Routine testing of close contacts of confirmed COVID-19 cases - National COVID-19 Contact Management Programme, Ireland, May to August 2020.

OBJECTIVES: The objective of this study was to inform public health practitioners who are designing, adapting and implementing testing and tracing strategies for Coronavirus disease (COVID-19) control. STUDY DESIGN: The study design is monitoring and evaluation of a national public health protection programme. METHODS: All close contacts of laboratory-confirmed cases of COVID-19 identified between the 19th May and 2nd August were included; secondary attack rates and numbers needed to test were estimated. RESULTS: Four thousand five hundred eighty six of 7272 (63%) close contacts of cases were tested with at least one test. The secondary attack rate in close contacts who were tested was 7% (95% Confidence Interval [CI]: 6.3 - 7.8%). At the 'day 0' test, 14.6% (95% CI: 11.6-17.6%) of symptomatic close contacts tested positive compared with 5.2% (95% CI: 4.4-5.9%) of asymptomatic close contacts. CONCLUSIONS: The application of additional symptom-based criteria for testing in this high-incidence population (close contacts) is of limited utility because of the low negative predictive value of absence of symptoms.

Chronic treatment with a tryptophan-rich protein hydrolysate improves emotional processing, mental energy levels and reaction time in middle-aged women.

Common pharmacological treatments of mood disorders aim to modulate serotonergic neurotransmission and enhance serotonin levels in the brain. Brain serotonin levels are dependent on the availability of its food-derived precursor essential amino acid tryptophan (Trp). We tested the hypothesis that delivery of Trp via food may serve as an alternative treatment, and examined the effects of a Trp-rich, bioavailable dietary supplement from egg protein hydrolysate on cognitive and emotional functions, mood state, and sleep quality. In a randomised, placebo-controlled, parallel trial, fifty-nine mentally and physically healthy women aged 45-65 years received placebo (n 30) or the supplement (n 29) (both as 0.5 g twice per d) for 19 d. Emotional processing was significantly changed by supplementation, exhibiting a shift in bias away from negative stimuli. The results for the Affective Go/No-Go Task exhibited a slowing of responses to negative words, suggesting reduced attention to negative emotional stimuli. The results for the Facial Emotional Expression Rating Task also supported a shift away from attention to negative emotions and a bias towards happiness. An increase in arousal-like symptoms, labelled 'high energy', shorter reaction times and a slight benefit to sustained attention were observed in the treated subjects. Finally, when the supplement was taken 60-90 min before bedtime, a feeling of happiness before going to bed was consistently reported. In summary, daily consumption of a low-dose supplement containing bioavailable Trp may have beneficial effects on emotional and cognitive functions.

Proboscis lateralis.

INTRODUCTION: Proboscis lateralis is a rare congenitally acquired facial abnormality characterized by a soft-tissue tube- or trunk-like appendage projecting from the surface of the face, most frequently rooted in the medial canthal region. Proboscis lateralis is generally associated with a wide range of concomitant craniofacial anomalies, giving rise to multiple theories describing the embryological pathogenesis and various classification systems to account for the pathological associations. RESULTS/CONCLUSION: This paper provides a literature review of the rare manifestations of proboscis lateralis and represents a summary of current literature related to embryological pathogenesis, definitive diagnosis, and surgical management approaches.

Effects of nitrogen enrichment on phosphatase activity and nitrogen : phosphorus relationships in Cladonia portentosa.

*Relationships between nitrogen deposition in the UK and phosphomonoesterase (PME) activity and nitrogen (N) and phosphorus (P) concentrations in Cladonia portentosa were quantified to understand factors limiting lichen growth and to further develop biomarkers for N pollution. *Lichen was collected from sites differing either in rates of wet N (NH(4)(+) + NO(3)(-)) deposition or in annual mean N concentration in rainfall based on both measured and modelled data sets. The PME activity, and total N and P concentrations were measured in specific horizontal strata in lichen mats and PME activity in the thallus was located using an enzyme-labelled fluorescent phosphatase substrate. *With an increase in modelled N deposition from 4.1 to 32.8 kg N ha(-1) yr(-1), PME activity, thallus N and N : P ratio increased by factors of 2.3, 1.4 and 1.8, respectively. Correlations with modelled data were generally stronger than with measured data and those with N deposition were stronger than those with N concentration in rainfall. The PME activity was located solely in the lichen fungus in outer regions of the thallus. *Nitrogen enrichment changes lichen N : P ratios from values typical of N limitation (for example, 10) to those indicative of P limitation (for example, 26) driving upregulation of PME activity.

