Proteomics of the nervous system

The recent success of large-scale industrialized genomic sequencing opens new doors in studies of biological systems. In the current post-genomic era we must ask how to translate this DNA sequence information into an understanding of living cells, tissues and organisms. One of the major goals is to characterize protein function, biochemical pathways and networks. Achieving this aim is greatly advanced by application of new proteomic tools combined with database mining. Neuroscience in particular is poised to benefit from these approaches in light of its high complexity and cross-talk between different neurotransmitter receptors within the same synapse or across the synaptic cleft. Little is known about the global in vivo protein interactions within synapses, and the knowledge of all proteins present in such structures will help in determining sub-complexes and the modular arrangement of proteins within them. This article reviews the impact of and outlines the application of proteomic analysis in the field of neuroscience, illustrating this with the example of NMDA receptor complexes.     

Immune suppression leading to hepatitis C virus re-emergence after sustained virological response

Sustained virological response SVR is defined as undetectable HCV RNA in plasma 6 months after therapy has been discontinued. Relapse or re-emergence of viremia after SVR is rare. We report two patients that relapsed when immune suppressive therapy was given within a few weeks of achieving SVR. Patient 1 received prednisone for bronchitis and patient 2 relapsed soon after immune suppression was started post renal transplantation. These data suggest that the early phase of SVR might be associated with incomplete protective immunity. They suggest that sterilizing immunity with complete elimination of virus is unlikely. The cases also caution against the use of immune suppressive therapy in the immediate aftermath of SVR.     

Digital image analysis of the distribution of proliferating cell nuclear antigen in hepatitis C virus-related chronic hepatitis,cirrhosis, and hepatocellular carcinoma

Viral dynamic studies in chronic hepatitis C virus (HCV) infection indicate a significantly shortened survival of virus-infected cells. Since at the steady state of chronic viral infection, the rate of infected cell elimination equals new cell regeneration, this would imply a high rate of hepatocyte turnover in chronic HCV liver disease. We estimated the fraction of regenerating hepatocytes in liver biopsy sections in chronic HCV liver disease, cirrhosis, and hepatocellular carcinoma (HCC). We used antibodies to proliferating cell nuclear antigen (PCNA) to detect proliferating cell nuclei in liver biopsy specimen from controls and patients with chronic hepatitis, cirrhosis, and HCC. We also used bis-benzimide to label fluorescently all hepatocyte nuclei simultaneously. Using digital image analysis, we calculated the area occupied by PCNA-stained hepatocyte nuclei, as a fraction of the total area occupied by fluorescently labeled hepatocyte nuclei (labeling index; LI). Antibody staining was negligible in the control specimen. The mean ± SE PCNA LI increased from 0.21 ± 0.1 in chronic hepatitis to 0.63 ± 0.15 in HCC. There was no significant difference between chronic hepatitis and cirrhosis. The fraction of cells undergoing regeneration is increased in chronic HCV liver disease, HCV-related cirrhosis, and HCC. Increased hepatocyte turnover could provide the link between chronic HCV liver disease and HCC.     

Separation of human vitamin K-dependent coagulation proteins using hydrophobic interaction chromatography

A rapid and simple method was developed to separate human vitamin K-dependent plasma proteins from each other, yielding virtually homogeneous pools. The purification technique is based on the single use of hydrophobic interaction chromatography, starting from prothrombin concentrate (PC or DEFIX, also termed factor IX concentrate) as initial material. Phenyl-sepharose HP demonstrated optimal separation by comparing several hydrophobic resins as well as resins used in standard procedures like immobilised heparin and Cibacron blue. Under ideal conditions, factor X could be separated in a single step as well as prothrombin. Factor IX co-eluted with other minor proteins. Focus was given only on these three proteins due to their relative abundance. Complete separation of all proteins present in the starting material was achieved by MonoQ anion-exchange chromatography following the phenyl-sepharose run. The resulting purified material could be demonstrated to be of equal or higher purity than using described methods. This strategy employing hydrophobic interaction chromatography for blood macromolecules could be of immense value for purifying the human vitamin K-dependent proteins and represents a considerable simplification over other purification schemes. It not only involves minimal sample handling but also can be readily up-scaled and is a cost-efficient alternative.     

Concomitant diffuse large B-cell lymphoma and hepatocellular carcinoma in chronic hepatitis C virus liver disease: a study of two cases

Chronic hepatitis C virus HCV infection progresses through liver fibrosis and cirrhosis to hepatocellular carcinoma HCC. It appears to be causally related to B-cell non-Hodgkin's lymphoma since regression after antiviral therapy has been described. Two cases are described of non-Hodgkin's lymphoma and HCC arising simultaneously in two patients. The first patient did not have cirrhosis on liver biopsy. HCV had been undetectable in plasma following successful therapy with interferon and ribavirin treatment 7 years earlier. The second patient developed an aggressive form of hepatocellular carcinoma HCC within weeks of stopping treatment with interferon and ribavirin. Therapy had induced complete viral suppression for over 40 weeks. The two cases suggest that non-Hodgkin's lymphoma and HCC can develop in the absence of detectable hepatitis C viremia and argues for continued surveillance even after sustained virological response to treatment.