Using lessons learned and advanced methods to design a 1,500 Ml/day DAF water treatment plant.

The paper describes the method that led to the design of the 1,500 Ml/day dissolved air flotation (DAF) water treatment plant for Boston's water supply. In particular, the topics of flocculation techniques, floated solids removal and DAF recycle as they relate to very large capacity plant design are covered in detail. The use of mathematical models, including computational fluid dynamics (CFD) software, to refine the design is described.

Crusted ("Norwegian") scabies in a specialist HIV unit: successful use of ivermectin and failure to prevent nosocomial transmission.

A nosocomial outbreak of scabies in a specialist inpatient HIV unit resulted from a patient admitted with crusted scabies. Treatment of his infestation with topical scabicides alone failed and he remained infectious for several weeks. His infestation was then eradicated with combined topical treatment and oral ivermectin. In total, 14 (88%) out of 19 ward staff became symptomatic, and 4 (21%) had evidence of scabies on potassium hydroxide examination of skin scrapings. The ward infection control policy was changed to distinguish patients with crusted scabies from those with ordinary scabies. A second patient with crusted scabies was treated with combined oral and topical therapy early in his admission and nursed with more stringent isolation procedures. No nosocomial transmission occurred and his infestation responded rapidly to treatment. Patients with crusted scabies require strict barrier nursing if nosocomial transmission is to be avoided. Ivermectin combined with topical scabicides may be a more efficacious treatment than topical scabicides alone in such patients.

Disseminated cutaneous Mycobacterium tuberculosis infection in a patient with AIDS.

Individuals infected with the human immunodeficiency virus (HIV) are at an increased risk of both pulmonary and extrapulmonary tuberculosis. Disseminated cutaneous tuberculosis is rare, but has been reported in four HIV-positive patients, all of whom also had pulmonary infection. In this report we describe an HIV-infected patient with a febrile illness and an abnormal chest radiograph who developed widespread cutaneous tuberculous pustules following a lymph node biopsy on the previous day.

Internal calcium release and activation of sea urchin eggs by cGMP are independent of the phosphoinositide signaling pathway.

We show that microinjecting cyclic GMP (cGMP) into unfertilized sea urchin eggs activates them by stimulating a rise in the intracellular free calcium ion concentration ([Ca2+]i). The increase in [Ca2+]i is similar in both magnitude and duration to the transient that activates the egg at fertilization. It is due to mobilization of calcium from intracellular stores but is not prevented by the inositol trisphosphate (InsP3) antagonist heparin. Furthermore, cGMP does not stimulate the eggs Na+/H+ antiport when the [Ca2+]i transient is blocked by the calcium chelator bis-(O-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), suggesting that cGMP does not activate eggs by interacting with the their phosphoinositide signaling pathway. However, the [Ca2+]i increase and activation are prevented in eggs in which the InsP3-sensitive calcium stores have been emptied by the prior microinjection of the InsP3 analogue inositol 1,4,5-trisphosphorothioate. These data indicate that cGMP activates eggs by stimulating the release of calcium from an InsP3-sensitive calcium store via a novel, though unidentified, route independent of the InsP3 receptor.

Phosphoprotein inhibition of calcium-stimulated exocytosis in sea urchin eggs.

We have investigated the role of protein phosphorylation in the control of exocytosis in sea urchin eggs by treating eggs with a thio-analogue of ATP. ATP gamma S (adenosine 5'-O-3-thiotriphosphate) is a compound which can be used as a phosphoryl donor by protein kinases, leading to irreversible protein thiophosphorylation (Gratecos, D., and E.H. Fischer. 1974. Biochem. Biophys. Res. Commun. 58:960-967). Microinjection of ATP gamma S inhibits cortical granule exocytosis, but has no effect on the sperm-egg signal transduction mechanisms which normally cause exocytosis by generating an increase in [Ca2+]i. ATP gamma S requires cytosolic factors for its inhibition of cortical granule exocytosis: it does not affect exocytosis when applied directly to the isolated exocytotic apparatus. Our data suggest that ATP gamma S irreversibly inhibits exocytosis via thiophosphorylation of proteins associated with the egg cortex. We have identified two thiophosphorylated proteins (33 and 27 kD) that are associated with the isolated exocytotic apparatus. They may mediate the inhibition of exocytosis by ATP gamma S. In addition, we show that okadaic acid, an inhibitor of phosphoprotein phosphatases, prevents cortical granule exocytosis at fertilization without affecting calcium mobilization. Like ATP gamma S, okadaic acid has no effect on exocytosis in vitro. Our results suggest that an inhibitory phosphoprotein can obstruct calcium-stimulated exocytosis in sea urchin eggs; on the other hand, they do not readily support the idea that a protein phosphatase is an essential component of the mechanism controlling exocytosis.

