C5a stimulates secretion of tumor necrosis factor from human mononuclear cells in vitro. Comparison with secretion of interleukin 1 beta and interleukin 1 alpha.

We have demonstrated that purified C5a is a potent stimulus to human PBMC secretion of TNF-alpha, IL-1 beta, and IL-1 alpha, which proceeds in a dose-dependent fashion. At a given concentration of C5a, TNF-alpha and IL-1 beta secretion did not differ significantly; both were secreted in significantly greater quantity than IL-1 alpha. Clinical conditions such as Gram-positive and Gram-negative bacterial infections, trauma, and immune complex diseases activate complement. Through the mediation of TNF and IL-1 secreted in response to C5a, these diverse disorders can share common features of fever, coagulopathy, acute phase protein production, and disordered metabolism.

Ultrastructural studies on the lymphatic anchoring filaments.

The fine structure of the lymphatic capillary and the surrounding tissue areas was investigated. Instead of a continuous basal lamina (basement membrane) surrounding the capillary wall, these observations revealed the occurrence of numerous fine filaments that insert on the outer leaflet of the trilaminar unit membrane of the lymphatic endothelium. These filaments appear as individual units, or they are aggregated into bundles that are disposed parallel to the long axis of the lymphatic capillary wall and extend for long distances into the adjoining connective tissue area among the collagen fibers and connective tissue cells. The filaments measure about 100 A in width and have a hollow profile. They exhibit an irregular beaded pattern along their long axis and are densely stained with uranyl and lead. These filaments are similar to the microfibrils of the extracellular space and the filaments observed in the peripheral mantle of the elastic fibers. Infrequently, connections between these various elements are observed, suggesting that the lymphatic anchoring filaments may also contribute to the filamentous units of the extracellular space. It is suggested that these lymphatic anchoring filaments connect the small lymphatics to the surrounding tissues and represent the binding mechansim that is responsible for maintaining the firm attachment of the lymphatic capillary wall to the adjoining collagen fibers and cells of the connective tissue area.

Wound tissue can utilize a polymeric template to synthesize a functional extension of skin.

Prompt and long-term closure of full-thickness skin wounds is guinea pigs and humans is achieved by applying a bilayer polymeric membrane. The membrane comprises a top layer of a silicone elastomer and a bottom layer of a porous cross-linked network of collagen and glycosaminoglycan. The bottom layer can be seeded with a small number of autologous basal cells before grafting. No immunosuppression is used and infection, exudation, and rejection are absent. Host tissue utilizes the sterile membrane as a culture medium to synthesize neoepidermal and neodermal tissue. A functional extension of skin over the entire wound area is formed in about 4 weeks.

Functional analysis of the transcriptional control regions of the copia transposable element.

The introduction of copia-based vectors in Drosophila hydei cells results in their high-level transient expression and the subsequent establishment of stably transformed cell lines containing multiple copies of vector integrated into host genomic DNA. Using transformation frequency and transient expression analysis as assays of promoter strength, we have defined the regions of copia essential for expression. We find that the essential sequences reside within the long terminal repeat, but 3' to the site of initiation of copia RNA. Deletion of the consensus enhancer-like sequences from copia appears to have no effect on vector expression.

Critical analysis of a computer-assisted tutorial on ECG interpretation and its ability to determine competency.

BACKGROUND: We developed a computer-based tutorial and a posttest on ECG interpretation for training residents and determining competency. METHODS: Forty residents, 6 cardiology fellows, and 4 experienced physicians participated. The tutorial emphasized recognition and understanding of abnormal ECG features. Active learning was promoted by asking questions prior to the discussion of ECGs. Interactivity was facilitated by providing rapid and in-depth rationale for correct answers. Responses to questions were recorded and extensively analyzed to determine the quality of questions, baseline knowledge at different levels of training and improvement of grades in posttest. Posttest grades were used to assess improvement and to determine competency. RESULTS: The questions were found to be challenging, fair, appropriate and discriminative. This was important since the quality of Socratic questions is critical for the success of interactive programs. The information on strengths and weakness in baseline knowledge at different levels of training were used to adapt our training program to the needs of residents. The posttest revealed that the tutorial contributed to marked improvement in feature recognition. Competency testing distinguished between residents with outstanding grades and those who needed remediation. CONCLUSIONS: The strategy for critical evaluation of our computer program could be applied to any computer-based educational program, regardless of topic.

