Physicochemical properties of cartilage proteoglycans extracted by lanthanum chloride

Bovine nasal cartilage was extracted with 0.5 M LaCl₃ and the extract then diluted with nine volumes of water. The resulting precipitate (P_LaCl3) contained the proteoglycan subunits, together with minor protein components, but was essentially free from hyaluronic acid. The properties of P_LaCl3 were investigated by chemical analysis, electrophoresis, viscometry and analytical ultracentrifugation, and the results compared with those for proteoglycan obtained by caesium chloride density gradient centrifugation of 2 M CaCl₂ cartilage extracts. Proteoglycan subunits (A1D1) prepared from P_LaCl3 showed identical properties to those obtained from other high ionic strength cartilage extracts.     

Antibody-induced conformational restriction as basis for new separation-free enzyme immunoassay

Under acid denaturing conditions, hologlucose oxidase labeled with 2,4-dinitrophenyl (DNP) was dissociated into flavin adenine dinucleotide (FAD) and DNP-labeled apoglucose oxidase (DNP-AG). Both lacked catalytic activity. The activity was restored by combining FAD and DNP-AG at about pH 7. If, on the other hand, anti-DNP serum was preincubated with the DNP-AG prior to the addition of FAD, activity was not restored. Furthermore, added DNP-aminocaproic acid counteracted the effects of the antibody in inhibiting the recombining of DNP-AG and FAD to form active enzyme. The anti-DNP serum probably prevented the DNP-AG from combining with FAD to form an active holoenzyme by restricting the mobility of the polypeptide chain of DNP-AG from folding into a catalytically active conformation. Based on such an antibody-induced conformational restriction of the DNP-AG, we developed a separation-free (homogeneous) enzyme immunoassay called AICREIA.     

An optimum method for the introduction or removal of permeable cryoprotectants: Isolated cells

On the basis of a nonequilibrium model for the transport of water and permeable solute across cell membranes, an optimum method has been devised for the introduction and the removal of a permeable cryoprotectant from single, isolated cells so that potentially lethal drastic alterations in cellular volume are minimized. The method involves the simultaneous variation of both the permeable (an initial step change, followed by a linear variation with time which overshoots the terminal value, and a final step change to the terminal value) and impermeable (an initial step change in the opposite direction of the permeable solute concentration change, followed by a period where the concentration remains constant, and a final step change back to the initial value) extracellular solute concentrations in a prescribed manner such that both the cellular water content and the intracellular electrolyte concentration remain constant as the intracellular permeable solute (CPA) concentration is either raised or lowered. The results of our theoretical analysis indicate that the osmotic stresses and strains usually imposed upon cells during the introduction and the removal of permeable cryoprotectants can be minimized and that the resulting protocols are clinically the most efficient.     

An NADH-coupled assay for femtogram or nanogram quantities of chymotrypsin

Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.     

Beta-Galactosidase immunoassay for the measurement of cyclic AMP

The previously described method for phenotyping of alpha₁-antitrypsin (alpha₁-protease inhibitor, Pi) that utilizes separator isoelectric focusing on thin-layer agarose gel (A. R. Qureshi and H. H. Punnett, in Electrophoresis '81, 3rd International Conference on Electrophoresis, pp. 83–87 (1981)) has been improved to give a better resolution of Pi pattern. A shallow pH gradient in the region of the isoelectric point of Pi pattern was obtained by the use of N-(2-acetamido)-2 aminoethanesulfonic acid (1%) and serine (0.8%). The present technique can resolve the Pi alleles. The patterns of Pi phenotypes were found to be similar to those observed on acrylamide gels. The method is fast, reliable, and reproducible.     

Quantification of two cytochrome P-450 isoenzymes by an enzyme-linked immunosorbent assay (ELISA)

An enzyme linked immunosorbent assay (ELISA) using monoclonal and polyclonal antibodies has been developed to quantify individual cytochrome P-450 isoenzymes in microsomal preparations, namely UT-A and PB-B. This very sensitive method can be used for the rapid processing of large quantities of determinations and requires only limited amounts of antibodies.     