Guanosine 5'-thiotriphosphate may stimulate phosphoinositide messenger production in sea urchin eggs by a different route than the fertilizing sperm.

We show that microinjecting guanosine-5'-thiotriphosphate (GTP gamma S) into unfertilized sea urchin eggs generates an intracellular free calcium concentration [( Ca]i) transient apparently identical in magnitude and duration to the calcium transient that activates the egg at fertilization. The GTP gamma S-induced transient is blocked by prior microinjection of the inositol trisphosphate (InsP3) antagonist heparin. GTP gamma S injection also causes stimulation of the egg's Na+/H+ antiporter via protein kinase C, even in the absence of a [Ca]i increase. These data suggest that GTP gamma S acts by stimulating the calcium-independent production of the phosphoinositide messengers InsP3 and diacylglycerol (DAG). However, the fertilization [Ca]i transient is not affected by heparin, nor can the sperm cause calcium-independent stimulation of protein kinase C. It seems that the bulk of InsP3 and DAG production at fertilization is triggered by the [Ca]i transient, not by the sperm itself. GDP beta S, a G-protein antagonist, does not affect the fertilization [Ca]i transient. Our findings do not support the idea that signal transduction at fertilization operates via a G-protein linked directly to a plasma membrane sperm receptor.

Bombesin stimulation of inositol 1,4,5-trisphosphate generation and intracellular calcium release is amplified in a cell line overexpressing the N-ras proto-oncogene.

Bombesin stimulation of T15 cells in which the inducible N-ras oncogene is overexpressed caused elevated production of inositol phosphates compared to uninduced cells [Wakelam, Davies, Houslay, McKay, Marshall & Hall (1986) Nature (London) 323, 173-176]. This elevated response is shown here to result from increased generation of inositol 1,4,5-trisphosphate leading to an elevated release of intracellular stored Ca2+. Single-cell analysis of Ca2+ release showed that the elevated response is not a consequence of an increased fraction of responding cells. These amplifications are consistent with p21N-ras acting like a guanine nucleotide coupling protein in this cell line.

Calcium-induced chromatin condensation and cyclin phosphorylation during chromatin condensation cycles in ammonia-activated sea urchin eggs.

Ammonia-activated sea urchin eggs undergo repeated cycles of DNA synthesis, nuclear envelope breakdown (NEB) and chromatin condensation. No mitotic spindle forms, nor do the eggs undergo cytokinesis. Ammonia-activated eggs exhibit a form of the cell cycle in which the nuclear cycle proceeds without segregation of the chromatin into daughter cells. We discuss here experiments that demonstrate that intracellular free calcium concentration controls the S phase-M phase transition in ammonia-activated eggs, as it does in fertilized embryos. Cyclins are proteins that are synthesized throughout the cell cycle and destroyed abruptly during each round of chromatin condensation. We find that cycles of cyclin phosphorylation and destruction occur coincident with chromatin condensation in ammonia-activated eggs. Cyclin phosphorylation also occurs in eggs treated with the tumour promoter, phorbol myristate acetate (PMA). There is no accompanying NEB or chromatin condensation, however, and the nucleus is insensitive to exogenously-generated calcium transients. These latter data indicate that cyclin synthesis and phosphorylation is not a sufficient condition for calcium-induced NEB in sea urchin embryos. PMA must fail to induce one of the necessary cell cycle initiation signals. We suggest that the missing signal is the activation of the cell cycle control protein p34cdc2, which we have shown to be phosphorylated at fertilization and which is phosphorylated in ammonia-activated eggs.

Structural modifications induced by TPA (12-O-tetradecanoyl phorbol-13-acetate) in sea urchin eggs.