Response of phosphomonoesterase activity in the lichen Cladonia portentosa to nitrogen and phosphorus enrichment in a field manipulation experiment.

*Effects of nitrogen (N) enrichment on the heathland lichen Cladonia portentosa were quantified to test the hypothesis that modified N : phosphorus (P) relationships observed in this species in N-polluted natural environments are a direct effect of increased N deposition, and to evaluate potential confounding effects of N form and P availability. *Cladonia portentosa was harvested from experimental plots in lichen-rich peatland vegetation (background total N deposition of 8 kg N ha(-1) yr(-1)) treated for 4 yr with additional wet N deposition at 0, 8, 24 and 56 kg N ha(-1) yr(-1) as either NH(4)(+) or NO(3)(-), and with or without P added at either 0.6 or 4 kg P ha(-1) yr(-1). *Nitrogen enrichment increased thallus N concentration, N : P mass ratio and phosphomonoesterase (PME) activity by factors of up to 1.3, 1.4 and 1.7, respectively, effects being independent of N form. Phosphomonoesterase activity was tightly related to thallus N : P ratio with additions of P at 4 kg ha(-1) yr(-1) depressing PME activity by a factor of 0.4. *Nitrogen enrichment induces P-limitation in C. portentosa with attendant changes in chemical and physiological characteristics that could be used as sensitive biomarkers with which to detect low levels of N pollution.

Chromosome condensation and sister chromatid pairing in budding yeast.

We have developed a fluorescent in situ hybridization (FISH) method to examine the structure of both natural chromosomes and small artificial chromosomes during the mitotic cycle of budding yeast. Our results suggest that the pairing of sister chromatids: (a) occurs near the centromere and at multiple places along the chromosome arm as has been observed in other eukaryotic cells; (b) is maintained in the absence of catenation between sister DNA molecules; and (c) is independent of large blocks of repetitive DNA commonly associated with heterochromatin. Condensation of a unique region of chromosome XVI and the highly repetitive ribosomal DNA (rDNA) cluster from chromosome XII were also examined in budding yeast. Interphase chromosomes were condensed 80-fold relative to B form DNA, similar to what has been observed in other eukaryotes, suggesting that the structure of interphase chromosomes may be conserved among eukaryotes. While additional condensation of budding yeast chromosomes were observed during mitosis, the level of condensation was less than that observed for human mitotic chromosomes. At most stages of the cell cycle, both unique and repetitive sequences were either condensed or decondensed. However, in cells arrested in late mitosis (M) by a cdc15 mutation, the unique DNA appeared decondensed while the repetitive rDNA region appeared condensed, suggesting that the condensation state of separate regions of the genome may be regulated differently. The ability to monitor the pairing and condensation of sister chromatids in budding yeast should facilitate the molecular analysis of these processes as well as provide two new landmarks for evaluating the function of important cell cycle regulators like p34 kinases and cyclins. Finally our FISH method provides a new tool to analyze centromeres, telomeres, and gene expression in budding yeast.

Monoclonal antibodies that recognize a polypeptide antigenic determinant shared by multiple Caenorhabditis elegans sperm-specific proteins.

