Ancient duplications of the human proglucagon gene

The human proglucagon gene (GCG) is encoded within a finished 576-kb DNA sequence generated by the Human Genome Project. GCG is flanked by 18 kb and 65 kb of DNA, 5' and 3', respectively, that do not encode genes. The genomic sequence that includes GCG was found to have a long history of gene duplication events. Some members of the glucagon-like family of genes, GCG on chromosome 2 and GIP on chromosome 17, may be products of ancient genome duplications on the early vertebrate lineage. A large genomic tandem duplication event that included DPP4-like and GCG genes occurred before the amphibian-mammal divergence, but one of the duplicated copies of GCG has been lost on the human lineage. Recently, a processed pseudogene of the X-chromosome-linked gene TIMM8A was inserted downstream of GCG. Some ancient duplicates of GCG may retain physiological functions in other vertebrates.     

Alanine is the main second amino acid in vertebrate proteins and its coding entails increased use of the rare codon GCG

The occurrence of a main amino acid at the start of proteins from various vertebrate species is reported. Computer analyses of protein-coding sequences showed that alanine occurs in one out of five cases as the second amino acid in proteins from seven mammals (including man), one amphibian, one bird and two fishes. We also show that the alanine codon GCG occurs >3-fold more often as second codon than in general, i.e. GCG was overrepresented. Options to explain the abundance of alanine as second amino acid may already exist, but the overrepresentation of GCG was harder to explain because GCG was classically a rare codon. However, based on similarities with the published translation-initiation enhancer sequence CCGGCGG, which has complementarity with 18S ribosomal RNA, a theoretical role of GCG in translation initiation is suggested. Namely, GCG is proposed to be part of a sequence with potential for base-pairing with 18S ribosomal RNA.     

Discovering Implicit Entity Relation with the Gene-Citation-Gene Network

In this paper, we apply the entitymetrics model to our constructed Gene-Citation-Gene (GCG) network. Based on the premise there is a hidden, but plausible, relationship between an entity in one article and an entity in its citing article, we constructed a GCG network of gene pairs implicitly connected through citation. We compare the performance of this GCG network to a gene-gene (GG) network constructed over the same corpus but which uses gene pairs explicitly connected through traditional co-occurrence. Using 331,411 MEDLINE abstracts collected from 18,323 seed articles and their references, we identify 25 gene pairs. A comparison of these pairs with interactions found in BioGRID reveal that 96% of the gene pairs in the GCG network have known interactions. We measure network performance using degree, weighted degree, closeness, betweenness centrality and PageRank. Combining all measures, we find the GCG network has more gene pairs, but a lower matching rate than the GG network. However, combining top ranked genes in both networks produces a matching rate of 35.53%. By visualizing both the GG and GCG networks, we find that cancer is the most dominant disease associated with the genes in both networks. Overall, the study indicates that the GCG network can be useful for detecting gene interaction in an implicit manner.     

The Geodia cydonium galectin exhibits prototype and chimera-type characteristics and a unique sequence polymorphism within its carbohydrate recognition domain

The ancestral galectin from the sponge Geodia cydonium (GCG) is classified on a structural basis to the prototype subfamily, whereas its carbohydrate-binding specificity is related to that of the mammalian chimera-type galectin-3. This dual coordination reveals GCG as a potential precursor of the later evolved galectin subfamilies, which is reflected in the primary structure of the protein. This study provides evidence that GCG is the LECT1 gene product, while neither a previously described LECT2 gene nor a functional LECT2 gene product was found in the specimen under investigation. The electrophoretically separated protein isomers with apparent molecular masses of 13, 15, and 16 kDa correspond to variants of the LECT1 protein-exhibiting peptide sequence polymorphisms that concern critical positions of the carbohydrate recognition domain (13 kDa: Leu51, Asn55, His130, Gly137; 15 kDa: Ser51, Asn55, Asn130, Gly137; 16 kDa: Ser51, Tyr55, Asn130, Glu137). Four residues, highly conserved in the galectin family, are substituted. None of the residues claimed to be involved in interactions with GalNAcalpha1-3 moieties at an extended binding subsite of galectin-3 was identified in the corresponding positions of GCG. Apparently, the substitutions do not confer distinct binding characteristics to the GCG variants as evidenced by binding studies with a recombinantly expressed 15-kDa isoform. The natural isoforms as well as the recombinant 15-kDa isoform oligomerize by the formation of non-covalent heteromeric or homomeric complexes. A phosphorylation of the galectin was confirmed neither by mass spectrometry nor by alkaline phosphatase treatment combined with isoelectric focusing.     

