Collagen synthesis by regenerating rabbit ear tissue

The principal collagen types synthesized during two distinct phases of regeneration in rabbit ears have been investigated, in order to relate altered phenotypic expression in connective tissue cells to regeneration of cartilage. To do this, radioactively labeled collagens synthesized in short-term culture by selected regenerating ear tissues were analyzed by ion-exchange chromatography and SDS-gel electrophoresis of the intact collagens and of the cyanogen bromide peptides derived from them. Prior to the appearance of cartilage, rabbit ear holes are filled by an outgrowth of mesenchyme-like cells derived locally from adjacent tissues. These cells produce a mixture of collagens including type I, [α1(I)]₂α2, and the type I trimer, [α1(I)]₃, but not type II collagen. Trimer production represents about one-fourth of the collagen synthesized in either a 4-, 10-, or a 24-hr incubation. Trimer is not made by tissues from healing skin wounds nor is it present in normal, uninjured ear tissues. Type II collagen synthesis was detected in tissues taken from late regenerates containing histologically recognizable cartilage, and direct analysis of regenerated cartilage confirmed the presence of type II collagen in the matrix. Thus, regenerated cartilage in the rabbit ear system contains the normal cartilage collagen, type II, while the proliferating cell mass from which the cartilage develops synthesizes the unusual collagen, [α1(I)]₃.     

Studies on glycosaminoglycans of regenerating rabbit ear cartilage

Cartilage regeneration in the adult rabbit ear was examined with respect to glycosaminoglycan (GAG) synthesis at various stages of the regeneration process. Increased hyaluronic acid and chondroitin sulfate synthesis was first seen 31 days after wounding, when a metachromatic cartilage matrix could be distinguished from blastemal cells. Analysis of cartilage and the overlying skin separately showed that 90% of the labeled chondroitin sulfate was found in the cartilage being regenerated. DEAE-cellulose chromatography of GAG preparations from 35-day regenerating cartilages showed hyaluronic acid and chondroitin sulfate peaks eluting in the same position as those isolated from normal cartilages. The identity of the hyaluronic acid and chondroitin sulfate peaks was confirmed by their susceptibility to Streptomyces hyaluronidase and chondroitinase ABC, respectively. Although the degree of sulfation in normal and regenerated cartilages was similar, the ratio of chondroitin 6-sulfate to chondroitin 4-sulfate was increased in regenerated cartilages. GAG preparations from unlabeled cartilages were digested with chondroitinase ABC and the disaccharide digestive products were identified and quantitiated. Normal cartilage had a $\text{Δ}\text{Di-6S}\text{Δ}\text{Di-4S}$ ratio of 0.27; the same ratio for the regenerated cartilage was 1.58.     

Electron paramagnetic and electron nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase

We have collected electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectra from the hydrogen peroxide compound of yeast cytochrome c peroxidase, termed ES, employing EPR microwave frequencies of 9.6 and 11.6 GHz. We have measured and analyzed the temperature dependence of the spin-lattice relaxation rate (1/T1) of the paramagnetic center of ES over the temperature range 1.9 to 4 K. In addition, an upper bound to exchange coupling between the ferryl heme and EPR-visible centers of ES has been calculated and expressions for the dipolar interaction between a ferryl heme and a free radical have been derived. These results all confirm that the EPR signal of ES is not associated with an aromatic amino acid radical, and in particular not with a tryptophanyl radical. This conclusion has led us to consider an explanation of the EPR signal in terms of a nucleophilically stabilized methionyl radical.     

Measurement of membrane potentials (ψ) of erythrocytes and white adipocytes by the accumulation of triphenylmethylphosphonium cation

Summary The accumulation of the lipophilic cation, triphenylmethylphosphonium, has been employed to determine the resting membrane potential in human erythrocytes, turkey erythrocytes, and rat white adipocytes. The triphenylmethylphosphonium cation equilibrates rapidly in human erythrocytes in the presence of low concentrations of the hydrophobic anion, tetraphenylborate. Tetraphenylborate does not accelerate the uptake of triphenylmethylphosphonium ion by adipocytes. The cell associatedvs. extracellular distribution of the triphenylmethylphosphonium ion is proportional to changes in membrane potential. The distribution of this ion reflects the membrane potential determining concentration of the ion with dominant permeability in a Nernst fashion. The resting membrane potentials for the human erythrocyte, turkey erythrocyte, and rat white adipocyte were found to be –8.4±1.3, –16.8±1.1, and –58.3±5.0 mV, respectively, values which compare favorably with values obtained by other methods. In addition, changes in membrane potential can be assessed by following triphenylmethylphosphonium uptake without determining the intracellular water space. The method has been successfully applied to a study of hormonally induced changes in membrane potential of rat white adipocytes.     

