De Novo Construction of Redox Active Proteins.

Relatively simple principles can be used to plan and construct de novo proteins that bind redox cofactors and participate in a range of electron-transfer reactions analogous to those seen in natural oxidoreductase proteins. These designed redox proteins are called maquettes. Hydrophobic/hydrophilic binary patterning of heptad repeats of amino acids linked together in a single-chain self-assemble into 4-alpha-helix bundles. These bundles form a robust and adaptable frame for uncovering the default properties of protein embedded cofactors independent of the complexities introduced by generations of natural selection and allow us to better understand what factors can be exploited by man or nature to manipulate the physical chemical properties of these cofactors. Anchoring of redox cofactors such as hemes, light active tetrapyrroles, FeS clusters, and flavins by His and Cys residues allow cofactors to be placed at positions in which electron-tunneling rates between cofactors within or between proteins can be predicted in advance. The modularity of heptad repeat designs facilitates the construction of electron-transfer chains and novel combinations of redox cofactors and new redox cofactor assisted functions. Developing de novo designs that can support cofactor incorporation upon expression in a cell is needed to support a synthetic biology advance that integrates with natural bioenergetic pathways.

Bile duct brushings cytology--improving sensitivity of diagnosis using the ThinPrep technique: a review of 113 cases.

OBJECTIVE: Common bile duct (CBD) brushings have been recognized as a technique of moderate sensitivity and high specificity in identifying carcinoma of the ampulla and pancreatico-biliary regions. This study evaluated the increase in sensitivity of this technique using the ThinPrep technique of specimen preparation when compared with conventional cytology smears. METHODS: A total of 113 bile duct brushings were included in the study (38 conventional smears and 75 slides prepared using the ThinPrep technique). All slides were reviewed by one cytologist. Five categories of reporting were used: inadequate, negative, atypia, suspicious and malignant. RESULTS: The inadequate category of reporting disappeared in the ThinPrep group with improved specimen fixation and preparation and hence reduced artefact. Sensitivity of diagnosis of malignancy increased from 39% in conventional smears to 53% in the ThinPrep group. Specificity, positive and negative predictive values and accuracy were 100%, 100%, 60% and 68% for conventional smears and were 100%, 100%, 60% and 72%, respectively, for ThinPrep specimens. CONCLUSIONS: ThinPrep technique was associated with increased sensitivity of diagnosis, in part due to improved specimen fixation and reduced artefact. Cytology of bile duct brushings is an important diagnostic tool for sites from which it can be difficult to obtain a histology biopsy. It may therefore provide the only opportunity for tissue diagnosis of carcinoma from these sites, hence the importance of optimizing sensitivity.

Dual polarisation interferometry characterisation of DNA immobilisation and hybridisation detection on a silanised support.

Dual polarisation interferometry is an analytical technique that allows the simultaneous determination of thickness, density and mass of a biological layer on a sensing waveguide surface in real time. We evaluated, for the first time, the ability of this technique to characterise the covalent immobilisation of single stranded probe DNA and the selective detection of target DNA hybridisation on a silanised support. Two immobilisation strategies have been evaluated: direct attachment of the probe molecule and a more complex chemistry employing a 1,2 homobifunctional crosslinker molecule. With this technique we demonstrate it was possible to determine probe orientation and measure probe coverage at different stages of the immobilisation process in real time and in a single experiment. In addition, by measuring simultaneously changes in thickness and density of the probe layer upon hybridisation of target DNA, it was possible to directly elucidate the impact that probe mobility had on hybridisation efficiency. Direct covalent attachment of an amine modified 19 mer resulted in a thickness change of 0.68 nm that was consistent with multipoint attachment of the probe molecule to the surface. Blocking with BSA formed a dense layer of protein molecules that absorbed between the probe molecules on the surface. The observed hybridisation efficiency to target DNA was approximately 35%. No further significant reorientation of the probe molecule occurred upon hybridisation. The initial thickness of the probe layer upon attachment to the crosslinker molecule was 0.5 nm. Significant reorientation of the probe molecule surface normal occurred upon hybridisation to target DNA. This indicated that the probe molecule had greater mobility to hybridise to target DNA. The observed hybridisation efficiency for target DNA was approximately 85%. The results show that a probe molecule attached to the surface via a crosslinker group is better able to hybridise to target DNA due to its greater mobility.

Identification and characterization of a lysis module present in a large proportion of bacteriophages infecting Streptococcus thermophilus.