Prenatal enzymatic diagnosis of Krabbe disease (globoid-cell leukodystrophy) using chorionic villi

Summary Sixteen pregnancies in families with children enzymatically diagnosed as having Krabbe disease (KD) were monitored for prenatal KD using the assay of galactosyl ceramide -galactosidase (GCG) in uncultured chorionic villi (CV), cultured CV, or cultured amniotic fluid cells (AFC). Prenatal KD diagnoses were made for 5 pregnancies on the basis of lower than 10% normal GCG activity in cultured CV or AFC. Uncultured CV were studied in 3 out of the 5 KD embryos, although the GCG activities of 14%–23% as compared with control villi were diagnostically inconclusive; the relatively high activities were considered to be caused by maternal GCG contamination of these very small villus samples. Although the villi from 6 of the other pregnancies yielded more conclusive results, the use of uncultured CV alone is not recommended for prenatal KD diagnosis, this material being subject to possible uncontrolled contamination with maternal enzyme.     

A hypertext-like approach to navigating through the GCG sequence analysis package

Programs of the recently released Unix version of the GeneticsComputing Group (GCG) sequence analysis package can now be accessedvia a user-friendly hypertext-like navigation system, HYGCG.The resultant system organizes the diverse suite of programsinto logical groups, and provides a guide and explanation ofcommands. In addition, for users unfamiliar with the Unix operatingsystem, the program also provides a similar interface to commonlyused Unix commands. Options for personal customization and expansionto accommodate GCG extensions and other software are also provided.This system should be useful especially to the inexperiencedor infrequent user as context-sensitive on-line help is providedwithin this simple and consistent approach. Written in the Clanguage and using the curses and termcap libraries, the systemis easily portable to most Unix environments and has been madefreely available via anonymous file transfer protocol (FTP)through the Internet global computer network. No modificationof the GCG package is needed.     

Doxycycline attenuates and delays toxicity of the oculopharyngeal muscular dystrophy mutation in transgenic mice

The muscular dystrophies are a heterogeneous group of disorders for which there are currently no cures. Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant late-onset, progressive disease that generally presents in the fifth or sixth decade with dysphagia, ptosis and proximal limb weakness. OPMD is caused by the abnormal expansion of a (GCG)n trinucleotide repeat in the coding region of the poly-(A) binding protein nuclear 1 (PABPN1) gene. In unaffected individuals, (GCG)6 codes for the first six alanines in a homopolymeric stretch of ten alanines. In most individuals with OPMD this (GCG)6 repeat is expanded to (GCG)8-13, leading to a stretch of 12-17 alanines in mutant PABPN1. PABPN1 with an expanded polyalanine tract forms aggregates consisting of tubular filaments within the nuclei of skeletal muscle fibers. We have developed a transgenic mouse model of OPMD that manifests progressive muscle weakness accompanied by intranuclear aggregates and TUNEL-stained nuclei in skeletal muscle fibers. The onset and severity of these abnormalities were substantially delayed and attenuated by doxycycline treatment, which may exert its therapeutic effect by reducing aggregates and by distinct antiapoptotic properties. Doxycycline may represent a safe and feasible therapeutic for this disease.     

基于GCG的生物信息学WEB系统的构建

利用APACHE服务器、PHP脚本,从生物学研究人员使用角度出发,开发了一套通过web界面来使用GCG软件包的系统。该系统的成功开发,提供给局域网内用户方便、安全、稳定的GCG的在线分析服务,大大降低了生物学研究人员使用GCG的难度。     

A shell program for the design of PCR primers using genetics computer group (GCG) software (7.1) on VAX/VMS systems.

