An endogenous green fluorescent protein-photoprotein pair in Clytia hemisphaerica eggs shows co-targeting to mitochondria and efficient bioluminescence energy transfer.

Green fluorescent proteins (GFPs) and calcium-activated photoproteins of the aequorin/clytin family, now widely used as research tools, were originally isolated from the hydrozoan jellyfish Aequora victoria. It is known that bioluminescence resonance energy transfer (BRET) is possible between these proteins to generate flashes of green light, but the native function and significance of this phenomenon is unclear. Using the hydrozoan Clytia hemisphaerica, we characterized differential expression of three clytin and four GFP genes in distinct tissues at larva, medusa and polyp stages, corresponding to the major in vivo sites of bioluminescence (medusa tentacles and eggs) and fluorescence (these sites plus medusa manubrium, gonad and larval ectoderms). Potential physiological functions at these sites include UV protection of stem cells for fluorescence alone, and prey attraction and camouflaging counter-illumination for bioluminescence. Remarkably, the clytin2 and GFP2 proteins, co-expressed in eggs, show particularly efficient BRET and co-localize to mitochondria, owing to parallel acquisition by the two genes of mitochondrial targeting sequences during hydrozoan evolution. Overall, our results indicate that endogenous GFPs and photoproteins can play diverse roles even within one species and provide a striking and novel example of protein coevolution, which could have facilitated efficient or brighter BRET flashes through mitochondrial compartmentalization.

The molecular basis of lactose intolerance.

A staggering 4000 million people cannot digest lactose, the sugar in milk, properly. All mammals, apart from white Northern Europeans and few tribes in Africa and Asia, lose most of their lactase, the enzyme that cleaves lactose into galactose and glucose, after weaning. Lactose intolerance causes gut and a range of systemic symptoms, though the threshold to lactose varies considerably between ethnic groups and individuals within a group. The molecular basis of inherited hypolactasia has yet to be identified, though two polymorphisms in the introns of a helicase upstream from the lactase gene correlate closely with hypolactasia, and thus lactose intolerance. The symptoms of lactose intolerance are caused by gases and toxins produced by anaerobic bacteria in the large intestine. Bacterial toxins may play a key role in several other diseases, such as diabetes, rheumatoid arthritis, multiple sclerosis and some cancers. The problem of lactose intolerance has been exacerbated because of the addition of products containing lactose to various foods and drinks without being on the label. Lactose intolerance fits exactly the illness that Charles Darwin suffered from for over 40 years, and yet was never diagnosed. Darwin missed something else--the key to our own evolution--the Rubicon some 300 million years ago that produced lactose and lactase in sufficient amounts to be susceptible to natural selection.

A large-conductance (BK) potassium channel subtype affects both growth and mineralization of human osteoblasts.

The pharmacology of the large-conductance K(+) (BK) channel in human osteoblasts is not well defined, and its role in bone is speculative. Here we assess BK channel properties in MG63 cells and primary human osteoblasts and determine whether pharmacological modulation affects cell function. We used RT-PCR and patch-clamp methods to determine the expression of BK channel subunits and cell number assays in the absence and presence of BK channel modulators. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3, and KCNMB4 subunits. The BK channel was voltage dependent, with a mean unitary conductance of 228.8 pS (n = 10) in cell-attached patches (140 mM K(+)/140 mM K(+)) and a conductance of 142.5 pS (n = 16) in excised outside-out and 155 pS (n = 6) in inside-out patches in 3 mM K(+)/140 mM K(+). The selectivity ratio (ratio of K(+) to Na(+) permeability) was 15:1. The channel was blocked by tetraethylammonium (TEA, 0.3 mM), iberiotoxin (5-60 nM), tetrandrine (5-30 microM), and paxilline (10 microM) and activated by isopimaric acid (20 microM). BK channel modulators affected MG63 cell numbers: TEA and tetrandrine significantly increased cell numbers at low concentrations (3 mM and 3 microM, respectively) and reduced cell numbers at higher concentrations (>10 mM and >10 microM, respectively). Neither iberiotoxin (20-300 nM) nor slotoxin (300 nM) affected cell numbers. The increase in cell numbers by TEA was blocked by isopimaric acid. TEA (0.1-3.0 mM) significantly increased mineralization in primary osteoblasts. In conclusion, the BK channel has a distinctive pharmacology and is thus a target for therapeutic strategies aimed at modulating osteoblast proliferation and function.