A lysis module encoded by the temperate bacteriophage phiO1205 was identified. This lysis module contains a lysin gene, designated lyt51, and two putative holin-encoding genes, designated lyt49 and lyt50. lyt51 encodes a lytic enzyme specifically directed against streptococcal cell walls. Similar to other phage-encoded lysins, Lyt51 appears to have a modular design in which the N-terminal portion corresponds to its enzymatic activity while the C-terminal region is responsible for its substrate binding specificity. The two putative holin-encoding genes, lyt49 and lyt50, located immediately upstream of lyt51, were identified on the basis of their homology to other identified holin-encoding genes. Expression of lyt49 or lyt50 in Escherichia coli was shown to cause cell death and leakage of the intracellular enzyme isocitrate dehydrogenase into the growth medium without apparent lysis of the cells. Southern blotting experiments demonstrated that at least one of the three components of the identified lysis module is present in all members of a large collection of bacteriophages, indicating that components of this lysis module are widespread among bacteriophages infecting Streptococcus thermophilus.

Detection of cytomegalovirus (CMV) in HIV+ patients: comparison of cytomorphology, immunocytochemistry and in situ hybridization.

CMV is regarded as an important pathogen in immunocompromised patients. This study compares three cytological methods of diagnosis of CMV in alcohol-fixed smears from bronchoalveolar lavage (BAL) specimens from 40 HIV+ patients, using cytomorphology (CM), immunocytochemical staining (ICC) and in situ hybridization (ISH). The results of CM are compared with virological detection methods using the detection of early fluorescent foci (DEAFF) 48-h culture technique and virus isolation studies (VISO 1). ICC was the most sensitive technique, identifying CMV in 13 cases, six of which were also positive on ISH. Cytomorphology was the least sensitive, with only one case showing diagnostic features of CMV cytopathic effect. One additional case showed morphological features suggesting viral infection but not specific for CMV. Both of these cases were confirmed by ICC and ISH. Virology studies identified CMV in all 13 cases and in an additional five cases. ICC detected two cases which were negative on the DEAFF test but which were later detectable by the VISO 1 technique. These findings support the usefulness of ISH and ICC in confirming CMV in cases where the infection was suspected on cytomorphological features. ISH and ICC also increase the detection of CMV in BAL smears not showing morphological features on CM.

The lytic enzyme of the pneumococcal phage Dp-1: a chimeric lysin of intergeneric origin.

We have localized, cloned and characterized the genes coding for the lytic system of the pneumococcal phage Dp-1. The lytic enzyme of this phage (Pal), previously identified as an N-acetyl-muramoyl-L-alanine amidase, shows a modular organization similar to that described for the lytic enzymes of Streptococcus pneumoniae and its bacteriophages. The construction of chimeric enzymes between pneumococcus and bacteria (or phages) that belong to different Gram-positive families has shown that the interchange of functional domains switches enzyme specificity. Interestingly, Pal appears to be a natural chimeric enzyme of intergeneric origin, that is the N-terminal domain was highly similar to that of the murein hydrolase coded by a gene found in the phage BK5-T that infects Lactococcus lactis, whereas the C-terminal domain was homologous to those found in the lytic enzymes of the pneumococcal system that is responsible for the binding to the choline residues present in the cell wall substrate. Biochemical analysis of Pal revealed that this enzyme shares important properties with those of the major LytA101 autolysin found in an atypical, clinical pneumococcal isolate. These peculiar characteristics have been ascribed to a modified C-terminal domain. The natural chimeric enzyme described here provides further support for the theory of modular evolution of proteins and its characteristics also furnish interesting clues on the molecular mechanisms involved in the more invasive types of atypical pneumococci.

Hepatitis C virus liver disease in women infected with contaminated anti-D immunoglobulin.

Screening for hepatitis C virus (HCV) infection is carried out by detection of antibodies to the virus (enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA)) with confirmation by identification of HCV RNA genome in serum (polymerase chain reaction (PCR)). We describe the histological features on liver biopsy in 88 women with chronic HCV infection (serum positive on ELISA, RIBA and PCR) acquired from virus contaminated anti-D immunoglobulin. For the majority of these patients the time interval from virus infection to presentation was between 17 and 18 years. We separately assessed necroinflammatory disease activity and architectural features on liver biopsy and applied a scoring system which permitted semi-quantitative documentation of abnormal features. Only three women showed liver biopsies within normal limits (+/-focal steatosis). The remaining 85 cases showed a predominantly mild or moderate degree of disease activity with interface hepatitis (56.8% of cases), spotty necrosis, apoptosis and focal inflammation (88.6% of cases) and portal inflammation (90.9% of cases). Confluent necrosis was an uncommon finding (2.3% of cases). Assessment of architectural features showed normal appearance in 35.2% of biopsies. The predominant architectural abnormality noted was portal tract fibrosis. Ten per cent of cases, however, showed significant fibrous band and/or nodule formation.

