Medicine at the centre of the nation's affairs.

The medical profession was shaped a century or so ago by the interaction of three forces. These were a class structure in which doctors were largely held to be gentlemen to whom deference was due, a society dominated by the activity of production (hence the label of the working class for the majority), and the doctrines of liberalism as the guiding star in politics. Prime Minister Gladstone defined his task as "opening doors and windows." The outcome was a minimum of government interference and control with the belief that professional self regulation was the way to ensure that practice matched principle. The state (the word was hardly ever used) was self effacing almost to the point of non-existence. These are ghosts of the past, but it is a comparatively recent past. Within memory, the major domestic preoccupation of politicians of all parties was how, and to what ends, the working class could be absorbed into the political system. Health had a crucial part to play in this task, as Lloyd George and others saw early on. The "panel" was very much a forerunner of the NHS. Indeed, Bevan based part of his case in 1946 on the claim that 21 million people were already on the "panel," clear evidence of the degree to which society was still dominated by production. While speaking of Nye Bevan, we might examine his claim that the NHS was "pure socialism." In fact, it was rather closer to being "impure liberalism" in the consideration with which general practitioners and consultants were treated, the considerable freedom enjoyed by local administrations, and the low profile of government itself. That is why many remember the period as something of a golden age. It suited almost everyone very well.     

Steady-state,Pre-steady-state,and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, k_off). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (k_off). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (<1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E>S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.     

HOMEOSTATIC ADJUSTMENTS AFTER EXERCISE : I. ACID-BASE EQUILIBRIUM OF THE BLOOD

The rate at which displacement and recovery of the acid-base equilibrium of the blood occur in young adult males subjected to short periods of maximal exertion has been determined. Displacement of acid-base equilibrium produced by severe exercise is along the fixed acid path, similar to the path of displacement produced by ingestion of acidifying agents such as ammonium chloride. Maximum displacement of the acid-base equilibrium is not reached until 7 to 10 minutes after the cessation of exercise. By this time over 50 per cent of the displacement in oxygen consumption, respiratory volume, and blood pressure have disappeared. A much greater metabolic acidosis was produced by exercise than could be induced by the oral administration of ammonium chloride. Recovery from the metabolic acidosis produced by exercise was much more rapid (10 times) than was recovery from the acidosis produced by ammonium chloride. After exercise the pH, returned to normal values more rapidly than did the bicarbonate content of the serum.     

A PHYSICAL THEORY OF ACID ANION DISPLACEMENT AND RECOVERY FOLLOWING EXERCISE

A simple theoretical model has been presented whose behavior duplicates the variation in bicarbonate ion concentration in the blood following exercise. Methods for the evaluation of the constants of rational equations to describe the concentration in muscle cells, in blood plasma, and in removal cells, of the anions produced in exercise have been devised. These methods have been applied to experimental data from 23 experiments, and a close agreement between the observed and theoretically predicted values for blood plasma has been found. From the mathematical analysis of the data values for permeability of acid anions produced in exercise have been estimated as 75 x 10^–5 and 5.9 x 10^–5 cm. per sec. between muscle cell and blood (extracellular fluid) and between blood plasma and removal cells respectively.     

Spatial and temporal variability of biomarkers and microbial diversity reveal metabolic and community flexibility in Streamer Biofilm Communities in the Lower Geyser Basin,Yellowstone National Park

Detailed analysis of 16S rRNA and intact polar lipids (IPLs) from streamer biofilm communities (SBCs), collected from geochemically similar hot springs in the Lower Geyser Basin, Yellowstone National Park, shows good agreement and affirm that IPLs can be used as reliable markers for the microbial constituents of SBCs. Uncultured Crenarchaea are prominent in SBS, and their IPLs contain both glycosidic and mixed glyco‐phospho head groups with tetraether cores, having 0–4 rings. Archaeal IPL contributions increase with increasing temperature and comprise up to one‐fourth of the total IPL inventory at >84 °C. At elevated temperatures, bacterial IPLs contain abundant glycosidic glycerol diether lipids. Diether and diacylglycerol (DAG) lipids with aminopentanetetrol and phosphatidylinositol head groups were identified as lipids diagnostic of Aquificales, while DAG glycolipids and glyco‐phospholipids containing N‐acetylgycosamine as head group were assigned to members of the Thermales. With decreasing temperature and concomitant changes in water chemistry, IPLs typical of phototrophic bacteria, such as mono‐, diglycosyl, and sulfoquinovosyl DAG, which are specific for cyanobacteria, increase in abundance, consistent with genomic data from the same samples. Compound‐specific stable carbon isotope analysis of IPL breakdown products reveals a large isotopic diversity among SBCs in different hot springs. At two of the hot springs, ‘Bison Pool’ and Flat Cone, lipids derived from Aquificales are enriched in ¹³C relative to biomass and approach values close to dissolved inorganic carbon (DIC) (approximately 0‰), consistent with fractionation during autotrophic carbon fixation via the reversed tricarboxylic acid pathway. At a third site, Octopus Spring, the same Aquificales‐diagnostic lipids are 10‰ depleted relative to biomass and resemble stable carbon isotope values of dissolved organic carbon (DOC), indicative of heterotrophy. Other bacterial and archaeal lipids show a similar variance, with values resembling the DIC or DOC pool or a mixture thereof. This variance cannot be explained by hot spring chemistry or temperature alone, but instead, we argue that intermittent input of exogenous organic carbon can result in metabolic shifts of the chemotrophic communities from autotrophy to heterotrophy and vice versa.     