ATP regulates calcium efflux and growth in E. coli.

Escherichia coli regulates cytosolic free Ca(2+) in the micromolar range through influx and efflux. Herein, we show for the first time that ATP is essential for Ca(2+) efflux and that ATP levels also affect generation time. A transcriptome analysis identified 110 genes whose expression responded to an increase in cytosolic Ca(2+) (41 elevated, 69 depressed). Of these, 3 transport proteins and 4 membrane proteins were identified as potential Ca(2+) transport pathways. Expression of a further 943 genes was modified after 1 h in growth medium containing Ca(2+) relative to time zero. Based on the microarray results and other predicted possible Ca(2+) transporters, the level of cytosolic free Ca(2+) was measured in selected mutants from the Keio knockout collection using intracellular aequorin. In this way, we identified a knockout of atpD, coding for a component of the F(o)F(1) ATPase, as defective in Ca(2+) efflux. Seven other putative Ca(2+) transport proteins exhibited normal Ca(2+) handling. The defect in the DeltaatpD knockout cells could be explained by a 70% reduction in ATP. One millimolar glucose or 1 mM methylglyoxal raised ATP in the DeltaatpD knockout cells to that of the wild type and restored Ca(2+) efflux. One millimolar 2,4-dinitrophenol lowered the ATP in wild type to that in the DeltaatpD cells. Under these conditions, a similar defect in Ca(2+) efflux in wild type was observed in DeltaatpD cells. Ten millimolar concentration of Ca(2+) resulted in a 30% elevation in ATP in wild type and was accompanied by a 10% reduction in generation time under these conditions. Knockouts of pitB, a potential Ca(2+) transporter, atoA, the beta subunit of acetate CoA-transferase likely to be involved in polyhydroxybutyrate synthesis, and ppk, encoding polyphosphate kinase, all indicated no defect in Ca(2+) efflux. We therefore propose that ATP is most likely to regulate Ca(2+) efflux in E. coli through an ATPase.

pH and monovalent cations regulate cytosolic free Ca(2+) in E. coli.

The results here show for the first time that pH and monovalent cations can regulate cytosolic free Ca(2+) in E. coli through Ca(2+) influx and efflux, monitored using aequorin. At pH 7.5 the resting cytosolic free Ca(2+) was 0.2-0.5 microM. In the presence of external Ca(2+) (1 mM) at alkaline pH this rose to 4 microM, being reduced to 0.9 microM at acid pH. Removal of external Ca(2+) caused an immediate decrease in cytosolic free Ca(2+) at 50-100 nM s(-1). Efflux rates were the same at pH 5.5, 7.5 and 9.5. Thus, ChaA, a putative Ca(2+)/H(+)exchanger, appeared not to be a major Ca(2+)-efflux pathway. In the absence of added Na(+), but with 1 mM external Ca(2+), cytosolic free Ca(2+) rose to approximately 10 microM. The addition of Na(+)(half maximum 60 mM) largely blocked this increase and immediately stimulated Ca(2+) efflux. However, this effect was not specific, since K(+) also stimulated efflux. In contrast, an increase in osmotic pressure by addition of sucrose did not significantly stimulate Ca(2+) efflux. The results were consistent with H(+) and monovalent cations competing with Ca(2+) for a non-selective ion influx channel. Ca(2+) entry and efflux in chaA and yrbG knockouts were not significantly different from wild type, confirming that neither ChaA nor YrbG appear to play a major role in regulating cytosolic Ca(2+) in Escherichia coli. The number of Ca(2+) ions calculated to move per cell per second ranged from <1 to 100, depending on conditions. Yet a single eukaryote Ca(2+) channel, conductance 100 pS, should conduct >6 million ions per second. This raises fundamental questions about the nature and regulation of Ca(2+) transport in bacteria, and other small living systems such as mitochondria, requiring a new mathematical approach to describe such ion movements. The results have important significance in the adaptation of E. coli to different ionic environments such as the gut, fresh water and in sea water near sewage effluents.

