27-day cycles in human mortality: Traute and Bernhard Düll.

This tribute to her parents by one co-author (NDP) is the fruit of a more than a decade-long search by the senior author (FH) for the details of the lives of Bernhard and Gertraud ("Traute") Düll. These pioneers studied how space/terrestrial weather may differentially influence human mortality from various causes, the 27-day mortality pattern being different whether death was from cardiac or respiratory disease, or from suicide. FH is the translator of personal information about her parents provided by NDP in German. Figuratively, he also attempts to "translate" the Dülls' contribution in the context of the literature that had appeared before their work and after their deaths. Although the Dülls published in a then leading journal, among others (and FH had re-analyzed some of their work in a medical journal), they were unknown to academies or libraries (where FH had inquired about them). The Dülls thoroughly assembled death certificates to offer the most powerful evidence for an effect of solar activity reflected in human mortality, as did others before them. They went several steps further than their predecessors, however. They were the first to show possibly differential effects of space and/or Earth weather with respect to suicide and other deaths associated with the nervous and sensory systems vs. death from cardiac or respiratory disease as well as overall death by differences in the phase of a common 27-day cycle characterizing these mortality patterns. Furthermore, Bernhard Düll developed tests of human visual and auditory reaction time to study effects of weather and solar activity, publishing a book (his professorial dissertation) on the topic. His unpublished finding of an increased incidence of airplane crashes in association with higher solar activity was validated after his death, among others, by Tatiana Zenchenko and A. M. Merzlyi.

Rationale and design of the enoximone clinical trials program.

BACKGROUND: Chronic heart failure is a disease syndrome characterized in its advanced stages by a poor quality of life, frequent hospitalizations, and a high risk of mortality. In advanced and ultra-advanced chronic heart failure, many treatment options, such as cardiac transplantation and mechanical devices, are severely limited by availability and cost. Short-term Phase II clinical trials suggest that low-dose oral inotropic therapy with enoximone may improve hemodynamics and exercise capacity, without adversely affecting mortality, in selected subjects with advanced chronic heart failure. Based on these data, the ability of enoximone to deliver safe and efficacious palliative treatment of advanced/ultra-advanced chronic heart failure is being evaluated in Phase III clinical trials. METHODS AND RESULTS: The Enoximone Clinical Trials Program is a series of 4 clinical trials designed to evaluate the safety and efficacy of oral enoximone in advanced chronic heart failure. ESSENTIAL I and II (The Studies of Oral Enoximone Therapy in Advanced Heart Failure) will investigate the effects of oral enoximone on all-cause mortality and cardiovascular hospitalization, submaximal exercise capacity, and quality of life in subjects with New York Heart Association Class III/IV chronic heart failure. EMOTE (Oral Enoximone in Intravenous Inotrope-Dependent Subjects) will evaluate the potential of oral enoximone to wean subjects with ultra-advanced chronic heart failure from chronic intravenous inotropic therapy to which they have been shown to be dependent. EMPOWER (Enoximone Plus Extended-Release Metoprolol Succinate in Subjects with Advanced Chronic Heart Failure) will explore the potential of enoximone to increase the tolerability of continuous release metoprolol in subjects shown previously to be hemodynamically intolerant to beta-blocker treatment. CONCLUSION: These studies are Phase III, multicenter, randomized, double-blinded, placebo-controlled trials designed to test the general hypothesis that chronic oral administration of low doses of enoximone can produce beneficial effects in subjects with advanced or ultra-advanced chronic heart failure.

Effects of metabolite uptake on proton-equivalent elimination by two species of deep-sea vestimentiferan tubeworm, Riftia pachyptila and Lamellibrachia cf luymesi: proton elimination is a necessary adaptation to sulfide-oxidizing chemoautotrophic symbionts.

