A new method for purification of mature elastin.

Stable low-noise dual-beam spectrophotometric detection systems have been built to measure protein in the effluent from chromatographic columns. Measurements are carried out at the magnesium 285.2 nm atomic resonance line isolated either by the technique of selective modulation or by the use of a narrow bandpass interference filter. The noise level is about 0.0005-0.001 absorbance units and the drift rate is ±0.001 absorbance units in 24 hr. While slightly better noise performance could be obtained using the interference filter, selective modulation gives better linearity at higher absorbance values (up to 2.0).     

Stimulation of ion transport by ascorbic acid through inhibition of 3′:5′-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues

Ascorbic acid stimulates active transport of Cl^? by the isolated intact corneas. The effect is not present in corneas previously stimulated by theophylline, an inhibitor of 3′: 5′-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3′: 5′-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using ³H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46 %. This compares with 58 % inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45 % is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50 %) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12 %, rat brain (caudate nucleus) 48 %, rabbit brain 14 %, rabbit liver 16 %. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3′: 5′-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.     

Plasma dihydroxyphenylglycol (DHPG) in the in vivo assessment of human neuronal norepinephrine metabolism

We investigated the utility of deaminated norepinephrine (NE) metabolites in the study of human sympathetic nervous pathophysiology. Plasma levels of the NE metabolite dihydroxyphenylglycol (DHPG) appear to be related to intraneuronal NE stores. Plasma DHPG increases when sympathetic nervous activity or circulating NE increase and decreases when neuronal NE is depleted or neuronal NE reuptake is blocked. Changes in plasma dihydroxymandelic acid (DOMA) related less closely to changes in plasma NE. The coupling of measurements of plasma NE with its deaminated metabolites and DHPG may improve understanding of human NE metabolism and neuronal NE reuptake.     

Dexamethasone-resistant cystic fibrosis fibroblasts show cross-resistance to sex steroids

Diploid skin fibroblasts derived from individuals with the autosomal recessive disease, cystic fibrosis (CF), were shown previously to be significantly more resistant to the cytotoxicity of dexamethasone, a glucocorticoid hormone, than were normal human fibroblasts. Here cystic fibrosis fibroblasts are also shown to be more resistant than normal human fibroblasts to the cytotoxic effects of the sex hormones, 17 beta-estradiol, dihydrotestosterone and progesterone. Since cells are believed to contain different receptors for each of the steroid hormones, it is not probable than the resistance of CF cells to these hormones results from a receptor deficiency. This was shown by the fact that CF cells were found to exhibit the same receptor activity as normal cells for 3-H-dexamethasone. Furthermore, neither normal human nor CF fibroblasts could be demonstrated to contain detectable receptor activity for 3H-17 beta-estradiol. In addition, the studies of fibroblast killing by hormones led to the further interesting observation that normal human diploid fibroblasts, regardless of the sex of the tissue donor, are sensitive to killing by each of the sex hormones. These findings suggest that the cytotoxic effects of the steroid hormones may be observed independently of the specific hormone receptors. The studies reported here thus suggest that the resistance of CF cells to the different steroid hormones is probably the result of a defect in a pathway in cellular steroid hormone metabolism other than that involving receptors.     

Muscarinic cholinergic binding sites in the developing avian heart.

The potent muscarinic cholinergic antagonist 3-quinuclidinyl benzylate (QNB) has been used to detect and quantify muscarinic receptors in the developing chick heart. Specific binding in microsomal pellets prepared from hearts ranging in age from 70 hr in ovo to adulthood was examined and was found to increase from 4 × 10^?13 moles of [³H]QNB bound/mg of protein at the earliest stage tested to 5 × 10^?12 moles of [³H]QNB/mg of protein at birth and then to drop slightly to 2 × 10^?12 moles of [³H]QNB/mg of protein at the latest age tested. The developmental significance of these results is discussed.     

Sensitivity of pigmented mucosa and skin to freezing injury.

Pigmented areas of canine skin and oral mucosa were subjected to freezing in situ at various temperatures for the purpose of investigating possible differences in the sensitivity of epidermal cells to cold injury. Destruction of melanocytes occurred in the range of ?4 to ?7 °C, while squamous cells resisted injury even at ?20 °C. Replacement of the lost pigmented cells occurred from the normal tissue at the periphery of the injured area. The experiments suggest that selective destruction of pigmented lesions in clinical conditions may be achieved by freezing tissue to ?4 to ?7 °C or colder, but not to exceed ?20 °C in order to avoid destruction of squamous cells. The experiments also support wider use of cryosurgery for pigmented lesions of the skin and oral cavity.     

Soluble age-related factors from skeletal muscle which influence muscle development

Successful regeneration of damaged striated muscle in adult mice is dependent on the regeneration of newly differentiated myofibers from proliferating satellite cells and inhibition of scar tissue formation by fibroblasts. As with most tissues, the ability of skeletal muscle to regenerate decreases in older animals. In this study, we have analysed soluble extracts from intact and regenerating skeletal muscle from mice of different ages for their ability to affect avian myogenesis in tissue culture. We were interested in determining whether an age-dependent difference could be detected with this tissue culture bioassay system. Total cell proliferation in the cultures, measured by [3H]thymidine incorporation was increased equally by muscle extracts from both young and older mice but the resulting cell populations differed in proportion of cell types. The ratio of myoblasts to fibroblasts was significantly greater in cultures exposed to extracts from younger mouse muscle as compared with cultures exposed to extracts from older animals. This age-related activity was found to reside in a low molecular weight (MW) (greater than 12 kD) component of the extract. This fraction had dissimilar effects on myoblasts and fibroblasts. Relative to saline controls, myoblast proliferation was increased and fibroblast proliferation decreased. The low MW fraction from younger mouse muscle extracts stimulated myogenic cell proliferation and myotube formation to a greater extent than the similar fraction prepared from older mouse muscle. Conversely, younger mouse muscle fractions had significantly greater inhibitory activity against fibroblast proliferation than did older mouse muscle fractions.     

