Ratios derived from an array of standard haematological indices predict the oncological outcome in colon cancer.

AIM: The interaction between inflammation and cancer is well established. Surrogate markers of systemic inflammation, such as the neutrophil/lymphocyte ratio (NLR), may be associated with the long-term oncological outcome. The present study aimed to characterize the relationship between several ratios derived from haematological indices using a classification and regression tree analysis. METHOD: Haematological white-cell ratios were established for all patients undergoing colonic cancer resection with curative intent (n = 436) in a regional cancer centre. The optimal ratios associated with overall survival (OS) were established in a training set (n = 386) using a classification and regression tree (CRT) technique. The association between ratios and OS was assessed in a separate test set (n = 50). Within the test set, two groups were generated based on each ratio (one group above and one group below the cut-off value identified in the training set). The association between ratios and OS was assessed using a stepwise Cox proportional-hazards regression model. RESULTS: The following ratios, identified by the CRT, were associated with adverse OS in the test set: an NLR of ≥ 3.4 [hazard ratio (HR) = 3.4, P < 0.001]; and a white-cell-count/lymphocyte ratio (WLR) of ≥ 5.28 (HR = 4.1, P = 0.03). CONCLUSION: This is the first study to apply recursive partitioning in determining the relationship between haematological ratios and OS in colon cancer. Haematological ratios were predictive of oncological outcome. What does this paper add to the literature? This study suggests an association between systemic inflammation and oncological outcome.

Postoperative nausea and vomiting in laparoscopic versus open cholecystectomy at two major hospitals in Jamaica.

Postoperative nausea and vomiting (PONV) is a distressing and potentially dangerous complication of general anaesthesia with volatile agents. The internationally reported average risk is 20 to 30%. It has been suggested that Jamaicans have a generally low risk of PONV and this is plausible since ethnic-based differences in response to emetogenic stimuli have been identified. It has also been suggested that laparoscopy, by stretching and irritation of the peritoneum during gas insufflation, may be a risk factor for PONV but it has become increasingly difficult to test this hypothesis as fewer comparable open abdominal operations are being performed. This retrospective cohort study of PONV after laparoscopic versus open cholecystectomy was designed to answer these two questions. Data were collected on 356 cases performed at two major hospitals in Jamaica. The risk of PONV after laparoscopic and open cholecystectomy was 28.7% and 28.6% respectively. As these are at the upper end of the internationally reported average range, the impression that PONV risk is generally low in Jamaicans is not supported The finding that 81.4% of cases of PONV occurred only after discharge from the recovery room may explain the misconception. There was no significant difference between the risk of PONV after laparoscopic versus open cholecystectomy and the effect of laparoscopy remained insignificant after risk-adjustment in a generalized linear regression model. Laparoscopy is not a major risk factor for PONV in this study.

Sclerotherapy of idiopathic hydroceles and epididymal cysts: a historical comparison trial of 5% phenol versus tetracycline

Operating time for idiopathic hydroceles and epididymal cysts is scarce as these conditions compete with an increasing caseload of more consequential surgical disease. Therapy is often relegated to repeated aspiration. Sclerotherapy appears to be effective in a majority of published trials, but comparative effectiveness, efficacy and safety of most agents, including phenol versus tetracycline, has not been established A deliberate strategy of re-treatment until cure is not universally practised, with surgery still being offered after single-treatment failures. Two trials, the first consisting of 53 scrotal cysts treated with 5% phenol-in-water and the second, 42 cysts treated with tetracycline, are compared for effectiveness, efficacy and safety of sclerotherapy per se and of re-treatment. Intention-to-treat analysis yields similar cure rates (no re-accumulation three months after last injection) for phenol and tetracycline (83% and 81% respectively, p = 0.8). Per-protocol analysis also yields similar cure rates (100% and 97% respectively, p = 0.26) and mean number of injections to cure (1.34 and 1.12 respectively, p = 0.069), with range 1-4 and 1-3 respectively. Severe pain following tetracycline injection required administration of pre-injection cord block. Other complications occurred equally (25% and 25.7% respectively, p = 0.94) and were trivial except for one case of chronic haematocele treated by orchiectomy in the tetracycline group. Phenol (5%) and tetracycline are equally efficacious sclerosants for idiopathic scrotal cysts, achieving almost 100% cure with re-treatment and matching the efficacy of surgery. Concern about post-treatment fertility applies equally to surgery and demands informed consent for both modalities.