We investigated the effect of the phorbol ester TPA (12-O-tetradecanoyl phorbol 13-acetate) on the egg morphology of the sea urchin Arbacia lixula. Our study indicates that TPA alters the cortical region of the egg: the pigment granules migrate toward the surface, while cortical granules detach from the plasma membrane. Cortical granule exocytosis did not occur but the endocytosis process was turned on. Prolonged treatment of the eggs by TPA partially inhibits the cortical granule exocytosis normally triggered by fertilization. We discuss the effects of TPA in terms of its interaction with the Ca2+ pool and cytoskeletal structures. In order to discern the respective roles of pHi and protein kinase C activity in endocytosis process activation, we compared the ultrastructural effects of TPA and ammonia. Finally, the role of pigment vesicles in egg metabolism activation is discussed.

Activation of sea urchin eggs by inositol phosphates is independent of external calcium.

We investigated the contribution of external calcium ions to inositol phosphate-induced exocytosis in sea urchin eggs. We show that: (a) inositol phosphates activate eggs of the sea urchin species Lytechinus pictus and Lytechinus variegatus independently of external calcium ions; (b) the magnitude and duration of the inositol phosphate induced calcium changes are independent of external calcium; (c) in calcium-free seawater, increasing the volume of inositol trisphosphate solution injected decreased the extent of egg activation; (d) eggs in calcium-free sea water are more easily damaged by microinjection; microinjection of larger volumes increased leakage from eggs pre-loaded with fluorescent dye. We conclude that inositol phosphates do not require external calcium ions to activate sea urchin eggs. This is entirely consistent with their role as internal messengers at fertilization. The increased damage caused to eggs in calcium-free seawater injected with large volumes may allow the EGTA present in the seawater to enter the egg and chelate any calcium released by the inositol phosphates. This may explain the discrepancy between this and earlier reports.

Ethanol metabolism in the generation of new antigenic determinants on liver cells.

Antibodies directed against ethanol altered liver cell components have been detected in the serum of nearly 50% of patients with alcoholic liver disease although the pathogenetic mechanisms are unclear. The importance of ethanol metabolism in the generation of new antigenic determinants on liver cells was investigated by in vivo inhibition of alcohol or acetaldehyde dehydrogenase and an induced cytotoxicity assay. There was a significant reduction in cytotoxicity to hepatocytes isolated from rabbits treated with ethanol 1 g/kg when the metabolism of ethanol to acetaldehyde by alcohol dehydrogenase was inhibited. In contrast when the oxidation of acetaldehyde was inhibited by disulfiram cytotoxicity was significantly enhanced. These results show that ethanol metabolism is integral to the expression of the ethanol related determinant and suggest that an impaired ability to metabolism acetaldehyde could lead to the development of immunological reactions to alcohol altered liver membrane antigens.

Effects of ethanol administration on rat liver plasma membrane-bound enzymes.

Changes in the properties of rat liver plasma membranes were examined in studies designed to differentiate between direct and metabolic effects of acute and chronic ethanol ingestion. One hour after a single dose of ethanol (3 g/kg body weight) there were increases in Na+,K+-ATPase (32%) and 5'-nucleotidase (36%), and hepatic concentrations of ethanol and acetaldehyde were approximately 23 mM and 50 microM, respectively. Na+,K+-ATPase and 5'-nucleotidase activities in liver plasma membranes from control rats were not significantly changed by in vitro addition of 30 microM acetaldehyde or 50 mM ethanol. Increases in Na+,K+-ATPase (approximately 20%) and 5'-nucleotidase (approximately 30%) were also observed in liver plasma membranes isolated from rats 16 hr after feeding ethanol or sucrose supplements for 17 days. The intake of calories from dietary protein and lipid was decreased by about 25% in both the ethanol and sucrose-fed animals. Na+,K+-ATPase activities in liver plasma membranes isolated from control rats were inhibited (approximately 20%) by 100 mM ethanol in vitro, whereas no inhibition was observed using membrane preparations from rats fed ethanol or sucrose supplements. Our results show that changes in liver plasma membrane enzyme activities associated with a single dose of ethanol are not a direct effect correlated with blood, hepatic or plasma membrane concentrations of ethanol or acetaldehyde. Chronic ingestion of ethanol or sucrose supplements had similar effects on liver plasma membrane enzyme characteristics and parallel changes in nutrient intake may be a more feasible explanation of these results than any analogous direct effects of the two compounds.

