The radioprotection management of a PET department with a cyclotron and radiopharmacy laboratory, in accordance with Italian legislation.

The possibility of setting up a positron emission tomography (PET) facility with a cyclotron and radiopharmaceutical laboratory in situ, at a feasible price and in a very restricted space, has led to a steady increase both in the use of the PET technique in diagnostic clinical routine imaging and in the number of cyclotrons for drug production. Owing to the progress made in the PET procedures, it is now possible to have not only a highly innovative system of diagnostic examination, with a remarkable improvement in the diagnostic quality and patient care, but also a considerable increase in the number of daily examinations. In this paper, the authors show how the acquired know-how, with respect to radioprotection, has applied to the planning, running and management of the PET/CT unit, installed in the Imaging Diagnostic Department of the Policlinico Tor Vergata (PTV), at Tor Vergata University, Rome.

Kinetic basis for activation of CDK2/cyclin A by phosphorylation.

The activation of most protein kinases requires phosphorylation at a conserved site within a structurally defined segment termed the activation loop. A classic example is the regulation of the cell cycle control enzyme, CDK2/cyclin A, in which catalytic activation depends on phosphorylation at Thr(160) in CDK2. The structural consequences of phosphorylation have been revealed by x-ray crystallographic studies on CDK2/cyclin A and include changes in conformation, mainly of the activation loop. Here, we describe the kinetic basis for activation by phosphorylation in CDK2/cyclin A. Phosphorylation results in a 100,000-fold increase in catalytic efficiency and an approximate 1,000-fold increase in the overall turnover rate. The effects of phosphorylation on the individual steps in the catalytic reaction pathway were determined using solvent viscosometric techniques. It was found that the increase in catalytic power arises mainly from a 3,000-fold increase in the rate of the phosphoryl group transfer step with a more moderate increase in substrate binding affinity. In contrast, the rate of phosphoryl group transfer in the ATPase pathway was unaffected by phosphorylation, demonstrating that phosphorylation at Thr(160) does not serve to stabilize ATP in the ATPase reaction. Thus, we hypothesize that the role of phosphorylation in the kinase reaction may be to specifically stabilize the peptide phosphoacceptor group.

Polysubstance abuse and traumatic brain injury: quantitative magnetic resonance imaging and neuropsychological outcome in older adolescents and young adults.

Few studies have examined the consequences of alcohol and drug abuse on TBI though they commonly co-occur. Both TBI and substance abuse independently result in neuropathological changes in the brain such as ventricular enlargement and cortical atrophy, thus it is reasonable to hypothesize that the combination of the two would result in more significant cerebral damage. In this study, 3 groups of patients--traumatically brain injured (TBI) with substance abuse (N = 19), TBI without substance abuse (N = 19), and substance abuse with no TBI (N = 16)--were compared with normal controls (N = 20) on several quantitative MRI (QMRI) measures. Since TBI most frequently occurs in older adolescents and young men, we examined only male participants between 16 and 30 years of age. Comparing young substance abusers to controls resulted in no QMRI differences. When controlling for head injury severity, the effects of substance abuse in combination with TBI resulted in greater atrophic changes than seen in any other group. TBI and substance abuse patients' neuropsychological test performances also were examined, and no differences were found among patient groups on any measures. These findings have implications for the deleterious interaction of substance abuse combining with TBI to result in greater neuropathological changes that can be detected by QMRI techniques.

[Multicenter study on the prevalence of hepatitis C virus infection in blood donors in the city of Curitiba, Brazil]. / Estudo multicêntrico da prevalência de infecção pelo vírus da hepatite C em doadores de sangue na cidade de Curitiba, Brasil.

In order to check the presence of anti-HCV in the blood of voluntary blood donors in four of the most active blood banks of Curitiba, PR, Brazil a third generation immunoassay (Murex--version III, ABBOTT AxSYM HCV version 3.0) was used. The study detected 347 positive cases, i.e. a median prevalence of 0.80% (range 0.64%-1.11%). Only 44 from the 347 anti-HCV positive blood donors had elevated values of serum aminotransferases (12.68%). Such low values of ALT suggest that the confirmatory RIBA should be applied to the remnant 303 anti-HCV positive cases (87.32%) which did not show elevation of ALT, with the expectation that 30% to 40% of them might be positive. The prevalence of anti-HCV in blood donors in Curitiba seems similar to published data in the Brazilian medical literature.

