Studies on the mechanisms of neurulation in the chick: morphometric analysis of the relationship between regional variations in cell shape and sites of motive force generation

Microfilaments, which are organized into bundles in the apical ends of neuroepithelial cells, are generally thought to play a major role in generating the driving forces for neural tube closure. Because of their proximity to the luminal surface, the contractile activity of these microfilament bundles results in conspicuous changes in the overall shape of neuroepithelial cells, most notably apical constriction and apical surface folding. In the present study, we have used morphometric methods and computer-assisted image analysis to reveal the distribution of microfilament-mediated forces in the developing midbrain during initial contact of apposing neural folds in chick embryos at Hamburger and Hamilton stage 8+ of development (Hamburger and Hamilton (1951) J. Morphol., 88:49-92). The degree of apical constriction, apical surface folding, and bending of the neuroepithelium was used as a barometer of local microfilament activity. Results indicate that cells forming the floor and midlateral walls of the developing midbrain consistently show a higher degree of apical constriction and surface folding than those at other locations. These same regions of the neuroepithelium also exhibit the greatest degree of bending. We conclude that the principal driving forces for closure of the neural tube, at the level of the midbrain, are concentrated in certain regions of the neuroepithelium (i.e., the floor and midlateral walls of the forming neural tube) rather than uniformly distributed.     

Verapamil inhibits serotonin uptake of endothelial cells in culture by a mechanism unrelated to Ca2+ channel blockade

Verapamil inhibited Na+-dependent uptake of serotonin (5-HT) by bovine pulmonary artery endothelial cells in culture both exposed to room air and stimulated by prior exposure to anoxia. The effect of verapamil occurred even in the absence of Ca2+ from the assay medium. Although absence of Ca2+ from the medium moderately reduced 5-HT uptake, stimulation of uptake was nevertheless observed for cells previously exposed to anoxia. Verapamil altered the Km, but not the Vmax, of 5-HT uptake. There was no change in 45Ca2+ uptake or release by cells previously exposed to anoxia as compared to those exposed to room air and verapamil did not influence 45Ca2+ fluxes by either set of cells. It is concluded that verapamil inhibits 5-HT uptake by endothelial cells through a mechanism other than Ca2+ channel blockade; the results are consistent with competitive inhibition of a 5-HT carrier. The stimulatory effect of anoxia on 5-HT uptake does not occur through a change in Ca2+ fluxes.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Equilibrium binding parameters of an autoimmune monoclonal antibody specific for double-stranded DNA

Two techniques have been developed to estimate binding parameters for Jel 241 under equilibrium conditions. Jel 241 is an autoimmune monoclonal antibody derived from an NZB/NZW mouse which binds to double-stranded DNA. Thermal denaturation profiles of poly[d(AT)] were measured in the presence of increasing concentrations of IgG Jel 241. From these data it was estimated that the IgG occludes 12 base-pairs on duplex DNA, and the binding to double-stranded DNA was at least four orders of magnitude greater than to single-stranded DNA. In addition, intrinsic association constants (K(O)) were measured by a gel filtration technique for the interaction of both Fab and IgG Jel 241 to native calf thymus DNA. K(O) for the IgG was only 60-fold greater than for the Fab fragment for which K(O) was 4.4 X 10(4) M-1 at an NaCl concentration of 150 mM. Also, K(O) for the Fab increased dramatically with decreasing ionic strength, suggesting that there are four phosphates involved in the interaction. These techniques should be applicable to most autoimmune antibodies which bind to nucleic acid polymers.     

Effect of auranofin treatment on aberrant splenic interleukin production in adjuvant arthritic rats

Adjuvant-induced arthritis (AA) in rats is associated with a number of immunologic abnormalities which include a marked decrease in spleen cell mitogenic responses. In this study we investigated the altered production of interleukins in arthritic rats and evaluated the effects of auranofin treatment on disease progression and aberrant interleukin production. The capacity of the AA rat spleen cells to produce interleukin (IL) 2 and IL-3 was found to decrease during the development of the arthritic lesion, with maximum suppression occurring 16 to 17 days after adjuvant injection. In contrast, the production of IL-1 by splenic adherent cells from arthritic rats was markedly increased. Prophylactic treatment of AA rats with auranofin resulted in a slight reduction in paw edema, a complete normalization of the depressed IL-2 production, and a reduction of the elevated IL-1 production, but had no effect on the depressed IL-3 production. In contrast, auranofin administered to normal rats, in the same dosing regimen, did not affect interleukin production. Therapeutic administration of auranofin to AA rats with established disease resulted in normalization of IL-1 production without affecting the suppressed IL-2 and IL-3 levels. In contrast, while indomethacin treatment effectively decreased paw edema, it did not appreciably affect the systemic aberrant interleukin production. Taken together, these results suggest that disease-associated abnormalities in interleukin production may be mediated by different mechanisms with differential sensitivity to the effects of the disease-modifying drug auranofin. Furthermore, defining the relationship between drug-mediated normalization of aberrant immune parameters and clinical improvement will provide a basis for the elucidation of the mechanism of action of disease-modifying antiarthritic drugs as well as for assessment of clinical efficacy of drug treatment.     