El tiempo de operación para los hidroceles y los quistes epididimales es escaso, ya que estas condiciones triviales compiten con una creciente carga de casos de enfermedades quirúrgicas de mayores consecuencias. La terapia es a menudo relegada a una aspiración repetida. La escleroterapiaparece ser efectiva en la mayoría de los ensayos publicados, pero no se han establecido la seguridad, eficacia y efectividad comparativa de la mayor parte de los agentes, incluyendo el fenol, frente a la tetraciclina. No se practica universalmente una estrategia deliberada de re-tratamiento hasta la cura, ofreciéndose todavía la cirugía, luego de fracasos con tratamientos individuales. Dos ensayos, el primero consistente en 53 quistes escrotales tratados con fenol acuoso al 5%, y el segundo, en 42 quistes tratados con tetraciclina, se comparan en cuanto a efectividad, eficacia y seguridad para laescleroterapia per se y para el re-tratamiento. El análisis de intención de tratamiento produce tasas de curación similares (no hay re-acumulación 3 meses después de la última inyección) para el fenol y la tetraciclina (83% y 81% respectivamente, p = 0.8). El análisis por protocolo también produce tasas de curación similares (100% y 97% respectivamente, p = 0.26) y el número medio de inyecciones paracurar (1.34 y 1.12 respectivamente, p = 0.069), con rangos de 1–4 y 1–3 respectivamente. El dolor severo tras la inyección de tetraciclina requirió hacer un bloqueo espinal de pre-inyec-ción. Asimismo ocurrieron otras complicaciones (25% y 25.7% respectivamente, p = 0.94) y fueron triviales, con excepción de un caso de hematoceles crónicos tratado mediante orquiectomía en el grupo de tetraciclina. El fenol (5%) y la tetraciclina poseen igual eficacia como esclerosantes de los quistes escrotales idiopáticos, ya que logran una curación de casi 100% con el re-tratamiento, e igualan la eficacia de la cirugía...

Mesh tuck repair of ventral hernias of the abdomen: a new, simplified technique for sublay herniorrhaphy

There is biomechanical advantage to placing mesh in the retro-myofascial plane for repair of ventral abdominal hernias. Intra-abdominal pressure applied to the periphery of the mesh increases apposition to the abdominal wall rather than causing distraction and this translates, in general, into lowerrecurrence rates than after “inlay” and “onlay” mesh placement. Traditionally, retro-myofascial meshis placed in the pre-peritoneal or retro-muscular space. Both traditional techniques require extensive dissection and placement of large sheets of mesh which can cause symptomatic impairment of abdominal wall compliance. Pre-peritoneal dissection can be particularly tedious due to pathological adherence of peritoneum to the posterior abdominal wall in longstanding primary and incisionalhernias. In the technique described, mesh is tucked into the retro-myofascial plane without any dissection into pre-peritoneal, retro-muscular or peritoneal spaces. The operation is less tedious, takes less time to perform, can often be done under local anaesthesia, demands less mesh and achieves similar recurrence rates to traditional retro-myofascial mesh repairs. Sixty-one operations have been performed by the author using this technique, with a recurrence rate of 8.2% after 13 years to 3 months of follow-up (median, 3.75 years) and 9.3% if patients with less than one year of follow-up are excluded. Factors predisposing to recurrence after mesh repair of ventral hernias are numerous and complex. A fair comparison of recurrence rates between this technique and traditional retro-myofascial repairs requires a randomized controlled trial but the crude recurrence rate for this operation falls well within the range reported for traditional repairs from other studies.