A number of software analysis packages for the design of PCR primers are available for PCs; however, software for users that depend on VAX/VMS operating systems is not available. By treating oligonucleotides as RNA molecules, I have designed an alternative means toward studying oligonucleotide interactions using software that is currently available from The Genetics Computer Group (GCG, Madison, WI). The oligonucleotide interactions with self and non-self are analyzed by the GCG FOLD program, a program which finds a secondary structure of minimum free energy for an RNA molecule. This approach allows the identification of self-priming primer pairs, and the interaction energies provide a guideline for the prediction of optimal PCR primers.     

Rearrangement of side-chains in a Zif268 mutant highlights the complexities of zinc finger-DNA recognition.

Structural and biochemical studies of Cys(2)His(2) zinc finger proteins initially led several groups to propose a "recognition code" involving a simple set of rules relating key amino acid residues in the zinc finger protein to bases in its DNA site. One recent study from our group, involving geometric analysis of protein-DNA interactions, has discussed limitations of this idea and has shown how the spatial relationship between the polypeptide backbone and the DNA helps to determine what contacts are possible at any given position in a protein-DNA complex. Here we report a study of a zinc finger variant that highlights yet another source of complexity inherent in protein-DNA recognition. In particular, we find that mutations can cause key side-chains to rearrange at the protein-DNA interface without fundamental changes in the spatial relationship between the polypeptide backbone and the DNA. This is clear from a simple analysis of the binding site preferences and co-crystal structures for the Asp20-->Ala point mutant of Zif268. This point mutation in finger one changes the specificity of the protein from GCG TGG GCG to GCG TGG GC(G/T), and we have solved crystal structures of the D20A mutant bound to both types of sites. The structure of the D20A mutant bound to the GCG site reveals that contacts from key residues in the recognition helix are coupled in complex ways. The structure of the complex with the GCT site also shows an important new water molecule at the protein-DNA interface. These side-chain/side-chain interactions, and resultant changes in hydration at the interface, affect binding specificity in ways that cannot be predicted either from a simple recognition code or from analysis of spatial relationships at the protein-DNA interface. Accurate computer modeling of protein-DNA interfaces remains a challenging problem and will require systematic strategies for modeling side-chain rearrangements and change in hydration.     

Analysis of the mechanism of inhibition of human matrix metalloproteinase 7 (MMP-7) activity by green tea catechins

Green tea catechins inhibit human matrix metalloproteinase 7 (MMP-7) activity non-competitively, and the galloyl group is essential for potent inhibition (Oneda et al., J. Biochem., 133, 571-576 (2003)). In this study, we analyzed the mechanism of this inhibition. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2), the inhibitory effects of (-)-epigallocatechin-3-gallate (EGCG), (-)-gallocatechin-3-gallate (GCG), (-)-epicatechin-3-gallate (ECG), and (-)-catechin-3-gallate (CG) increased with increasing pH levels from 7.0 to 8.5. The inhibitory effects of EGCG and GCG were more potent than those of ECG and CG, and increased with increasing CaCl(2) concentrations from 10 to 50 mM. The fluorescence of EGCG and GCG decreased with increasing CaCl(2) concentrations and with the addition of MMP-7, while those of ECG and CG did not. Our results suggest that these differences result from that in the B ring, EGCG and GCG have phenol hydroxyl groups at the 3', 4', and 5' positions, while ECG and CG have them at the 3' and 4' positions.     

Synthesis and characterization of oligodeoxynucleotides containing 4-O-methylthymine

The carcinogenicity of N-nitroso compounds is believed to result from the alkylation of DNA, particularly on O-6 of the guanine and O-4 of the thymine residues. In order to study the base-pairing properties of 4-O-methylthymidine (T*) residues and the structural changes produced in DNA by the presence of this alkylated nucleoside, the oligodeoxyribonucleotides T*GCG, CGCAAGCTT*GCG, CGCGAGCTT*GCG, and CGCAAGCTTGCG were synthesized by the phosphotriester approach in solution. The 4-O-methylthymidine required for oligonucleotide synthesis was prepared by treating the 4-(3-nitro-1,2,4-triazolo) derivative of 3',5'-bis-O-(methoxyacetyl)thymidine with 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in methanol solution. The susceptibility of the 4-O-methyl group of T* toward nucleophiles enables this group of 4-O-methylthymidine-containing oligomers to be labeled by a direct exchange reaction with [13C]- or [14C]methanol in the presence of DBU. Although it has been previously suggested that 4-O-methylthymine forms stable base pairs with guanine, the thermal melting profiles of the double helices formed by these dodecamers suggest that the presence of 4-O-methylthymine paired to either adenine or guanine destablizes the helix. The melting curve of the sequence containing a 4-O-methylthymine residue base paired to guanine was biphasic and similar to that of an analogous sequence containing 6-O-methylguanine paired to thymine.     

