Biomedical subjects
Publications and source records attributed to E Cheah.
Varices of the angular vein may arise anywhere along its length that may give rise to different clinical presentations. We report a case of a 56 year old male who presented with a medical canthal mass of 6 months duration. Surgical exploration and excison biopsy showed varix of the angular vein.
We report two female patients with gonadal dysgenesis and sex chromosome mosaicism involving the Y chromosome. Conventional karyotyping was supplemented with fluorescent in situ hybridisation techniques in order to confirm the presence of Y chromosomes. One patient is a phenotypic female with karyotype 45,X/46,X,idic(Y)(q11.2). She underwent a laparoscopic gonadectomy at which streak ovaries without evidence of gonadoblastoma were removed. The second patient presented as a virilised female with karyotype 45,X/47,XYY. At laparoscopy, she was found to have mixed gonadal dysgenesis with a gonadoblastoma in situ. We recommend early gonadectomy in female children presenting with gonadal dysgenesis and the presence of a Y chromosome although once the gonadoblastoma locus on Y chromosome gene has been cloned it may be possible to identify those patients who have a low risk of developing gonadoblastoma.
GlnK is a recently discovered homologue of the PII signal protein, an indicator of the nitrogen status of bacteria. PII occupies a central position in the dual cascade that regulates the activity of glutamine synthetase and the transcription of its gene. The complete role of Escherichia coli GlnK is yet to be determined, but already it is known that GlnK behaves like PII and can substitute for PII under some circumstances thereby adding to the subtleties of nitrogen regulation. There are also indications that the roles of the two proteins differ; the expression of PII is constitutive while that of GlnK is linked to the level of nitrogen in the cell. The discovery of GlnK begs the question of why E. coli has both GlnK and PII. Clearly, the structural similarities and differences of GlnK and PII will lead to a better understanding of how PII-like proteins function in E. coli and other organisms. We have crystallised and solved the X-ray structure of GlnK at 2.0 A resolution. The asymmetric unit has two independent copies of the GlnK subunit and both pack around 3-fold axes to form trimers. The trimers have a barrel-like core with recognition loops (the T-loops) that protrude from the top of the molecule. The two GlnK molecules have similar core structures to PII but differ significantly at the C terminus and the loops. The T-loops of the two GlnK molecules also differ from each other; one is disordered while the conformation of the other is stabilised by lattice contacts. The conformation of the ordered T-loop of GlnK differs from that observed in the PII structure despite the fact that their sequences are very similar. The structures suggest that the T-loops do not have a rigid structure and that they may be flexible in solution. The presence of a turn of 310 helix in the middle of the T-loop suggests that secondary structure could form when it interacts with soluble receptor enzymes.Co-crystals of GlnK and ATP were used to determine the structure of the complex. In these crystals, GlnK occupies a position of 3-fold symmetry. ATP binds in a cleft on the side of the molecule. The cleft is suitably positioned for ATP to influence the flexible T-loops. It is found at the junction of two beta sheets and is formed by two peptides one of which contains a variant of the "Gly-loop" found in other mononucleotide binding proteins. This sequence, Thr-Gly-X-X-Gly-Asp-Gly-Lys-Ile-Phe, forms part of the B-loop and is conserved in a wide variety of organisms that include bacteria, algae and archeabacteria. This sequence is more highly conserved than the functional T-loop, suggesting that ATP has an important role in PII-like proteins.
The trimeric signal-transduction protein GlnK, from Escherichia coli, has been over-expressed, purified to homogeneity and crystallized. The crystals belong to space group P213 with a = 85.53 A and have two subunits in the asymmetric unit. The complex of GlnK with ATP crystallized in space group P63 with a = 57.45 and c = 54.79 A. These crystals have a single subunit in the asymmetric unit. High-quality diffraction data from crystals of GlnK and the GlnK complex have been collected to 2.0 A.
IND, a redox flavoprotein from Chromobacterium violaceum has been crystallized in the presence and absence of NADH. The crystals belong to the space group P41212 or its enantiomorph P43212 with a = 73.9 and c = 153.6 A. There is one molecule per asymmetric unit and the crystals diffract beyond 2.1 A resolution.
Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the "mutant ali-esterase hypothesis"). In the sheep blowfly, Lucilia cuprina, the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly137 --> Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp137 substitution alone is responsible for both the loss of E3's carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp137 in the homologous position in acetylcholinesterase suggests that Asp137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.