Existe una ventaja biomecánica en colocar una malla en el plano retro-miofascial para reparar las hernias abdominales ventrales. La presión intra-abdominal aplicada a la periferia de la malla, aumenta la aposición en la pared abdominal, en lugar de causar distracción, lo cual se traduce generalmente en tasas de recurrencia más bajas que cuando se colocan mallas “inlay” y “onlay”. Tradicionalmente la malla retro-miofascial se coloca en el espacio-pre-peritoneal o retro-muscular. Ambas técnicas tradicionales requieren disección extensa y la colocación de grandes láminas de malla que pueden causar afectación sintomática de la distensibilidad de la pared abdominal. La disección pre-peritoneal puede ser particularmente tediosa debido a la adherencia patológica del peritoneo a lapared abdominal posterior en las hernias primarias de larga duración e incisionales. En la técnica descrita, la malla se instala en el plano retro-miofascial sin disección alguna de los espacios preperitoneales, retro-musculares o peritoneales. La operación es menos tediosa, toma menos tiempo,puede a menudo hacerse con anestesia local, requiere menos malla, y logra tasas de recurrencia al menos similares a las tradicionales reparaciones retro-miofasciales con malla. Sesenta y una operaciones han sido realizadas por el autor usando esta técnica, con una tasa de recurrencia de 8.2% luego prede13 años a 3 meses de seguimiento (mediana, 3.75 años), y 9.3% si se excluyen los pacientes con menos de un año de seguimiento. Los factores que predisponen a la recurrencia después de una reparación con malla son numerosos y complejos. La comparación justa de las tasas de recurrencia entre esta técnica y las reparaciones retro-miofasciales tradicionales requiere un ensayo controlado randomizado, pero la tasa cruda de recurrencia para esta operación cae sin objeciones dentro delrango de reparaciones tradicionales reportado por otros estudios.

Mesh tuck repair of ventral hernias of the abdomen: a new, simplified technique for sublay herniorrhaphy.

There is biomechanical advantage to placing mesh in the retro-myofascial plane for repair of ventral abdominal hernias. Intra-abdominal pressure applied to the periphery of the mesh increases apposition to the abdominal wall rather than causing distraction and this translates, in general, into lower recurrence rates than after "inlay" and "onlay" mesh placement. Traditionally, retro-myofascial mesh is placed in the pre-peritoneal or retro-muscular space. Both traditional techniques require extensive dissection and placement of large sheets of mesh which can cause symptomatic impairment of abdominal wall compliance. Pre-peritoneal dissection can be particularly tedious due to pathological adherence of peritoneum to the posterior abdominal wall in longstanding primary and incisional hernias. In the technique described, mesh is tucked into the retro-myofascial plane without any dissection into pre-peritoneal, retro-muscular or peritoneal spaces. The operation is less tedious, takes less time to perform, can often be done under local anaesthesia, demands less mesh and achieves similar recurrence rates to traditional retro-myofascial mesh repairs. Sixty-one operations have been performed by the author using this technique, with a recurrence rate of 8.2% after 13 years to 3 months of follow-up (median, 3.75 years) and 9.3% if patients with less than one year of follow-up are excluded Factors predisposing to recurrence after mesh repair of ventral hernias are numerous and complex. A fair comparison of recurrence rates between this technique and traditional retro-myofascial repairs requires a randomized controlled trial but the crude recurrence rate for this operation falls well within the range reported for traditional repairs from other studies.

Sclerotherapy of idiopathic hydroceles and epididymal cysts: a historical comparison trial of 5% phenol versus tetracycline.

Operating time for idiopathic hydroceles and epididymal cysts is scarce as these conditions compete with an increasing caseload of more consequential surgical disease. Therapy is often relegated to repeated aspiration. Sclerotherapy appears to be effective in a majority of published trials, but comparative effectiveness, efficacy and safety of most agents, including phenol versus tetracycline, has not been established A deliberate strategy of re-treatment until cure is not universally practised, with surgery still being offered after single-treatment failures. Two trials, the first consisting of 53 scrotal cysts treated with 5% phenol-in-water and the second, 42 cysts treated with tetracycline, are compared for effectiveness, efficacy and safety of sclerotherapy per se and of re-treatment. Intention-to-treat analysis yields similar cure rates (no re-accumulation three months after last injection) for phenol and tetracycline (83% and 81% respectively, p = 0.8). Per-protocol analysis also yields similar cure rates (100% and 97% respectively, p = 0.26) and mean number of injections to cure (1.34 and 1.12 respectively, p = 0.069), with range 1-4 and 1-3 respectively. Severe pain following tetracycline injection required administration of pre-injection cord block. Other complications occurred equally (25% and 25.7% respectively, p = 0.94) and were trivial except for one case of chronic haematocele treated by orchiectomy in the tetracycline group. Phenol (5%) and tetracycline are equally efficacious sclerosants for idiopathic scrotal cysts, achieving almost 100% cure with re-treatment and matching the efficacy of surgery. Concern about post-treatment fertility applies equally to surgery and demands informed consent for both modalities.

