The interrelationship of complement-activation fragments and angiogenesis-related factors in early pregnancy and their association with pre-eclampsia.

OBJECTIVE: To determine the interrelationships during early pregnancy of complement-activation fragments Bb, C3a and sC5b-9, and angiogenesis-related factors placental growth factor (PiGF), soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng), and their associations with pre-eclampsia. DESIGN: Prospective cohort study. SETTING: Denver complement study (June 2005-June 2008). POPULATION: A total of 668 pregnant women with singleton gestations, recruited between 10 and 15 weeks of gestation. METHODS: Using univariable and multivariable logistic regression analysis, concentrations of complement-activation fragments and angiogenesis-related factors were compared between 10 and 15 weeks of gestation in women who subsequently did or did not develop pre-eclampsia. Interrelationships between these variables were tested using the non-parametric Spearman rank correlation coefficient. MAIN OUTCOME MEASURE: Pre-eclampsia. The association of complement-activation fragments and angiogenesis-related factors with obesity was also examined. RESULTS: The mean (+/-SD) levels of complement Bb in early pregnancy among women who did and did not develop pre-eclampsia were 0.84 (+/-0.26) microg/ml and 0.69 (+/-0.2) microg/ml, respectively (P = 0.001). Concentrations of PiGF were significantly (P = 0.01) lower (31 +/- 12 pg/ml) in early pregnancy in the pre-eclamptic group of women, as compared with the normotensive group (39 +/- 32 pg/ml). The adjusted odds ratio (AOR) of Bb and PiGF were 2.1 (CI = 1.4-3.1, P < 0.0003) and 0.2 (CI = 0.07-0.7, P = 0.01), respectively. There was no significant difference in the levels of C3a, sC5b-9, sFlt-1 and sEng in early pregnancy among women who developed pre-eclampsia, compared with women who remained normotensive during pregnancy. Higher levels of Bb (P = 0.0001) and C3a (P = 0.03), and lower levels of sFlt-1 (P = 0.0002) and sEng (P = 0.0001) were found among women with obesity, compared with non-obese controls. No meaningful relationships were found between the complement-activation fragments and the angiogenesis-related factors. CONCLUSIONS: In this cohort during early pregnancy, increased concentrations of complement-activation factor Bb and lower concentrations of PiGF were associated with the development of pre-eclampsia later in pregnancy.

Paramyosin in invertebrate muscles. I. Identification and localization.

By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunodiffusion, we identified paramyosin in two smooth invertebrate "catch" muscles (Mytilus anterior byssus retractor and Mercenaria opaque adductor) and five invertebrate striated muscles (Limulus telson levator, Homarus claw muscle, Balanus scutal depressor, Lethocerus air tube retractor, and Aequipecten striated adductor). We show that (a) the paramyosins in all of these muscles have the same chain weights and (b) they are immunologically similar. We stained all of these muscles with specific antibody to Limulus paramyosin using the indirect fluorescent antibody technique. Paramyosin was localized to the A bands of the glycerinated striated muscles, and diffus fluorescence was seen throughout the glycerinated fibers of the smooth catch muscles. The presence of paramyosin in Homarus claw muscle, Balanus scutal depressor, and Lethocerus air tube retractor is shown here for the first time. Of the muscles in this study, Limulus telson levator is the only one for which the antiparamyosin staining pattern has been previously reported.

Electron microscopic and optical diffraction analysis of the structure of scorpion muscle thick filaments.

We rapidly and gently isolated thick filaments from scorpion tail muscle by a modification of the technique previously described for isolating Limulus thick filaments. Images of negatively stained filaments appeared to be highly periodic, with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrograph images were detailed and similar to optical diffraction patterns from Limulus and tarantula thick filaments. Analysis of the optical diffraction patterns and computed Fourier transforms, together with the appearance of the filaments in the micrographs, suggested a model for the filaments in which the myosin cross-bridges were arranged on four helical strands with 12 cross-bridges per turn of each strand, thus giving the observed repeat every third cross-bridge level. Comparison of the scorpion thick filaments with those isolated from the closely related chelicerate arthropods, Limulus and tarantula, revealed that they were remarkably similar in appearance and helical symmetry but different in diameter.

Three-dimensional reconstruction of thick filaments from Limulus and scorpion muscle.

