Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Dopamine D₃ receptors are not involved in the induction of c-<em>fos</em> mRNA by neuroleptic drugs: comparison of the dopamine D₃ receptor antagonist GR103691 with typical and atypical neuroleptics

The effect of acute and chronic administration of dopamine receptor antagonists on the expression of mRNA encoding the cellular immediate-early gene c-fos was investigated in rat brain by in situ hybridization using 35S-labelled oligonucleotide probes. The selective dopamine D3 receptor antagonist GR103691 had no effect on the level of c-fos mRNA after acute or chronic treatment. Acute treatment with haloperidol increased the level of c-fos mRNA in the caudate-putamen, nucleus accumbens shell and core, olfactory tubercle and parietal cortex. After chronic treatment with haloperidol increases in the level of c-fos mRNA in the caudate-putamen and nucleus accumbens core were no longer observed. The increase in the level of c-fos mRNA in the nucleus accumbens shell was attenuated but still significantly elevated above the level measured in vehicle-treated animals. In the olfactory tubercle, parietal cortex, frontal cortex and cingulate cortex the level of c-fos mRNA was decreased after chronic haloperidol treatment. Acute sulpiride treatment reduced the level of c-fos mRNA in the olfactory tubercle, parietal cortex and cingulate cortex. After chronic treatment with sulpiride the level of c-fos mRNA was reduced in the dorsal caudate-putamen only. Acute clozapine treatment increased the level of c-fos mRNA in the nucleus accumbens shell and islands of Calleja. After chronic treatment with clozapine the level of c-fos mRNA remained elevated in the islands of Calleja but not in the nucleus accumbens shell. These results indicate that acute and chronic blockade of dopamine D3 receptors does not cause induction of c-fos transcription in limbic, striatal or cortical regions of rat brain. This study also demonstrated that acute blockade of dopamine receptors with haloperidol, sulpiride and clozapine induced different regionally specific patterns of c-fos expression which were altered after chronic blockade.</p

D3 receptor expression within the basal ganglia is not affected by Parkinson's disease

Quantitative receptor autoradiography and in situ hybridization were used to investigate the expression of dopamine D3 receptors in sections of human brain containing limbic (nucleus accumbens) and striatal (caudate nucleus, putamen) regions. High levels of dopamine D3 receptor mRNA and specific [3H](+/-)7-hydroxy-N,N-di-N-propyl-2-aminotetralin ([3H]7-OH-DPAT) binding sites were detected in the nucleus accumbens with lower levels in the caudate nucleus and putamen. No difference in D3 receptor expression was observed between normal and parkinsonian brain. These results indicate that D3 receptor expression is not altered in Parkinson's disease. In addition, they suggest that dopamine release in striatal and limbic areas is not necessary for maintenance of D3 receptor expression.</p

Effect of chronic treatment with typical and atypical neuroleptics on the expression of dopamine D₂ and D₃ receptors in rat brain

The effect of chronic treatment (21 days) with typical and atypical neuroleptics on the expression of striatal and limbic D2 and D3 dopamine receptors was investigated in rat brain by in situ hybridization and receptor autoradiography. Haloperidol and sulpiride increased D2 receptor expression in striatal and limbic areas. In contrast, clozapine had no effect on D2 receptor expression. Haloperidol decreased D3 receptor expression in limbic areas, with the exception of the islands of Calleja where an increase occurred. Sulpiride and clozapine increased D3 receptor expression in limbic and striatal regions but decreased D3 receptor expression in the islands of Calleja. This study demonstrates that chronic treatment with typical and atypical neuroleptics produces different regionally specific changes in limbic and striatal D2 and D3 receptor expression. The alterations in dopamine receptor expression were different for each drug, but a distinction between the effects of atypical and typical neuroleptics could be made. Comparison of mRNA levels in animals which were not withdrawn from drug treatment with those that were withdrawn, demonstrated that some changes in receptor expression occurred during drug treatment, whilst others only manifested when drug treatment had ceased. The different regulation of dopamine D2 and D3 receptor expression by typical and atypical neuroleptics may have relevance to the ability of these drugs to cause extrapyramidal side-effects.</p