Lipid interactions with bacterial channels: fluorescence studies.

Interactions between a membrane protein and the lipid molecules that surround it in the membrane are important in determining the structure and function of the protein. These interactions can be pictured at the molecular level using fluorescence spectroscopy, making use of the ability to introduce tryptophan residues into regions of interest in bacterial membrane proteins. Fluorescence quenching methods have been developed to study lipid binding separately on the two sides of the membrane. Lipid binding to the surface of the mechanosensitive channel MscL is heterogeneous, with a hot-spot for binding anionic lipid on the cytoplasmic side, associated with a cluster of three positively charged residues. The environmental sensitivity of tryptophan fluorescence emission has been used to identify the residues at the ends of the hydrophobic core of the second transmembrane alpha-helix in MscL. The efficiency of hydrophobic matching between MscL and the surrounding lipid bilayer is high. Fluorescence quenching methods can also be used to study binding of lipids to non-annular sites such as those between monomers in the homotetrameric potassium channel KcsA.

Surgical complications of cocaine body-packing: a survey of Jamaican hospitals.

Body-packing is a common method of smuggling cocaine. Complications requiring surgery do not occur with sufficient frequency to allow any individual surgeon to determine patterns of presentation and the best approach to the conduct of surgery. A survey of all surgical units in Jamaica was conducted. A case was any patient requiring surgery for cocaine body-packing since and including the first reported case in 1987. Seventeen cases were identified. There were 11 cases of bowel obstruction, two of delayed passage of pellets, three of ruptured pellets with cocaine toxicity and one patient panicked and requested surgery. The distal ileum was the commonest site of obstruction in the normal gastrointestinal tract. In all three cases with cocaine poisoning, the ruptured packets were encountered in the upper gastrointestinal tract and several other partially ruptured packets were also found, implying that poor packaging was the cause of rupture. Obstructing packets should be removed but non-obstructing, unruptured packets encountered in the colon may safely be allowed to pass spontaneously. All cases of packet rupture with cocaine toxicity should have immediate surgery.

Surgical complications of cocaine body-packing: a survey of Jamaican hospitals / Complicaciones Quirúrgicas del Empaquetamiento Corporal de Cocaína

Body-packing is a common method of smuggling cocaine. Complications requiring surgery do not occur with sufficient frequency to allow any individual surgeon to determine patterns of presentation and the best approach to the conduct of surgery. A survey of all surgical units in Jamaica was conducted. A case was any patient requiring surgery for cocaine body-packing since and including the first reported case in 1987. Seventeen cases were identified. There were 11 cases of bowel obstruction, two of delayed passage of pellets, three of ruptured pellets with cocaine toxicity and one patient panicked and requested surgery. The distal ileum was the commonest site of obstruction in the normal gastrointestinal tract. In all three cases with cocaine poisoning, the ruptured packets were encountered in the upper gastrointestinal tract and several other partially ruptured packets were also found, implying that poor packaging was the cause of rupture. Obstructing packets should be removed but non-obstructing, unruptured packets encountered in the colon may safely be allowed to pass spontaneously. All cases of packet rupture with cocaine toxicity should have immediate surgery

El empaquetamiento dentro del organismo de seres humanos es un método común del contrabando la cocaína. Las complicaciones que requieren cirugía no ocurren con frecuencia suficiente como para permitir al cirujano determinar patrones de presentación de los casos, y el mejor enfoque en cuanto a la conducta quirúrgica a seguir. En este sentido, se llevó a cabo una investigación en todas las unidades quirúrgicas de Jamaica. Se tomó como caso cualquier paciente que hubiere requerido cirugía por haber empaquetado cocaína dentro de su cuerpo, desde que ocurriera el primer caso en 1987, e incluyendo éste. Se identificaron diecisiete casos. Hubo 11 casos de obstrucción intestinal, dos pasajes retardados de cápsulas, tres por cápsulas rotas con intoxicación por cocaína, y un paciente que preso de pánico, pidió ser intervenido quirúrgicamente. El íleo distal fue el sitio más común de la obstrucción en el tracto gastrointestinal normal. En los tres casos de envenenamiento por cocaína envenenar, las paquetes rotos se encontraban en el tracto gastrointestinal superior, y se encontraron también varios otros paquetes parcialmente rotos, lo cual indicaba que el pobre empaquetamiento fue la causa del rompimiento. Los paquetes que causan obstrucción deben ser retirados, pero los que se hallan en el colon sin causar obstrucción y sin estar rotos, puede dejárseles con seguridad ser excretados espontáneamente. Todos los casos de rompimiento de paquetes con intoxicación por cocaína deben ser sometidos a cirugía de manera inmediata

The potassium channel KcsA and its interaction with the lipid bilayer.

