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Thromboxane B2 (TXB2) levels in bleeding time blood and in serum were measured in 13 elderly patients with cardiovascular disease, seven of whom were receiving continuous treatment with low dose acetylsalicylic acid (ASA, 125 mg every second day--250 mg daily) for prevention of stroke. Blood sampling was performed openly, but assays of TXB2 were performed by a blinded investigator. In patients treated with ASA, median serum TXB2-levels were 4% and TXB2-levels in bleeding-time blood were less than 16% of the corresponding levels in patients without ASA (P less than 0.01). The results show that in elderly atherosclerotic patients very low doses of ASA substantially suppress TXB2 formation, not only in serum but also at the site of local haemostasis. The extent of suppression is comparable to that previously reported from young healthy subjects.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.

Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide.

Comparisons were made among different variants of ninhydrin reaction to determine overall titres of free alpha amino acids in body fluids. Included were para-benzoquinone, 2,4,6-trinitrobenzene sulphonic acid, and 2,4-dinitro-1-fluorobenzene (DNFB). The best reproducibility of results was recorded from DNFB which can be used, as well, for analysis of milk or rumen juice.

The short-term effect of gamma-irradiation (single dose, 100 Gy = 10 000 rad) on tth monoamine oxidase activity in the rat brain was investigated. In contrast to other investigations virtually no changes in MAO-activity were found.

In this paper, we have analysed cells which are in the process of enucleation in mouse spleen. We have provided additional support for the hypothesis that erythroid cells can autonomously undergo enucleation. The presence of a constriction in the nucleus and in the plasma membrane during this process has bee confirmed. Microfilaments have been described for the first time in the cytoplasm of maturing erythroid cells. Their position during enucleation is suggestive of an active role for these elements in this process.

We present observations on the relative location of the centriole and keratin filament cap in motile PtK1 cells. Subconfluent cells were double labeled with anticentriole and antikeratin sera. These preparations revealed that the centriole is separate from, but neighboring, the keratin filament cap. Serial ultrathin sections confirm this observation. These observations are consistent with the idea that the microtubule organizing center and intermediate filament distribution center are not identical or concentric in PtK1 cells.

Treatment of PtKl cells with 5 mM acrylamide for 4 hr results in alterations in the distribution of keratin filaments within the cells. This effect is reversible within 18 hr. Labeling of PtKl cells with 32P demonstrates that there are four phosphorylated keratins, having Mr of 56 k, 53 k, 45 k, and 40 k. Phosphate associated with these polypeptides appears to turn over with a t1/2 of 12 hr. Incubation of labeled cells in 5 mM acrylamide results in approximately 50% dephosphorylation of keratins within 2 hr, which is 3 times faster than normal turnover. Recovery of cells from acrylamide is accompanied by rephosphorylation of keratins within 18 hr. Analysis by 31P NMR spectroscopy shows that acrylamide treatments are accompanied by a transient decrease in soluble inorganic phosphate. This is followed by a rapid increase in Pi which gradually returns to normal levels. These studies show a strong correlation between phosphorylation of PtKl cell keratins and morphological response of keratin filaments to acrylamide. These observations suggest that normal distribution of keratin filaments may be, in part, mediated by protein phosphorylation.

Antibody prepared against avian smooth muscle actin has been used to localize actin in the slime mold, Dictyostelium discoideum. The distribution of actin in migrating cells is different from that in feeding cells. Migrating amebas display fluorescence primarily in advancing regions whereas feeding amebas show uniform fluorescence throughout. The reaction is specific for actin since the fluorescence observed is blocked when the antibody is absorbed by actin purified from avian skeletal muscle, human platelets, and Dictyostelium. These results, in addition to describing the distribution of actin in D. discoideum, demonstrate that actins from these diverse sources share at least one common antigenic determinant.

