A Rosenthal fiber encephalomyelopathy resembling Alexander's disease in 3 sheep.

We report an encephalomyelopathy in three 18-month-old Merino sheep with features of adult-onset Alexander's disease (AD), a human primary astrocytic disorder. The signature histologic finding was the presence of numerous hypereosinophilic, intra-astrocytic inclusions (Rosenthal fibers), mainly in perivascular, subpial, and subependymal sites, especially in the caudal brain stem and spinal cord. Although AD usually results from mutations in the glial fibrillary acidic protein (GFAP) gene, no such mutation was detected in these sheep. However, the annual clinical presentation of this disorder in a few sheep in the affected flock is suggestive of a familial pattern of occurrence.

Serous carcinoma of the uterus-determination of HER-2/neu status using immunohistochemistry, chromogenic in situ hybridization, and quantitative polymerase chain reaction techniques: its significance and clinical correlation.

Uterine serous papillary carcinoma (USPC) are high-grade tumors with Her2 gene expression and poor prognosis. The human gene Her2 is a proto-oncogene that encodes a protein with tyrosine kinase activity. The objective of this study was to determine Her2 protein expression and gene amplification in USPC using three methods: immunohistochemistry (IHC), chromogenic in situ hybridization (CISH), and quantitative polymerase chain reaction (Q-PCR), to compare the three techniques, and to correlate Her2 expression and amplification with clinical outcome. Clinical data were obtained from the records of the patients provided by the database of the Gynaecological Cancer Unit at the Royal Adelaide Hospital. Paraffin-embedded tissues of 45 cases were examined using three techniques. Her2 positive rate was 40%. About 13% was strongly positive by all three methods. About 67% Her2 positive patients had advanced-stage disease. Relapse rate was 61% (P = 0.6). Stages I and II had a better survival with negative receptor. Age and stage were major prognostic variables in Cox analysis. Marker status did not reach statistical significance in overall survival (OS) and relapse-free survival (RFS), but had a hazard ratio (HR) of 1.5 in RFS. Five-year OS with Her2 negative was 39%. HR was 0.97 (95% CI 0.46-2.1). RFS was 39% and HR was 1.4 (95% CI 0.65-2.9). The three methods have strong correlation. IHC, 3+ positive cases should be regarded as exhibiting evidence of gene amplification and do not require further testing. Equivocal results require further testing by CISH or PCR. Age and stage are strong prognostic variables and receptor status has a HR of 1.5 in RFS. The therapeutic role of Trastuzumab should be tested in clinical trial setting.

Structural basis for potent inhibition of the Aurora kinases and a T315I multi-drug resistant mutant form of Abl kinase by VX-680.

The small molecule inhibitor of the Aurora-family of protein kinases VX-680 or MK-0457, demonstrates potent anti-cancer activity in multiple in vivo models and has recently entered phase II clinical trials. Although VX-680 shows a high degree of enzyme selectivity against multiple kinases, it unexpectedly inhibits both Flt-3 and Abl kinases at low nanomolar concentrations. Furthermore VX-680 potently inhibits Abl and the Imatinib resistant mutant (T315I) that is commonly expressed in refractory CML and ALL. We describe here the crystal structure of VX-680 bound to Aurora-A and show that this inhibitor exploits a centrally located hydrophobic pocket in the active site that is only present in an inactive or "closed" kinase conformation. A tight association of VX-680 with this hydrophobic pocket explains its high affinity for the Aurora kinases and also provides an explanation for its selectivity profile, including its ability to inhibit Abl and the Imatinib-resistant mutant (T315I).

Ratio of male to female births in the offspring of BRCA1 and BRCA2 carriers.

A recent report based on 68 families, including 17 with mutations in BRCA1, suggested that there was an excess of female offspring born to BRCA1 mutation carriers. We have examined the gender ratio among offspring of 511 mutation carriers from 116 BRCA1 families, 77 and 39 from Australia and the United States, respectively. We found no evidence for a significant deviation from the expected proportion of female offspring in the Australian pedigrees, but there was an excess of female offspring in pedigrees from the USA. Ascertainment bias probably explains this bias, rather than a link with X-chromosome inactivation as previously suggested, because the families from the USA were ascertained for the purposes of linkage studies whereas those from Australia were ascertained through Familial Cancer Clinics to which they had been referred for clinical genetic counseling and mutation testing.

Insights into transcription: structure and function of single-subunit DNA-dependent RNA polymerases.

Single-subunit RNA polymerases are widespread throughout prokaryotic and eukaryotic organisms, and also viruses. T7 RNA polymerase is one of the simplest DNA-dependent enzymes, capable of transcribing a complete gene without the need for additional proteins. During the past two years, three illuminating crystal structures of T7 RNA polymerase complexed to either T7 lysozyme, which is a transcription inhibitor, an open promoter DNA fragment or a promoter DNA fragment being transcribed into RNA at initiation have been determined. For the first time, these structures describe in detail the intricate mechanism of transcription initiation by T7 RNA polymerase, which is likely to be a general model for other related RNA polymerases.

Structure of a transcribing T7 RNA polymerase initiation complex.