An experimental study of the somitomeric organization of the avian segmental plate

The segmental plate mesoderm of chicken and Japanese quail embryos HH stages 9 to 16 was studied with scanning electron microscopy (SEM) imaging. The segmental plates were found to exhibit a metameric pattern consisting of tandemly stacked somitomeres. It was found that the numbers of somitomeres in segmental plates removed from the same embryo were nearly identical. Furthermore, the number of somitomeres in a segmental plate was found to be quite consistent (10.0 ± 1.5) and independent of the length of the segmental plate. These results are very similar to those obtained in previous experimental studies in which “prospective somites” were detected in avian segmental plates. Further experiments showed that for each somite that is formed by a cultured segmental plate-containing explant, the somitomere complement of the segmental plate is reduced by one. It was concluded that the segmental plate mesoderm is already organized into a metameric pattern consisting of somitomeres and that the somitomeres undergo further morphogenesis to become somites. The specification of the somite pattern in birds may occur at the level of Hensen's node and the cephalic primitive streak.     

An ultrastructural immunocytochemical localization of hyalin in the sea urchin egg

The fertilized sea urchin egg is invested by the hyaline layer, a thick extracellular coat which is necessary for normal development. On the basis of ultrastructural studies and the fact that hyalin is released during the time of the cortical reaction, it has been generally accepted that hyalin is derived from the cortical granules. However, this has never been proven definitely, and recently, it has been reported that hyalin is a membrane and/or cell surface protein. To determine where hyalin is stored, we carried out an ultrastructural immunocytochemical localization of hyalin in the unfertilized egg. Hyalin purified from isolated hyaline layers was used to immunize rabbits. Antisera so obtained were shown to be hyalin specific following absorption with a combination of sea urchin proteins. Immunocytochemical localizations were carried out on sections of Epon-embedded material using protein A-coated gold particles as an antibody marker. Our results demonstrate that, prior to fertilization, hyalin is stored in the homogeneous component of the cortical granule in Strongylocentrotus droebachiensis and Strongylocentrotus purpuratus. Labeling of small cortical vesicles in both unfertilized and fertilized eggs, suggests that these vesicles may contain a secondary reservoir of hyalin.     

An examination of the rate assay to determine the active site normality of papain

Data are presented which demonstrate that the α-N-benzoyl-l-argine ethyl ester rate assay procedure, based on a burst titration with N-benzyloxy-carbonyl-l-tyrosine p-nitrophenyl ester as previously desribed (1), is an accurate and reliable method for determining the normality of papain in solution.     

Deaminating enzyme labels for potentiometric enzyme immunoassay

Adenosine deaminase, asparaginase, and urease are examined as possible enzyme labels for immunoassays using potentiometric detection with the ammonia gas-sensing membrane electrode. Considerations of binding ability, retained activity, and stability reveal asparaginase to be the most effective enzyme label for immunoassay purposes. The utility of the potentiometric approach with this enzyme label is demonstrated via model hapten assays for dinitrophenyl groups and for cortisol.     

An analysis of the fate of the chick wing bud apical ectodermal ridge in culture

Stages 20 and 25 chick apical ectodermal ridge have been cultured in nutrient medium containing fetal bovine serum and the tissues have been examined for dying cells at 0, 6, 12, 18, and 24 hr. By 12 hr, an average of 43% of the cells were dying. By 24 hr, stage 20 ridge had lost its integrity and stage 25 ridge contained an average of 50% dying cells. These results are in agreement with the observations of R. L. Searls and E. Zwilling (1964, Dev. Biol. 9, 38-55) on isolated stage 20 ridge. In subsequent experiments, ridge ectoderm was cultured in serum-containing medium to which insulin (5 micrograms/ml), transferrin (5 micrograms/ml), and selenium (5 ng/ml) or insulin (5 micrograms/ml) had been added. Under these conditions the ectoderms remained viable even after 24 hr in vitro.     

Cycloheximide: An adrenergic agent

Cycloheximide, a widely used inhibitor of protein synthesis, stimulates glycogenolysis, gluconeogenesis and ureogenesis in isolated rat hepatocytes. The effects of cycloheximide were compared to those of norepinephrine. Both agents, cycloheximide and norepinephrine, produced slight increases in the levels of cyclic AMP (30% increases) which were blocked by propranolol. Interestingly, it was found that the metabolic actions of norepinephrine and cycloheximide (stimulation of glycogenolysis, gluconeogenesis and ureogenesis) were only slightly diminished by the β adrenergic antagonist propranolol but abolished by the selective α₁ adrenergic antagonist prazosin. The ability of cycloheximide to inhibit protein synthesis was not affected by either prazosin or propranolol. It is concluded that the stimulation of glycogenolysis, gluconeogenesis and ureogenesis by cycloheximide in rat hepatocytes, is an effect of the antibiotic independent of its ability to inhibit protein synthesis and that is mediated through activation of α₁ adrenoceptors. The adrenergic activity of cycloheximide should be considered when this drug is used as an inhibitor of protein synthesis.     