Gly40Ser Polymorphism of the Glucagon Receptor Gene Is Associated with Central Adiposity in Men

Objective: To study the association between the Gly40Ser polymorphism of the glucagon receptor gene (GCG‐R) and central adiposity. Research Methods and Procedures: Data from 985 working men (The Olivetti Heart Study) examined in 1994 were used in a cross‐sectional design. A complete anthropometry was performed; body mass index and waist circumference were taken as measures of total and central adiposity, respectively. The GCG‐R Gly40Ser polymorphism was characterized. Biochemical variables linked to energy metabolism were measured. Results: The GCG‐R Gly40Ser variant was present in 37 individuals only in heterozygous form and was significantly associated with anthropometric indices of central adiposity, accounting for age and body mass (odds ratio for waist circumference > 94 cm; 95% confidence interval: 3.14, 1.26 to 7.81), whereas no difference between the two groups was found with regard to biochemical indices of insulin resistance or plasma leptin levels. Discussion: The Gly40Ser polymorphism of the GCG‐R gene is associated with central adiposity independently from total body mass in men.     

Modified (−)-gallocatechin gallate-enriched green tea extract rescues age-related cognitive deficits by restoring hippocampal synaptic plasticity

Aging leads to cognitive impairments characterized by reduced hippocampal functions that are associated with impairment of long-term potentiation of CA1 synapses. Here, we assessed the safety and efficacy of modified (?)-gallocatechin gallate (GCG)-enriched green tea extract (HTP-GTE) in ameliorating the cognitive dysfunctions in late middle-aged murine model. We developed a novel HTP-GTE that was enriched with GCG via epimerization that involved heating. We compared the effects of oral administrations of conventional green tea and HTP-GTE in young and aged male C57/BL6 mice, and examined the changes in the hippocampal functions related to aging process. The functional outcome was assessed by the electrophysiological experiments to measure the long-term potentiation (LTP). HTP-GTE improved the age-related cognitive impairments via restoring long-term synaptic plasticity. We also identified that GCG was the main active component responsible for the HTP-GTE effect. The main molecular pathway in ameliorating the age-related cognitive dysfunctions involved protein kinase A (PKA) which was shown to be modulated by HTP-GTE. Thus, HTP-GTE has a therapeutic potential as a dietary supplement which may aid to rescue the impaired cognitive functions at the early phase of aging process through the modulation of LTP threshold.     

Flavonoid-mediated inhibition of SARS coronavirus 3C-like protease expressed in Pichia pastoris

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome associated coronavirus (SARS-CoV) is vital for SARS-CoV replication and is a promising drug target. Recombinant 3CL(pro) was expressed in Pichia pastoris GS115 as a 42?kDa protein that displayed a K ( m ) of 15?±?2?μM with Dabcyl-KTSAVLQSGFRKME-Edans as substrate. Purified 3CL(pro) was used for inhibition and kinetic assays with seven flavonoid compounds. The IC(50) of six flavonoid compounds were 47-381?μM. Quercetin, epigallocatechin gallate and gallocatechin gallate (GCG) displayed good inhibition toward 3CL(pro) with IC(50) values of 73, 73 and 47?μM, respectively. GCG showed a competitive inhibition pattern with K ( i ) value of 25?±?1.7?μM. In molecular docking experiments, GCG displayed a binding energy of -14?kcal?mol(-1) to the active site of 3CL(pro) and the galloyl moiety at 3-OH position was required for 3CL(pro) inhibition activity.     