Analysis of the catalytic domain of the lysin of the lactococcal bacteriophage Tuc2009 by chimeric gene assembling.

An active chimeric cell wall lytic enzyme (Tsl) has been constructed by fusing the region coding for the N-terminal half of the lactococcal phage Tuc2009 lysin and the region coding for the C-terminal domain of the major pneumococcal autolysin. The chimeric enzyme exhibited a glycosidase activity capable of hydrolysing choline-containing pneumococcal cell walls. This experimental approach demonstrated that the Tuc2009 lysin possesses a modular structure and further supports the hypothesis that many cell wall lytic enzymes have evolved by the fusion of preexisting catalytic and peptidoglycan-binding domains.

Fatal vertebral giant cell arteritis.

Death due to giant cell arteritis (GCA) is rare, and is usually caused by coronary or vertebral arteritis in the acute phase of the disease. A case of fatal GCA is reported in a woman with a normal erythrocyte sedimentation rate, who had been treated for temporal arteritis for eight months. Post mortem examination showed a dissection and thrombosis of the intracranial portion of the left vertebral artery caused by giant cell arteritis. Focal coronary artery GCA was also found. As far as is known, this is the only case in which dissection of the vertebral artery attributable to GCA has been reported.

Placental chorioangiosis associated with foetal death--a case report.

A case of foetal death with retroplacental haemorrhage at 24 weeks gestation is reported, where the placenta histologically showed diffuse severe chorioangiosis. The latter is an infrequently reported placental vascular anomaly, which recent studies have indicated to be an important sign of neonatal morbidity and mortality.

The CNS-PNS transitional zone of rat cervical dorsal roots during development and at maturity. A morphological and morphometric study.

Each seventh cervical dorsal nerve root is attached to the spinal cord surface by four to eight rootlets. A tapering outgrowth of central nervous tissue, the central tissue projection, extends distally into the proximal part of each rootlet in the immediate postnatal period. The central ends of the most proximal peripheral internodes surround this projection. Thus a length of rootlet contains both CNS and PNS tissue. This is termed the transitional zone. Material was processed by standard preparative techniques for electron microscopy. Serial semithin and ultrathin sections were made over the entire extent of several transitional zones at ages ranging from 2 to 300 days postnatum. Central tissue projections were reconstructed in three dimensions and analysed morphometrically. The morphology of the central tissue projection varies during development. At first, it forms an irregular projection into the anterior portion of the rootlet. It than elongates and takes the form of a dorsoventrally flattened, distally tapering wedge. By 20 days postnatum it has attained its definitive form. This consists of three segments: a proximal wedge-shaped portion, similar to that described above; continuous with this is a distally tapering, dorsoventrally flattened, cone-shaped segment which generally branches into two or more slender projections of central tissue. The latter comprise the third segment. The projection comes to form a substantial proportion of the anterior, proximal and distal surfaces of the dorsal rootlet from an early stage. The mean length of the central tissue projection increases progressively over all intervals studied, except that between 12 and 30 days postnatum, when a reduction in length is associated with reorganisation of the morphology of the projection. Projection length varies considerably between rootlets and is relatively weakly correlated with rootlet cross sectional area. There is a great deal of overlap between the distributions of projection lengths at all stages between 20 and 300 days.

Dental anaesthesia for a child with complete amelia.

A four year-old boy born without limbs (amelia) presented for dental restorations under general anaesthesia as an outpatient. Following intramuscular atropine administration anaesthesia was induced using halothane, oxygen and nitrous oxide inhaled by mask. Next, intravenous access was secured by external jugular vein catheterization. Because of his small mouth, hypognathic mandible, arched palate and anterior-superiorly located larynx, oral intubation under deep anaesthesia during spontaneous ventilation was difficult. The epiglottis was noted to be inverted on subsequent laryngoscopic inspection after intubation but was reduced mechanically to anatomic position. Despite being unable to accurately monitor the blood pressure the intraoperative period was uneventful. Postoperatively the patient was extubated and was able to return home the same day.