Dopamine D3 receptors in the basal ganglia of the common marmoset and following MPTP and L-DOPA treatment

The distribution of the dopamine D3 receptor was studied by receptor autoradiography using [3H]7-OH-DPAT in striatal and extrastriatal brain regions of the common marmoset (Callithrix jacchus). Saturation studies demonstrated that [3H]7-OH-DPAT bound with similar affinity to different regions of marmoset brain. In normal marmosets, specific [3H]7-OH-DPAT binding was found in both striatal and extrastriatal regions. Very high levels of specific [3H]7-OH-DPAT binding were detected in the islands of Calleja and nucleus accumbens but in addition high levels of binding were detected in rostral caudate nucleus and putamen. In common marmosets treated with the selective nigral neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), the levels of specific [3H]7-OH-DPAT binding in striatal and extrastriatal regions were not different to those in normal animals. Chronic treatment of MPTP-treated marmosets with L-DOPA/ carbidopa did not alter the levels of specific [3H]7-OH-DPAT binding in any brain region. These results demonstrate that in common marmosets D3 receptors are located in both striatal and limbic regions. The receptor density is not altered by dopaminergic denervation or by chronic L-DOPA administration. The D3 receptor may, therefore, be important in both the therapeutic and adverse effects of drugs used to treat Parkinson's disease.</p

Conserved cystatin segments as models for designing specific substrates and inhibitors of cysteine proteinases

Peptide segments derived from consensus sequences of the inhibitory site of cystatins, the natural inhibitors of cysteine proteinases, were used to develop new substrates and inhibitors of papain and rat liver cathepsins B, H, and L. Papain hydrolyzed Abz-QVVAGA-EDDnp and Abz-LVGGA-EDDnp at about the same rate, with specificity constants in the 10(7) M(-1) sec(-1) range; cathepsin L also hydrolyzes both substrates with specificity constants in the 10(5) M(-1) sec(-1) range due to lower k(cat) values, with the K-m's being identical to those with papain. Only Abz-LVGGA-EDDnp was rapidly hydrolyzed by cathepsin B, and to a lesser extent by cathepsin H. Peptide substrates that alternate these two building blocks (LVGGQVVAGAPWK and QVVAGALVGGAPWK) discriminate the activities of cathepsins B and L and papain. Cathepsin L was highly selective for cleavage at the G-G bond of the LVGG fragment in both peptides. Papain and cathepsin B cleaved either the LVGG fragment or the QVVAG fragment, depending an their position within the peptide. While papain was more specific for the segment located C-terminally, cathepsin B was specific for that in N-terminal position. Peptidyl diazomethylketone inhibitors based an these two sequences also reacted differently with papain and cathepsins. GlcA-QVVA-CHN, was a potent inhibitor of papain and reacted with papain 60 times more rapidly (k(+0) = 1,100,000 M(-1) sec(-1)) than with cathepsin L, and 220 times more rapidly than with cathepsin B. Cathepsins B and L were preferentially inhibited by Z-RLVG-CHN2. Thus cystatin-derived peptides provide a valuable framework for designing sensitive, selective substrates and inhibitors of cysteine proteinases.UNIV TOURS,FAC MED,CNRS URA 1334,LAB ENZYMOL & CHIM PROT,F-37032 TOURS,FRANCEESCOLA PAULISTA MED,INFAR,DEPT BIOFIS,BR-04044020 São Paulo,BRAZILCNRS,CTR BIOPHYS MOLEC,LAB BIOCHIM GLYCOCONJUGUES & LECTINES ENDOGENES,F-45071 ORLEANS 2,FRANCEESCOLA PAULISTA MED,INFAR,DEPT BIOFIS,BR-04044020 São Paulo,BRAZILWeb of Scienc