Linear aggregation and the turbidimetric lag phase: type I collagen fibrillogenesis in vitro.

Using classical light scattering theory it is shown that the turbidimetric lag phase is a property of macromolecular systems that form linear aggregates during the initial period of self-assembly. This analysis and previous observations on Type I collagen suggests that initiation occurs by the conversion of single molecules into linear dimers in which neighboring molecules are staggered by 4·0 D (D = 67 nm). Linear.growth of dimers occurs by 4 D addition of either single molecules or linear aggregates until the aggregate is about 60 to 70 molecules long. Linear growth appears to involve charged binding sites in the amino and carboxy termini. The same sites have been shown to be involved in crosslink formation and in the attachment of disaccharides.Lateral growth occurs via the rapid formation of several discretely sized intermediates which lead to the formation of narrow fibrils ～ 25 nm wide. Narrow fibrils individually wrap around each other increasing the diameter to 100 nm or greater.     

Changes in conformation and nucleotide binding of Ca, Mn, or MgG-actin upon removal of the bound divalent cation. Studies of ultraviolet difference spectra and optical rotation

The ultraviolet difference spectra of EDTA-induced denaturation of dithiothreitoltreated actin prepared with either Ca²⁺, Mn²⁺, or Mg²⁺ as the strongly bound cation showed no appreciable difference, nor could any difference be found in the change of optical rotation. However, at different wavelengths the changes in the spectra have different rates and these rates do differ significantly depending on the bivalent cation bound to G-actin. The nucleotide and the cation appear to be removed simultaneously and at the fastest rate; about 50–80% is released within 1 min. The spectral changes have two phases: a fast change whose detailed kinetics have not been investigated in this paper, followed by a slower rate with first-order kinetics. The changes of optical rotation follow a single-phase first-order kinetics. The rates depend on the divalent cation, the sequence being Mn²⁺ > Ca²⁺ > Mg²⁺. ATP release is partially reversible upon Ca²⁺ addition; the reversibility is diminished as the time of incubation with EDTA is increased. On rebinding of ATP and Ca²⁺, the spectral and optical rotatory changes are not reversed, but no further changes occur. Such an EDTA-treated actin is polymerizable after addition of Ca²⁺, and the G-actin obtained after polymerization and depolymerization shows the same spectral change on a second addition of EDTA as the original actin. On the basis of these observations a scheme is suggested for the denaturation of G-actin.     

The solubility of fibrinogen in dilute salt solutions

Highly purified human fibrinogen was dialyzed versus eleven different sodium salts at ionic strengths of 0.005–0.3 and pH values of 4.5–8.0. After equilibration and centrifugation of the protein solutions, fibrinogen solubilities were determined spectrophotometrically and were analyzed as functions of pH, ionic strength, and specific anion. Bell-shaped curves are obtained when fibrinogen solubility is plotted as a function of pH. The solubility exhibits a minimum at a given pH and rises at acid and alkaline values. As the ionic strength is increased, the solubility curves shift toward more acid pH values. At constant pH values between 6 and 7, fibrinogen solubility increases with an increase in ionic strength. At constant pH values below pH 6, a decrease in solubility occurs as the ionic strength is increased. The isoionic pH of a saturated aqueous fibrinogen solution has been determined to be 6.25, meaning that fibrinogen in pure water behaves as a weak acid with a mean net charge of ?0.9. At pH values acid to 6.25, the anions solubilize fibrinogen in the following order of increasing efficacy: thiocyanate, perchlorate, sulfate, citrate, bromide, nitrate, phosphate, chloride, acetate, fluoride, and formate. This order is reversed at pH values alkaline to 6.25. Anion binding parameters calculated from the solubility data indicate that those anions which most effectively solubilize fibrinogen at alkaline pH and precipitate it at acid pH have the highest apparent binding affinities for the protein. Anions with less pronounced solubility effects have lower binding affinities.     

Reduction and Subsequent Binding of Ruthenium Ions Catalyzed by Subcellular Components

The reduction of Cl(NH₃)₅Ru(III) and subsequent binding of heterocyclic ligands by the resultant (H₂O)(NH₃)₅Ru(II) ion is shown to be catalyzed by components of rat-liver cells. The presence of air significantly decreases the rate of heterocyclic ligand binding. In the case of microsome and soluble component catalysis, this is probably due to oxidation of the Ru(II) ion prior to complexation. Various inhibitors of electron-transfer proteins were employed in an effort to determine the preferred reducing species. These results lend support to the hypothesis that the antitumor activity of acido ruthenium(III) ammine complexes involves activation by reduction in vivo prior to metal coordination to nucleic acids. Anticancer drugs functioning by this mechanism may be preferentially toxic to or may localize in hypoxic areas of tumors.     

Long-term skin preservation by combined use of tissue culture and freezing techniques.