Cytoskeletons of primitive erythrocytes have been isolated from the embryos of day 12 pregnant C57/Bl mice and examined by transmission electron microscopy, immunofluorescence microscopy, and SDS-polyacrylamide gel electrophoresis. Microtubules are the most prominent cytoskeletal component. They are found either singly or organized into loose bundles just under the plasma membrane, but do not form classical marginal bands in most cells. Immunofluorescence with a polyclonal tubulin antiserum confirms this distribution and further reveals numerous mitotic figures among cells. Rhodamine-conjugated phalloidin and heavy meromyosin labeling reveal that actin is localized in the cortex of the primitive erythrocyte in the form of 6 nm filaments. Antibody directed against avian erythrocyte alpha spectrin demonstrates that spectrin is also found in the cortex. Occasional 10-nm intermediate filaments, observed in the primitive erythrocytes by electron microscopy, are believed to be of the vimentin class based on positive reaction of the cells with vimentin-specific antiserum. In addition, a band in erythrocyte cytoskeletons comigrates in SDS-polyacrylamide gels with vimentin isolated from mouse kidney. Spectrin and actin were also found to be associated with the membrane of primitive erythrocytes when membrane ghost preparations were analyzed by SDS-polyacrylamide gel electrophoresis.

The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position.

Reorganization of intermediate filaments during cell spreading is examined by immunofluorescence, electron microscopy, and time-lapse video microscopy. A juxtanuclear cap, believed to correspond to the intermediate filament distribution center, was observed to be spatially related to the organization of the intermediate filament network as cells spread. A keratin cap was observed, which appeared spontaneously in motile PtK1 cells. Cap formation may be a consequence of retraction of intermediate filaments from the cytoplasm as cells move. The position of this juxtanuclear cap is related to the direction of movement, located on the side of the nucleus near the advancing edge of the cell. As the cell spreads, the cap disappears as the keratin filament network returns to the cytoplasm. Evidence presented here is consistent with the hypothesis that the distribution center mediates keratin filament organization during cell shape change.

The results of epineural microneurorrhaphy with use of 10/0 monofilament absorbable (Vicryl and Dexon) and nonabsorbable ( Dermalon , Ethilon, and Prolene) microsuture were compared in 150 isogeneic male Sprague-Dawley rats. After sciatic nerve transection and epineural repair, the animals were observed clinically and reexplored before death at intervals from 2 days to 20 weeks. Half of the animals were randomly selected for electrodiagnostic studies at 6, 12, and 20 weeks before sacrificed. We found no significant clinical, electrodiagnostic, or histologic differences affecting axonal regeneration that were attributable to any of the suture types used. All sutures incited moderate zones of localized inflammation acutely. After dissolution, the absorbable group was essentially free of inflammation, whereas the nonabsorbable sutures persisted in small local granulomas. A possible advantage may be suggested in the use of monofilament absorbable sutures for microneural repairs in certain situations.

PtK1 cells were treated with low concentrations of acrylamide resulting in disruption of intermediate filament networks. An optimum treatment, 5 mM acrylamide in culture medium for 4 h, resulted in formation of a juxtanuclear aggregate containing both keratin and vimentin intermediate filaments. Actin-containing stress fibers and microtubules appeared normal after this treatment. Cells recovered when acrylamide was washed out of the cultures, and normal keratin and vimentin networks reappeared. These cells were capable of proliferation and grew to confluence. Acrylamide-treated cells appeared to locomote normally, showing membrane ruffling and changes in shape, but cytoplasmic organelles did not appear to move normally throughout the cell but remained at the cell center. These observations indicate that acrylamide is a useful intermediate filament inhibitor that does not affect other cytoskeletal elements.