Measurement of breath hydrogen and methane, together with lactase genotype, defines the current best practice for investigation of lactose sensitivity.

BACKGROUND: Currently, there is no 'gold standard' for detecting patients with sensitivity to lactose. Biochemical investigation by a breath hydrogen test alone detects <50% cases. Breath methane and symptoms are not recorded as standard practice. The clinical value of analysing C/T(13910) and G/A(22018) polymorphisms, strongly associated with lactose sensitivity, has not been established. METHODS: Two hundred and ten patients with unexplained gut and systemic symptoms and controls were challenged with 50 g lactose. Breath hydrogen and methane were measured and symptoms recorded. All were genotyped for two polymorphisms, C/T(13910) and G/A(22018). RESULTS: CC(13910)/GG(22018) in 14.5%, CT(13910)/GA(22018) in 39% and TT(13910)/AA(22018) in 46.5%. One hundred percent of CC(13910)/GG(22018) were lactose sensitive having a breath hydrogen >20 ppm within 6 h and symptoms. But the breath hydrogen test lacked sensitivity and specificity in the other groups. There was elevated breath hydrogen in 21% of CT(13910)/GA(22018) and 15% of TT(13910)/AA(22018) by 6 h, whereas 17 and 30.9% had elevated breath methane alone. Breath methane and breath hydrogen with clinical symptoms improved sensitivity and specificity, increasing detection of lactose sensitivity in genotypes CT/GA and TT/AA from <50 to >75%. CONCLUSIONS: The data presented define the current best practice for the clinical identification of lactose sensitivity. Patients were first genotyped. Those identified as CC with symptoms should immediately undertake a 12-week lactose-free diet. Those identified as CT or TT should undertake a breath hydrogen and methane test. Those positive for hydrogen or methane along with symptoms or with symptoms only, should also undertake a lactose-free diet. Those with high hydrogen without symptoms should be investigated for causes other than lactose sensitivity.

Methylglyoxal and other carbohydrate metabolites induce lanthanum-sensitive Ca2+ transients and inhibit growth in E. coli.

The results here are the first demonstration of a family of carbohydrate fermentation products opening Ca2+ channels in bacteria. Methylglyoxal, acetoin (acetyl methyl carbinol), diacetyl (2,3 butane dione), and butane 2,3 diol induced Ca2+ transients in Escherichia coli, monitored by aequorin, apparently by opening Ca2+ channels. Methylglyoxal was most potent (K(1/2) = 1 mM, 50 mM for butane 2,3 diol). Ca2+ transients depended on external Ca2+ (0.1-10 mM), and were blocked by La3+ (5 mM). The metabolites affected growth, methylglyoxal being most potent, blocking growth completely up to 5 h without killing the cells. But there was no affect on the number of viable cells after 24 h. These results were consistent with carbohydrate products activating a La3+-sensitive Ca2+ channel, rises in cytosolic Ca2+ possibly protecting against certain toxins. They have important implications in bacterial-host cell signalling, and where numbers of different bacteria compete for the same substrates, e.g., the gut in lactose and food intolerance.

Cytosolic Ca2+ regulates protein expression in E. coli through release from inclusion bodies.

The results here are the first clear demonstration of a physiological role for cytosolic Ca(2+) in Escherichia coli by releasing a Ca(2+) binding protein, apoaequorin, from inclusion bodies. In growth medium LB the cytosolic free Ca(2+) was 0.1-0.3 microM. Addition of EGTA reduced this to <0.1 microM, whereas addition of Ca(2+) (10mM) resulted in a cytosolic free Ca(2+) of 1-2 microM for at least 2h. Ca(2+) caused a 1.5- to 2-fold increase in the level of apoaequorin induced by IPTG. Whereas EGTA induced a 50% decrease. The effect of a Ca(2+) was explained by release of protein from the inclusion bodies, together with a stabilisation of apoaequorin against degradation. Ca(2+) also reduced the generation time by 4-5 min. These results have important implications for unravelling the physiological role of cytosolic Ca(2+) in bacteria, particularly where several species are competing for the same nutrients, such as in the gut.