Intracellular symbiosis requires that the host satisfy the symbiont's metabolic requirements, including the elimination of waste products. The hydrothermal vent tubeworm Riftia pachyptila and the hydrocarbon seep worm Lamellibrachia cf luymesi are symbiotic with chemolithoautotrophic bacteria that produce sulfate and protons as end-products. In this report, we examine the relationship between symbiont metabolism and host proton equivalent elimination in R. pachyptila and L. cf luymesi, and the effects of sulfide exposure on proton-equivalent elimination by Urechis caupo, an echiuran worm that lacks intracellular symbionts (for brevity, we will hereafter refer to proton-equivalent elimination as 'proton elimination'). Proton elimination by R. pachyptila and L. cf luymesi constitutes the worms' largest mass-specific metabolite flux, and R. pachyptila proton elimination is, to our knowledge, the most rapid reported for any metazoan. Proton elimination rates by R. pachyptila and L. cf luymesi correlated primarily with the rate of sulfide oxidation. Prolonged exposure to low environmental oxygen concentrations completely inhibited the majority of proton elimination by R. pachyptila, demonstrating that proton elimination does not result primarily from anaerobic metabolism. Large and rapid increases in environmental inorganic carbon concentrations led to short-lived proton elimination by R. pachyptila, as a result of the equilibration between internal and external inorganic carbon pools. U. caupo consistently exhibited proton elimination rates 5-20 times lower than those of L. cf luymesi and R. pachyptila upon exposure to sulfide. Treatment with specific ATPase inhibitors completely inhibited a fraction of proton elimination and sulfide and inorganic carbon uptake by R. pachyptila, suggesting that proton elimination occurs in large part via K(+)/H(+)-ATPases and Na(+)/H(+)-ATPases. In the light of these results, we suggest that protons are the primary waste product of the symbioses of R. pachyptila and L. cf luymesi, and that proton elimination is driven by symbiont metabolism, and may be the largest energetic cost incurred by the worms.

A paradox resolved: sulfide acquisition by roots of seep tubeworms sustains net chemoautotrophy.

Vestimentiferan tubeworms, symbiotic with sulfur-oxidizing chemoautotrophic bacteria, dominate many cold-seep sites in the Gulf of Mexico. The most abundant vestimentiferan species at these sites, Lamellibrachia cf. luymesi, grows quite slowly to lengths exceeding 2 meters and lives in excess of 170-250 years. L. cf. luymesi can grow a posterior extension of its tube and tissue, termed a "root," down into sulfidic sediments below its point of original attachment. This extension can be longer than the anterior portion of the animal. Here we show, using methods optimized for detection of hydrogen sulfide down to 0.1 microM in seawater, that hydrogen sulfide was never detected around the plumes of large cold-seep vestimentiferans and rarely detectable only around the bases of mature aggregations. Respiration experiments, which exposed the root portions of L. cf. luymesi to sulfide concentrations between 51-561 microM, demonstrate that L. cf. luymesi use their roots as a respiratory surface to acquire sulfide at an average rate of 4.1 micromol x g(-1) x h(-1). Net dissolved inorganic carbon uptake across the plume of the tubeworms was shown to occur in response to exposure of the posterior (root) portion of the worms to sulfide, demonstrating that sulfide acquisition by roots of the seep vestimentiferan L. cf. luymesi can be sufficient to fuel net autotrophic total dissolved inorganic carbon uptake.

Biological sequences integrated: a relational database approach.

Over the last decade the modeling and the storage of biological data has been a topic of wide interest for scientists dealing with biological and biomedical research. Currently most data is still stored in text files which leads to data redundancies and file chaos. In this paper we show how to use relational modeling techniques and relational database technology for modeling and storing biological sequence data, i.e. for data maintained in collections like EMBL or SWISS-PROT to better serve the needs for these application domains. For this reason we propose a two step approach. First, we model the structure (and therefore the meaning of the) data using an Entity-Relationship approach. The ER model leads to a clean design of a relational database schema for storing and retrieving the DNA and protein data extracted from various sources. Our approach provides the clean basis for building complex biological applications that are more amenable to changes and software ports than their file-base counterparts.

Use of human preconditioned phagocytes for extracorporeal immune support: introduction of a concept.

