Into the Blue: First in Man Data on Diving Physiology in Fontan Patients.

Swimming and diving are popular recreational activities. As congenital heart disease, especially patients with univentricular hearts after Fontan palliation are thought to have reduced physiologic capacities for compensation of submersion-associated physiologic demands, current guidelines put restraints on this group of patients. Although these restrictions on doctoral advice place a significant burden on affected patients, it is especially interesting that these guideline recommendations are merely based on physiologic assumptions, i.e., expert consensus. A recent study by Paech et al. presented the first in vivo data on the effects of immersion in Fontan patients, stating no major adverse events in their study group as well as comparable physiologic adaption as reported in the literature for healthy people. Yet, submersion was not reflected in this study, and the current study therefore aimed to conduct a first study for the evaluation of the effects of submersion and apnea diving in Fontan patients. A control group of healthy adults as well as patients recruited from the Heart Center Leipzig, Department of pediatric cardiology underwent a standardized diving protocol including a static as well as dynamic apnea phase. Physiologic data were recorded. This study presents the first structured data on diving physiology in Fontan patients compared to healthy probands. There were no adverse events. The physiologic response to diving seems to be comparable between healthy probands and Fontan patients. Although, healthy probands did reach a much better performance, the basic mechanisms of physiologic adaption seem comparable.

A recombinant amino terminal fragment of bactericidal/permeability-increasing protein inhibits the induction of leukocyte responses by LPS.

Bactericidal/permeability-increasing protein (BPI) is a major component of the granules of polymorphonuclear neutrophils (PMNs) and is involved in the killing of gram-negative bacteria. A 23-kd recombinant protein, corresponding to the NH2-terminal fragment of human BPI (rBPI23), has been shown to bind lipid A and antagonize some lipopolysaccharide (LPS)-mediated effects. In this study the ability of rBPI23 to prevent a wide range of cellular responses to LPS was investigated. In vitro assays were carried out using human blood to more closely approximate in vivo conditions. The release of proinflammatory cytokines [tumor necrosis factor (TNF), interleukin-1 beta (IL-1 beta), IL-6, IL-8], induced by E. coli O113 LPS, was markedly reduced by rBPI23 in a concentration-dependent fashion. The production of the anti-inflammatory protein IL-1ra (IL-1 receptor antagonist) was triggered by lower LPS concentrations than those necessary for the other cytokines. Furthermore, prevention of IL-1ra release required higher rBPI23 concentrations than for other cytokines. The LPS-induced production of oxygen-derived free radicals by phagocytic cells (resulting in chemiluminescence) was also prevented by rBPI23. The inhibition was specific for LPS because the activation of leukocytes by phorbol myristate acetate, zymosan, or TNF was unaffected by BPI. The ability of rBPI23 to antagonize specifically the effects of endotoxin in the complex environment of human blood along with its bactericidal activity suggests that rBPI23 may be a novel therapeutic agent in the treatment of gram-negative infections.

Protective effect of a recombinant amino-terminal fragment of bactericidal/permeability-increasing protein in experimental endotoxemia.

Bactericidal/permeability-increasing protein (BPI), a cationic protein found in neutrophil granules, binds with high affinity to gram-negative bacterial lipopolysaccharide (LPS) and can inhibit its actions in vitro. The in vivo efficacy of a recombinant 23-kDa amino-terminal LPS-binding fragment of BPI (rBPI23) was assessed in a mouse model of lethal endotoxemia. Systemic administration of rBPI23 protected actinomycin D-sensitized mice from lethal LPS (Escherichia coli O111:B4) challenge in a dose-dependent manner, with almost complete protection at the highest dose (10 mg/kg; 93% survival vs. 13% in vehicle-treated controls). Surviving rBPI23-treated animals did not show histopathologic signs of tissue damage evident in control animals that had died after LPS challenge. rBPI23 also attenuated the LPS-induced elevation in serum levels of tumor necrosis factor-alpha and interleukin-1 alpha, mediators believed to be involved in the pathogenesis of endotoxemia and sepsis. Thus, rBPI23 may be a potential new therapeutic agent for the treatment of gram-negative bacterial infection and sepsis.

Modification of the C terminus of cecropin is essential for broad-spectrum antimicrobial activity.