Alternative complement pathway activation increases mortality in a model of burn injury in mice.

We have studied the role of the complement system in burn injury in an experimental model in mice. A 25% body surface area, full-thickness scald wound was produced in anesthetized animals. Massive activation of the alternative complement pathway, but not the classical pathway, was seen. This activation was associated with the generation of neutrophil aggregating activity in the plasma, neutrophil aggregates in the lungs, increased pulmonary vascular permeability, and increased lung edema formation. Decomplementation with cobra venom factor (CVF) or genetic C5 deficiency diminished these pathologic changes, and CVF pretreatment substantially reduced burn mortality in the first 24 h. Preliminary data show that human burn patients have a similar pattern of complement activation involving predominantly the alternative pathway, indicating the possible relevance of the murine model to human disease.

Staphylococcus epidermidis induces complement activation, tumor necrosis factor and interleukin-1, a shock-like state and tissue injury in rabbits without endotoxemia. Comparison to Escherichia coli.

Tumor necrosis factor (TNF) and IL-1 are thought to mediate many of the pathophysiologic changes of endotoxemia and Gram-negative bacteremia. In these studies, heat-killed Staphylococcus epidermidis were infused into rabbits to determine whether an endotoxin (LPS)-free microorganism also elicits cytokinemia and the physiologic abnormalities seen in Gram-negative bacteremia. S. epidermidis induced complement activation, circulating TNF and IL-1, and hypotension to the same degree as did one-twentieth the number of heat-killed Escherichia coli. Circulating IL-1 beta levels had a greater correlation coefficient (r = 0.81, P less than 0.001) with the degree of hypotension than TNF levels (r = 0.48, P less than 0.02). Leukopenia, thrombocytopenia, diffuse pulmonary capillary aggregation of neutrophils, and hepatic necrosis with neutrophil infiltration were observed to the same extent after either S. epidermidis or E. coli infusion. However, S. epidermidis infusion did not induce significant (less than 60 pg/ml) endotoxemia, whereas E. coli infusion resulted in high (11,000 pg/ml) serum endotoxin levels. S. epidermidis, E. coli, LPS, or S. epidermidis-derived lipoteichoic acid (LTA) induced TNF and IL-1 from blood mononuclear cells in vitro. E. coli organisms and LPS were at least 100-fold more potent than S. epidermidis or LTA. Thus, a shock-like state with similar levels of complement activation as well as circulating levels of IL-1 and TNF were observed following either S. epidermidis or E. coli. These data provide further evidence that host factors such as IL-1 and TNF are common mediators of the septic shock syndrome regardless of the organism.

Structural and functional studies on hemoglobin Bethesda (alpha2beta2 145His), a varient associated with compensatory erythrocytosis.

Studies have been performed on a 12-yr-old Chinese girl with compensatory erythrocytosis due to the presence of hemoglobin Bethesda comprising about 45% of the red cell hemoglobin. Her parents and three siblings were normal. The oxygen affinity of her blood was markedly increased: under physiological conditions (pH 7.40, 37 degrees C). P(50) was 12.8 mm Hg (normal = 26.5 mm Hg). The red cell 2,3-diphosphoglycerate (2.3-DPG) level was normal. The abnormal hemoglobin could not be separated from hemoglobin A by zone electrophoresis at pH 8.6 or isoelectric focusing on polyacrylamide gel. However, after the hemoglobin was split into free alpha and beta chains by treatment with p-hydroxymercuribenzoate (PMB) or 6 M urea, an abnormal beta chain was readily demonstrated having a higher isoelectric point (more positive net charge) than normal beta(A). Structural analysis of the variant beta chain demonstrated the substitution of histidine for tyrosine at position 145: hemoglobin Bethesda (alpha(2)beta(2) (145His)). From earlier chemical and crystallographic studies, it has been postulated that this residue is a critical determinant of hemoglobin function. Hemoglobin Bethesda was separated from hemoglobin A by column chromatography. Oxygen equilibria of purified hemoglobin Bethesda revealed an extremely high oxygen affinity (exceeding that of isolated alpha and beta chains), and markedly reduced cooperativity. The Bohr effect of hemoglobin Bethesda was 1/3 that of hemoglobin A. However, hemoglobin Bethesda showed a significant interaction with 2.3-DPG and inositol hexaphosphate.