We have produced three dimensional reconstructions, at a nominal resolution of 5 nm, of thick filaments from scorpion and Limulus skeletal muscle, both of which have a right-handed four-stranded helical arrangement of projecting subunits. In both reconstructions there was a distinct division of density within projecting subunits consistent with the presence of two myosin heads. Individual myosin heads appeared to be curved, with approximate dimensions of 16 X 5 X 5 nm and seemed more massive at one end. Our reconstructions were consistent with the two heads in a projecting subunit being arranged either antiparallel or parallel to each other and directed away from the bare zone. Although we cannot exclude the second of these interpretations, we favor the first as being more consistent with both filament models and also because it would enable easy phosphorylation of light chains. The antiparallel interpretation requires that the two heads within a subunit derive from different myosin molecules. In either interpretation, the two heads have different orientations relative to the thick filament shaft.

An electron microscopic and optical diffraction analysis of the structure of Limulus telson muscle thick filaments.

Long, thick filaments (greater than 4.0 micrometer) rapidly and gently isolated from fresh, unstimulated Limulus muscle by an improved procedure have been examined by electron microscopy and optical diffraction. Images of negatively stained filaments appear highly periodic with a well-preserved myosin cross-bridge array. Optical diffraction patterns of the electron micrographs show a wealth of detail and are consistent with a myosin helical repeat of 43.8 nm, similar to that observed by x-ray diffraction. Analysis of the optical diffraction patterns, in conjunction with the appearance in electron micrographs of the filaments, supports a model for the filament in which the myosin cross-bridges are arranged on a four-stranded helix, with 12 cross-bridges per turn or each helix, thus giving an axial repeat every third level of cross-bridges (43.8 nm).

The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments.

Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends. Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3 microns) and thick filament (from 4.1 to 3.3 microns) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A- and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap filaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the I-bands by elastic recoil, in highly stretched fibers.

Arrangement of myosin heads on Limulus thick filaments.

The two myosin heads with a single surface subunit on thick filaments from chelicerate arthropod muscle may originate from the same, or from axially sequential molecules, as suggested by three-dimensional reconstructions. The resolution attained in the reconstructions, however, does not permit one to distinguish unequivocally between these two possible arrangements. We examined the effect of 0.6 M KCl on relaxed thick filaments separated from Limulus muscle and filaments in which nearest myosin heads were cross-linked by the bifunctional agent, 3,3'-dithio-bis[3'(2')-O-[6-propionylamino)hexanoyl]adenosine 5'-triphosphate (bis22ATP), in the presence of vanadate (Vi). In high salt, surface myosin dissolved from both native, relaxed filaments and those exposed to 1-2 mM dithiothreitol after cross-linking, but was retained on filaments with cross-linked heads. Since bis22ATP must form intermolecular bonds between myosin heads within each subunit to prevent myosin solubilization in high salt, we conclude that each of these heads originates from a different myosin molecule, as was previously predicted by the reconstructions.

Paramyosin in invertebrate muscles. II. Content in relation to structure and function.

By quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, paramyosin:myosin heavy chain molecular ratios were calculated for three molluscan muscles:Aequipecten striated adductor, Mercenaria opaque adductor, and Mytilus anterior byssus retractor; and four arthropodan muscles:Limulus telson, Homarus slow claw. Balanus scutal depressor, and Lethocerus air tube retractor. These ratios correlate positively with both thick filament dimensions and maximum active tension development in these tissues. The role of paramyosin in these muscles is discussed with respect to the following characteristics: force development, "catch," and extreme reversible changes in length.

Immunohistochemical localization of contractile proteins in limulus striated muscle.

Limulus paramyosin and myosin were localized in the A bands of glycerinated Limulus striated muscle by the indirect horseradish peroxidase-labeled antibody and direct and indirect fluorescent antibody techniques. Localization of each protein in the A band varied with sarcomere length. Antiparamyosin was bound at the lateral margins of the A bands in long ( approximately 10.0 micro) and intermediate ( approximately 7.0 micro) length sarcomeres, and also in a thin line in the central A bands of sarcomeres, 7.0- approximately 6.0 micro. Antiparamyosin stained the entire A bands of short sarcomeres (<6.0). Conversely, antimyosin stained the entire A bands of long sarcomeres, showed decreased intensity of central A band staining except for a thin medial line in intermediate length sarcomeres, and was bound only in the lateral A bands of short sarcomeres. These results are consistent with a model in which paramyosin comprises the core of the thick filament and myosin forms a cortex. Differential staining observed using antiparamyosin and antimyosin at various sarcomere lengths and changes in A band lengths reflect the extent of thick-thin filament interaction and conformational change in the thick filament during sarcomeric shortening.