Four monoclonal antibodies that are directed against antigens present in sperm and absent from other worm tissues were characterized. Antibody TR20 is directed against the major sperm proteins, a family of small, abundant, cytoplasmic proteins that have been previously described (Klass, M. R., and D. Hirsh, 1981, Dev. Biol., 84:299-312; Burke, D. J., and S. Ward, 1983, J. Mol. Biol., 171:1-29). Three other antibodies, SP56, SP150, and TR11, are all directed against the same set of minor sperm polypeptides that range in size from 29 to 215 kD. More than eight different sperm polypeptides are antigenic by both immunotransfer and immunoprecipitation assays. The three antibodies are different immunoglobulin subclasses, yet they compete with each other for antigen binding so they are directed against the same antigenic determinant on the multiple sperm proteins. This antigenic determinant is sensitive to any of six different proteases, is insensitive to periodate oxidation or N-glycanase digestion, and is detectable on a polypeptide synthesized in vitro. Therefore, the antigenic determinant resides in the polypeptide chain. However, peptide fragments of the proteins are not antigenic, thus the determinant is likely to be dependent on polypeptide conformation. The antigenic determinant shared by these proteins could represent a common structural feature of importance to the localization or cellular specificity of these proteins.

The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes.

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.

Centromere position in budding yeast: evidence for anaphase A.

Although general features of chromosome movement during the cell cycle are conserved among all eukaryotic cells, particular aspects vary between organisms. Understanding the basis for these variations should provide significant insight into the mechanism of chromosome movement. In this context, establishing the types of chromosome movement in the budding yeast Saccharomyces cerevisiae is important since the complexes that mediate chromosome movement (microtubule organizing centers, spindles, and kinetochores) appear much simpler in this organism than in many other eukaryotic cells. We have used fluorescence in situ hybridization to begin an analysis of chromosome movement in budding yeast. Our results demonstrate that the position of yeast centromeres changes as a function of the cell cycle in a manner similar to other eukaryotes. Centromeres are skewed to the side of the nucleus containing the spindle pole in G1; away from the poles in mid-M and clustered near the poles in anaphase and telophase. The change in position of the centromeres relative to the spindle poles supports the existence of anaphase A in budding yeast. In addition, an anaphase A-like activity independent of anaphase B was demonstrated by following the change in centromere position in telophase-arrested cells upon depolymerization and subsequent repolymerization of microtubules. The roles of anaphase A activity and G1 centromere positioning in the segregation of budding yeast chromosomes are discussed. The fluorescence in situ hybridization methodology and experimental strategies described in this study provide powerful new tools to analyze mutants defective in specific kinesin-like molecules, spindle components, and centromere factors, thereby elucidating the mechanism of chromosome movement.

Optical coherence tomography characteristics of different types of big bubbles seen in deep anterior lamellar keratoplasty by the big bubble technique.

PurposeTo define optical coherence tomography (OCT) characteristics of type-1, type-2, and mixed big bubbles (BB) seen in deep anterior lamellar keratoplasty.MethodsHuman sclero-corneal discs were obtained from UK (30) and Canada (16) eye banks. Air was injected into corneal stroma until a BB formed. UK samples were fixed in formalin before scanning with Fourier-domain (FD-OCT). One pair of each type of BB was scanned fresh. All BB obtained from Canada were scanned fresh with time-domain (TD-OCT). For each OCT machine used, type-1 BB from which Descemets membrane (DM) was partially peeled, were also scanned. The morphological characteristics of the scans were studied.ResultsFD-OCT of the posterior wall of type-1 (Dua's layer (DL) with DM) and type-2 BB (DM alone) both revealed a double-contour hyper-reflective curvilinear image with a hypo-reflective zone in between. The anterior line of type-2 BB was thinner than that seen with type-1 BB. In mixed BB, FD-OCT showed two separate curvilinear images. The anterior image was a single hyper-reflective line (DL), whereas the posterior image, representing the posterior wall of type-2 BB (DM) was made of two hyper-reflective lines with a dark space in between. TD-OCT images were similar with less defined component lines, but the entire extent of the BB could be visualised.ConclusionOn OCT examination the DM and DL present distinct features, which can help identify type-1, type-2, and mixed BB. These characteristics will help corneal surgeons interpret intraoperative OCT during lamellar corneal surgery.

Predicting pork loin chop yield using carcass and loin characteristics.

