An e.p.r. study of the non-equivalence of the copper sites of caeruloplasmin.

The two Type 1 (blue) copper-binding sites of caeruloplasmin were spectroscopically differentiated by the kinetic analysis of the e.p.r. spectra during the redox cycle. One blue copper, with a hyperfine splitting constant (A parallel) of 6.8 mT, which was rapidly reduced, was not reoxidized by oxygen, whereas it was reoxidized by H2O2. The other blue copper (A parallel = 5.8 mT), which was reduced slowly, was rapidly reoxidized by either oxygen or H2O2. A conformational change of the Type 2 copper was concomitant with the fast reduction of Type 1 copper, whereas its reduction occurred during the slow phase. This sequence of events was reversed in the reoxidation step, that is, the Type 2 copper reappeared rapidly as the species with altered conformation and reverted to the symmetry typical of the native state in the slow phase. The specific reaction of a blue-copper site with the H2O2 can tentatively be related to the established ability of caeruloplasmin to prevent 'oxidative' attack of proteins and lipids.     

L.D.H. and L.D.H. isoenzymes of the intra-uterine secretions of the cow during the estrous cycle

Uterine secretions were collected from 20 mature cows during estrus (day 0), metestrus (day 5), diestrus (day 10) and proestrus (day-1). Lactate dehydrogenase (LDH) and LDH isoenzymes activity were evaluated. No significant cyclic variations of LDH activity was found in the uterine secretions while the mean of the enzyme activity was higher during the estrogenic period of the cycle. The relative activity of LDH-1, LDH-2 and LDH-3 isoenzymes were higher during proestrus and estrus whereas LDH-5 activity was more important during metestrus. The LDH-3 seems to have the higher relative activity in uterine secretions of the cow.     

Stimulation of the salt receptor of the blowfly. I. NaCl

Application of NaCl solutions to the tip of a labellar sensillum of the blowfly elicited a repetitive neural response from the salt receptor. The response was examined with respect to reproducibility and adaptation. A threshold was observed for tests with dilute solutions. Above this, the response increased linearly with the logarithm of the molarity. The response was not significantly affected by the pH of stimulating solutions, buffered or not, between 3 and 10. Beyond this range, it was reversibly inhibited until, at greater extremes of pH, atypical stimulation independent of the presence of salt was seen. Receptor sensitivity increased with fly age. The results presented here may be due to effects at sites in the sensillum other than the receptor membrane.     

Chloroplast membranes of the green alga Acetabularia mediterranea. I. Isolation of the photosystem II.

1. In the presence of Triton X-100, chloroplast membranes of the green alga Acetabularia mediterranea were disrupted into two subchloroplast fragments which differed in buoyant density. Each of these fractions had distinct and unique complements of polypeptides, indicating an almost complete separation of the two fragments. 2. One of the two subchloroplast fractions was enriched in chlorophyll b. It exhibited Photosystem II activity, was highly fluorescent and was composed of particles of approx. 50 A diameter. 3. The light-harvesting chlorophyll-protein complex of the Photosystem II-active fraction had a molecular weight of 67 000 and contained two different subunits of 23 000 and 21 500. The molecular ratio of these two subunits was 2:1.     

Information from e.x.a.f.s. spectroscopy on the structures of different forms of molybdenum in xanthine oxidase and the catalytic mechanism of the enzyme.

In isolated perfused rat hearts, epidermal growth factor (EGF; 15 nM) increased cellular cyclic AMP (cAMP) content by 9.5-fold. In rat cardiac membranes, EGF also stimulated adenylate cyclase activity in a dose-dependent manner, with maximal stimulation (35% above control) being observed at 10 nM-EGF. Half-maximal stimulation of adenylate cyclase was observed at 40 pM-EGF. Although the beta-adrenergic-receptor antagonist propranolol markedly attenuated the isoprenaline-mediated increase in cAMP content of perfused hearts and stimulation of adenylate cyclase activity, it did not alter the ability of EGF to elevate tissue cAMP content and stimulate adenylate cyclase. The involvement of a guanine-nucleotide-binding protein (G-protein) in the activation of adenylate cyclase by EGF was indicated by the following evidence. First, the EGF-mediated stimulation of adenylate cyclase required the presence of the non-hydrolysable GTP analogue, guanyl-5'-yl-imidodiphosphate (p[NH]ppG). Maximal stimulation was observed in the presence of 10 microM-p[NH]ppG. Secondly, in the presence of 10 microM-p[NH]ppG, the stable GDP analogue guanosine 5'-[beta-thio]diphosphate at a concentration of 10 microM blocked the stimulation of the adenylate cyclase by 1 nM- and 10 nM-EGF. Third, NaF + AlCl3-stimulated adenylate cyclase activity was not altered by EGF. The ability of EGF to stimulate adenylate cyclase was not affected by pertussis-toxin treatment of cardiac membranes. However, in cholera-toxin-treated cardiac membranes, when the adenylate cyclase activity was stimulated by 2-fold, EGF was ineffective. Finally, PMA by itself did not alter the activity of cardiac adenylate cyclase, but abolished the EGF-mediated stimulation of this enzyme activity. The experimental evidence in the present paper demonstrates, for the first time, that EGF stimulates adenylate cyclase in rat cardiac membranes through a stimulatory GTP-binding regulatory protein, and this effect is manifested in elevated cellular cAMP levels in perfused hearts exposed to EGF.     