An in vitro study of the stability of the chicken intestinal cytosol 1,25-dihydroxyvitamin D3-specific receptor

The stability of the chick intestinal cytosol receptor for 1,25-dihydroxyvitamin D₃ has been examined using radiological binding studies and sucrose density gradient ultracentrifugation. Specific binding of 1,25-dihydroxyvitamin D₃ to the 3.7 S binding protein decreases in crude cytosol in a time- and temperature-dependent manner. Increased receptor instability is also observed outside a pH range of 6 to 10. Ionic strength does not seem to be a critical factor in preventing loss of specific 1,25-dihydroxyvitamin D₃ binding activity. However, when KCl is present at a concentration of 300 mm during cytosol preparation, quantitatively more specific binding per unit protein was obtained. Consistent with the idea that loss of specific binding might be due to enzymatic degradation or inactivation of receptor, dilution of cytosol was found to slow the rate of loss of specific 1,25-dihydroxyvitamin D₃ binding. The importance of maintaining a reducing environment for the 1,25-dihydroxyvitamin D₃ binding protein is demonstrated by the destruction of binding activity by n-ethylmaleimide and by the increased stability in the presence of 5.0 mm dithiothreitol. Likewise, 5.0 mm monothioglycerol was partially effective in preventing the loss of specific 1,25-dihydroxyvitamin D₃ binding during in vitro incubation. Several protease inhibitors were not able to exert a stabilizing influence on receptor integrity during in vitro incubations. Albeit, both tosylamide-phenylethylchloromethyl ketone and p-hydroxymercuribenzoate actually decreased specific 1,25-dihydroxyvitamin D₃ binding. This inhibition appeared to be reversible if samples were subsequently incubated in the presence of dithiothreitol. These results clearly demonstrate that the aporeceptor is extremely unstable and the integrity of sulfhydryl constituents is of primary importance.     

An efficient two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis method for simultaneous peptide mapping of protein contained in a mixture

A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.     

An electrophysiological study of parthenogenetic activation in mammalian oocytes

Using conventional electrophysiological techniques, we have investigated the electrical responses of mouse and hamster oocytes in metaphase of the second meiotic division to agents which induce parthenogenetic activation. Oocytes from MF1 mice responded to 8.7% ethanol and to 0.3% benzyl alcohol by a depolarization (sometimes preceded by a brief hyperpolarization). The response to ethanol did not "desensitize," and the membrane potential recovered completely when the exposure to ethanol was interrupted. The response was accompanied by a decrease in membrane input resistance (Rin) and had an equilibrium potential of about +5 mV in standard medium and of -10mV in Na-free medium. The oocytes responded to A23187 and to La3+ by an increased Rin, and usually lysed during or after treatment. Multiphasic responses were elicited by ethanol and by Ca-ionophore in metaphase II hamster oocytes; an early hyperpolarization accompanied by a decreased Rin was a common feature of the response to both activating agents. The early hyperpolarization was no longer elicited when the cells were exposed for a second time to ethanol or A23187. K+ and Cl- were the ions mainly involved in the hyperpolarizing potential elicited by A23187, and K+ (but not Cl-) was the ionic species mainly involved in ethanol response. The above responses were peculiar to metaphase II oocytes since mouse and hamster ovarian oocytes (in prophase I) and fertilized eggs either failed to respond to the activating agents, or responded by increasing Rin. The variety of electrical responses to parthenogenetic agents indicates that in mammalian oocytes parthenogenetic activation is not triggered by a "classical" activation potential.     

Noncompetitive enzyme immunoassay for the measurement of bronchial inhibitor in biological fluids

An enzyme-linked immunosorbent assay (ELISA) of bronchial inhibitor using rabbit antibronchial inhibitor antibody-coated polystyrene balls as the solid-phase antibody and peroxidase-labeled antibody as the conjugate is described. A crude antibody fraction is used for coating the solid phase. The assay can be run within 8 h and gives reproducible results in the range of 6 to 60 micrograms/l of bronchial inhibitor (mean within-run coefficient of variation, 7%). It can detect bronchial inhibitor concentrations as low as 2 micrograms/l (10(-10) M) and recovery of varying amounts of bronchial inhibitor added to bronchial liquids is greater than 90%. This enzyme immunoassay appears to be a convenient way to quantify bronchial inhibitor in biological fluids such as serum, sputum, or bronchoalveolar lavage fluid.