The crystal structure of the K+ channel KcsA explains many features of ion channel function. The selectivity filter corresponds to a narrow region about 12 Along and 3 A wide, lined by carbonyl groups of the peptide backbone, through which a K+ ion can only move ina dehydrated form. The selectivity filter opens into a central, water-filled cavity leading to a gating site on the intracellular side of the channel. The channel is tetrameric, each monomer containing two transmembrane a helices, M1 and M2. Helix M1 faces the lipid bi-layer and helix M2 faces the central channel pore; the M2 helices participate in subunit-subunit interactions. Helices M1 and M2 in each subunit pack as a pair of antiparallel coils with a heptad repeat, but the M2 helices of neighbouring subunits show fewer interactions, crossing at an angle of about -40 degrees. Trp residues at the ends of the transmembrane a helices form clear girdles on the two faces of the membrane, which, together with girdles of charged residues, define a hydrophobic thickness of about 37 A for the channel. Binding constants for phosphatidylcholines to KcsA vary with fatty acyl chain length, the optimum chain length being C22. A phosphatidylcholine with this chain length gives a bilayer of thickness about 34 A in the liquid crystalline phase, matching the hydrophobic thickness of the protein. However, a typical biological membrane has a hydrophobic thickness of about 27 A. Thus either the transmembrane a helices of KcsA are more tilted in the native membrane than they are in the crystal structure, or the channel is under stress in the native membrane. The efficiency of hydrophobic matching between KcsA and the surrounding lipid bilayer is high over the chain length range C10-C24. The channel requires the presence of some anionic lipids for function, and fluorescence quenching studies show the presence of two classes of lipid binding site on KcsA; at one class of site (nonannular sites) anionic phospholipids bind more strongly than phosphatidylcholine, whereas at the other class of site (annular sites) phosphatidylcholines and anionic phospholipids bind with equal affinity.

Artemisinins target the SERCA of Plasmodium falciparum.

Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron.

Mechanism of binding of warfarin enantiomers to recombinant domains of human albumin.

Domain fragments of human serum albumin corresponding to domains 1 and 2 (D12) and domains 2 and 3 (D23) were expressed in yeast. The kinetics of warfarin binding to these fragments were investigated using stopped-flow fluorescence spectroscopy. Binding can be characterized by a two-step process, a rapid diffusion-controlled step and a slower rate-limiting step in which a stable drug-protein complex is formed. The equilibrium constant for step 1 is greater for both D12 and D23 than for albumin, probably as a result of reduced steric hindrance offered by the domain fragments. Binding step 2, thought to be the result of a conformational change as warfarin is accommodated by the protein, is faster for D12 and D23. Albumin and the domain fragments show an increased preference for the R enantiomer, but the preference is particularly enhanced for domain fragment D12. These preferences can largely be explained by the domains having different rates for step 2 of the binding process.

Effects of phospholipid headgroup and phase on the activity of diacylglycerol kinase of Escherichia coli.

Diacylglycerol kinase (DGK) of Escherichia coli has been reconstituted into a variety of phospholipid bilayers and its activity determined as a function of lipid headgroup structure and phase preference. The anionic phospholipids dioleoylphosphatidic acid, dioleoylphosphatidylserine, and cardiolipin were all found to support activities lower than that supported by dioleoylphosphatidylcholine. In mixtures of dioleoylphosphatidylcholine and 20 mol % anionic phospholipids, the presence of anionic phospholipids all resulted in lower activities than in dioleoylphosphatidylcholine, except for dioleoylphosphatidylglycerol whose presence had little effect on activity. In some cases, the low activity in the presence of anionic phospholipid followed from a decrease in v(max); in some cases, it followed from an increase in the K(m) for diacylglycerol, and in the case of dioleoylphosphatidic acid, it followed from both. Activities in mixtures containing 80 mol % dioleoylphosphatidylethanolamine were lower than in dioleoylphosphatidylcholine at temperatures where both lipids adopted a bilayer phase; at higher temperatures where dioleoylphosphatidylethanolamine preferred a hexagonal H(II) phase, the differences in activity were greater. These experiments suggest that the presence of lipids preferring a hexagonal H(II) phase leads to low activities. Activities of DGK are low in a gel phase lipid.