Structure of Limulus striated muscle. The contractile apparatus at various sarcomere lengths.

The musculature of the telson of Limulus polyphemus L. consists of three dorsal muscles: the medial and lateral telson levators and the telson abductor, and one large ventral muscle; the telson depressor, which has three major divisions: the dorsal, medioventral, and lateroventral heads. The telson muscles are composed of one type of striated muscle fiber, which has irregularly shaped myofibrils. The sarcomeres are long, with discrete A and I and discontinuous Z bands. M lines are not present. H zones can be identified easily, only in thick (1.0 microm) longitudinal sections or thin cross sections. In lengthened fibers, the Z bands are irregular and the A bands appear very long due to misalignment of constituent thick filaments. As the sarcomeres shorten, the Z lines straighten somewhat and the thick filaments become more aligned within the A band, leading to apparent decrease in A band length. Further A band shortening, seen at sarcomere lengths below 7.4 microm may be a function of conformational changes of the thick filaments, possibly brought about by alterations in the ordering of their paramyosin cores.

Effects of phosphorylation by myosin light chain kinase on the structure of Limulus thick filaments.

The results discussed in the preceding paper (Levine, R. J. C., J. L. Woodhead, and H. A. King. 1991. J. Cell Biol. 113:563-572.) indicate that A-band shortening in Limulus muscle is a thick filament response to activation that occurs largely by fragmentation of filament ends. To assess the effect of biochemical changes directly associated with activation on the length and structure of thick filaments from Limulus telson muscle, a dually regulated tissue (Lehman, W., J. Kendrick-Jones, and A. G. Szent Gyorgyi. 1973. Cold Spring Harbor Symp. Quant. Biol. 37:319-330.) we have examined the thick filament response to phosphorylation of myosin regulatory light chains. In agreement with the previous work of J. Sellers (1981. J. Biol. Chem. 256:9274-9278), Limulus myosin, incubated with partially purified chicken gizzard myosin light chain kinase (MLCK) and [gamma 32P]-ATP, binds 2 mol phosphate/mole protein. On autoradiographs of SDS-PAGE, the label is restricted to the two regulatory light chains, LC1 and LC2. Incubation of long (greater than or equal to 4.0 microns) thick filaments, separated from Limulus telson muscle under relaxing conditions, with either intact MLCK in the presence of Ca2+ and calmodulin, or Ca2(+)-independent MLCK obtained by brief chymotryptic digestion (Walsh, M. P., R. Dabrowska, S. Hinkins, and D. J. Hartshorne. 1982. Biochemistry. 21:1919-1925), causes significant changes in their structure. These include: disordering of the helical surface arrangement of myosin heads as they move away from the filament backbone; the presence of distal bends and breaks, with loss of some surface myosin molecules, in each polar filament half; and the production of shorter filaments and end-fragments. The latter structures are similar to those produced by Ca2(+)-activation of skinned fibers (Levine, R. J. C., J. L. Woodhead, and H. A. King. J. Cell Biol. 113:563-572). Rinsing experimental filament preparations with relaxing solution before staining restores some degree of order of the helical surface array, but not filament length. We propose that outward movement of myosin heads and thick filament shortening in Limulus muscle are responses to activation that are dependent on phosphorylation of regulatory myosin light chains. Filament shortening may be due, in large part, to breakage at the filament ends.

Structure and paramyosin content of tarantula thick filaments.

