Macular and perimacular vascular remodelling sickling haemoglobinopathies

The posterior pole vasculature of 100 patients with different sickling haemoglobinopathies was studied prospectively over a period of three years. Various abnormalities of the posterior pole vasculature were seen in 29 per cent of the patients. Continuous remodelling of the macular and perimacular vasculature occurred. Visual acuity was variably affected and sometimes remained intact.     

Production of human normal adult and fetal hemoglobins in Escherichia coli

A hemoglobin expression system in Escherichia coli is described. In order to produce authentic human hemoglobin, we need to co-express both methionine aminopeptidase and globin genes under the control of a strong promoter. We have constructed three plasmids, pHE2, pHE4 and pHE7, for the expression of human normal adult hemoglobin and a plasmid, pHE9, for the expression of human fetal hemoglobin, in high yields. The globin genes can be derived from either synthetic genes or human globin cDNAs. The extra amino-terminal methionine residues of the expressed globins can be removed by the co-expressed methionine aminopeptidase. The heme is inserted correctly into the expressed alpha- globin from our expression plasmids. A fraction (approximately 25%) of the heme is not inserted correctly into the expressed beta- or gamma- globin. However, the incorrectly inserted hemes can be converted into the correct conformation by carrying out a simple oxidation-reduction process on the purified hemoglobin molecule. We have investigated the functional properties of the expressed hemoglobins by measuring their oxygen-binding properties and their structural features by obtaining their 1H-NMR spectra. Our results show that authentic human normal adult and fetal hemoglobins can be produced from our expression plasmids in E. coli and in high yields. Our expression system allows us to design and to produce any recombinant hemoglobins needed for our research on the structure-function relationship in hemoglobin.      

The current and residual value of superphosphate,Christmas Island C-grade ore,and Calciphos as fertilizers for a subterranean clover pasture

The effectiveness in the year of application of three phosphorus fertilizers, superphosphate, Christmas Island C-grade ore, and 500°C calcined Christmas Island C-grade ore (Calciphos), was measured for 5 consecutive years in a field experiment on a lateritic soil. The residual value of the phosphorus fertilizers was also measured for 6 years. Dry matter production of subterranean clover-based pasture and bicarbonate extractable soil phosphorus were used as indicators of fertilizer effectiveness.Despite the use of very large amounts of C-grade ore and Calciphos, the plateau of the pasture yield versus fertilizer applied curve for these fertilizers did not reach the yield plateau achieved with superphosphate in either the short or long term.C-grade ore and Calciphos were 3% and 8% as effective as superphosphate for dry matter production in the year of application. Relative to superphosphate applied in the current year the effectiveness of superphosphate decreased by about 70% between the first and second year after application and decreased by a further 14% from year 3 to year 6. C-grade ore and Calciphos remained about 2% and 9% as effective as currently applied superphosphate each year.The residual value of superphosphate as measured by bicarbonate-extracted soil phosphorus decreased by about 60% from year 2 to year 7. The residual value of Calciphos was very low for year 2, doubled from year 2–4 and thereafter decreased gradually to its original value by year 7. The residual value of C-grade ore was extremely low throughout the experiment. Thus after year 2, compared to pasture yield, bicarbonate extracted soil phosphorus overestimated the residual value of superphosphate and calciphos.It follows that neither C-grade ore or Calciphos are suitable replacement fertilizers for superphosphate for use on pastures growing on lateritic soils in south-western Australia.     

Determination of Ag ions in water, urine and blood

Isolated human polymorphonuclear leukocytes were submitted to long wave ultraviolet light (UVA) with and without preincubation of the cells with 8-methoxypsoralen (8-MOP). Leukocyte chemotaxis was determined in modified Boyden chambers using caseine as the attractant. Combined treatment (8-MOP + UVA) significantly inhibited the chemotactic activity with 8-MOP concentrations above 0.1 mug/ml and 2 J/cm(2). However, with high dosage of UVA (5 J/cm(2)) with and without 8-MOP still 25-30% cells migrated through the filters. Also, cell viability as determined by trypan blue exclusion was only moderately affected by combined treatment. The results indicate that these nonreplicating cells are comperatively insensitive to UVA and 8-MOP.     