Regulated expression of a Drosophila melanogaster heat shock locus after stable integration in a Drosophila hydei cell line.

DNA-mediated cotransformation has been used to transfer a Drosophila melanogaster heat shock locus into cultured Drosophila hydei cells by use of the copia-based selectable vector pCV2gpt and of pMH10A, a cloned 87A7 heat shock locus encoding a mutant heat shock protein (hsp). Transformed lines contain between 50 and 200 copies of both plasmids, each separately organized as a head-to-tail concatemer which is stably maintained in the transformed lines. Exposure of the cotransformants to heat shock temperatures induces the regulated expression of the hsp RNA and the mutant hsp in all the lines analyzed.

Inactivation of a transfected gene in human fibroblasts can occur by deletion, amplification, phenotypic switching, or methylation.

Plasmids containing the bacterial gpt gene under control of the simian virus 40 promoter were transfected into a simian virus 40-transformed human fibroblast line. Two transfectants, E2 and C10, which contain stably integrated single copies of the gpt gene, were isolated. These two lines produce Gpt- variants spontaneously with a frequency of about 10(-4). We carried out a detailed molecular analysis of the spectrum of alterations which gave rise to the Gpt- phenotype in these variants. DNA from 14 of 19 Gpt- derivatives of one of the cell lines (E2) contains deletions or rearrangements of gpt-containing sequences. In four of the remaining five lines, the Gpt- phenotype was correlated with reduced levels of expression rather than with changes in the gross structure of the gpt gene, and it was possible to reactivate the gpt gene. In one Gpt- line, gpt mRNA was present at normal levels, but no active enzyme was produced. Spontaneous Gpt- derivatives of the other cell line (C10) produced a completely different spectrum of alterations. Very few deletions were found, but several derivatives contained additional extrachromosomal gpt sequences, and, remarkably, in two other Gpt- lines, gpt-containing sequences were amplified more than 100-fold. The phenotypes of the majority of the Gpt- derivatives of C10 could be attributed to alterations in gene expression caused by methylation.

Web alert. Neural control.

A selection of World Wide Web sites relevant to reviews published in this issue of Current Opinion in Neurobiology.

Cell cycle progression, morphology and contact inhibition are regulated by the amount of SV40 T antigen in immortal human cells.

Expression of Simian Virus 40 (SV40) T antigen in human dermal fibroblasts over-rides the normal controls on cell division leading to changes in cellular proliferation and life span. These changes are accompanied by other changes in cell morphology, expression of cell specific functions, and altered cell-cell interactions. In this study, we have examined the effects of different amounts of T antigen on cell cycle progression, life span and morphology in human dermal fibroblasts and demonstrated T antigen to be a concentration dependent regulator of the cell cycle. Using a novel, metal inducible episomal expression vector (p735.6) which produces low basal levels of protein but high (greater than 100-fold) levels of induction, we have compared the effects of low and high levels of T antigen expression in a precrisis and immortalised human line (1BRMT1). The presence of inducing agent led to maximal levels of T antigen expression and resulted in cultures with a high rate of proliferation, an extended in vitro life span, a loss of contact inhibition of growth and a morphology characteristic of SV40-transformed cells. In the absence of inducing agent, read-through of the T antigen gene resulted in low but detectable levels of protein. The reduction in T antigen levels was accompanied by a 50% or greater reduction in the proliferative rate and restoration of cell morphology and contact inhibition similar to that found in non-transfected cells. The results presented here demonstrate that low amounts of T antigen are sufficient to maintain cell viability and prevent the re-expression of the senescent phenotype seen in the absence of T antigen. Similarly, the ability of T antigen to extend the in vitro life span is not dependent on high level expression of T antigen. In contrast, the rate of proliferation of human cells as well as the cell morphology and contact inhibition are dependent on the amount of T antigen present. Many of the cellular effects can be minimised or reversed by reducing T antigen expression.

Neuronal expression of an FMRFamide-gated Na+ channel and its modulation by acid pH.