Interleukin 1 secretion is not required for human macrophage support of T-cell proliferation

Interleukin 1 (IL-1) is a soluble factor secreted by stimulated monocytes (Mo) and animal macrophages (Mx). We have previously demonstrated that human Mo cultured in vitro for 1-6 days transform to Mx, and retain their ability to support concanavalin A (Con A)-driven T-cell proliferation. We have also shown that, paradoxically, these Mx do not secrete IL-1, when stimulated by endotoxin (LPS). In this study we examined two alternative hypotheses: T cells plus mitogen induce Mx IL-1 production, and human Mx deliver a second signal to T cells via a non-IL-1 mechanism. IL-1 was assayed in a mouse CD-1 thymocyte system without concanavalin A. Mo/Mx were cultured with T cells at low (2 X 10(4)/200 microliters) or high (1 X 10(5)/200 microliters) concentrations for 2 or 4 days, in the presence of Con A. Six hours prior to quantitation of proliferation, 50 microliters of supernatant was removed and assayed for IL-1. As expected both Mo and Mx enhanced T-cell proliferation eight- to tenfold. Mo secreted large amounts of IL-1; there was no demonstrable IL-1 activity present in supernatants from cultures containing either T cells and Mx, or Mx alone. Similar results were obtained by preincubating the cells (Mo, Mx, and T cells) with Con A for 12 hr and removing Con A prior to a 36-hr coculture. We examined the possibility that a small amount of IL-1 may be able to support Con A-stimulated T-cell proliferation and yet may not induce thymocyte proliferation. The highest dilutions of Mo supernatant (1:125) which supported T-cell proliferation also caused a fivefold increase in thymocyte proliferation. Supernatants from Mx failed to stimulate thymocyte proliferation or support Con A-driven T-cell proliferation. However, Mo and Mx lysates contain Il-1 activity. We conclude that human Mx support Con A-induced T-cell proliferation in the absence of IL-1 secretion. Mx may support T-cell proliferation by cell-bound IL-1 or by a non-IL-1 mechanism.     

Assessment of in situ host immunity to syngeneic tumors utilizing the multicellular spheroid model

By the 1g sedimentation method using discontinuous gradients of Ficoll solution (concentrations of 6 to 14%), keyhole limpet hemocyanin (KLH)-primed spleen cells of C3H/He or DBA/2 mice were fractionated into 4 to 10 populations after IgG antibody-coated erythrocytes (EA gamma) rosetting and then treatment with anti-Thy-1.2 + complement (C). No significant difference was observed in the distribution of isotype specificities of surface immunoglobulins on B cells in each population thus fractionated, when determined by indirect immunofluorescence staining. The mixture of the 12 and 14% Ficoll fractions contained 95% of B cells bearing Fc receptor for IgG (FcR+ gamma) and 3.58% of antigen-binding cells (ABC) for KLH, while the 8% Ficoll fraction included 15% of FcR+ gamma B cells and 1.53% of ABC. Nevertheless, the FcR- gamma B-cell-enriched populations caused intensive plaque-forming cell (PFC) responses to dinitrophenol (DNP), whereas FcR+ gamma B-cell-enriched populations generated weak responses. Noteworthy is that 4 days preculture of a population containing 95% FcR+ gamma B cells resulted in the appearance of precursor activity which was ascertained by a further 4 days culture of these cells with antigen, DNP-dextran. These findings suggest that FcR gamma bearing B cells intrinsically possess precursor activity for IgM/IgG antibody-forming cells, but lose it transiently by binding immune complexes (IC). Moreover, the titer of a factor suppressing anti-DNP PFC responses (suppressive B-cell factor, SBF) was higher in the 24-hr culture supernatants of the FcR+ gamma B-cell-enriched fraction than of the FcR- gamma B-cell-enriched fraction, suggesting that SBF is produced by FcR+ gamma B cells themselves. Thus, IC seems to play an important role for the negative feedback regulation of antibody production by stimulating FcR gamma bearing B cells.     

Target organ regulation of substance P in sympathetic neurons in culture

The distribution of the mRNA for one of the two mouse protamines, the cysteine-rich, tyrosine-containing protamine (MP1), was examined in the polysomal and nonpolysomal compartments of total testis and purified populations of round and elongating spermatids using Northern blots. In postmitochondrial supernatants prepared from total testis, about 10-15% of MP1-mRNA sediments with the small polysomes. The nonpolysomal molecules of MP1-mRNA are homogeneous in size, about 580 bases, while the polysomal molecules are heterogeneous with a mode of about 450 bases. Digestion with RNase H and thermal chromatography on poly(U) Sepharose reveals that the difference in size of polysomal and nonpolysomal MP1-mRNA is due to a shortening of the poly(A) from about 160 to 30 bases. In round spermatids, essentially all of MP1-mRNA is 580 bases long and is in the nonpolysomal fraction. Elongating spermatids contain roughly equal proportions of the homogeneous, 580 base form in the nonpolysomal compartment, and the heterogeneous 450 base form solely in the polysomal compartment. These results indicate that mRNA for one of the mouse protamines is stored as an untranslated RNP in round spermatids, and that it is partially deadenylated when it is translated in elongating spermatids.