occurred at the gaps in chromatin DNA and about 50% of the total synthesized DNA was found in the shorter strands having 6 to 297 bases in all species of nuclei. Based on these results, it was concluded that in nuclei isolated from non-dividing cells (neurons) and slowly dividing cells (glial and liver cells) DNA-ligation and -synthesis reactions proceeded in parallel at the breaks in single-stranded DNA, which was produced mainly by endogenous deoxyribonuclease during isolation and incubation processes.     

Effect of Nocardia rubra cell-wall skeleton treatment on tumor formation in two-stage chemical carcinogenesis of mouse skin

Summary In two-stage chemical carcinogenesis of mouse skin, Nocardia rubra cell-wall skeleton (N-CWS), a potent immunopotentiator, was injected SC at various times. The dorsal skin of C57BL/6 male mice (about 10 cm²) was painted with 20 g 7,12-dimethylbenz(a)anthracene (DMBA) in 0.1 ml acetone when the animals were 11 weeks old (initiation). Seven weeks later, they were painted with 2.5 g 12-0-tetradecanoyl-phorbol-13-acetate (TPA) in 0.1 ml acetone twice weekly for 30 weeks (promotion). The timing of N-CWS treatment was important. N-CWS treatment before initiation reduced the incidence of skin tumor and the mean number of skin tumors per mouse most effectively. It is speculated that the antitumor activity of N-CWS may be composed of at least two mechanisms, being achieved through the enhancement of immunological surveillance and through changes in the metabolism of chemical carcinogens.     

Isolation and characterization of cDNA clones encoding cdc2 homologues from Oryza sativa: a functional homologue and cognate variants.

Summary Using probes obtained by PCR amplification, we have isolated two cognate rice cDNAs (cdc2Os-1 andcdc2Os-2) encoding structural homologues of thecdc2 ⁺/CDC28(cdc2) protein kinase from a cDNA library prepared from cultured rice cells. Comparison of the deduced amino acid sequences of cdc2Os-1 and cdc2Os-2 showed that they are 83 % identical. They are 62 % identical toCDC28 ofSaccharomyces cerevisiae and much more similar to the yeast and mammalian p34^cdc2 kinases than to riceR2, acdc2-related kinase isolated previously by screening the same rice cDNA library with a different oligonucleotide probe. Southern blot analysis indicated that the three rice clones (cdc2Os-1,cdc2Os-2 andR2) are derived from distinct genes and are each found in a single copy per rice haploid genome. RNA blot analysis revealed that these genes are expressed in proliferating rice cells and in young rice seedlings.cdc2Os-1 could complement a temperature-sensitive yeast mutant ofcdc28. However, despite the similarity in structure, bothcdc2Os-2 andR2 were unable to complement the same mutant. Thus, the present results demonstrate the presence of structurally related, but functionally distinct cognates of thecdc2 cell cycle kinase in rice.The nucleotide sequence data in this paper have been deposited in the EMBL database under accession number X60374 (cdc2Os-1) and X60375 (cdc2Os-2)     

Small molecule inhibitors of the CCR2b receptor. Part 1: Discovery and optimization of homopiperazine derivatives

N,N'-Disubstituted homopiperazine derivatives have been discovered as CC-chemokine receptor 2b (CCR2b) inhibitors with submicromolar activity in the CCR2b binding assay. A 4-substituted benzyl group on one homopiperazine nitrogen was an important moiety for binding affinity to the CCR2b receptor. The SAR for CCR2b binding affinity correlated inversely with the sigma factor of the functional group on this benzyl moiety. Introduction of hydroxy groups to appropriate positions in the 3,3-diphenylpropyl group on the other homopiperazine nitrogen increased CCR2b binding activity. The synthesis of an informer library to search for alternative substructures is also described.     

Solubility properties of a 65-kDa peptide prepared by restricted digestion of myosin with astacin-like squid metalloprotease

Substructure of the myosin rod and its correlation to filament formation is largely based on studies of proteolytic digests and expressed proteins. However, tryptic digestion of myosin always produces polymorphous peptides. Consequently, it is difficult to determine the relation between myosin substructure and filament formation. Similarly, filament formation with recombinant myosin protein is also difficult to interpret because it is never clear whether the recombinant protein folds like the native protein. We recently reported a novel metal protease isolated from squid liver, astacin-like squid metalloprotease (ALSM), which can specifically hydrolyze in vitro myosin heavy chain. In the present study, we examined the solubility properties of the 65-kDa peptide and light meromyosin (LMM) prepared by ALSM isoform II and trypsin digestion, respectively. The 65-kDa peptide is much less soluble than LMM under physiological conditions, even though the length of 65-kDa peptide is shorter than that of LMM. These results suggest that a novel substructure of myosin drives filament assembly.     

Wnt4-transformed mouse embryonic stem cells differentiate into renal tubular cells

Embryonic stem (ES) cells have the potential to differentiate into various progenitor cells. Here we investigated the capacity of mouse ES cells to differentiate into renal tubular cells both in vitro and in vivo. After stably transfecting Wnt4 cDNA to mouse ES cells (Wnt4-ES cells), undifferentiated ES cells were incubated by the hanging drop culture method to induce differentiation to embryoid bodies (EBs). During culturing of the EBs derived from the Wnt4-ES cells, aquaporin-2 (AQP2) mRNA and protein were expressed within 15-20 days. The expression of AQP2 in Wnt4-EBs was enhanced in the presence of hepatocyte growth factor (HGF) and activin A. We next performed in vivo experiments by transplanting the Wnt4-EBs into the mouse renal cortex. Four weeks after transplantation, some portions of the EB-derived cells expressing AQP2 in the kidney assembled into tubular-like formations. In conclusion, our in vitro and in vivo experiments revealed two new findings: first, that cultured Wnt4-EBs have an ability to differentiate into renal tubular cells; and second, that Wnt4, HGF, and activin A may promote the differentiation of ES cells to renal tubular cells.