Neutrophils are critical effector cells in humoral and innate immunity and play a vital role in phagocytosis and bacterial killing. If they and/or their specific functions are lacking, then immunoparalysis may occur, and severe diseases like systemic inflammatory response syndrome (SIRS) or sepsis can take a fatal course. In this paper, we discuss the possibility of using preconditioned cells in an extracorporeal biohybrid immune support system. A human promyelocytic cell line was stimulated for different times with all-trans retinoic acid. The resulting cells displayed major signs and functions of mature neutrophilic granulocytes including oxygen radical production, phagocytosis of living and dead Escherichia coli, Staphylococcus aureus, Candida albicans, intracellular killing, and interleukin production. The cells can be expanded to yield a sufficient cell mass, and subsequent prestimulation results in an expression of specific neutrophil functions. Extracorporeal bioreactor experiments seem to be feasible to test the benefit in immunoparalysis-associated diseases like SIRS or sepsis.

Liver support by extracorporeal blood purification: a clinical observation.

Liver failure associated with excretory insufficiency and jaundice results in an endogenous accumulation of toxins involved in the impairment of cardiovascular, kidney, and cerebral function. Moreover, these toxins have been shown to damage the liver itself by inducing hepatocellular apoptosis and necrosis, thus creating a vicious cycle of the disease. We report a retrospective cohort study of 26 patients with acute or chronic liver failure with intrahepatic cholestasis (bilirubin level > 20 mg/dL) who underwent a new extracorporeal blood purification treatment. A synthetic hydrophilic/hydrophobic domain-presenting semipermeable membrane (pore size < albumin size, 100-nm thick) was used for extracorporeal blood detoxification using dialysis equipment. The opposite side was rinsed with ligandin-like proteins as molecular adsorbents that were regenerated online using a chromatography-like recycling system (molecular adsorbent recirculating system [MARS]). Bile acid and bilirubin levels, representing the previously described toxins, were reduced by 16% to 53% and 10% to 90% of the initial concentration by a single treatment of 6 to 8 hours, respectively. Toxicity testing of patient plasma onto primary rat hepatocytes by live/dead fluorescence microscopy showed cell-damaging effects of jaundiced plasma that were not observed after treatment. Patients with a worsening of Child-Turcotte-Pugh (CTP) index before the treatments showed a significant improvement of this index during a period of 2 to 14 single treatments with an average of 14 days. After withdrawal of MARS treatment, this improvement was sustained in all long-term survivors. Ten patients represented a clinical status equivalent to the United Network for Organ Sharing (UNOS) status 2b (group A1), and all survived. Sixteen patients represented a clinical status equivalent to UNOS status 2a, and 7 of these patients survived (group A2), whereas 9 patients (group B) died. We conclude that in acute excretory failure caused by a chronic liver disease, this treatment provides a therapy option to remove toxins involved in multiorgan dysfunction secondary to liver failure.

Improvement of hepatorenal syndrome with extracorporeal albumin dialysis MARS: results of a prospective, randomized, controlled clinical trial.

In hepatorenal syndrome (HRS), renal insufficiency is often progressive, and the prognosis is extremely poor under standard medical therapy. The molecular adsorbent recirculating system (MARS) is a modified dialysis method using an albumin-containing dialysate that is recirculated and perfused online through charcoal and anion-exchanger columns. MARS enables the selective removal of albumin-bound substances. A prospective controlled trial was performed to determine the effect of MARS treatment on 30-day survival in patients with type I HRS at high risk (bilirubin level, > or =15 mg/dL) compared with standard treatment. Thirteen patients with cirrhosis with type I HRS were included from 1997 to 1999. All were Child's class C, with Child-Turcotte-Pugh scores of 12.4 +/- 1. 0, United Network for Organ Sharing status 2A, and total bilirubin values of 25.7 +/- 14.0 mg/dL. Eight patients were treated with the MARS method in addition to hemodiafiltration (HDF) and standard medical therapy, and 5 patients were in the control group (HDF and standard medical treatment alone). None of these patients underwent liver transplantation or received a transjugular intrahepatic portosystemic shunt or vasopressin analogues during the observation period. In the MARS group, 5.2 +/- 3.6 treatments (range, 1 to 10 treatments) were performed for 6 to 8 hours daily per patient. A significant decrease in bilirubin and creatinine levels (P <.01) and increase in serum sodium level and prothrombin activity (P <.01) were observed in the MARS group. Mortality rates were 100% in the control group at day 7 and 62.5% in the MARS group at day 7 and 75% at day 30, respectively (P <.01). We conclude that the removal of albumin-bound substances with the MARS method can contribute to the treatment of type I HRS.