Muscle fibers of the tarantula femur exhibit structural and biochemical characteristics similar to those of other long-sarcomere invertebrate muscles, having long A-bands and long thick filaments. 9-12 thin filaments surround each thick filament. Tarantula muscle has a paramyosin:myosin heavy chain molecular ratio of 0.31 +/- 0.079 SD. We studied the myosin cross-bridge arrangement on the surface of tarantula thick filaments on isolated, negatively stained, and unidirectionally metal-shadowed specimens by electron microscopy and optical diffraction and filtering and found it to be similar to that previously described for the thick filaments of muscle of the closely related chelicerate arthropod, Limulus. Cross-bridges are disposed in a four-stranded right-handed helical arrangement, with 14.5-nm axial spacing between successive levels of four bridges, and a helical repeat period every 43.5 nm. The orientation of cross-bridges on the surface of tarantula filaments is also likely to be very similar to that on Limulus filaments as suggested by the similarity between filtered images of the two types of filaments and the radial distance of the centers of mass of the cross-bridges from the surfaces of both types of filaments. Tarantula filaments, however, have smaller diameters than Limulus filaments, contain less paramyosin, and display structure that probably reflects the organization of the filament backbone which is not as apparent in images of Limulus filaments. We suggest that the similarities between Limulus and tarantula thick filaments may be governed, in part, by the close evolutionary relationship of the two species.

Changes in thick filament length in Limulus striated muscle.

Here we describe the change in thick filament length in striated muscle of Limulus, the horseshoe crab. Long thick filaments (4.0 microns) are isolated from living, unstimulated Limulus striated muscle while those isolated from either electrically or K+-stimulated fibers are significantly shorter (3.1 microns) (P less than 0.001). Filaments isolated from muscle glycerinated at long sarcomere lengths are long (4.4 microns) while those isolated from muscle glycerinated at short sarcomere lengths are short (2.9 microns) and the difference is significant (P less than 0.001). Thin filaments are 2.4 microns in length. The shortening of thick filaments is related to the wide range of sarcomere lengths exhibited by Limulus telson striated muscle.

Histamine synthesis in man: inhibition by 4-bromo-3-hydroxybenzyloxyamine.

Oral administration of 4-bromo-3-hydroxybenzyloxyamine to normal humans resulted in decreased urinary excretion of histamine; the normal increase in urinary levels of histamine after oral histidine loading was prevented. In two patients having systemic mastocytosis, additional evidence of inhibition of biosynthesis of histamine included marked reduction in symptoms attributed to histamine, and prevention of symptomatic exacerbation associated with histidine loading.

Tyrosine metabolism in patients with liver disease.

Plasma levels of tyrosine were assayed in the fasting state and after oral administration of either tyrosine (tyrosine tolerance test) or phenylalanine (phenlyalanine conversion test) in normal subjects and in patients with hepatitis, biliary obstruction, or cirrhosis. Fasting tyrosine levels tended to be slightly increased in patients with hepatitis and biliary obstruction and markedly increased in patients with cirrhosis. Tyrosine tolerance tests in patients with cirrhosis were characterized by larger than normal increments in tyrosine levels and by delayed returns toward fasting levels. The results of phenylalanine conversion tests were abnormal in approximately one-half of patients with either hepatitis or biliary obstruction and four-fifths of patients with cirrhosis. Abnormalities were characterized by elevated fasting plasma tyrosine levels, or small and delayed increments in tyrosine levels, or both. Abnormal phenylalanine conversion test results in patients with cirrhosis did not correlate closely with any clinical feature of cirrhosis or with the results of any standard liver function test; there was positive correlation only with abnormal ammonia tolerance, a test of portalsystemic shunting. Tests of tyrosine metabolism do not appear to be useful for routine clinical assessment of liver function. Tyrosine tolerance tests and phenylalanine conversion tests done for purposes of diagnosis of other diseases may yield misleading results in patients with liver disease.

Ethics in cardiovascular medicine. Task Force IV: Scientific responsibility and integrity in medical research.

Ethical standards are a set of affirmative responsibilities to which the investigator must subscribe; behavior that is incompatible with these responsibilities should be presumed unethical, whether or not it is explicitly proscribed. This Task Force sought to present these standards as principles or guidelines. In undertaking research an investigator must accept that publicly funded or supported research is intended to yield public benefit; personal gain should be only incidental to and not at the expense of the public benefit. The responsibilities of the investigator are summarized as follows: Design of Research To develop a research design that effectively and efficiently addresses the scientific question while minimizing the likelihood of incorrect or misleading results. To protect the rights and welfare of human subjects, assure the humane use of laboratory animals and protect the safety of laboratory workers and the environment. Conduct of Research To ensure that accepted laboratory and research practices are followed and that all data are accurately collected and properly recorded; the investigator must participate in the review of original data. To carry out research in accordance with that approved by the institutional review board and ensure that fully informed consent is obtained, that the welfare of human subjects is protected and that animal welfare and laboratory safety procedures are carried out. To provide effective ongoing supervision of research trainees and technicians. In multidisciplinary collaborative research, to have at least an overview familiarity with the work outside his or her areas of expertise. In fixed protocol, multicenter collaborative research the investigator must be satisfied with the adequacy of the collaborative activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Structure of short thick filaments from Limulus muscle.