Curcumin, a molecule that inhibits the Ca2+-ATPase of sarcoplasmic reticulum but increases the rate of accumulation of Ca2+.

Curcumin, an important inhibitor of carcinogenesis, is an inhibitor of the ATPase activity of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR). Inhibition by curcumin is structurally specific, requiring the presence of a pair of -OH groups at the 4-position of the rings. Inhibition is not competitive with ATP. Unexpectedly, addition of curcumin to SR vesicles leads to an increase in the rate of accumulation of Ca(2+), unlike other inhibitors of the Ca(2+)-ATPase that result in a reduced rate of accumulation. An increase in the rate of accumulation of Ca(2+) is seen in the presence of phosphate ion, which lowers the concentration of free Ca(2+) within the lumen of the SR, showing that the effect is not passive leak across the SR membrane. Rather, simulations suggest that the effect is to reduce the rate of slippage on the ATPase, a process in which a Ca(2+)-bound, phosphorylated intermediate releases its bound Ca(2+) on the cytoplasmic rather than on the lumenal side of the membrane. The structural specificity of the effects of curcumin on ATPase activity and on Ca(2+) accumulation is the same, and the apparent dissociation constants for the two effects are similar, suggesting that the two effects of curcumin could follow from binding to a single site on the ATPase.

Self-association of model transmembrane alpha-helices is modulated by lipid structure.

We have developed a fluorescence quenching method using peptides containing 3,5-dibromotryrosine to measure oligomerization of model transmembrane alpha-helices in lipid bilayers. Peptides of the type Ac-LysLysGlyLeu(m)XLeu(n)LysLysAla-amide where X is tryptophan or 3,5-dibromotyrosine were found to form heterodimers in bilayers of phosphatidylcholine in the liquid-crystalline phase. The free energy of dimer formation changed little with increasing number of Leu residues from 16 to 22 but increased with increasing phospholipid fatty acyl chain length, with a slope of about 0.5 kJ mol(-1) per fatty acyl chain carbon. Peptides were excluded from lipid in the gel phase, resulting in increased levels of oligomerization. Addition of cholesterol to form the liquid-ordered state led to increased dimerization but without phase separation. The presence of phosphatidylethanolamine had little effect on dimerization.

Effects of bilayer thickness on the activity of diacylglycerol kinase of Escherichia coli.

We have developed a procedure for the reconstitution of Escherichia coli diacylglycerol kinase (DGK) into phospholipid bilayers containing diacylglycerol substrate. When DGK is reconstituted into a series of phosphatidylcholines containing monounsaturated fatty acyl chains, activity against dihexanoylglycerol (DHG) as a substrate was found to be markedly dependent on the fatty acyl chain length with the highest activity in dioleoylphosphatidylcholine [di(C18:1)PC] and a lower activity in bilayers with shorter or longer fatty acyl chains. Low activities in the short chain phospholipid dimyristoleoylphosphatidylcholine [di(C14:1)PC] followed from an increase in the K(m) value for DHG and ATP, with no effect on v(max). In contrast, in the long chain lipid dierucoylphosphatidylcholine [di(C24:1)PC], the low activity followed from a decrease in v(max) with no effect on K(m). In mixtures of two phosphatidylcholines with different chain lengths, the activity corresponded to that expected for the average chain length of the mixture. Cholesterol increased the activity in di(C14:1)PC but slightly decreased it in di(C18:1)PC or di(C24:1)PC, effects that could follow from changes in bilayer thickness caused by cholesterol.

What the structure of a calcium pump tells us about its mechanism.