Biosynthesis of lysosomal hydrolases: their synthesis in bound polysomes and the role of co- and post-translational processing in determining their subcellular distribution

By in vitro translation of mRNA’s isolated from free and membrane-bound polysomes, direct evidence was obtained for the synthesis of two lysosomal hydrolases, β-glucuronidase of the rat preputial gland and cathespin D of mouse spleen, on polysomes bound to rough endoplasmic reticulum (ER) membranes. When the mRNA’s for these two proteins were translated in the presence of microsomal membranes, the in vitro synthesized polypeptides were cotranslationally glycosylated and transferred into the microsomal lumen. Polypeptides synthesized in the absence of microsomal membranes were approximately 2,000 daltons larger than the respective unglycosylated microsomal polypeptides found after short times of labeling in cultured rat liver cells treated with tunicamycin. This strongly suggests that nascent chains of the lysosomal enzymes bear transient amino terminal signals which determine synthesis on bound polysomes and are removed during the cotranslational insertion of the polypeptides into the ER membranes. In the line of cultured rat liver cells used for this work, newly synthesized lysosomal hydrolases showed a dual destination; approximately 60 percent of the microsomal polypeptides detected after short times of labeling were subsequently processed proteolytically to lower molecular weight forms characteristic of the mature enzymes. The remainder was secreted from the cells without further proteolytic processing. As previously observed by other investigations in cultured fibroblasts (A. Gonzalez-Noriega, J.H. Grubbs, V. Talkad, and W.S. Sly, 1980, J Cell Biol. 85: 839-852; A. Hasilik and E.F. Neufeld, 1980, J. Biol. Chem., 255:4937-4945.) the lysosomotropic amine chloroquine prevented the proteolytic maturation of newly synthesized hydrolases and enhanced their section. In addition, unglycosylated hydrolases synthesized in cells treated with tunicamycin were exclusively exported from the cells without undergoing proteolytic processing. These results support the notions that modified sugar residues serve as sorting out signals which address the hydrolases to their lysosomal destination and that final proteolytic cleavage of hydrolase precursors take place within lysosome itself. Structural differences in the carbohydrate chains of intracellular and secreted precursors of cathespin D were detected from their differential sensitivity to digestion with endoglycosidases H and D. These observations suggest that the hydrolases exported into the medium follow the normal secretory route and that some of their oligosaccharides are subject to modifications known to affect many secretory glycoproteins during their passage through the Golgi apparatus.     

Molecular basis of CIB binding to the integrin alpha IIb cytoplasmic domain

Integrin adhesion receptors appear to be regulated by molecules that bind to their cytoplasmic domains. We previously identified a 22-kDa, EF-hand-containing protein, CIB, which binds to the alpha(IIb) cytoplasmic tail of the platelet integrin, alpha(IIb)beta(3). Here we describe regions within CIB and alpha(IIb) that interact with one another. CIB binding to alpha(IIb) cytoplasmic tail peptides, as measured by intrinsic tryptophan fluorescence, indicates a CIB-binding site within a hydrophobic, 15-amino acid, membrane-proximal region of alpha(IIb). This region is analogous to the alpha-helical targets of other EF-hand-containing proteins, such as calcineurin B or calmodulin. A homology model of CIB based upon calcineurin B and recoverin indicated a conserved hydrophobic pocket within the C-terminal EF-hand motifs of CIB as a potential integrin-binding site. CIB engineered to contain alanine substitutions in the implicated regions retained wild type secondary structure as determined by circular dichroism, yet failed to bind alpha(IIb) in 11 of 12 cases, whereas CIB mutated within the N terminus retained binding activity. Thus, specific hydrophobic residues in the C terminus of CIB appear necessary for CIB binding to alpha(IIb). The identification of essential interacting regions within alpha(IIb) and CIB provides tools for further probing potential interrelated functions of these proteins.