Monkey glutathione S-aryltransferases. II. Properties of the major enzyme purified from the liver.

1. The molecular and enzymatic properties of the major component (Fraction IV) of glutathione S-aryltransferases [EC 2.5.1.13] purified from the liver of monkey (mainly rhesus monkey) have been investigated. The enzyme had a molecular weight of about 48,000 and was composed of two subunits of apparently identical molecular weight (ca. 24,000) bound to each other non-covalently. Each subunit contained one SH group. The amino acid composition showed characteristic high contents of leucine and glutamic acid residues. No amino-terminal residue was detected by the dansyl method. 2. The enzyme showed a rather broad optimum pH range from 7.5 to 9 with 1,2-dichloro-4-nitrobenzene as a substrate. It was moderately stable below 40 degrees C at pH 7.5. However, it showed an anomalous instability at pH around 4.2. It was reversibly denatured at least partially by urea or guanidine hydrochloride and irreversibly denatured by sodium dodecyl-sulfate. It was significantly inhibited by Zn2+, Cd2+, and Hg2+, and also by benzene hexachloride. It was extensively inactivated by reaction with phenylglyoxal or 2,4,6-trinitrobenzene sulfonate, whereas several SH reagents were without marked effect on the activity under the reaction conditions employed.     

Studies of polysaccharide fractions from the lipopolysaccharide of Pseudomonas aeruginosa N.C.T.C. 1999.

Two polymeric water-soluble fractions were isolated by gel filtration after mild acid hydrolysis of the lipopolysaccharide from Pseudomonas aeruginosa N.C.T.C. 1999. The fraction of higher molecular weight retained the O-antigenic specificity of the lipopolysaccharide and may be 'side-chain' material. This fraction was rich in N (about 10%) and gave several basic amino compounds on acid hydrolysis; fucosamine (at least 2.8% w/w) was the only specifc component identified. The fraction of lower molecular weight was a phosphorylated polysaccharide apparently corresponding to 'core' material. The major components of this fraction and their approximate molar proportions were: glucose (3-4); rhamnose (1); heptose (2); 3-deoxy-2-octulonic acid (1); galactosamine (1); alanine (1-1.5); phosphorus (6-7). In the intact lipopolysaccharide this fraction was probably linked to lipid A via a second residue of 3-deoxy-2-octulonic acid, and probably also contained additional phosphate residues and ethanolamine. The residues of 3-deoxy-2-octulonic acid were apparently substituted in the C-4 or C-5 position, and the phosphorylated heptose residues in the C-3 position. The rhamnose was mainly 2-substituted, though a little 3-substitution was detected. The glucose residues were either unsubstituted or 6-substituted. Four neutral oligosaccharides were produced by partial acid hydrolysis and were characterized by chemical, enzymic, chromatographic and mass-spectrometric methods of analysis. The structures assigned were: Glcpalpha1-6Glc; Glcpbeta1-2Rha; Rhapalpha1-6Glc; Glcpbeta1-2Rhapalpha1-6Glc. The galactosamine was substituted in the C-3 or C-4 position, the attachment of alanine was indicated, and evidence that the amino sugar linked the glucose-rhamnose region to the 'inner core' was obtained.     

Studies on the maturation of immune responsiveness in the mouse. II. Role of the spleen.