The objective was to determine the predictive ability of carcass length for the number of equal-thickness chops obtained from a boneless pork loin. Longer pork carcasses are assumed to yield longer loins and, therefore, an increased number of chops. Loins were collected from pigs (1,238 total) raised under commercial conditions and marketed when the mean pig weight in a pen reached 138 kg. Pigs were slaughtered over 7 wk in a commercial facility. Carcass length was measured at 1 d postmortem on the left side of each carcass from the anterior edge of the symphysis pubis bone to the anterior edge of the first rib. Carcasses were fabricated, and boneless loins (North American Meat Processors number 414) were vacuum packaged and transported to the University of Illinois Meat Science Laboratory. Loins were stored at 4°C for 14 d. At the end of the aging period, loins were weighed, measured for stretched length (stretched to maximum length without distortion) and compressed length (compressed to minimum length without distortion), and sliced into 2.54-cm-thick chops. Boneless chops were counted and weighed. Carcass length ranged from a minimum of 78.2 cm to a maximum of 96.5 cm and the number of boneless chops ranged from a minimum of 13 to a maximum of 20 chops. Data were analyzed using the regression procedure of SAS. The dependent variable was the number of boneless chops. Coefficient of determination () was calculated for carcass length, boneless loin weight, compressed loin length, and stretched loin length. Carcass length explained 15% ( < 0.0001) of the variation in the number of loin chops. Loin weight explained 33% ( < 0.0001) of the variation in the number of loin chops. Compressed loin length and stretched loin length explained 28 and 8% ( < 0.0001), respectively, of the variation in the number of loin chops. Multiple linear regression was used to determine a predictive equation for the number of loin chops using the stepwise selection option of all independent variables. The combination of boneless loin weight, compressed loin length, 10th-rib carcass fat depth, and carcass length explained 45% of the variation ( < 0.0001; C(p) = 16.76) in the number of loin chops using a required statistic at the SLENTRY and SLSTAY level = 0.15. Overall, carcass length is a poor predictor of the number of equal-thickness loin chops that can be derived from a boneless pork loin.

Pork loin quality is not indicative of fresh belly or fresh and cured ham quality.

The objective was to characterize the relationship between fresh loin quality with fresh belly or fresh and cured ham quality. Pigs raised in 8 barns representing 2 seasons [cold ( = 4,290) and hot ( = 3,394)] and 2 production focuses [lean ( = 3,627) and quality ( = 4,057)] were used. Carcass characteristics and other meat quality data were collected on 7,684 carcasses. All of the carcasses were evaluated for HCW, LM depth, tenth rib fat depth, leg (ham primal) weight, instrumental color on the gluteus medius and gluteus profundus of the ham face, and subjective loin quality. Instrumental loin color and ultimate pH (≥ 22 h postmortem) were collected on the ventral side of loins along with dimensions and firmness scores of fresh bellies from 50% of the carcasses. Ten percent of the boneless loins and fresh hams were evaluated for slice shear force (SSF) or cured ham characteristics. Correlation coefficients between traits were computed using the CORR procedure of SAS and considered significantly different from 0 at ≤ 0.05. Temperature decline, beginning at 31 min postmortem and concluding at 22 h postmortem, for the longissimus dorsi and semimembranosus muscles were evaluated on 10% of the carcasses. Ultimate loin pH was correlated with dimensional belly characteristics ( ≥ |0.07|; < 0.0001) fresh ham instrumental color ( ≥ |0.03|; ≤ 0.05), and semimembranosus ultimate pH ( = 0.33; < 0.0001). Further, ultimate loin pH was correlated ( ≤ 0.01) with pump retention ( = 0.087) and cooked yield ( = 0.156) of cured hams. Instrumental L*on the ventral surface of the loin was related to L* on both muscles of the ham face ( ≤ 0.0001). Even though significant relationships between the loin, belly, and ham were detected, the variability in belly and ham quality explained by variability in loin quality was poor (≤ 22.09%). Compositional differences between the loin and belly may have contributed to those poor relationships. Additionally, differences in temperature declines during chilling between the loin and ham likely contributed to the weak nature of relationships. Equilibration of longissimus dorsi temperature to ambient cooler temperature occurred at 14 h postmortem ( = 0.0005), yet the semimembranosus had not equilibrated with ambient (equilibration bay) temperature ( < 0.0001) at 22 h postmortem. Using loin quality to draw conclusions about fresh belly and fresh and cured ham quality may be misleading.