Molecular adsorbent recycling system (MARS): clinical results of a new membrane-based blood purification system for bioartificial liver support.

The use of xenogenic or genetically engineered cell types in bioartificial liver support systems requires separation methods between the patients' blood and the liver support bioreactors that guarantee the sufficient transfer of pathophysiologically relevant substances but prevent complications. The present paper describes a new membrane separation system that is nearly impermeable to proteins but enables the exchange of water soluble and protein bound toxins by a special membrane and a recycled protein containing dialysate. Because the full range of toxins in hepatic failure has still not been identified, the value of this membrane separation method was evaluated clinically. Thirteen patients suffering from life threatening hepatic failure who had not responded to state of the art therapy were treated with this device, the molecular adsorbent recycling system (MARS). The overall survival rate was 69%. All patients showed positive response to the therapy, indicating that the presented membrane separator combines therapeutic effectivity with the highest safety criteria for the patient by cutting the exchange of substances below the level of proteins.

Large unilamellar lipid vesicles for use in therapeutic and diagnostic medicine.

Large unilamellar lipid vesicles are prepared by a detergent dialysis procedure using beta-D-octylglucoside as the detergent. This procedure is nondenaturing and allows for the encapsulation of sensitive biological molecules. Vesicles prepared with the composition of 2 mol phosphatidylcholine and 1 mol cholesterol have a mean diameter of 200 nm and allow for the encapsulation of 150 molecules of alkaline phosphatase per vesicle without loss of activity. Stability studies show that less than 4 per cent of the original enzyme leaks from the vesicles over a 250 day period upon storage at 4 degrees C. A mechanistic model for vesicle formation is described to provide a clear understanding of the events occurring during the encapsulation stages.

A simple method for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli.

A simple method was described for the conjugation of affinity-purified Fab' to horseradish peroxidase and beta-D-galactosidase from Escherichia coli. IgG was subjected to successive processes of pepsin digestion, reduction with 2-mercaptoethylamine, affinity-purification and reaction with maleimide groups introduced into the enzymes. In the present method, gel filtration was required only once to separate the conjugate from unconjugated components in the final step, while gel filtration had to be repeated 3-4 times in the previous methods. The conjugate preparations obtained by the present method contained less nonspecific conjugate and gave a lower background by immunoenzymometric assay technique than those obtained by the previous method.

Preparation of a monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate for immunoenzymometric assay.

A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.

An automated, affinity-column-mediated, enzyme-linked immunometric assay for digoxin on the Du Pont aca discrete clinical analyzer.

We describe an automated assay for digoxin that requires a 200-microL sample of serum. Total analysis time is 18 min. The method is extremely precise, with within-run CVs of 2.6, 1.6, and 4.9%, respectively, at 0.5, 1.5, and 3.5 micrograms of digoxin per liter (n = 20). The lower limit of detection is 0.2 micrograms of digoxin per liter. For patients' samples, the correlation with RIA (x) is excellent (r = 0.95; y = 0.95x - 0.14; standard error = 0.17 micrograms/mL). We saw no interferences in samples having high concentrations of rheumatoid factors, lipid, bilirubin, or hemoglobin. Cross reactivity with digoxin analogs and steroidal compounds is similar to that observed by RIA.

Asymmetric mass distribution of (Na+ + K+)-ATPase in membranes studied by freeze-fracture-etch electron microscopy.