Reductions in portal pressure by selective beta 2-adrenoceptor blockade in patients with cirrhosis and portal hypertension.

In order to elucidate the mechanisms by which beta-adrenoceptor blockade leads to a reduction in portal pressure, we have measured portal pressure, heart rate and cardiac index in 17 patients with cirrhosis and portal hypertension following the oral administration of the selective beta 2-adrenoceptor blocking agent, ICI 118551, in the two dosage ranges 10-20 mg and 50-100 mg. There was a fall in portal pressure in 14 of the 17 patients from a median of 17 mm Hg to a median of 15 mm Hg, P less than 0.01, that occurred only at 60 min following administration of the drug. Although there were early systemic haemodynamic changes, with a significant fall in heart rate and cardiac index from before to 30 min after the ingestion of ICI 118551 (median heart rates 86 and 80 beats/min respectively, P less than 0.01, median cardiac indices 3.5 and 3.31 min-1 m-2, P less than 0.05), these occurred before the fall in portal pressure and were unrelated to changes in portal pressure. This reduction in portal pressure independent of systemic haemodynamic changes is consistent with beta 2-adrenoceptor blockade within the splanchnic and hepatic arterial circulations; subsequent increases in splanchnic and hepatic arterial resistances with a concomitant fall in portal blood flow and hepatic portal resistance may lead to the reduction in portal pressure.

Effects of converting enzyme inhibitor on hepatic blood flow in man.

The acute effects of the oral angiotension converting enzyme inhibitor captopril on hepatic blood flow and systemic hemodynamics were studied in six patients with essential hypertension. Mean arterial pressure decreased from 141.9 +/- 6.9 mm Hg to 130.2 +/- 6.7 mm Hg (p less than 0.05) one hour after the administration of captopril. There was no significant change in other hemodynamic values, but hepatic blood flow decreased uniformly from 1,127 +/- 115 ml per minute to 841 +/- 93 ml per minute (p less than 0.001).

Antibodies to alcohol altered liver cell determinants in patients with alcoholic liver disease.

Circulating antibodies reacting specifically with hepatocytes isolated from ethanol pretreated rabbits have been demonstrated by two techniques - induced cytotoxicity and immunofluorescence. In the cytotoxicity assay antibodies were found in seven of 19 (39%) of patients with alcoholic fatty liver (with or without fibrosis), six of 13 (46%) of those with alcoholic hepatitis, 15 of 36 (43%) of those with cirrhosis, and seven of 14 patients (50%) of those with hepatitis and cirrhosis. In the immunofluorescence studies, nine of 15 sera induced a granular pattern of fluorescence on the ethanol pretreated hepatocytes; two sera which induced significant cytotoxicity did not induce immunofluorescence. No ethanol related antibodies were found in normal individuals or in patients with other types of acute or chronic liver disease. These results show that antibodies directed against ethanol altered liver cell determinants are present in the serum of 43% of patients with alcoholic liver disease, and suggest a mechanism whereby chronic alcohol consumption may, by inducing antigenic changes in hepatocyte membranes, trigger a cell damaging immune reaction.

Selective and non-selective beta receptor blockade in the reduction of portal pressure in patients with cirrhosis and portal hypertension.

To elucidate the mechanisms by which beta receptor blockade leads to a reduction of portal pressure, 18 patients with cirrhosis and portal hypertension were given comparable doses of propranolol or metoprolol. The fall in portal pressure was more marked with propranolol together with a significant reduction in hepatic blood flow, which was not seen with metoprolol. No correlation between the reduction in cardiac output and the decrease in portal pressure or changes in hepatic blood flow could be elicited in each group, but there was a direct relationship between the decrease in hepatic blood flow and fall in portal pressure in the propranolol treated patients. The difference observed may be related to blockade of beta 2 vasodilator receptors in the splanchnic circulation which will occur only with propranolol and lead to a greater fall in splanchnic blood flow than will be produced by a reduction in cardiac output alone. Metoprolol, by maintaining effective hepatic blood flow, may be preferable to propranolol in patients with severely impaired liver function.