The molluscan Phe-Met-Arg-Phe-amide (FMRFamide)-gated sodium channels (FaNaCs) show both structural and functional similarities to the mammalian acid-sensing ion channels (ASICs). Both channel types are related to the epithelial sodium channels and, although the neuropeptide FMRFamide directly gates the FaNaCs, it also modulates the proton-gating properties of ASICs. It is not yet known whether protons can alter the gating properties of the FaNaCs. We chose to examine this possibility at a site of FaNaC expression in the nervous system of the mollusk Lymnaea stagnalis. We cloned a putative L. stagnalis FaNaC (LsFaNaC) that exhibited a high degree of sequence identity to the Helix aspersa FaNaC (HaFaNaC, 60%), and a weaker homology to the ASICs (ASIC3, 22%). In situ hybridization was used to map the LsFaNaC expression pattern in the brain and to identify the right pedal giant1 (RPeD1) neuron as a site where the properties of the endogenous channel could be studied. In RPeD1 neurons isolated in culture, we demonstrated the presence of an FMRFamide-gated sodium current with features expected for a FaNaC: amiloride sensitivity, sodium selectivity, specificity for FMRFamide and Phe-Leu-Arg-Phe-amide (FLRFamide), and no dependency on G-protein coupling. The sodium current also exhibited rapid desensitization in response to repeated FMRFamide applications. Lowering of the pH of the bathing solution reduced the amplitude of the FMRFamide-gated inward current, while also activating an additional sustained weak inward current that was apparently not mediated by the FaNaC. Acidification also prevented the desensitization of the FMRFamide-induced inward current. The acid sensitivity of LsFaNaC is consistent with the hypothesis that FaNaCs share a common ancestry with the ASICs.

Restriction fragment primed phi X174 single-stranded DNA as template for DNA polymerase alpha and beta. Detection and partial purification of a polymerase alpha stimulating factor.

Template-primers constructed of phiX174 single-stranded viral DNA hybridized to a restriction fragment of phiX174 RF DNA can be used for extensive polymerization by DNA polymerase alpha. Polymerization is dependent upon a restriction fragment containing a 3'OH. The products of the reaction have been identified by agarose gel electrophoresis. Polymerization of 150--400 nucleotides can be obtained in 1h depending upon the restriction fragment used as primer. Synthesis may be limited by barriers in the primary or secondary structure of the template. A factor which stimulates the rate of alpha polymerase activity on these templates was partially purified. This factor does not stimulate alpha polymerase on activated DNA. The stimulating factor sediments at 5.5 S in glycerol gradients containing 0.4M potassium phosphate and has an apparent molecular weight of 70 000 on Sephadex G-100.

Isolated prolapse of the tricuspid valve.

Tricuspid valve prolapse has remained a poorly defined entity. Some authors have stated that prolapse isolated to the tricuspid valve has not been documented. This report contains three cases of isolated tricuspid valve prolapse including the first pathologically confirmed case. A review of worldwide literature including all reported cases of isolated tricuspid valve prolapse is also presented. Although signs and symptoms are similar to those found with mitral valve prolapse, tricuspid valve prolapse may occasionally be differentiated by auscultation. The diagnostic criteria of tricuspid valve prolapse are thoroughly discussed for each of the presently available invasive and noninvasive techniques. Right heart catheterization can define such prolapse but is invasive and requires meticulous technique. Two-dimensional echocardiography supersedes M-mode because of the superior spatial evaluation of the tricuspid leaflets in relation to the right atrium and ventricle. Multiple views including a long-axis view of the right ventricular inflow are often required. This parasternal echocardiographic window is often the only one which permits adequate visualization of the posterior leaflet. The pathologic findings of tricuspid valve prolapse are similar to those of mitral valve prolapse. This report concludes with a description of associated conditions. Severe tricuspid regurgitation has not been noted with tricuspid valve prolapse in the absence of superimposed disease, yet much remains undefined concerning the clinical significance of this condition.

Inhibition of human neutrophil and Pseudomonas elastases by the amyloid P-component: a constituent of elastic fibers and amyloid deposits.