The purpose of this study was to determine if marginal bands, such as those present in mature nucleated red blood cells of other non-mammalian vertebrates and in primitive mammalian erythrocytes are present in definitive mammalian erythroblasts. In a small number of erythroblasts examined from mouse spleen, a bundle of 5-8 microtubules could be seen. These microtubules appeared similar to those previously identified by others as marginal band microtubules in liver and marrow erythroblasts. However, it was difficult to distinguish these bundles from remnants of mitotic spindle microtubules, or bundles of microtubules which extend to the midbody, a structure which is seen quite frequently in sections of erythroid cells. Triton extraction, a process which renders cytoskeletal elements such as microtubules more visible, also failed to confirm the presence of conventional marginal bands in these cells. It is suggested that use of the term "marginal band" be restricted to those cases in which it can be unequivocally demonstrated that a bundle of microtubules encircles the perimeter of the cell.

Human neutrophils contain intermediate filaments of the vimentin type. A cytoskeletal preparation, produced by high-salt and Triton X-100 extraction of human neutrophils, reveals a major band at 57000 Mr that comigrates with 3T3 cell vimentin on one-dimensional gels. Two-dimensional gel electrophoresis of whole neutrophils illustrates the presence of vimentin but not desmin- or keratin-filament subunits. The presence of vimentin in neutrophils is also shown by its specific staining with avian vimentin antiserum by two-dimensional gel immunoautoradiography. Indirect immunofluorescence studies show that vimentin antiserum labels an area on one side of the nucleus in spreading neutrophils. This bright area appears as a loose knot of vimentin filaments; a few filaments may radiate from the knot. In contrast to spreading neutrophils, those undergoing random locomotion contain a fine network of filaments that are located in the cytoplasm between the nucleus and the trailing end of the cell. Similarly, in chemoattractant-treated neutrophils, vimentin filaments are bundled in the uropod. Transmission electron microscopy of human neutrophil monolayers confirms the intracellular distribution of intermediate filaments as shown by immunofluorescence in spreading and randomly locomoting cells.

A controlled laboratory study using matched groups fo pure-bred Sprague-Dawley rats evaluated the histologic microvascular response to contrast angiography. The femoral vessels were studied by both light and transmission electron microscopy (TEM) following injection of the infrarenal aorta with Renografin-60 or saline at intervals from 2 hours to 56 days. None of the specimens studied showed significant cellular, interstitial, or intravascular injury. TEM demonstrated electron-dense particles which migrated from the vessel lumen into the endothelial cytoplasm and basal lamina and subsequently diffused back into the cytoplasm. This study demonstrates the absence of microvessel damage from a radiopaque contrast medium administered by techniques designed to parallel the clinical model.

The process of nuclear extrusion continues when erythroid cells of mouse spleen are placed in vitro, although the process may be considerably retarded. This allows visualization of unusually large numbers of enucleating cells after 30 min in vitro. With this system, the mechanism of enucleation can be analyzed both qualitatively and quantitatively. We have used this system to study the effects of cytochalasin B and have found it to be a potent inhibitor of enucleation. These results suggest that microfilaments are involved in the process of enucleation.

A number of patients with silicone rubber implants performed by us and other surgeons initially had excellent results; however, they returned with swelling and discomfort. We studied 18 patients ranging in age from 16 years to 57 years who presented 8 to 78 months (average, 31.7 months) after silicone arthroplasty (four scaphoid, six lunate, one scapholunate, four finger, two wrist, one trapezium, and one ulnar head for metacarpal hemiarthroplasty). Erosive osteolysis was seen on x-ray films, with progressive destruction evident in patients followed serially. None of the patients' conditions responded to conservative care. The severity of the proliferative, inflammatory synovitis and the foreign material in the multinucleated giant cells correlated with the interval since arthroplasty. Implant surface analysis by scanning electron microscope and x-ray spectrometer showed that silicone microparticles were the result of implant degeneration and erosion. All joint cultures were negative. Silicone particulate synovitis and destruction were arrested by the removal of the implant, a synovectomy, and curettage of the lytic lesions at salvage (resection arthroplasty or arthrodesis). Patients who have had silicone arthroplasties should be followed indefinitely, at regular intervals, by x-ray films and clinical examination.