A polymorphism in the growth hormone (GH)-releasing hormone (GHRH) receptor gene is associated with elevated response to GHRH by human pituitary somatotrophinomas in vitro

A previous study has suggested that a G to A base change at position 169 of the GHRH-receptor gene in human somatotrophinomas is a mutation and confers hypersensitivity to GHRH. The alternative base converts codon 57 from GCG to AGC, resulting in replacement of alanine (Ala) with threonine (Thr). In the present study, two of five human GH-secreting somatotrophinomas were found to possess the codon 57 AGC sequence. The GCG allele was also detected, indicating heterozygosity. However, the patients' normal blood-derived DNA also yielded the same sequence pattern, indicating that the Ala --> Thr amino acid change is a normal polymorphism, and not a somatic mutation. Nevertheless, in vitro, the tumors possessing the Ala --> Thr amino acid change responded very strongly to GHRH in terms of cAMP formation, being increased 40- and 200-fold, in comparison to the 2-fold increases by tumors without the alternative GHRH-receptor sequence. Likewise, the in vitro response of GH secretion to GHRH was elevated. One of the two tumors with the alternative Thr residue, and the highest responder to GHRH, possessed a gsp mutation, despite the fact that these defects are thought to reduce responsiveness to GHRH. These results fail to confirm that the GCG --> AGC at codon 57 of the GHRH-receptor gene is a mutation, but do support the concept that the alternative form with Thr confers increased sensitivity to GHRH.     

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.     

Treadmill exercise after social isolation increases the levels of NGF,BDNF, and synapsin I to induce survival of neurons in the hippocampus,and improves depression-like behavior

[Purpose]

We investigated the effects of 8 weeks of treadmill exercise on nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and synapsin I protein expression and on the number of 5-bromo-2''-deoxyuridine-5''-mono-phosphate (BrdU)-positive cells in the dentate gyrus of the hippocampus in socially isolated rats. Additionally, we examined the effects of exercise on the number of serotonin (5-HT)- and tryptophan hydroxylase (TPH)-positive cells in the raphe nuclei and on depression behaviors induced by social isolation.

[Methods]

Forty male Sprague-Dawley rats were divided into four groups: (1) group housing and control group (GCG, n = 10); (2) group housing and exercise group (GEG, n = 10); (3) isolated housing and control group (ICG, n = 10); and (4) isolated housing and exercise group (IEG, n = 10). After 1 week of housing under the normal condition of 3 animals per cage, rats were socially isolated via transfer to individual cages for 8 weeks. Rats were then subjected to treadmill exercise for 5 days per week for 8 weeks during which time the speed of the treadmill was gradually increased.

[Results]

Compared to the GCG, levels of NGF, BDNF, and synapsin I were significantly decreased in the ICG and significantly increased in the IEG (p < 0.001 respectively). Significantly more BrdU-positive cells in the GEG were present as compared to the GCG and ICG, and more BrdU-positive cells were found in the IEG as compared to the ICG (p < 0.001). 5-HT-positive cells in the GEG were significantly increased compared to the GCG and ICG, and more of these cells were found in the IEG as compared to the ICG (p < 0.01). TPH-positive cells in the GEG were significantly increased compared to those in the GCG and ICG (p < 0.05). In the forced swim test, immobility time was significantly increased in the ICG and significantly decreased in the IEG as compared to the ICG (p < 0.01).

[Conclusion]

These results showed that regular treadmill exercise following social isolation not only increased the levels of NGF, BDNF, and synapsin I to induce survival of neurons in the hippocampus but also improved depression by increasing the number of serotonergic cells in the raphe nuclei.     

A database of 32 DNA triplets to study triple helices by molecular mechanics and dynamics

We present here a database of 32 deoxyribonucleotide triplets, that can be used as building blocks of triple helix forming deoxyribonucleotides on a computer. This database is made of all the pairing schemes of the triplets ATT, GCC⁺, ATA and GCG where the third base forms two hydrogen bonds with the purine of the first two Watson-Crick strands. The essential features of the known triple helices were preserved in the resulting structures. A triple helix can be easily built from any combination of these basic triplets. Four homogeneous and alternate triple helices thus obtained were studied by molecular mechanics and dynamics in vacuo. The results are in agreement with known experimental observations for ATT and suggest a possible structure for the GCG triple helix. In order to characterize the geometry of the structures obtained, the definitions of nucleic acid structure parameters (R.E. Dickerson et al., EMBO J. 8 (1989) 1–4) have been extended to triple helical polynucleotides.