In vivo function of Hsp90 is dependent on ATP binding and ATP hydrolysis.

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

Structural basis for inhibition of the cyclin-dependent kinase Cdk6 by the tumour suppressor p16INK4a.

The cyclin-dependent kinases 4 and 6 (Cdk4/6) that control the G1 phase of the cell cycle and their inhibitor, the p16INK4a tumour suppressor, have a central role in cell proliferation and in tumorigenesis. The structures of Cdk6 bound to p16INK4a and to the related p19INK4d reveal that the INK4 inhibitors bind next to the ATP-binding site of the catalytic cleft, opposite where the activating cyclin subunit binds. They prevent cyclin binding indirectly by causing structural changes that propagate to the cyclin-binding site. The INK4 inhibitors also distort the kinase catalytic cleft and interfere with ATP binding, which explains how they can inhibit the preassembled Cdk4/6-cyclin D complexes as well. Tumour-derived mutations in INK4a and Cdk4 map to interface contacts, solidifying the role of CDK binding and inhibition in the tumour suppressor activity of p16INK4a.

Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities.

Cell cycle progression is controlled by the sequential functions of cyclin-dependent kinases (cdks). Cdk activation requires phosphorylation of a key residue (on sites equivalent to Thr-160 in human cdk2) carried out by the cdk-activating kinase (CAK). Human CAK has been identified as a p40(MO15)/cyclin H/MAT1 complex that also functions as part of transcription factor IIH (TFIIH) where it phosphorylates multiple transcriptional components including the C-terminal domain (CTD) of the large subunit of RNA polymerase II. In contrast, CAK from budding yeast consists of a single polypeptide (Cak1p), is not a component of TFIIH, and lacks CTD kinase activity. Here we report that Cak1p and p40(MO15) have strikingly different substrate specificities. Cak1p preferentially phosphorylated monomeric cdks, whereas p40(MO15) preferentially phosphorylated cdk/cyclin complexes. Furthermore, p40(MO15) only phosphorylated cdk6 bound to cyclin D3, whereas Cak1p recognized monomeric cdk6 and cdk6 bound to cyclin D1, D2, or D3. We also found that cdk inhibitors, including p21(CIP1), p27(KIP1), p57(KIP2), p16(INK4a), and p18(INK4c), could block phosphorylation by p40(MO15) but not phosphorylation by Cak1p. Our results demonstrate that although both Cak1p and p40(MO15) activate cdks by phosphorylating the same residue, the structural mechanisms underlying the enzyme-substrate recognition differ greatly. Structural and physiological implications of these findings will be discussed.

Structure of the retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV E7.

The pocket domain of the retinoblastoma (Rb) tumour suppressor is central to Rb function, and is frequently inactivated by the binding of the human papilloma virus E7 oncoprotein in cervical cancer. The crystal structure of the Rb pocket bound to a nine-residue E7 peptide containing the LxCxE motif, shared by other Rb-binding viral and cellular proteins, shows that the LxCxE peptide binds a highly conserved groove on the B-box portion of the pocket; the A-box portion appears to be required for the stable folding of the B box. Also highly conserved is the extensive A-B interface, suggesting that it may be an additional protein-binding site. The A and B boxes each contain the cyclin-fold structural motif, with the LxCxE-binding site on the B-box cyclin fold being similar to a Cdk2-binding site of cyclin A and to a TBP-binding site of TFIIB.

Crystal structure of an Hsp90-geldanamycin complex: targeting of a protein chaperone by an antitumor agent.

The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.

Structural basis of cyclin-dependent kinase activation by phosphorylation.