The 3D structure of PII, the central protein that controls the level of transcription and the enzymatic activity of glutamine synthetase in enteric bacteria revealed that residues 37-55 form the "T' loop, part of which protrudes from the core of the protein. Within this loop are the only two tyrosine residues that occur in the polypeptide, and one of them, Tyr-51, has been shown by chemical modification studies to be the site of uridylylation. Since tyrosine at position 46 is conserved in all known PII proteins, oligonucleotide directed mutagenesis was used to investigate the role of the two residues. Changing Tyr-51 to phenylalanine or serine abolished uridylylation. Altering tyrosine at position 46 to phenylalanine affected the rate of uridylylation of the protein. This latter mutation does not alter the structure of PII but the reduction in the uridylylation efficiency suggests a role for this residue in recognition and binding of the sensor enzyme uridylyl transferase.
New crystals of the signal-transducing protein P(II) have been obtained in the presence of a number of different effector ligands. Various crystal forms are observed depending on the nature of the ligand(s). Co-crystallization with 2-ketoglutarate, glutamate and pyrophosphate produces hexagonal crystals similar to the wild type, ATP yields cubic crystals and ATP in conjunction with 2-ketoglutarate or glutamate yields orthorhombic crystal forms. All of the above crystals have been characterized by X-ray diffraction analysis. The hexagonal crystals belong to space group P6(3), cubic crystals to either I23 or I2(1)3 and orthorhombic crystals to I222. A molecular-replacement solution for the P(II)/ATP/2-ketoglutarate crystals has been obtained giving us an initial model for a trimer in the orthorhombic crystal form.
The structure of the bacterial signal transduction protein P(II) has been refined to an R factor of 13.2% using 3sigma data between 10 and 1.9 A. The crystals exhibited twinning by merohedry and X-ray intensities were corrected using the method of Fisher & Sweet [Fisher & Sweet (1980). Acta Cryst. A36, 755-760] prior to refinement. Our earlier 2.7 A structure [Cheah, Carr, Suffolk, Vasudevan, Dixon & Ollis (1994). Structure, 2, 981-990] served as a starting model. P(II) is a trimeric molecule, each subunit has a mass of 12.4 kDa and contains 112 amino-acid residues. The refined model includes all 1065 protein atoms per subunit plus 312 water molecules. The high-resolution refinement confirms the correctness of our 2.7 A model, although it leads to a redefinition of the extent of various secondary-structural elements. The monomeric structure of P(II) exhibits an interlocking double betaalphabeta fold. This is a stable fold found in a number of proteins with diverse functions. The association of the protein into a trimer leads to a new structure which we describe in detail. The effects of crystal packing forces are discussed and potential interaction sites with other proteins and effector molecules are identified.
BACKGROUND: In Gram-negative proteobacteria, the nitrogen level in the cell is reflected by the uridylylation status of a key signal transducing protein, PII. PII modulates the activity of glutamine synthetase (GS) through its interaction with adenylyl transferase and it represses the expression of GS by acting in concert with nitrogen regulatory protein II. RESULTS: The three-dimensional structure of the Escherichia coli PII trimer has been determined at 2.7 A resolution. PII shows a low level of structural similarity to a broad family of alpha/beta proteins and contains a double beta alpha beta motif. The PII trimer contains three beta-sheets, each of which is composed of strands from each of the three monomers. These are surrounded by six alpha-helices. CONCLUSIONS: The structure of PII suggests potential regions of interaction with other proteins and serves as an initial step in understanding its signal transducing role in nitrogen regulation.
The Escherichia coli signal transduction protein PII, product of the glnB gene, was overproduced and purified. The predicted molecular weight of the protein based on the correct nucleotide sequence is 12,427 and is very close to the value 12,435 obtained by matrix-assisted laser desorption mass spectrometry. Hexagonal crystals of the unuridylylated form of PII with dimensions 0.2 x 0.2 x 0.3 mm were grown and analysed by X-ray diffraction. The crystals belong to space group P6(3) with a = b = 61.6 A, c = 56.3 A and Vm of 2.5 for one subunit in the asymmetric unit. A low-resolution electron density map showed electron density concentrated around a three-fold axis, suggesting the molecule to be a trimer. A sedimentation equilibrium experiment of the meniscus depletion type was used to estimate a molecular weight of 35,000 +/- 1,000 for PII in solution. This result is consistent with the native protein being a homotrimer.