Cecropin A is a naturally occurring peptide with bactericidal activity against gram-negative and gram-positive bacteria. Production of large quantities of bactericidal peptides that are similar in structure and activity to cecropin A has been achieved by combining recombinant DNA techniques and techniques and chemical modification. Expression of the bactericidal peptide in Escherichia coli was accomplished through the formation of a fusion protein. The 5' end of the L-ribulokinase gene was fused to a single copy of a synthetic gene encoding cecropin A. A methionine codon was engineered between the two genes, and a methionylglycine extension was introduced at the C terminus of cecropin A. Cyanogen bromide treatment of the fusion protein yielded cecropin A with a C-terminal homoserine. The recombinant cecropin A with a homoserine at the C terminus did not kill most gram-positive bacteria tested. However, recombinant cecropin A with a chemically modified C terminus has antimicrobial activity similar to that of cecropin produced by cecropia pupae.

X-ray analysis of new crystal forms of the sweet protein thaumatin.

Thaumatin is a plant protein that in the mature form contains 8 disulfide bonds and 207 amino acids. Several forms of this protein occur naturally and each elicits an intense sweetness sensation when tasted in microgram quantities. The two major forms of thaumatin are easily separable by ion exchange chromatography. Crystals of the two proteins (designated here A and B) have been grown by vapor equilibration from solutions containing polyethylene glycol and examined by X-ray diffraction. The thaumatin A crystals are of space group P2(1)2(1)2(1) with a = 44.3 A, b = 63.7 A and c = 72.7 A. The crystals of thaumatin B are of space group C2 with a = 117.7 A, b = 44.9 A, and c = 38.0 A and beta = 94.0 degrees. Both crystals diffract to well beyond 2.3 A and appear suitable for high resolution structure analysis. Four heavy atom derivatives of thaumatin B have been generated and diffraction data to 4 A resolution have been collected. This work is designed to provide a basis for studying the 3-dimensional structure of more than 100 genetically generated thaumatin derivatives, several of which show enhanced stability and improved taste characteristics.

Expression of synthetic thaumatin genes in yeast.

Thaumatin is a plant protein that contains 8 disulfides and 207 amino acids in the mature form. The protein is of potential commercial interest since microgram quantities elicit an intense sweetness sensation. Two major variants of thaumatin have been identified in our laboratory by using sequence data obtained from thaumatin tryptic peptides. These differ by one amino acid at position 46 (asparagine or lysine), and both proteins differ from previously published sequences. We have synthesized DNA-coding sequences for three of these thaumatin variants using yeast preferred codons. The genes were inserted into an expression vector that contained a yeast 3-phosphoglycerate kinase promoter and terminator, and the vectors were transformed into yeast for expression of the recombinant protein. Upon lysis of the yeast cells, all thaumatin was localized in the insoluble cell fraction. Analysis of the sodium dodecyl sulfate solubilized yeast extracts by gel electrophoresis and Western blotting showed that thaumatin represented about 20% of the insoluble yeast protein. Although expressed at high levels, none of the thaumatins was biologically active (sweet). Preliminary protein folding experiments showed that two of three thaumatin variants could be folded to the sweet conformation.

Specificity of cotranslational amino-terminal processing of proteins in yeast.

Polypeptides synthesized in the cytoplasm of eukaryotes are generally initiated with methionine, but N-terminal methionine is absent from most mature proteins. Many proteins are also N alpha-acetylated. The removal of N-terminal methionine and N alpha-acetylation are catalyzed by two enzymes during translation. The substrate preferences of the methionine aminopeptidase (EC 3.4.11.x) and N alpha-acetyltransferase (EC 2.3.1.x) have been partially inferred from the distribution of amino-terminal residues and/or mutations found for appropriate mature proteins, but with some contradictions. In this study, a synthetic gene corresponding to the mature amino acid sequence of the plant protein thaumatin, expressed in yeast as a nonexported protein, i.e., lacking a signal peptide, has been used to delineate the specificities of these enzymes with respect to the penultimate amino acid. Site-directed mutagenesis, employing synthetic oligonucleotides, was utilized to construct genes encoding each of the 20 amino acids following the initiation methionine codon, and each protein derivative was isolated and characterized with respect to its amino-terminal structure. All four possible N-terminal variants--those with and without methionine and those with and without N alpha-acetylation--were obtained. These results define the specificity of these enzymes in situ and suggest that the nature of the penultimate amino-terminal residue is the major determinant of their selectivity.