Simple but effective methods for shipping newly excised rabbit skin to a distant central laboratory for in vitro culture on a pigskin base, followed by freezing in a cryoprotective agent (DMSO) for frozen storage and subsequent reshipment to the originating laboratory while still frozen are described. At a suitable time the frozen tissue was rapidly thawed and transplanted to the autologous recipient rabbit. Of 12 cultures, seven indicated good to excellent cell growth and maturation on the host. The successful method combined the use of in vitro tissue culture and freezing to permit a central laboratory to grow the skin and ship it to the originating center for autografting at a convenient time.     

Zymosan activated plasma infusion in sheep: Effects of ethanol administration

Zymosan activated plasma infusion induces pulmonary sequestration of neutrophils and the release of TXA₂ into the pulmonary vascular bed causing profound and transient pulmonary hypertension.Since ethanol (ETOH) inhibits several inflammatory functions of neutrophils, including adherence and aggregation, we examined the ability of anesthetic doses of ETOH to alter the hemodynamic and cellular response to the infusion of zymosan activated plasma (ZAP) in vivo. Twenty five ml of autologous ZAP was intravenously infused into five control and seven (ETOH-treated sheep during mechanical ventillation. In control sheep the mean pulmonary artery pressure (PAP) transiently increased from 14.7±1.4 mm Hg (mean±SEM) to a pead of 38+8 mm Hg by three minutes after beginning the infusion of ZAP. Blood leukocyte concentration transiently decreased 19% below the baseline value due to pulmonary sequestration of polymorphonuclear leukocytes (PMN). Plasma TXB₂ levels measured by radioimmunoassay (RIA) increased from 0.2 to 5.4 ng/ml six minutes after the initiation of ZAP infusion.In five sheep, intravenous infusion of 200 ml of 96% ETOH yielded very high plasma concentrations (882±101 mg%) and completely inhibited both the rise of PAP and the increase of plasma TXB₂ levels after ZAP infusion. However, blood leukocytes transiently decreased 58% below the baseline value. Lower plasma levels of ETOH (200 and 400 mg%) did not prevent either the increase of PAP or the elevation of plasma TXB₂ after ZAP infusion.     

Cell polarity in precartilage mouse limb mesenchyme cells

The patterns of orientation of individual mesenchyme cells have been evaluated in the hindlimb of the mouse embryo during the period of transition from early aggregation (Day 12) to cartilage formation (Day 13). Orientation was measured by determining the angular relationship between the Golgi-nucleus axis of each cell relative to either the longitudinal limb axis or the center of the cartilaginous aggregate. Patterns were assessed qualitatively and quantitatively in horizontal, vertical, and transverse sections of the proximal, middle, and distal precartilage mesenchyme. These analyses showed that the mesenchyme cells are oriented predominantly toward the longitudinal axes of both the early (Day 12) and late (Day 13) aggregates.     

Early mouse embryos: Growth and differentiation in vitro

Mouse blastocysts grown in a simple, easily controlled culture for 5 days developed into egg cylinder-like structures. The removal of zona pellucida and treatment with proteolytic enzymes promoted the development of blastocysts in vitro. Egg cylinders similarly cultured grew and showed signs of early organogenesis, i.e., beating heart, blood islands, somites and cephalization.     

Progesterone stimulates running wheel activity in adrenalectomized-ovariectomized rats.

Daily treatment with progesterone (5 mg) increased running wheel activity, food intake, and body weight of adrenalectomized-ovariectomized rats. These effects of progesterone are quite similar to those of various corticosteroid treatments in adrenalectomized rats reported previously. In addition, the activity-stimulating action of progesterone is just the opposite of its effect in intact and estradiol-primed ovariectomized rats. These observations are consistent with the hypothesis that the principal role of progesterone in the regulation of body weight is to antagonize the actions of estradiol and that the actions of excessive doses of progesterone in adrenalectomized-ovariectomized rats are simply a by-product of its corticosteroidlike, health-promoting properties.     

Effect of acute stress on the absorption and distribution of zinc and on Zn-metallothionein production in the liver of the chick.

A study has been made of the effects of chloroform inhalation, Escherichia coli endotoxin injection and hydrocortisone injection on the absorption of a single intragastric dose of 65Zn by the chick. Injection of hydrocortisone increased the absorption of the 65Zn by 30-55% in both Zn-deficient and Zn-supplemented chicks. The influence of chloroform and endotoxin was less consistent; the former treatment only increased 65Zn absorption and endotoxin was less consistent; the former treatment only increased 65Zn absorption in Zn-supplemented chicks fed ad libitum whereas endotoxin only increased that in Zn-supplemented chicks on a restricted food intake. Injection of endotoxin increased the hepatic uptake of the absorbed 65Zn in both Zn-deficient and Zn-supplemented chicks, whereas hydrocortisone had a similar effect in the Zn-supplemented birds only. Chloroform inhalation increased hepatic 65Zn uptake in Zn-deficient chicks only. The increase in hepatic Zn concentrations in the stressed chicks was mainly associated with a protein in the cytosol identified as metallothionein. Both endotoxin and hydrocortisone decreased total plasma Zn concentrations in Zn-supplemented and Zn-deficient chicks; chloroform decreased plasma 65Zn content only.     