Catecholamine uptake, accumulation, and release in acute porphyria

Hypertension and tachycardia are well known features of acute porphyria and have been shown to be related to increased circulating catecholamines. The mechanism by which circulating catecholamines are increased was studied using the isolated perfused rat heart and human platelets as a model of adrenergic neuronal function. It was found that neither delta-aminolevulinate (ALA) nor porphobilinogen (PBG) blocked uptake or caused release in the isolated perfused rat heart. Platelets from six patients with acute prophyria, three in remission and three latent, with matching normal controls were studied with regard to their uptake of [(3)H]norepinephrine in the presence of ALA or PBG. It was found that ALA and PBG significantly reduced uptake and accumulation of [(3)H]-norepinephrine in patients with acute porphyria; however, no similar reduction in uptake and accumulation was observed in the platelets of normal controls. Therefore, it appears that there is a latent defect in the catecholamine uptake and (or) accumulation of platelets of patients with acute prophyria which only manifests itself in the presence of ALA or PBG. If platelet uptake serves as a model of adrenergic neuron uptake, this suggests that elevated circulating catecholamine levels during acute attacks of acute porphyria are caused at least partially by blockade of re-uptake into the sympathetic neurons.     

Subaponeurotic haemorrhage of the newborn

Five newborn infants who developed subaponeurotic haemorrhage are described. Three infants were delivered by vacuum extraction, and 3 infants died. The importance of early diagnosis and prompt treatment is emphasized.     

Membrane topology of the Rickettsia prowazekii ATP/ADP translocase revealed by novel dual pho-lac reporters

Here, we report the construction and characterization of dual reporters, consisting of both an Escherichia coli alkaline phosphatase (AP) gene and an alpha-fragment of the beta-galactosidase (BG) gene, for studying membrane protein topology by the gene fusion approach. Each of the reporters, when fused to periplasmic domains of polytopic proteins, produces fusions with high AP activity and, when fused to cytoplasmic domains, produces fusions with high BG activity in E. coli strains capable of alpha-complementation. The dual nature of these reporters simplifies interpretation of data obtained with poorly expressed fusions and allows one to evaluate the reliability of topological data. Deleterious effects resulting from the cell's attempt to export the full-length BG are eliminated in this approach. We describe dual indicator plates that allow for discrimination between colonies bearing cytoplasmic fusions, periplasmic fusions, and no fusions. We have generated a set of fusions to the topologically well-studied lactose permease of E. coli and demonstrated that topological information generated by these new reporters is in good agreement with the existing model. We used this new methodology for the determination of membrane topology of the Rickettsia prowazekii ATP/ADP translocase (Tlc). Our results were in agreement with the proposed in silico topological model in which Tlc traverses the cytoplasmic membrane of E. coli 12 times with its N and C termini facing the cytoplasm.     

DNA polymerase fidelity: from genetics toward a biochemical understanding

This review summarizes mutagenesis studies, emphasizing the use of bacteriophage T4 mutator and antimutator strains. Early genetic studies on T4 identified mutator and antimutator variants of DNA polymerase that, in turn, stimulated the development of model systems for the study of DNA polymerase fidelity in vitro. Later enzymatic studies using purified T4 mutator and antimutator polymerases were essential in elucidating mechanisms of base selection and exonuclease proofreading. In both cases, the base analogue 2-aminopurine (2AP) proved tremendously useful-first as a mutagen in vivo and then as a probe of DNA polymerase fidelity in vitro. Investigations into mechanisms of DNA polymerase fidelity inspired theoretical models that, in turn, called for kinetic and thermodynamic analyses. Thus, the field of DNA synthesis fidelity has grown from many directions: genetics, enzymology, kinetics, physical biochemistry, and thermodynamics, and today the interplay continues. The relative contributions of hydrogen bonding and base stacking to the accuracy of DNA synthesis are beginning to be deciphered. For the future, the main challenges lie in understanding the origins of mutational hot and cold spots.