Primer-directed mutagenesis of linearized plasmids.

Two novel mutagenesis techniques to specifically alter the sequence of plasmid DNA have been developed. In contrast to other primer-directed mutagenesis methods which require a single-stranded, closed-circular template, a linearized single strand was used as the mutagenesis template. The template is prepared by restriction enzyme digestion of covalently-closed-circular plasmid DNA. These methods are simple, require small amounts of plasmid DNA, and can result in a relatively high frequency of mutagenesis.

Immunological assay of pancreatic ribonuclease in serum as an indicator of pancreatic cancer.

Serum levels of RNase activity, presumed to originate in the pancreas, have been suggested to be of use in the diagnosis of pancreatic cancer. We have used a radioimmunological assay of human pancreatic-like RNase to quantitate this protein in serum from normal blood donors and patients with a variety of diseases. Serum pancreatic-like RNase rises gradually with age, and its level is usually higher in males than females. Although many patients with pancreatic cancer show elevated serum levels of immunologically cross-reactive enzyme, others are apparently normal. In several other types of cancer, a similar pattern of elevated RNase is apparent. However, in kidney or bladder carcinoma and in patients with severe kidney disease, RNase levels are almost always greater than normal. Regardless of the nature of the disease, an elevated level of pancreatic-like enzyme is usually accompanied by above-normal levels of serum urea nitrogen. Hence, elevated circulating levels of pancreatic-like RNase are best related to kidney function and do not serve as a specific marker for cancers of the pancreas or other organs.

The amino acid sequence of human pancreatic ribonuclease.

The primary structure of human (Homo sapiens) pancreatic ribonuclease has been determined by automatic sequencing of the native protein and by analysis of peptides obtained by cleavage with proteolytic enzymes, cyanogen bromide, and hydroxylamine. The following sequence was deduced: (sequence in text). Human pancreatic ribonuclease differs at 37 positions from bovine pancreatic ribonuclease. In addition the human enzyme has three more residues at the C-terminus. About half of the enzyme molecules contain carbohydrate attached to the sequence Asn-Met-Thr (34-36). Two other Asn-X-Ser/Thr sequences are carbohydrate free. Human pancreatic ribonuclease contains many positively charged residues, especially near the N-terminus, while negatively charged residues are more concentrated near the C-terminus.

Human ribonucleases. Quantitation of pancreatic-like enzymes in serum, urine, and organ preparations.

Antibodies against pure human pancreatic ribonuclease (RNase) were used to study ribonuclease levels in human tissues and body fluids. The antibodies completely inhibit the activity of purified RNase as well as ribonuclease activity in crude pancreatic extracts. RNase activity is inhibited by 70-80% in serum and urine, indicating that a significant proportion of the RNases in these preparations are structurally like the pancreatic enzyme. In contrast, inhibition of RNase activities from spleen (8%) and liver (30%) was inefficient suggesting that most of the RNases in these tissues are structurally unlike the pancreatic enzyme. A competitive binding radioimmunoassay (RIA), sensitive in the range of 1-100 ng of RNase, was developed to quantitate the pancreatic like enzymes. The RIA of crude tissue preparations and samples fractionated by gel filtration was compatible with inhibition results. Enzymes structurally like pancreatic RNase could be quantitated despite the presence of other RNase activities. Immunological quantitation of pancreatic like RNases was also found to be much more simple and precise than enzymatic assays comparing RNA and polycytidylate substrates. We suggest the immunological assays will be useful in the quantitation and definition of tissue of origin of RNases in serum of patients with pancreatic carcinoma.

Purification and characterization of human pancreatic ribonuclease.

A ribonuclease (RNase) has been isolated from normal human pancreas obtained upon autopsy. About 5 mg of RNase is normally recovered per kilogram of pancreas, equivalent to ca. 70% of the total activity and a 700-fold purification from the initial acidified extract. The specific activity of the purified enzyme is identical with that of bovine pancreatic ribonuclease, and a single component is found in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and reversed-phase high-pressure liquid chromatography. Aggregation of the protein is found upon ultracentrifugation under native and denaturing conditions, and several bands of equal specific activity are seen in polyacrylamide gel electrophoresis of the native protein. At least two components are glycoproteins. A molecular weight of 15 000 is estimated from sodium dodecyl sulfate gel electrophoresis, gel filtration, and amino acid and peptide analyses. The enzyme is related to bovine pancreatic RNase, but distinguishable by amino acid analysis, tryptic peptide maps, and low cross-reactivity of antibodies with the heterologous enzymes. The human enzyme is also inactivated by treatment with iodoacetic acid at pH 5.5 and is essentially identical with bovine RNase in its far-ultraviolet circular dichroism spectrum. The human RNase is like bovine pancreatic RNase catalytically; RNA is cleaved at pyrimidine residues, and activity against poly(cytidylic acid) is high.