Ectoprotein kinase activity of the isolated rat adipocyte.

Intact adipocytes exhibit ectoprotein kinase activity as reflected by their ability to catalyze the transfer of the terminal phosphate of (γ-³²P) ATP to histone added to a cell suspension. This activity is substrate, time and cell number dependent. Lineweaver-Burk plots gave Km and Vmax values for ATP of 5 × 10^?5 M and 7.14 pmoles/min/1.5 × 10⁵ cells. Cyclic AMP but not cyclic GMP in μM concentrations stimulates ectoprotein kinase activity. The controlled tryptic digestion of intact cells results in reduction of ectoprotein kinase activity. This activity is not due to leakage of intracellular protein kinases during the preparative procedure nor to penetration of histone into the cells. Additional phosphoproteins not accessible to endogenous protein kinase activity are also localized on the external surface of the intact fat cell.     

The amino acid sequence of chick skin collagen α1-CB7: The presence of a previously unrecognized triplet

Because alignment of the amino acid sequences of chick skin collagen α2-CB3 (1) with the relevant portion of chick skin collagen α1-CB7 (2) suggested that a Gly-X-Y triplet may have been missed in the latter, the peptide TM-2, produced by tryptic digestion of maleylated α1-CB7, was reinvestigated. Cleavage by trypsin at the unblocked lysine at position 18, and isolation of the resulting COOH-terminal peptide, showed this to be a 15-residue peptide containing a previously unrecognized Gly-Pro-Hyp triplet. Sequencing of the peptide showed this to occupy positions 4 through 6, or 56 through 58 of α1-CB7. The latter thus has 271 instead of 268 residues, and the α1[I] chain 1055 instead of 1052.     

Bronchial reactivity to histamine before and after sodium cromoglycate in bronchial asthma.

Out of 19 patients with extrinsic bronchial asthma challenged with 123 mug histamine acid phosphate by intravenous infusion only 13 responded with a fall in FEV1 of over 10% (mean 16%). Seventeen of these patients were given histamine 2 mg/ml by aerosol, and all responded with a mean decrease in FEV1 of 37.8%. When challenged with allergen extract by aerosol the mean decrease in FEV1 was 37.5%. After 40 mg sodium cromoglycate 15 of the 17 patients showed significant protection against allergen challenge with a mean decrease in FEV1 of only 23.6%. Inhalation of 40 mg sodium cromoglycate, however, failed to protect against histamine given by either the intravenous or aerosol route. Histamine given intravenously to asthmatic patients produces less of a bronchial response than when given by aerosol, even though the intravenous route produces many more systemic symptoms, such as flushing and throbbing headache. The protection of sodium cromoglycate against an allergen inhalation challenge is not due to histamine antagonsim.     

Interaction of a chick skin collagen fragment (alpha1-CB5) with human platelets. Biochemical studies during the aggregation and release reaction.

The denatured alpha1(I) chain and the cyanogen bromide peptide, alpha1(I)-CB5, of chick skin collagen cause the relaese of serotonin and leakage of lactic dehydrogenase from human platelets in a manner similar to the release reaction mediated by adenosine diphosphate and native collagen. These peptides also cause a decrease in the level of adenosine 3':5'-monophosphate (cAMP) in platelets. Adenylate cyclase activity of platelets is partially inhibited by these peptides as well as by native collagen, ADP, and epinephrine, but cAMP phosphodiesterase activity is unaltered by these substances. In contrast, the level of platelet guanosine 3':5'-monophosphate (cGMP) is increased by the collagen peptides as well as the other aggregating agents. The increase is associated with increased guanylate cyclase, but normal cGMP phosphodiesterase activities of platelets. Optical rotatory and viscometric measurements of the alpha1 chains and alpha1-CB5 of chick skin in 0.01 M phosphate/0.15 M sodium chloride, pH 7.4, at various temperatures as a function of time indicate that no detectable renaturation occurs at 37 degrees for at least 30 min of observation. Molecular sieve chromatography of alpha1-CB5 in the phosphate buffer at 37 degrees shows that its elution position is identical to that performed under denaturing conditions (at 45 degrees) with no evidence of higher molecular weight aggregates, and the alpha1-CB5 glycopeptide fraction eluting from the column at the position of its monomer retains the platelet aggregating activity. Additionally, electron microscopic examination of the platelet-rich plasma that had been reacted with these peptides fail to show any ordered collagen structures. These data indicate that the denatured alpha1 chain and alpha1-CB5 glycopeptide of chick skin collagen mediate platelet aggregation through the "physiologic" release reaction in a manner similar to that induced by other aggregating agents such as ADP, epinephrine, or native collagen, and support the conclusion that the aggregating activity of the alpha1 chain and alpha1-CB5 is not likely to be due to the formation of polymerized products.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.