Increased accumulation of the glycoxidation product Nepsilon-(carboxymethyl)lysine in hearts of diabetic patients: generation and characterisation of a monoclonal anti-CML antibody

Heart failure is a condition closely linked to diabetes. Hyperglycaemia amplifies the generation of a major advanced glycation end product Nepsilon-(carboxymethyl)lysine (CML), which has been associated with the development of vascular and inflammatory complications. An increased accumulation of CML in hearts of diabetic patients may be one of the mechanisms related to the high risk of heart failure. Therefore, we investigated the localization of CML in diabetic hearts. To investigate the presence and accumulation of CML in tissues, a monoclonal anti-CML antibody was generated and characterised. With this novel monoclonal antibody against CML, the localization of CML was investigated by immunohistochemistry, in heart tissue of controls (n = 9) and heart tissue of diabetic patients (n = 8) without signs of inflammation or infarction. In addition, in the same subjects we studied the presence of CML in renal and lung tissues. CML staining was approximately sixfold higher in hearts from diabetic patients as compared to control hearts (2.0 +/- 0.3 and 0.3 +/- 0.2 A.U., respectively, P < 0.01). CML deposition was localized in the small intramyocardial arteries in endothelial cells and smooth muscle cells, but not in cardiomyocytes. These arteries did not show morphological abnormalities. The intensity of staining between arteries at the epicardial, midcardial and endocardial side did not vary significantly within patients. In renal tissues, CML staining was most prominent in tubules and in atherosclerotic vessels, without differences in intensity between controls and diabetic patients. In non-infected lungs, no CML was detected. In conclusion, CML adducts are abundantly present in small intramyocardial arteries in the heart tissue of diabetic patients. The accumulation of CML in diabetic hearts may contribute to the increased risk of heart failure in hyperglycaemia.     

The cytomegalovirus DNA polymerase subunit UL44 forms a C clamp-shaped dimer

The human cytomegalovirus DNA polymerase consists of a catalytic subunit, UL54, and a presumed processivity factor, UL44. We have solved the crystal structure of residues 1-290 of UL44 to 1.85 A resolution by multiwavelength anomalous dispersion. The structure reveals a dimer of UL44 in the shape of a C clamp. Each monomer of UL44 shares its overall fold with other processivity factors, including herpes simplex virus UL42, which is a monomer that binds DNA directly, and the sliding clamp, PCNA, which is a trimer that surrounds DNA, although these proteins share no obvious sequence homology. Analytical ultracentrifugation and gel filtration measurements demonstrated that UL44 also forms a dimer in solution, and substitution of large hydrophobic residues along the homodimer interface with alanine disrupted dimerization and decreased DNA binding. UL44 represents a hybrid processivity factor as it binds DNA directly like UL42, but forms a C clamp that may surround DNA like PCNA.     

Linear diffusion on DNA despite high-affinity binding by a DNA polymerase processivity factor.

The oligomeric "sliding clamp" processivity factors, such as PCNA, are thought to rely on a loose, topological association with DNA to slide freely along dsDNA. Unlike PCNA, the processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer and has an intrinsic affinity for dsDNA that is remarkably high for a sequence-independent DNA binding protein. Using a DNase footprinting assay, we demonstrate that UL42 translocates with the catalytic subunit of the polymerase during chain elongation. In addition, footprinting and electrophoretic mobility shift assays show that, despite its tight DNA binding, UL42 is capable of linear diffusion on DNA at a rate of between 17 and 47 bp/s. Our results thus suggest that, despite profound biochemical differences with the sliding clamps, UL42 can freely slide downstream with the catalytic subunit during DNA replication.     