Dienelactone hydrolase (DLH), an enzyme from the beta-ketoadipate pathway, catalyzes the hydrolysis of dienelactone to maleylacetate. Our inhibitor binding studies suggest that its substrate, dienelactone, is held in the active site by hydrophobic interactions around the lactone ring and by the ion pairs between its carboxylate and Arg-81 and Arg-206. Like the cysteine/serine proteases, DLH has a catalytic triad (Cys-123, His-202, Asp-171) and its mechanism probably involves the formation of covalently bound acyl intermediate via a tetrahedral intermediate. Unlike the proteases, DLH seems to protonate the incipient leaving group only after the collapse of the first tetrahedral intermediate, rendering DLH incapable of hydrolyzing amide analogues of its ester substrate. In addition, the triad His probably does not protonate the leaving group (enolate) or deprotonate the water for deacylation; rather, the enolate anion abstracts a proton from water and, in doing so, supplies the hydroxyl for deacylation.
The Cys-His-Asp catalytic triad found in dienelactone hydrolase (DLH) is unusual for several reasons. It has not been observed in other hydrolytic enzymes and it is virtually inactive when it is produced by site-directed mutagenesis in the proteases. We propose a model to explain why this triad is catalytically active in DLH but not in the proteases. In the resting state of DLH, His202 forms an ion pair with Asp171 and Cys123 exists as a thiol. The resting state thiol does not interact with His202 in the active site but instead forms a hydrogen bond with Glu36 in the interior of the molecule. In the absence of substrate, Glu36 is also ion paired with Arg206. When substrate binds, Arg206 forms a second ion pair with the anionic substrate and the Arg206/Glu36 ion pair weakens. The destabilized Glu36 carboxylate shifts towards and deprotonates the Cys123 thiol, thereby activating the nucleophile. As the thiolate anion is not energetically favoured in the hydrophobic interior of the enzyme, it swings into the active site where it can be stabilized by the His202 imidazolium and the dipole of helix C. The Cys123 thiolate which now lies adjacent to the acyl carbon of the substrate, is thus generated only in the presence of substrate. The mode of thiolate activation reduces the susceptibility of DLH towards thiol alkylating agents.
We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
A case is reported where a cystic basal cell adenoma was found synchronously with two foci of adenolymphoma within the superficial lobe of a parotid gland. A review of this rare occurrence, as quoted in the literature, has been undertaken.
A great variety of Intraocular Lenses (IOLs) made by various manufacturers are now available to the Implant Surgeon. The Surgeon must decide which IOLs are better than others, and be aware of physical characteristics of IOLs that would be potentially dangerous to the intraocular structures. The Scanning Electron-Microscope (SEM) is now recognised as a powerful tool in providing detailed information of the physical characteristics of Intraocular lenses. We randomly selected five posterior chamber intraocular lenses from each of five different manufacturers and subjected them to scanning with the SEM to evaluate their physical qualities as well as to assess the consistency of manufacture. Striking differences in quality of finish of the intraocular lenses were revealed. The consistency of quality of good or of defective characteristics was high amongst manufacturers.
In order to assess the likely clinical value of AFP and beta-hCG levels as markers of the extent of spread of infiltrating duct mammary carcinoma, we compared (Mann-Whitney U test) the immunoreactive tumour tissue concentrations of the respective antigens, measured by specific RIA, after mastectomy in Chinese women at stages I (n = 9), II (n = 13) and III (n = 7) with that of normal breast tissue (n = 11). Elevated concentrations of the oncofetal antigens were found at all stages of malignancy. The discovery of an hCG-like material in the extract of normal breast tissues indicates limited value of beta-hCG assay in mammary tumours. However, our findings generally suggest that measurement of the tumour tissue AFP levels warrant consideration as a useful biochemical marker for in-vitro evaluation of tumour spread at all stages of malignancy.
Fifty-five Chinese healthy asymptomatic Hepatitis Bs Antigen carriers, 49 males and 6 females, were studied over a six year period, 1978 to 1984. Seventeen patients (30.9%) had normal transaminases, and 38 patients (69.1%) had fluctuating levels of transaminases throughout the period of study. 83.6% of patients were below 40 years, and mean age was 29.5 years (range 18 to 49 years). Twenty three patients (41.8%) had liver biopsy, and a spectrum of chronic liver diseases was seen, with Chronic Lobular Hepatitis (CLH) and Chronic Persistent Hepatitis (CPH) being the commonest. Six patients (26.1%) had normal histology, seven patients (30.4%) had CLH, seven patients (30.4%) had CPH, two patients (8.7%) had Chronic Active Hepatitis (CAH), and one patient (4.4%) with Active Cirrhosis (AC). An attempt to correlate the severity of histological changes with the levels of transaminases at the time of biopsy failed to show any definite correlation. However, three patients with transaminases of more than four times normal had severe liver diseases (2 CAH and 1 AC).