The report of the crystal structure of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum in its Ca(2+)-bound form [Toyoshima, Nakasako and Ogawa (2000) Nature (London) 405, 647-655] provides an opportunity to interpret much kinetic and mutagenic data on the ATPase in structural terms. There are no large channels leading from the cytoplasmic surface to the pair of high-affinity Ca(2+) binding sites within the transmembrane region. One possible access pathway involves the charged residues in transmembrane alpha-helix M1, with a Ca(2+) ion passing through the first site to reach the second site. The Ca(2+)-ATPase also contains a pair of binding sites for Ca(2+) that are exposed to the lumen. In the four-site model for transport, phosphorylation of the ATPase leads to transfer of the two bound Ca(2+) ions from the cytoplasmic to the lumenal pair of sites. In the alternating four-site model for transport, phosphorylation leads to release of the bound Ca(2+) ions directly from the cytoplasmic pair of sites, linked to closure of the pair of lumenal binding sites. The lumenal pair of sites could involve a cluster of conserved acidic residues in the loop between M1 and M2. Since there is no obvious pathway from the high-affinity sites to the lumenal surface of the membrane, transport of Ca(2+) ions must involve a significant change in the packing of the transmembrane alpha-helices. The link between the phosphorylation domain and the pair of high-affinity Ca(2+) binding sites is probably provided by two small helices, P1 and P2, in the phosphorylation domain, which contact the loop between transmembrane alpha-helices M6 and M7.

Expression and functional characterization of a Plasmodium falciparum Ca2+-ATPase (PfATP4) belonging to a subclass unique to apicomplexan organisms.

We have obtained a full-length P type ATPase sequence (PfATP4) encoded by Plasmodium falciparum and expressed PfATP4 in Xenopus laevis oocytes to study its function. Comparison of the hitherto incomplete open reading frame with other Ca(2+)-ATPase sequences reveals that PfATP4 differs significantly from previously defined categories. The Ca(2+)-dependent ATPase activity of PfATP4 is stimulated by a much broader range of [Ca(2+)](free) (3.2-320 micrometer) than are an avian SERCA1 pump or rabbit SERCA 1a (maximal activity < 10 micrometer). The activity of PfATP4 is resistant to inhibition by ouabain (200 micrometer) or thapsigargin (0.8 micrometer) but is inhibited by vanadate (1 mM) or cyclopiazonic acid (1 microM). We used a quantitative polymerase chain reaction to assay expression of mRNA encoding PfATP4 relative to that for beta-tubulin in synchronized asexual stages and found variable expression throughout the life cycle with a maximal 5-fold increase in meronts compared with ring stages. This analysis suggests that PfATP4 defines a novel subclass of Ca(2+)-ATPases unique to apicomplexan organisms and therefore offers potential as a drug target.

Selectivity in lipid binding to the bacterial outer membrane protein OmpF.

The outer membrane porin OmpF from Escherichia coli has been reconstituted into lipid bilayers of defined composition, and fluorescence spectroscopy is used to characterize its interaction with the surrounding lipid. OmpF is a trimer within the membrane. It contains two Trp residues per monomer, Trp(214) at the lipid-protein interface and Trp(61) at the trimer interface. The fluorescence of Trp-214 in the mutant W61F is quenched by dibromostearoylphosphatidylcholine (di(Br(2)C18:0)PC), whereas the fluorescence of Trp(61) in the mutant W214F is not quenched by di(Br(2)C18:0)PC when fluorescence is excited directly through the Trp rather than through the Tyr residues. Measurements of relative fluorescence quenching for OmpF reconstituted into mixtures of lipid X and di(Br(2)C18:0)PC have been analyzed to give the binding constant of lipid X for OmpF, relative to that for dioleoylphosphatidylcholine (di(C18:1)PC). The phosphatidylcholine showing the strongest binding to OmpF is dimyristoyloleoylphosphatidylcholine (di(C14:1)PC) with binding constants decreasing with either increasing or decreasing fatty acyl chain length. Comparison with various theories for hydrophobic matching between lipids and proteins suggests that in the chain length range from C14 to C20, hydrophobic matching is achieved largely by distortion of the lipid bilayer around the OmpF, whereas for chains longer than C20, distortion of both the lipid bilayer and of the protein is required to achieve hydrophobic matching. Phosphatidylcholine and phosphatidylethanolamine bind with equal affinity to OmpF, but the affinity for phosphatidylglycerol is about half that for phosphatidylcholine.