Arginine deiminase from Mycoplasma arthritidis. Properties of the enzyme from log phase cultures.

Hydrodynamic, chemical, and optical properties of arginine deiminase (EC 3.5.3.6) from Mycoplasma arthritidis are reported for the enzyme isolated from log phase cells. The S020,w and D020,w values of the enzyme are 5.48 S and 5.87 X 10(-7) cm3/s, respectively; the molecular weight is 87,300. Determination of the amino acid composition shows that about 45% of the residues are nonpolar. Another unique feature of the composition is the presence of 36 half-cystine residues. The state of oxidation of the half-cystines appears to be well established as 16 disulfide and 4 sulfhydryl groups. The reaction of 1 sulfhydryl group with 0.3 mM 5,5'-dithiobis(2-nitrobenzoic acid) has a half-life of about 50 min at pH 8. The modified enzyme retains full activity. Two -SH groups are accessible to this reagent in 2 M guanidine hydrochloride, whereas all 4 -SH groups react immediately in 4 M guanidine hydrochloride. Reduction of disulfide bonds with dithiothreitol occurs only to a limited extent in 8 M urea, but is complete in 4 M guanidine hydrochloride. The enzyme loses activity immediately at pH 2.5, but retains full activity upon standing 8 h at pH 9.5 in several buffers. The large number of cystine residues leads to a complex near ultraviolet circular dichroism spectrum with cystine contributions apparently superimposed on contributions from aromatic residues. The far ultraviolet spectrum suggests that the molecule contains about 18% alpha helix. At pH 2.5, beta conformation and disulfide contributions are dominant. Aromatic and alpha bands are reduced considerably at pH 9.5.

Arginine deiminase from Mycoplasma arthritidis. Evidence for multiple forms.

Arginine deiminase (EC 3.5.3.6) from Mycoplasma arthritidis ATCC 14152 has been purified 6-fold by a new procedure, protamine sulfate fractionation and DEAE-agarose chromatography. The yield was 75 to 85%. The homogeneity of the final preparation was demonstrated by gel filtration, sodium dodecyl sulfate-gel electrophoresis, NH2-terminal analysis, and polyacrylamide gel electrophoresis at two pH values. The enzyme has a molecular weight of 80,000 as measured by gel filtration. The dimeric nature of the enzyme is suggested by the molecular weight of 49,000 from sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing in polyacrylamide gels showed a major band corresponding to an isoelectric point of 7.0 and sometimes minor bands having lower isoelectric points. The ultraviolet spectrum exhibits a maximum at 278 nm. The enzyme has high affinity for L-arginine, with a Km value of 4 +/- 1 micronM at pH 7.2, 25 degrees. Mycoplasma arthritidis produces two distinct forms of arginine deiminase. Deiminase I is isolated from cells harvested during logarithmic phase; deiminase II is obtained from late logarithmic or early stationary phase cells. The two forms are resolved by DEAE-agarose chromatography and by polyacrylamide gel electrophoresis. Deiminase II elutes later from a DEAE-agarose column and moves toward the anode faster than deiminase I at pH 9.5 The two forms also have different specific activities and 280:260 spectral ratios. Each form has the same Km and molecular weight. A third form of the enzyme, deiminase III, can be generated by incubating deiminase II at pH 9.8, or in 50% saturated ammonium sulfate, pH 7.0, at 25 degrees. The transformation can be followed by chromatography and is completed within 10 h. The specific activity of deiminase III is 1.3 times that of deiminase II. No change in molecular weight or subunit dissociation was observed during the transformation. Deiminiase III has the same specific activity, absorbance ratio A280:A260, and electrophoretic properties as deiminase I. Deiminase I undergoes no change upon incubation at pH 9.8 for several days.