Effects of marketing group on the quality of fresh and cured hams sourced from a commercial processing facility.

The objective was: 1) to characterize the effect of marketing group on fresh and cured ham quality, and 2) to determine which fresh ham traits correlated to cured ham quality traits. Pigs raised in 8 barns representing 2 seasons (hot and cold) and 2 production focuses (lean and quality) were used. Three groups were marketed from each barn. A total of 7,684 carcasses were used for data collection at the abattoir. Every tenth carcass was noted as a select carcass for in-depth ham quality analyses. Leg primal weight and instrumental color were measured on 100% of the population. On the select 10% of the population, hams were fabricated into sub-primal pieces, and 3-piece hams were manufactured to evaluate cured ham quality and processing yield. Data were analyzed as a split-plot design in the MIXED procedure of SAS with production focus as the whole-plot factor, and marketing group as the split-plot factor. Pearson correlation coefficients between fresh and cured ham traits were computed. There were no differences ( ≥ 0.15) in instrumental color or ultimate pH ( ≥ 0.14) among fresh ham muscles from any marketing group. The only exception was the semimembranosus of marketing group 2 was lighter than marketing group 1 ( = 0.03) and the dark portion of the semitendinosus muscle from group 1 was lighter than from group 3 ( = 0.01). There were no differences ( ≥ 0.33) in ultimate pH of fresh ham muscles between production focuses, but several muscles from quality focus pigs were lighter in color than ham muscles from lean focus pigs. The lack of differences in fresh ham quality lead to few differences in cured ham quality. Cured hams from the quality focus pigs had greater lipid content ( < 0.01) than hams from lean focus pigs. Cured lightness values of hams from marketing group 1 and 2 were 1.52 units lighter than hams from marketing group 3 ( 0.01). Overall, marketing group did not impact ham quality. Fresh ham quality was not strongly related to cured ham quality. Some correlations were present between fresh and cured ham traits, but those relationships were likely not strong enough to be used as a sorting tool for fresh hams to generate high quality cured hams.

Characterization of two gangliosides of the paragloboside series from chicken skeletal muscle.

Two glucosamine-containing gangliosides, a ceramide pentasaccharide and ceramide heptasaccharide, have been purified from the gangliosides of chicken skeletal muscle. The lipids were extracted with a mixture of tetrahydrofuran and aqueous KCl and purified by anion-exchange and silicic acid column chromatography. Their structures were determined by specific enzymatic hydrolysis and methylation analysis. The proposed structures are sialosyl derivates of: (a) paragloboside, and (b) paragloboside with an additional lactosamine disaccharide: (a) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer, (b) NeuAc alpha 2 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcNAc beta 1 leads to 3Gal beta 1 leads to 4GlcCer. The long base was predominantly sphingosine and the acyl groups were mainly palmitate, stearate and oleate.

Sialylation of lacto-N-neohexaosylceramide by sialyltransferase from embryonic chicken muscle.

A sialyltransferase which catalyzes the in vitro biosynthesis of N-acetylneuraminosyllacto-N-neohexaosylceramide from lacto-N-neohexaosylceramide and CMP-NeuAc has been examined in embryonic chicken breast muscle. The maximum enzyme activity was observed in 11-12-day-old embryos. The enzyme has optimum activity at pH 6.8 in the presence of Triton CF-54 and Mg2+. The apparent Km values for lacto-N-neohexaosylceramide and CMP-NeuAc were 0.9 and 0.67 mM, respectively. The enzymic product was characterized by TLC, neuraminidase hydrolysis and permethylation analysis. The structure was identical to authentic N-acetylneuraminosyllacto-N-neohexaosylceramide from chicken muscle. In addition, a disialo derivative has been detected that constitutes 15% of the total radioactivity incorporated. The two sialic acids connected by sialosyl-sialosyl linkage were attached to the terminal galactose residue. To our knowledge, this is the first report of biosynthesis of this disialo compound.