The structure of a T7 RNA polymerase (T7 RNAP) initiation complex captured transcribing a trinucleotide of RNA from a 17-base pair promoter DNA containing a 5-nucleotide single-strand template extension was determined at a resolution of 2.4 angstroms. Binding of the upstream duplex portion of the promoter occurs in the same manner as that in the open promoter complex, but the single-stranded template is repositioned to place the +4 base at the catalytic active site. Thus, synthesis of RNA in the initiation phase leads to accumulation or "scrunching" of the template in the enclosed active site pocket of T7 RNAP. Only three base pairs of heteroduplex are formed before the RNA peels off the template.

Structural basis for initiation of transcription from an RNA polymerase-promoter complex.

Although the single-polypeptide-chain RNA polymerase from bacteriophage T7 (T7RNAP), like other RNA polymerases, uses the same mechanism of polymerization as the DNA polymerases, it can also recognize a specific promoter sequence, initiate new RNA chains from a single nucleotide, abortively cycle the synthesis of short transcripts, be regulated by a transcription inhibitor, and terminate transcription. As T7RNAP is homologous to the Pol I family of DNA polymerases, the differences between the structure of T7RNAP complexed to substrates and that of the corresponding DNA polymerase complex provides a structural basis for understanding many of these functional differences. T7RNAP initiates RNA synthesis at promoter sequences that are conserved from positions -17 to +6 relative to the start site of transcription. The crystal structure at 2.4 A resolution of T7RNAP complexed with a 17-base-pair promoter shows that the four base pairs closest to the catalytic active site have melted to form a transcription bubble. The T7 promoter sequence is recognized by interactions in the major groove between an antiparallel beta-loop and bases. The amino-terminal domain is involved in promoter recognition and DNA melting. We have also used homology modelling of the priming and incoming nucleoside triphosphates from the T7 DNA-polymerase ternary complex structure to explain the specificity of T7RNAP for ribonucleotides, its ability to initiate from a single nucleotide, and the abortive cycling at the initiation of transcription.

Crystal structures of a rat anti-CD52 (CAMPATH-1) therapeutic antibody Fab fragment and its humanized counterpart.

The CAMPATH-1 family of antibodies are able systematically to lyse human lymphocytes with human complement by targeting the small cell-surface glycoprotein CD52, commonly called the CAMPATH-1 antigen. These antibodies have been used clinically for several years, providing therapy for patients with a variety of immunologically mediated diseases. We report here the first X-ray crystallographic analyses of a Fab fragment from a rat antibody, the original therapeutic monoclonal CAMPATH-1G and its humanized counterpart CAMPATH-1H, into which the six complementarity-determining regions of the rat antibody have been introduced. These structures have been refined at 2.6 A and 3.25 A resolution, respectively. The VL domains of adjacent molecules of CAMPATH-1H form a symmetric dimer within the crystals with an inter-molecular extended beta-sheet as seen in light chain dimers of the kappa class. Crystals of CAMPATH-1G have translational pseudo-symmetry. Within the antibody-combining sites, which are dominated by the protrusion of LysH52b and LysH53 from hypervariable loop H2, the charge distribution and overall integrity are highly conserved, but large changes in the position of loop H1 are observed and an altered conformation of loop H2. The major determinants of this are framework residues H71 and H24, whose identity differs in these two antibodies. These structures provide a detailed structural insight into the transplantation of an intact antibody-combining site between a rodent and a human framework, and provide an increased understanding of the specificity and antigen affinity of this pair of CAMPATH-1 antibodies for CD52. This study forms the structural basis for future modification and design of more effective antibodies to this important antigen.

Three dimensional structure of human C-reactive protein.

The structure of the classical acute phase reactant human C-reactive protein provides evidence that phosphocholine binding is mediated through calcium and a hydrophobic pocket centred on Phe 66. The residue Glu 81 is suitably positioned to interact with the choline group. A cleft on the pentameric face opposite to that containing the calcium site may have an important functional role. The structure provides insights into the molecular mechanisms by which this highly conserved plasma protein, for which no polymorphism or deficiency state is known, may exert its biological role.

Synchrotron radiation laue diffraction for the time-resolved study of a transformation in crystals of p(4)n(4)cl(8).

Experiments are described to show some of the potential of the synchrotron radiation Laue method for the study of structural change within single crystals. In the metastable tetragonal crystals of P(4)N(4)Cl(8) the eight-membered P(4)N(4) ring is in a boat conformation, with symmetry {\bar 4}. On heating to ca 340 K the crystals transform, slowly to a second tetragonal form in which the ring conformation is a chair, its symmetry {\bar 1}. Both structures are known [Hazekamp, Migchelsen & Vos (1962). Acta Cryst. 15, 539-543; Wagner & Vos (1968). Acta Cryst. B24, 707-713]. In the transformation the molecular packing, unit-cell dimensions and crystal quality remain almost unchanged. To study this transformation, series of Laue diffraction patterns were recorded at 2-3 min intervals over a period of 30-40 min, while the temperature was raised to 373 K. For two series, reflection intensities were measured and they allowed determination and refinement of the fraction of boat and chair molecules present in a mixed boat/chair model of the structure. No significant change in the crystal occurs below ca 340 K; at or above 340 K, 40-50% of the molecules are converted from boat to chair conformations within 5 min, but the remainder of the conversion is much slower, even when the temperature is raised towards 370 K.