Synthesis and biochemical studies of analogs of platelet-activating factor bearing a methyl group at C2 of the glycerol backbone

Two platelet-activating factor (PAF) analogs containing a methyl group at C2 of the glycerol moiety were synthesized, and some of their biochemical properties were investigated. 1-O-Hexadecyl-2-C,O-dimethyl-rac-glycero-3-phosphocholine (2-methyl-2-methoxy PAF) was prepared in a synthetic scheme beginning with the etherification of 2-methylpropen-1-ol. A reaction sequence involving hydroxylation, tritylation, alkylation, and detritylation afforded 1-O-hexadecyl-2-C,O-dimethyl-rac-glycerol, which was converted into the phosphocholine. A 2-lyso derivative of this PAF analog (2-methyl-lyso PAF) was synthesized from 1-O-hexadecyl-2-C-methyl-3-O-trityl-rac-glycerol. Benzylation followed by detritylation gave 1-O-hexadecyl-2-C-methyl-2-O-benzyl-rac-glycerol, which was converted into the phosphocholine compound. Hydrogenolysis afforded 1-O-hexadecyl-2-C-methyl-rac-glycero-3-phospholine (2-methyl-lyso PAF). The 2-methyl-lyso PAF analog served as a substrate for the acetyl-CoA-dependent acetyltransferase that acetylates 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine. However, 2-methyl-lyso PAF did not have a significant effect on the activities of a CoA-independent transacylase or of the acetylhydrolase that inactivates PAF, and thus does not appear to be a substrate or an inhibitor, respectively, for these enzymes. In addition, this analog exhibited only one-half of the antitumor activity of rac-1-O-alkyl-2-methoxy-rac-glycero-3-phosphocholine in human leukemic (HL-60) cells, and elicited no hypotensive response in rats and no platelet-activating activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fine needle aspiration of the breast. Importance of the aspirator

All 503 fine needle aspirations (FNAs) of the breast performed over a three-year period at the Medical Center Hospital of Vermont were analyzed. There were 93 aspirates diagnosed as "positive," all of which were from patients eventually shown to have cancer. However, there were 38 patients with primary carcinoma in which the FNA was not diagnosed as positive, for a diagnostic failure rate of 31.4%. In order to determine the possible effect of technique as practiced by an experienced aspirator in diminishing such diagnostic failures, we compared 190 aspirates obtained by a single individual with 193 aspirates obtained by 15 individuals in the same community. For the single experienced aspirator, the technical failure rate was 9.8% whereas in the group with many aspirators it was 45.9%. This finding confirms that, although fine needle aspiration of the breast is considered easy to perform, skill on the part of the aspirator is important for satisfactory results.     

Purification and spectroscopic properties of 124-kDa oat phytochrome

A simplified procedure for the isolation and purification of 124-kDa phytochrome from etiolated Avena seedlings has been developed using the method of ammonium sulfate back-extraction. After hydroxyapatite chromatography of seedling tissue extracts, the pooled phytochrome was subjected to ammonium sulfate back-extraction instead of the usual application to an Affi-Gel Blue column. The resulting phytochrome had specific absorbance ratios (SAR = A666/A280) ranging from 0.85 to 0.95. Subsequent Bio-Gel filtration chromatography yielded highly pure 124-kDa phytochrome with SAR values ranging from 0.99 to 1.13. The absorption maxima of 124-kDa phytochrome were at 280, 379, and 666 nm for the red absorbing form of phytochrome (Pr) and at 280, 400 and 730 nm for the far-red absorbing form (Pfr). The A730/A673 ratio in Pfr was found to be 1.5 to 1.6. The mole fraction of Pfr under red light photoequilibrium was 0.88. No dark reversion was detected within 5 h at 3 degrees C. A photoreversible far-uv-circular dichroism was observable with all phytochrome preparations examined. Fluorescence and phosphorescence lifetimes were measured to further characterize the differences between the phytochromes prepared under different conditions. The Trp fluorescence and phosphorescence lifetimes of Pr and Pfr with the chromophore "X", probably polyphenolic in nature, were significantly shorter than those of phytochrome without the contaminant X. The short lifetime of the fluorescence of the Pr chromophore is attributable to X in the former.     

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Relationships between Biovolume and Biomass of Naturally Derived Marine Bacterioplankton

Microscopic estimation of bacterial biomass requires determination of both biovolume and biovolume-to-biomass conversion. Both steps have uncertainty when applied to the very small bacteria typically found in natural seawater. In the present study, natural bacterioplankton assemblages were freshly collected, passed through 0.6-μm-pore-size Nuclepore filters to remove larger particulate materials, and diluted for growth in 0.22-μm-pore-size Millipore filter-sterilized unenriched seawater. This provided cells comparable in size and morphology to those in natural seawater, but the cultures were free of the interfering particulate detritus naturally present. Cells were collected on glass-fiber GF/F filters, and biovolumes were corrected for cells passing these filters; C and N were measured with a CHN analyzer. Our criteria for size measurement by epifluorescence photomicrography were confirmed with fluorescent microspheres of known diameters. Surprisingly, in six cultures with average per-cell biovolumes ranging from 0.036 to 0.073 μm³, the average per-cell carbon biomass was relatively constant at 20 ± 0.08 fg of C (mean ± standard error of the mean). The biovolume-to-biomass conversion factor averaged 0.38 ± 0.05 g of C cm⁻³, which is about three times higher than the value previously estimated from Escherichia coli, and decreased with increasing cell volume. The C:N ratio was 3.7 ± 0.2. We conclude that natural marine bacterial biomass and production may be higher than was previously thought and that variations in bacterial size may not reflect variations in biomass per cell.