Fermentation product butane 2,3-diol induces Ca2+ transients in E. coli through activation of lanthanum-sensitive Ca2+ channels.

The results here are the first demonstration of a physiological agonist opening Ca2+ channels in bacteria. Bacteria in the gut ferment glucose and other substrates, producing alcohols, diols, ketones and acids, that play a key role in lactose intolerance, through the activation of Ca2+ and other ion channels in host cells and neighbouring bacteria. Here we show butane 2,3-diol (5-200mM; half maximum 25mM) activates Ca2+ transients in E. coli, monitored by aequorin. Ca2+-transient magnitude depended on external Ca2+ (0.1-10mM). meso-Butane 2,3-diol was approximately twice as potent as 2R,3R (-) and 2S,3S (+) butane 2,3-diol. There were no detectable effects on cytosolic free Ca2+ of butane 1,3-diol, butane 1,4-diol and ethylene glycol. The glycerol fermentation product propane 1,3-diol only induced significant Ca2+ transients in 10mM external Ca2. Ca2+ butane 2,3-diol Ca2+ transients were due to activation of Ca2+ influx, followed by activation of Ca2+ efflux. The effect of butane 2,3-diol was abolished by La3+, and markedly reduced as a function of growth phase. These results were consistent with butane 2,3-diol activating a novel La3+-sensitive Ca2+ channel. They have important implications for the role of butane 2,3-diol and Ca2+ in bacterial-host cell signalling.

Darwin's illness revealed.

After returning from the Beagle in 1836, Charles Darwin suffered for over 40 years from long bouts of vomiting, gut pain, headaches, severe tiredness, skin problems, and depression. Twenty doctors failed to treat him. Many books and papers have explained Darwin's mystery illness as organic or psychosomatic, including arsenic poisoning, Chagas' disease, multiple allergy, hypochondria, or bereavement syndrome. None stand up to full scrutiny. His medical history shows he had an organic problem, exacerbated by depression. Here we show that all Darwin's symptoms match systemic lactose intolerance. Vomiting and gut problems showed up two to three hours after a meal, the time it takes for lactose to reach the large intestine. His family history shows a major inherited component, as with genetically predisposed hypolactasia. Darwin only got better when, by chance, he stopped taking milk and cream. Darwin's illness highlights something else he missed--the importance of lactose in mammalian and human evolution.

The molecular basis of lactose intolerance.

A staggering 4000 million people cannot digest lactose, the sugar in milk, properly. All mammals, apart from white Northern Europeans and few tribes in Africa and Asia, lose most of their lactase, the enzyme that cleaves lactose into galactose and glucose, after weaning. Lactose intolerance causes gut and a range of systemic symptoms, though the threshold to lactose varies considerably between ethnic groups and individuals within a group. The molecular basis of inherited hypolactasia has yet to be identified, though two polymorphisms in the introns of a helicase upstream from the lactase gene correlate closely with hypolactasia, and thus lactose intolerance. The symptoms of lactose intolerance are caused by gases and toxins produced by anaerobic bacteria in the large intestine. Bacterial toxins may play a key role in several other diseases, such as diabetes, rheumatoid arthritis, multiple sclerosis and some cancers. The problem of lactose intolerance has been exacerbated because of the addition of products containing lactose to various foods and drinks without being on the label. Lactose intolerance fits exactly the illness that Charles Darwin suffered from for over 40 years, and yet was never diagnosed. Darwin missed something else--the key to our own evolution--the Rubicon some 300 million years ago that produced lactose and lactase in sufficient amounts to be susceptible to natural selection.

Lactose causes heart arrhythmia in the water flea Daphnia pulex.