Biosynthesis of GM1b and similar neolactoseries gangliosides by a partially purified chicken skeletal muscle sialyltransferase. Effect of sphingomyelin and acetylcholine.

An alpha 2----3 glycolipid galactosyl sialyltransferase (SAT3/4) has been partially purified from embryonic chicken skeletal muscle. It is preserved in 50 mM Hepes buffer (pH 6.8) containing 1% Triton CF-54 and 20% glycerol at -70 degrees C for a period of 6 months without loss of activity. The SAT3+4 preparation transfers sialic acid to nLcOse4Cer, nLcOse6Cer and GgOse4Cer with respective Km values of 1.4, 0.83 and 0.45 mM. The activity is stimulated 2-3-fold at high substrate concentration and 6-8-fold at low substrate concentration; 0.01 and 0.005 mumol for asialo GM1 and 0.025 and 0.01 mumol for other glycolipids in the presence of phosphatidylcholine (PC) and sphingomyelin (SM) at an optimum concentration 0.75%. A higher concentration is inhibitory. SM from chicken muscle is more effective than that from bovine brain and the stimulation is qualitatively proportional to that of the saturated fatty acyl content of SM. Free fatty acids (palmitic and stearic), their sodium salts, other choline compounds including choline chloride, phosphorylcholine and acetylcholine either do not have any effect or are inhibitory. Acetylcholine, even in the presence of SM and PC, is strongly inhibitory (70%).

Regulation of the calcium-activated neutral proteinase (CANP) of bovine brain by myelin lipids.

Since calcium-activated neutral proteinase (CANP; calpain) activation occurs at the plasmalemma and the enzyme is found in myelin, we examined myelin lipid activation of brain CANP. Purified lipids were dried, sonicated and incubated with purified myelin CANP. The CANP was assayed using [14C]azocasein as substrate and the Ca2+ concentration ranged from 2 microM for muCANP to 5 mM for mCANP. Phosphatidylinositol (PI), phosphatidylserine (PS) and dioleoylglycerol stimulated the mCANP activity by 193, 89 and 78%, respectively. PI stimulated both m- and muCANP in a concentration-dependent manner, while phosphatidylcholine was least effective. Cerebroside and sulfatide at higher concentrations (750 microM) were stimulatory. The phospholipid (PL)-mediated activation was inhibited by the PL-binding drug trifluoperazine. PI reduced the Ca2+ requirement for CANPs significantly (20-fold). These results suggest that acidic lipids and particularly acidic phospholipids activate membrane CANP.

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons. I. Base efflux.

We used microelectrodes to monitor the recovery (i.e., decrease) of intracellular pH (pHi) after using internal dialysis to load squid giant axons with alkali to pHi values of 7.7, 8.0, or 8.3. The dialysis fluid (DF) contained 400 mM K+ but was free of Na+ and Cl-. The artificial seawater (ASW) lacked Na+, K+, and Cl-, thereby eliminating effects of known acid-base transporters on pHi. Under these conditions, halting dialysis unmasked a slow pHi decrease caused at least in part by acid-base transport we refer to as "base efflux." Replacing K+ in the DF with either NMDG+ or TEA+ significantly reduced base efflux and made membrane voltage (Vm) more positive. Base efflux in K(+)-dialyzed axons was stimulated by decreasing the pH of the ASW (pHo) from 8 to 7, implicating transport of acid or base. Although postdialysis acidifications also occurred in axons in which we replaced the K+ in the DF with Li+, Na+, Rb+, or Cs+, only with Rb+ was base efflux stimulated by low pHo. Thus, the base effluxes supported by K+ and Rb+ appear to be unrelated mechanistically to those observed with Li+, Na+, or Cs+. The combination of 437 mM K+ and 12 mM HCO3- in the ASW, which eliminates the gradient favoring a hypothetical K+/HCO3- efflux, blocked pHi recovery in K(+)-dialyzed axons. However, the pHi recovery was not blocked by the combination of 437 mM Na+, veratridine, and CO2/HCO3- in the ASW, a treatment that inverts electrochemical gradients for H+ and HCO3- and would favor passive H+ and HCO3- fluxes that would have alkalinized the axon. Similarly, the recovery was not blocked by K+ alone or HCO3- alone in the ASW, nor was it inhibited by the K-H pump blocker Sch28080 nor by the Na-H exchange inhibitors amiloride and hexamethyleneamiloride. Our data suggest that a major component of base efflux in alkali-loaded axons cannot be explained by metabolism, a H+ or HCO3- conductance, or by a K-H exchanger. However, this component could be mediated by a novel K/HCO3- cotransporter.