SDS-purified porcine kidney (Na+ + K+)-ATPase was studied by thin-section and freeze-etch electron microscopy. Freeze-fracturing of resealed membrane fragments shows no difference in the distribution of intramembranous particles of approx. 9.0 nm in diameter between convex and concave fracture faces. However, two types of convex face are found: FA, which shows a rather smooth background with many intramembranous particles, and FB, which shows a textured background with very few or no intramembranous particles. Etching the fractured samples further reveals that FA faces are covered with many intramembranous particles, while the etched external faces (EA) are either irregularly granulated or reveal many particles half the size of intramembranous particles. FB faces are covered with distinct pits of 9 nm or larger. The etched external surfaces (EB) are covered with many particles of intramembranous particle size. These results suggest that there are two vesicle orientations in our resealed purified membrane preparation: right-side-out, as in vivo, and inside-out. The majority of the protein mass is distributed only on one side of the membranes. Right-side-out resealed membrane vesicles after fracturing and etching show particulated FA convex fracture faces and irregularly granulated or smooth etched EA surfaces, indicating that the FA face is the protoplasmic fracture face and that the majority of the protein mass of the (Na+ + K+)-ATPase is located on the cytoplasmic half of the membrane.

Highly sensitive immunoassays based on use of liposomes without complement.

We describe a novel liposome-based immunoassay in which covalently linked hapten-cytolysin conjugates are used instead of complement and surface-immobilized immunoreagents. Stable, unilamellar liposomes containing entrapped alkaline phosphatase as a marker enzyme were prepared by dialysis of octyl glucoside from suspensions of cholesterol and egg yolk lecithin. The resulting vesicles could be immediately lysed by addition of either bee venom melittin or hapten-melittin conjugates. Using ouabain, an analog of digoxin, we synthesized conjugates that were more lytic than mellitin alone but that were inhibited in the presence of antibody. This inhibition was affected by adding competing free digoxin at various concentrations to obtain standard curves. The same liposome preparations could be lysed with a biotin-melittin conjugate, which was inhibited by avidin. The latter system was affected by free biotin and might be used to couple this approach to various heterogeneous immunoassays.

Affinity-column-mediated immunoenzymometric assays: influence of affinity-column ligand and valency of antibody-enzyme conjugates.

We describe an affinity-column-mediated, enzyme-linked immunometric assay that is highly sensitive and adaptable to automation. Digoxin is the model test analyte. A comparison of digoxin with its analog, ouabain, for use as the immobilized ligand on the affinity column showed ouabain to be superior. We also report the effect of column elution rate. Antibody-enzyme conjugates prepared with the monovalent Fab'-fragment and the divalent F(ab')2-fragment coupled to beta-galactosidase are compared in terms of their overall assay performance. Although the monovalent Fab'--beta-galactosidase conjugate yields a more sensitive assay and dose-response curves that are linear over a wider range, the divalent F(ab')2--beta-galactosidase conjugate provides an assay with adequate sensitivity and extremely good precision, and is generally easier to synthesize reproducibly. This fully automated, rapid, and precise assay for digoxin is compatible with the Du Pont aca discrete clinical analyzer.

Liposome-mediated immunoassays for small haptens (digoxin) independent of complement.

A lipid vesicle-mediated immunoassay for small haptens (digoxin) is described. Using a detergent removal procedure for vesicle formation, a water-soluble marker system like alkaline phosphatase is stably entrapped within 150-200 nm unilamellar lipid vesicles composed of egg yolk lecithin and cholesterol. Specific lysis of the lipid vesicles is achieved upon addition of a hapten-melittin conjugate. Inhibition or modulation of this lysis by the hapten-melittin conjugate can then be achieved by adding stoichiometric amounts of high affinity antibody. Finally, the antibody inhibition of hapten-melittin lysis can be modulated by the addition of competing amounts of free hapten. This assay approach is simple, fast, highly sensitive, and versatile such that it can be carried out in either a homogeneous or heterogeneous mode. Furthermore, unlike all other liposome-mediated immunoassays, complement is not required for lysis.