Cyclin-dependent kinase (CDK)-cyclin complexes require phosphorylation on the CDK subunit for full activation of their Ser/Thr protein kinase activity. The crystal structure of the phosphorylated CDK2-CyclinA-ATP gamma S complex has been determined at 2.6 A resolution. The phosphate group, which is on the regulatory T-loop of CDK2, is mostly buried, its charge being neutralized by three Arg side chains. The arginines help extend the influence of the phosphate group through a network of hydrogen bonds to both CDK2 and cyclinA. Comparison with the unphosphorylated CDK2-CyclinA complex shows that the T-loop moves by as much as 7 A, and this affects the putative substrate binding site as well as resulting in additional CDK2-CyclinA contacts. The phosphate group thus acts as a major organizing centre in the CDK2-CyclinA complex.

Crystal structure of the p27Kip1 cyclin-dependent-kinase inhibitor bound to the cyclin A-Cdk2 complex.

The crystal structure of the human p27Kip1 kinase inhibitory domain bound to the phosphorylated cyclin A-cyclin-dependent kinase 2 (Cdk2) complex has been determined at 2.3 angstrom. p27Kip1 binds the complex as an extended structure interacting with both cyclin A and Cdk2. On cyclin A, it binds in a groove formed by conserved cyclin box residues. On Cdk2, it binds and rearranges the amino-terminal lobe and also inserts into the catalytic cleft, mimicking ATP.

Traumatic brain injury, alcohol and quantitative neuroimaging: preliminary findings.

Magnetic resonance (MR) quantitative neuroimaging analysis was undertaken with a large group of normal (n = 197) and traumatically brain injured (TBI, n = 99) adults. Of the TBI subjects 18 patients were identified with a history of substance-related abuse (TBI/Abuse group). Both the TBI/ Abuse group and the remaining sample of TBI patients (n = 81, TBI/Non-abuse group) without a history of substance-related abuse differed significantly from the control group on most quantitative MR imaging analyses. The TBI/Abuse group displayed the greatest degree of atrophic change. However, the TBI/Abuse group had a significantly lower Glasgow Coma Scale (GCS) score, ostensibly suggesting that those with substance-related abuse suffered more severe brain injury than non-abuse TBI patients. When a subset (n = 18) of the TBI/Non-abuse group was matched by GCS, gender and age to the TBI/Abuse group, both groups differed significantly from the control group on most morphometric measures, but did not differ from one another. Results are discussed in terms of the potential adverse role that substance-related abuse, particularly alcohol, plays in the individual who sustains traumatic injury to the brain.

Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex.

The crystal structure of the human cyclinA-cyclin-dependent kinase2 (CDK2)-ATP complex has been determined at 2.3 A resolution. CyclinA binds to one side of CDK2's catalytic cleft, inducing large conformational changes in its PSTAIRE helix and T-loop. These changes activate the kinase by realigning active site residues and relieving the steric blockade at the entrance of the catalytic cleft.

Risk-taking behaviors and AIDS knowledge: experiences and beliefs of minority adolescent mothers.

Using a qualitative focus-group methodology, this study investigated risk-taking behaviors and AIDS knowledge among minority pregnant and parenting adolescents at risk for heterosexual and perinatal transmission of HIV. Seven focus groups were conducted with a total of 48 young women recruited from alternative schools and residential facilities for pregnant adolescents and young mothers in Southern California. Participants also completed a background questionnaire soliciting sociodemographic information and an AIDS knowledge test. The sample included 33 Latinas and 15 African-Americans, ranging in age from 12 to 19 years. There were bipolar findings regarding risk-taking behaviors. At one end of the continuum were young women with a history of one of more of the following behaviors: multiple sex partners, drug and alcohol use, carrying weapons, and participating in gang-related activities. Contrasting with these, were those who had one or two sex partners and no history of alcohol or drug abuse. A majority of the participants were having unprotected sex. A variety of factors affected condom use, including gender inequality, embarrassment, and personal preferences and values. Risk-taking was also influenced by lack of security and safety in daily living, emotion-focused coping and peer pressure.

The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication.