The cladoceran Daphnia pulex is well established as a model for ecotoxicology. Here, we show that D. pulex is also useful for investigating the effects of toxins on the heart in situ and the toxic effects in lactose intolerance. The mean heart rate at 10 degrees C was 195.9+/-27.0 beats/min (n=276, range 89.2-249.2, >80% 170-230 beats/min). D. pulex heart responded to caffeine, isoproteronol, adrenaline, propranolol and carbachol in the bathing medium. Lactose (50-200 mM) inhibited the heart rate by 30-100% (K(1/2)=60 mM) and generated severe arrhythmia within 60 min. These effects were fully reversible by 3-4 h. Sucrose (100-200 mM) also inhibited the heart rate, but glucose (100-200 mM) and galactose (100-200 mM) had no effect, suggesting that the inhibition by lactose or sucrose was not simply an osmotic effect. The potent antibiotic ampicillin did not prevent the lactose inhibition, and two diols known to be generated by bacteria under anaerobic conditions were also without effect. The lack of effect of l-ribose (2 mM), a potent inhibitor of beta-galactosidase, supported the hypothesis that lactose and other disaccharides may affect directly ion channels in the heart. The results show that D. pulex is a novel model system for studying effects of agonists and toxins on cell signalling and ion channels in situ.

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca2+ channels in the membrane.

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca(2+) homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca(2+) in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca(2+)polyP) complexes. PHB(Ca(2+)polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA(-) strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.

DNA damage-induced [Zn(2+)](i) transients: correlation with cell cycle arrest and apoptosis in lymphoma cells.

Reactive changes in free intracellular zinc cation concentration ([Zn(2+)](i)) were monitored, using the fluorescent probe Zinquin, in human lymphoma cells exposed to the DNA-damaging agent VP-16. Two-photon excitation microscopy showed that Zinquin-Zn(2+) forms complexes in cytoplasmic vesicles. [Zn(2+)](i) increased in both p53(wt) (wild type) and p53(mut) (mutant) cells after exposure to low drug doses. In p53(mut) cells noncompetent for DNA damage-induced apoptosis, elevated [Zn(2+)](i) was maintained at higher drug doses, unlike competent p53(wt) cells that showed a collapse of the transient before apoptosis. In p53(wt) cells, the [Zn(2+)](i) rise paralleled an increase in p53 and bax-to-bcl-2 ratio but preceded an increase in p21(WAF1), active cell cycle arrest in G(2), or nuclear fragmentation. Reducing [Zn(2+)](i), using N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine, caused rapid apoptosis in both p53(wt) and p53(mut) cells, although cotreatment with VP-16 exacerbated apoptosis only in p53(wt) cells. This may reflect changed thresholds for proapoptotic caspase-3 activation in competent cells. We conclude that the DNA damage-induced transient is p53-independent up to a damage threshold, beyond which competent cells reduce [Zn(2+)](i) before apoptosis. Early stress responses in p53(wt) cells take place in an environment of enhanced Zn(2+) availability.

A mathematical model predicts that calreticulin interacts with the endoplasmic reticulum Ca(2+)-ATPase.

A robust mathematical model developed from single cell calcium (Ca(2+)) dynamics has enabled us to predict the consequences of over-expression of endoplasmic reticulum-located chaperones. Model predictions concluded that calreticulin interacts with the lumenal domain of the sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) pump, altering pump affinity for Ca(2+) (K(1/2) switches from 247 to 431 nM) and hence generating Ca(2+) oscillations. Expression of calreticulin in the ER generated an average of six transient-decline oscillations during the Ca(2+) recovery phase, upon exposure to maximal levels of the agonist ATP. In contrast, normal cells produced a single Ca(2+) transient with few or no oscillations. By conditioning the model to experimental data, parameters for generation and decay of IP(3) and SERCA pump kinetics were determined. To elucidate the possible source of the oscillatory behavior three possible oscillators, 1) IP(3), 2) IP(3)R, and 3) SERCA pump, were investigated and parameters constrained by experimental data to produce the best candidate. Each of the three oscillators generated very good fits with experimental data. However, converting a normal exponential recovery to a transient-decline oscillator predicted that the SERCA pump is the most likely candidate for calreticulin-mediated Ca(2+) release, highlighting the role of this chaperone as a signal protein within the endoplasmic reticulum.