K(+)- and HCO3(-)-dependent acid-base transport in squid giant axons II. Base influx.

We used microelectrodes to determine whether the K/HCO3 cotransporter tentatively identified in the accompanying paper (Hogan, E. M., M. A. Cohen, and W. F. Boron. 1995. Journal of General Physiology. 106:821-844) can mediate an increase in the intracellular pH (pHi) of squid giant axons. An 80-min period of internal dialysis increased pHi to 7.7, 8.0, or 8.3; the dialysis fluid was free of K+, Na+, and Cl-. Our standard artificial seawater (ASW), which also lacked Na+, K+, and Cl-, had a pH of 8.0. Halting dialysis unmasked a slow pHi decrease. Subsequently introducing an ASW containing 437 mM K+ and 0.5% CO2/12 mM HCO3- had two effects: (a) it caused membrane potential (Vm) to become very positive, and (b) it caused a rapid pHi decrease, because of CO2 influx, followed by a slower plateau-phase pHi increase, presumably because of inward cotransport of K+ and HCO3- ("base influx"). Only extracellular Rb+ substituted for K+ in producing the plateau-phase pHi increase in the presence of CO2/HCO3-. Mean fluxes with Na+, Li+, and Cs+ were not significantly different from zero, even though Vm shifts were comparable for all monovalent cations tested. Thus, unless K+ or Rb+ (but not Na+, Li+, or Cs+) somehow activates a conductive pathway for H+, HCO3-, or both, it is unlikely that passive transport of H+, HCO3-, or both makes the major contribution to the pHi increase in the presence of K+ (or Rb+) and CO2/HCO3-. Because exposing axons to an ASW containing 437 mM K+, but no CO2/HCO3-, produced at most a slow pHi increase, K-H exchange could not make a major contribution to base influx. Introducing an ASW containing CO2/HCO3-, but no K+ also failed to elicit base influx. Because we observed base influx when the ASW and DF were free of Na+ and Cl-, and because the disulfonic stilbene derivatives SITS and DIDS failed to block base influx, Na(+)-dependent Cl-HCO3 exchange also cannot account for the results. Rather, we suggest that the most straightforward explanation for the pHi increase we observed in the simultaneous presence of K+ and CO2/HCO3- is the coupled uptake of K+ and HCO3-.

Intracellular Cl- dependence of Na-H exchange in barnacle muscle fibers under normotonic and hypertonic conditions.

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl-. The purpose of the present study was to determine (a) whether Cl- plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl-]i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl-- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na+ and 0-194 mM Cl-. The artificial seawater bathing the BMF initially contained no Na+. After dialysis was halted, adding 50 mM Na+ to the artificial seawater caused an amiloride-sensitive pHi increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (JNa-H) increased with increasing [Cl-]i under both normo- and hypertonic conditions, with similar apparent Km values ( approximately 120 mM). However, the maximal JNa-H increased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pHi requires Cl- under both normo- and hypertonic conditions, but at any given [Cl-]i, JNa-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl-]i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl--dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl--free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl-, dialyzing with GTPgammaS or AlF3, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl--depleted fibers, the Cl--dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF3 indicates that the G protein is a heterotrimeric G protein.