DNA polymerase alpha is the only enzyme in eukaryotic cells capable of starting DNA chains de novo and is required for the initiation of SV40 DNA replication in vitro. We have cloned the 70 kDa subunit of human DNA polymerase alpha (hereafter referred to as the B subunit) and expressed it as a fusion protein in bacteria. The purified fusion protein forms a stable complex with SV40 T antigen, both in solution and when T antigen is bound to the SV40 origin of DNA replication. Analysis of mutant forms of the B subunit indicates that the N-terminal 240 amino acids are sufficient to mediate complex formation. The B subunit fusion protein promotes formation of a complex containing T antigen and the catalytic subunit (subunit A) of DNA polymerase alpha, suggesting that it serves to tether the two proteins. These physical interactions are functionally significant, since the ability of T antigen to stimulate the activity of the catalytic subunit of DNA polymerase alpha is highly dependent upon the B subunit. We suggest that the interactions mediated by the B subunit play an important role in SV40 DNA replication by promoting DNA chain initiation at the origin and/or facilitating the subsequent priming and synthesis of DNA chains on the lagging strand template. The protein may play similar roles in cellular DNA replication.

Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding.

A mutant simian virus 40 (SV40) large tumor (T) antigen bearing alanine instead of threonine at residue 124 (T124A) failed to replicate SV40 DNA in infected monkey cells (J. Schneider and E. Fanning, J. Virol. 62:1598-1605, 1988). We investigated the biochemical properties of T124A T antigen in greater detail by using purified protein from a baculovirus expression system. Purified T124A is defective in SV40 DNA replication in vitro, but does bind specifically to the viral origin under the conditions normally used for DNA replication. The mutant protein forms double-hexamer complexes at the origin in an ATP-dependent fashion, although the binding reaction requires somewhat higher protein concentrations than the wild-type protein. Binding of T124A protein results in local distortion of the origin DNA similar to that observed with the wild-type protein. These findings indicate that the replication defect of T124A protein is not due to failure to recognize and occupy the origin. Under some conditions T124A is capable of unwinding short origin DNA fragments. However, the mutant protein is almost completely defective in unwinding of circular plasmid DNA molecules containing the SV40 origin. Since the helicase activity of T124A is essentially identical to that of the wild-type protein, we conclude that the mutant is defective in the initial opening of the duplex at the origin, possibly as a result of altered hexamer-hexamer interactions. The phenotype of T124A suggests a possible role for phosphorylation of threonine 124 by cyclin-dependent kinases in controlling the origin unwinding activity of T antigen in infected cells.

Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A.

The catalytic subunit of protein phosphatase 2A (PP2Ac) stimulates the initiation of replication of simian virus 40 DNA in vitro by dephosphorylating T antigen at specific phosphoserine residues (K. H. Scheidtmann, D. M. Virshup, and T. J. Kelly, J. Virol. 65:2098-2101, 1991). To better define the biochemical mechanism responsible for this stimulation, we investigated the effect of PP2Ac on the interaction of T antigen with wild-type and mutant origins of replication. Analysis of the binding of T antigen to the wild-type origin as a function of protein concentration revealed that binding occurs in two relatively discrete steps: the assembly of a T-antigen hexamer on one half-site of the origin, followed by the assembly of the second hexamer on the other half-site. The major effect of PP2Ac was to stimulate binding of the second hexamer, so that the binding reaction became much more cooperative. This observation suggests that dephosphorylation of T antigen by PP2Ac primarily affects interactions between the two hexamers bound to the origin. Pretreatment with PP2Ac increased the ability of the bound T antigen to unwind the origin of replication but had no effect on the intrinsic helicase activity of the protein. Thus, dephosphorylation of PP2Ac appears to increase the efficiency of the initial opening of the origin by T antigen. An insertion mutation at the dyad axis in the simian virus 40 origin, which altered the structural relationship of the two halves of the origin, abolished the effect of the phosphatase on the cooperativity of binding and completely prevented origin unwinding. These findings suggest that the ability of T antigen to open the viral origin of DNA replication is critically dependent on the appropriate functional interactions between T-antigen hexamers and that these interactions are regulated by the phosphorylation state of the viral initiator protein.