The Pre-NH2-Terminal Domain of the Herpes Simplex Virus 1 DNA Polymerase Catalytic Subunit Is Required for Efficient Viral Replication

The catalytic subunit of herpes simplex virus 1 DNA polymerase (HSV-1 Pol) has been extensively studied; however, its full complement of functional domains has yet to be characterized. A crystal structure has revealed a previously uncharacterized pre-NH₂-terminal domain (residues 1 to 140) within HSV-1 Pol. Due to the conservation of the pre-NH₂-terminal domain within the herpesvirus Pol family and its location in the crystal structure, we hypothesized that this domain provides an important function during viral replication in the infected cell distinct from 5′-3′ polymerase activity. We identified three pre-NH₂-terminal Pol mutants that exhibited 5′-3′ polymerase activity indistinguishable from that of wild-type Pol in vitro: deletion mutants PolΔN43 and PolΔN52 that lack the extreme N-terminal 42 and 51 residues, respectively, and mutant PolA₆, in which a conserved motif at residues 44 to 49 was replaced with alanines. We constructed the corresponding pol mutant viruses and found that the polΔN43 mutant displayed replication kinetics similar to those of wild-type virus, while polΔN52 and polA₆ mutant virus infection resulted in an 8-fold defect in viral yield compared to that achieved with wild type and their respective rescued derivative viruses. Additionally, both polΔN52 and polA₆ viruses exhibited defects in viral DNA synthesis that correlated with the observed reduction in viral yield. These results strongly indicate that the conserved motif within the pre-NH₂-terminal domain is important for viral DNA synthesis and production of infectious virus and indicate a functional role for this domain.     

Quantification and analysis of thymidine kinase expression from acyclovir-resistant G-string insertion and deletion mutants in herpes simplex virus-infected cells

To be clinically relevant, drug-resistant mutants must both evade drug action and retain pathogenicity. Many acyclovir-resistant herpes simplex virus mutants from clinical isolates have one or two base insertions (G8 and G9) or one base deletion (G6) in a homopolymeric run of seven guanines (G string) in the gene encoding thymidine kinase (TK). Nevertheless, G8 and G9 mutants express detectable TK activity and can reactivate from latency in mice, a pathogenicity marker. On the basis of studies using cell-free systems, ribosomal frameshifting can explain this ability to express TK. To investigate frameshifting in infected cells, we constructed viruses that express epitope-tagged versions of wild-type and mutant TKs. We measured TK activity by plaque autoradiography and expression of frameshifted and unframeshifted TK polypeptides using a very sensitive immunoprecipitation-Western blotting method. The G6 mutant expressed ～0.01% of wild-type levels of TK polypeptide. For the G9 mutant, consistent with previous results, much TK expression could be ascribed to reversion. For the G8 mutant, from these assays and pulse-labeling studies, we determined the ratio of synthesis of frameshifted to unframeshifted polypeptides to be 1:100. The effects of stop codons before or after the G string argue that frameshifting can initiate within the first six guanines. However, frameshifting efficiency was altered by stop codons downstream of the string in the 0 frame. The G8 mutant expressed only 0.1% of the wild-type level of full-length TK, considerably lower than estimated previously. Thus, remarkably low levels of TK are sufficient for reactivation from latency in mice.     

Historical ecology with real numbers: past and present extent and biomass of an imperilled estuarine habitat

Historic baselines are important in developing our understanding of ecosystems in the face of rapid global change. While a number of studies have sought to determine changes in extent of exploited habitats over historic timescales, few have quantified such changes prior to late twentieth century baselines. Here, we present, to our knowledge, the first ever large-scale quantitative assessment of the extent and biomass of marine habitat-forming species over a 100-year time frame. We examined records of wild native oyster abundance in the United States from a historic, yet already exploited, baseline between 1878 and 1935 (predominantly 1885-1915), and a current baseline between 1968 and 2010 (predominantly 2000-2010). We quantified the extent of oyster grounds in 39 estuaries historically and 51 estuaries from recent times. Data from 24 estuaries allowed comparison of historic to present extent and biomass. We found evidence for a 64 per cent decline in the spatial extent of oyster habitat and an 88 per cent decline in oyster biomass over time. The difference between these two numbers illustrates that current areal extent measures may be masking significant loss of habitat through degradation.