Shortened Limulus thick filaments, isolated from stimulated muscle, are structurally similar to long filaments, isolated from unstimulated muscle, except for length. Both have 3-fold screw symmetry with a helical repeat at approximately 43 nm, axial spacing of 14.5 nm between successive crowns of crossbridges and 4-fold rotational symmetry as estimated from the Bessel argument, by analysis of optical transforms of electron micrograph negatives of negatively stained samples. Both short and long filaments also have similar radii for the location of their crossbridges, thus similar diameters. Equal numbers of subunits/helical strand are also apparent on images of metal-shadowed long and short filaments. Since these data argue against molecular reorganization during filament shortening, it is suggested that the change in length of Limulus thick filaments may occur by reversible disaggregation of constituent protein molecules.

Some ethical considerations in clinical trials.

The ethical norms established in various codes and regulations are inadequate to resolve some of the ethical problems presented by clinical trials. They are stated too vaguely to provide unequivocal answers to many specific questions. In order to remedy this situation, many commentators have proposed the development of more specific and complex regulations. We propose that a more fruitful approach would be to examine the ethical principles underlying the norms and to apply these principles to the specific problems. We apply this approach to two questions: (1) Is it ethical to select subjects for a randomized clinical trial (RCT) exclusively from Veterans Administration (VA) hospitals? (2) In the conduct of a RCT is it necessary to disclose the fact that therapy will be determined by chance? We conclude that problems of justice arise not only because of the vulnerability of patients in VA hospitals but also because of the loss of the physician-patient relationship in an RCT. However, the use of patients in a VA hospital is not always unjust; in most cases such use can be made more just through various modifications in design. We also conclude that the fact of randomization should be disclosed in any situation in which it might materially affect the prospective subject's decision, and that the values and preferences of the subjects should be taken into account in determining what information might be material. This work is only a preliminary step toward analyzing ethical issues in clinical trials. While some would challenge our conclusions, we hope that our methods will facilitate clarity about the locus of disagreement in current controversies and about the value questions that must be answered in order to set an ethical context for the conduct of clinical trials.

Stability of immunoglobulin E (IgE) in stored obstetric sera.

OBJECTIVE: To determine the stability of immunoglobulin E levels in obstetric sera. METHODS: AlaSTAT(R) and AlaTOP(R) (Diagnostic Products) were used to assay total and specific IgE levels in obstetric sera collected in Memphis, TN and Portland, OR. The samples were collected from the Collaborative Perinatal Project (CPP) between 1959 and 1965 and stored at -20 degrees C. The assay results were compared with IgE levels found in sera collected at the same locations for the Calcium for Pre-eclampsia Prevention Study (CPEP) and stored since 1992 at -70 degrees C. The samples were also assayed for cockroach (CR) and mouse urine specific IgE using the AlaSTAT(R) assay (Diagnostic Products). RESULTS: Total IgE and specific IgE to CR and mouse urine were detectable in older and recent samples. The median total IgE for the recent and older Portland samples was 26 IU/ml and 65 IU/ml, respectively. The median total IgE was identical (40 IU/ml) in the recent and older Memphis samples. CONCLUSION: Long-term storage does not diminish the ability to measure serum IgE. Levels of IgE in sera stored 32-37 years were equal to or greater than levels in sera stored for 5 years. reserved.

Antibody binding by native and denatured myosin and paramyosin.

By the techniques of immunodiffusion and fluorescent immunohistochemistry we show that antibodies to both the native and the SDS-denatured forms of the proteins, paramyosin and myosin, react with the native, SDS-denatured and glutaraldehyde-fixed forms of their respective antigens. Anti-denatured myosin also binds to both native and denatured forms of the proteolytic subfragments of myosin: globular subfragment-1 and alpha-helical LMM. Anti-native myosin, on the other hand, while able to bind to both native and denatured LMM or rod and to native and glutaraldehyde-fixed S-1, does not bind to SDS-denatured S-1.