Membrane associations between subsurface cisternae of endoplasmic reticulum and the plasma membrane of rat Sertoli cells

Close membrane associations between the endoplasmic reticulum and the plasma membrane (ER-PM) occur in specialized regions of the rat Sertoli cell cytoplasm. They are characterized, in freeze fracture replicas, as mesa-like modifications of E membrane fracture faces or as corresponding discoid depressions on P membrane fracture faces. When these structures lie along transitional regions in the membrane fracture plane, they are seen to be complementary, and the space between them to be greatly reduced. These specialized close membrane associations may represent adhesive sites between the endoplasmic reticulum and the plasma membrane. However, their resemblance to vascular endothelial fenestrae which are known to be sites of increased membrane permeability may suggest other functional roles.     

Effects of tricyclic antidepressants on muscarinic cholinergic receptor binding in mouse brain

Tricyclic antidepressants (TADs) were administered (10 mg/kg/day, i.p.) to mice for 2 or 4 weeks. Tolerance to the antimuscarinic effects of these agents was demonstrated by comparing their ability to supress oxotremorine-induced tremors in treated and in control animals. ED50's increased nearly three-fold after four weeks of treatment. CNS muscarinic acetylcholine receptor binding was also examined after 2 to 7 weeks of treatment by measurement of 3H-quinuclidinyl benzilate (QNB) binding. No change was found in either density or affinity of these receptors. The development of tolerance to the antimuscarinic effects of TADs is not due to alteration of either the number or the conformation of central muscarinic receptors. Evidence is presented that this phenomenon may instead be the result of an unidentified mechanism by which the post-synaptic effect of a single receptor-agonist interaction is magnified.     

Freeze-fracture analysis of gap junction disruption in rat ovarian granulosa cells

The effects of chemical dissociation on rat ovarian granulosa cell gap junctions has been studied using freeze-fracture electron microscopy. Sequential exposure of granulosa cells within follicles to solutions containing 6·8 mM EGTA [ethylene-bis-(β-aminoethyl ether)-N,N′-tetra acetic acid] and 0·5 M sucrose results in extensive cellular dissociation of the follicular epithelium. Freeze-fracture replicas made from fixed, control or EGTA-treated ovarian follicles exhibit extensive gap junctions between granulosa cells that are characterized by a range of packing order of constituent P-face particles or E-face pits. In contrast, exposure to 0·5 M sucrose containing 1·8 mM EGTA for as little as 1 min results in a consistently close packing of particles or pits which is accompanied by splitting of gap junctions between granulosa cells. The process of junction splitting was studied in detail in replicas prepared from follicles treated sequentially for various periods of time with EGTA and sucrose solutions. Initially, large gap junctions lose their regular shape and fragment into numerous tightly packed aggregates of P-face particles or E-face pits which are separated by unspecialized areas of plasma membrane. Subsequent to junction fragmentation, individual junction plaques separate at sites of cell contact and generate hemijunctions that border the intercellular space, Hemijunctions undergo particle dispersion of the P fracture face which results in an increased density of large intramembrane particles; no corresponding change in E-face pits is discernible at this stage. Morphometric analysis of replicas of tissue undergoing junction splitting indicates that junctional surface area decreases to 10–20% of control levels during this same treatment and so further supports the qualitative observations on junction fragmentation. Viabilities of granulosa cells obtained by these techniques also agree with the sequence observed in the morphometric analysis of the replicas. Finally, within 15 min after placing ovaries in isotonic, Ca²⁺-containing salt solutions, gap junction reformation occurs by aggregation of particles at sites of intercellular contact. These sites are distinguished by the appearance of short surface protrusions or indentations on their respective P and E fracture faces. The data suggest a mechanism for EGTA-sucrose mediated cellular dissociation in the follicular epithelium in which gap junctional particles are free to move in the plane of the plasma membrane and may be re-utilized to form gap junctions in the presence of extracellular calcium.     

Trace element uptake in liver cells. 2. Effect of different proteins in the medium on the uptake of copper and zinc by hepatoma cells

Cultured rat hepatoma cells (HTC-cells) were used to study the uptake of copper and zinc from a minimal salt-glucose medium, supplemented with albumin from different species or with ovalbumin. Competitive equilibrium dialysis showed that at low molar ratios of metal/protein (less than 1) the affinity for copper of human and bovine albumin was about equal, but that of dog albumin or ovalbumin was much lower. Only a small difference in affinity for zinc could be detected between human albumin and ovalbumin. Supplementing the medium with the different proteins the rate of copper uptake in the cell at a given molar Cu/protein ratio increased as follows: human albumin congruent to bovine albumin less than dog albumin less than ovalbumin. When the molar Cu/protein ratio was increased, a discontinuity was seen with all three albumin species at a ratio of about 1. In contrast, the zinc uptake mimics that of Cu/ovalbumin, and no discontinuity was observed using different molar Zn/protein ratios. These results indicate that the rate of copper and zinc uptake depends strongly on its affinity for the protein: a low affinity leads to a high uptake. The results suggest further that at physiologic concentrations zinc is taken up by a mechanism different from that for copper.