The amyloid P-component (AP), a ubiquitous component of amyloid fibrils, is also a plasma protein and a connective tissue constituent. Its proximity to elastin, in particular, suggested that AP might serve to protect elastic tissue from hydrolytic enzymes. The inhibition of pancreatic elastase by AP has been reported. In the present study, the effects of AP on human neutrophil elastase and Pseudomonas elastase were investigated, and AP was shown to interfere with the cleavage of soluble elastin. As indicated by Michaelis-Menten analysis, AP is acting as a noncompetitive inhibitor. C-reactive protein, which is structurally similar to AP, had no effect on either elastase. AP was also found to inhibit the degradation of secondary amyloid fibrils by neutrophil elastase when these structures were first partially purified and then reexposed to AP. AP's ability to inhibit elastase was compared with alpha-1 antitrypsin in the presence and absence of oxidizing agents. These substances, which are released by inflammatory cells, are known to abrogate alpha-1 antitrypsin's anti-protease capacity. This contributes to elevated levels of free proteases in the circulation and extravascular spaces during severe inflammation. AP is not susceptible to oxidation and remains a functional inhibitor under these conditions. The potential role of AP as an elastase inhibitor is discussed.

Doxorubicin hydrochloride-associated renal failure.

Doxorubicin hydrochloride (Adriamycin) therapy was associated with renal failure in a 78-year-old man. The pathophysiologic findings in this patient were similar to those seen following administration of structural analogues of doxorubicin. Daunorubicin hydrochloride, particularly, is known to cause renal failure in experimental animals. Renal function should be monitored in patients receiving doxorubicin.

The retention mechanism of technetium-99m-HM-PAO: intracellular reaction with glutathione.

Preparations of d,l- and meso-hexamethylpropyleneamine oxime (HM-PAO) labeled with technetium-99m were added to rat brain homogenates diluted with phosphate buffer (1:10). The conversion of d,l-HM-PAO to hydrophilic forms took place with an initial rate constant of 0.12 min-1. Incubation of the brain homogenate with 2% diethyl maleate for 5 h decreased the homogenate's measured glutathione (GSH) concentration from 160 to 16 microM and decreased the conversion rate to 0.012 min-1. Buffered aqueous solutions of glutathione rapidly converted the HM-PAO tracers to hydrophilic forms having the same chromatographic characteristics as found in the brain homogenates. The rate constant for the conversion reaction of d,l-HM-PAO in GSH aqueous solution was 208 and 317 L/mol/min in two different assay systems and for meso-HM-PAO the values were 14.7 and 23.2 L/mol/min, respectively. Rat brain has a GSH concentration of about 2.3 mM and the conversion of the d,l-HM-PAO due to GSH alone should proceed with a rate constant of 0.48 to 0.73 min-1 and be correspondingly 14-fold slower for meso-HM-PAO. In human brain, the in vivo data of Lassen et al. show a conversion rate constant of 0.80 min-1. This correspondence of values supports the notion that GSH may be important for the in vivo conversion of 99mTc-labeled HM-PAO to hydrophilic forms and may be the mechanism of trapping in brain and other cells. A kinetic model for the trapping of d,l- and meso-HM-PAO in tissue is developed that is based on data of GSH concentration in various organs.(ABSTRACT TRUNCATED AT 250 WORDS)

High-sensitivity S1 mapping with single-stranded [32P]DNA probes synthesized from bacteriophage M13mp templates.

A method is described by which high-specific-activity single-stranded (ss) [alpha-32P]DNA of a defined size complementary to sequences cloned into bacteriophage M13 is synthesized. The ss DNA template is annealed with a universal sequencing primer, the primer extended with DNA polymerase I Klenow fragment and the DNA duplex cut at a unique site 5' to the multiple cloning sites in the M13 phage. The reaction products are denatured and the ss alpha-32P probe fragment complementary to the cloned sequence is separated from the template by electrophoresis. The utility of such probes for S1 mapping is shown by mapping the 3' ends of transcripts from a mutant Drosophila heat-shock gene. The method described here is up to 300 times more sensitive than conventional S1 mapping techniques.

Transient assay, by [3H]guanine incorporation of Escherichia coli xanthine-guanine phosphoribosyl transferase (GPT) in transfected human fibroblasts.

We have used [3H]guanine incorporation as a rapid and sensitive assay of xanthine-guanine phosphoribosyl transferase (GPT) activity in SV40 transformed human fibroblasts. The SV40 early promoter is more efficient than the Rous sarcoma virus long terminal repeat for transient expression of the gpt gene. The assay works well in a derivative of AT5BIVA which lacks hypoxanthine-guanine phosphoribosyl transferase (hprt-) and we show here how the assay has been adapted to work in the hprt+ AT5BIVA parent.