Experiments were designed to investigate the role of the spleen in the development of the murine immune system. By using mice splenectomized within 24 hr of birth, as well as mice with a hereditary, congenital absence of the spleen, the primary immune response to sheep erythrocytes was examined. The immunocompetence of lymph node cells from spleenless or control mice was assessed in vitro, in organ and in cell suspension cultures, and in vivo, by transfer into lethally irradiated syngeneic recipients followed by antigenic stimulation. The immunologic capacities of thymus and bone marrow cells were similarly tested by injection separately or in combination into irradiated syngeneic mice. Lymph node cells from spleenless animals appeared fully competent both in vitro and in transfer experiments. Neither neonatal splenectomy nor congenital absence of the spleen significantly reduced the capacity of bone marrow or thymus cells to participate in the immune response to sheep erythrocytes.     

Mechanism of liquefaction of the human ejaculate. I. Changes of the ejaculate proteins.

The spontaneous liquefaction of the human ejaculate in vitro was studied by macroscopic inspection, disc electrophoresis of the ejaculate proteins, free amino acid and nitrogen determinations and by electron microscopy. Liquefaction occurred in three phases. Phase 1 was characterized by the macroscopic solubilization of the gelatinous material and by disappearance of its granular ultrastructure. Zn++, Hg++ and Cu++ completely inhibited the first step of the liquefraction, which could be reversed by Na2EDTA. The factor(s) involved is most probably a proteolytic enzyme(s) which is tightly incorporated into the gelatinous material. In Phase 2 of liquefaction solubilized proteins were degraded to peptides and in Phase 3 these peptides were degraded to amino acids. Factor(s) responsible for these two steps of liquefaction could be removed by washing the gelatinous material.     

Bioelectrorheological model of the cell. 3. Viscoelastic shear deformation of the membrane.

An analytical electromechanical model of a spherical cell exposed to an alternating electric field was used to calculate shear stress generated in the cellular membrane. Shape deformation of Neurospora crassa (slime) spheroplasts was measured. Statistical analysis permitted empirical evaluation of creep of the cellular membrane within the range of infinitesimal stress. Final results were discussed in terms of various rheological models.     

Cytodifferentiation of the chick pancreas. II. Ultrastructure of the acinar cells

The ultrastructural changes occurring during the differentiation of the pancreatic acinar cell were studied in White Leghorn chick embryos from the onset of pancreatic morphogenesis on day 3 of incubation (day 3) to hatching. Generally, the changes included a loss of some structures, the addition of others and modification of existing structures. Numerous cytoplasmic filaments which were present in the early migrating cells of the pancreatic bud were no longer present on day 5. The nucleoli enlarged temporarily on days 5–6 and then resumed a reduced size. The Golgi apparatus enlarged by day 6 and remained this way throughout the embryonic period. Associated with these changes was the initial appearance of the zymogen granules on day 5. The endoplasmic reticulum was present initially in both the smooth and the rough forms. The rough form and the outer nuclear membrane were both initially studded intermittently with aggregates of ribosomes. Subsequently, there was an increase in the number of attached ribosomes, an increase in the amount of rough reticulum and a decrease in smooth membranes. The ribosomes attached to the membranes appeared to augment the large free ribosome population characteristic of the early cells. Mitochondria did not appear to increase in number but there was an increase in size. The granules varied in kind, number and size with developmental age. The first granules formed (days 3–5) appeared to be miniatures of the mature type. Subsequently, a heterogeneity of granule morphologies was present.     

Genetics of esterases inDrosophila. IX. Characterization of the JH-esterase inD. virilis

The kinetic characteristics of the main isozymes of Drosophila virilis esterase were studied and Km values of esterase-2, -4, and -6 and p-esterase for alpha- and beta-naphthyl acetate were obtained. Juvenile hormone (JH) was shown to inhibit the p-esterase activity when in competition with beta-naphthyl acetate and the general esterase inhibitor, diisopropylphosphofluoridate (DFP), was shown to inhibit all the components of the D. virilis esterase patterns except p-esterase. While studying the changes of p-esterase activity in D. virilis ontogenesis, the increase in p-esterase activity in the wandering larvae, prepupae, and early pupae was found to correlate with a decrease in JH titer at these stages. The decrease in JH level in a temperature-sensitive lethal mutant larvae of D. virilis at high temperatures was shown to correlate with increased p-esterase activity. These results confirm that p-esterase of D. virilis is JH-esterase.     

Stimulation of the salt receptor of the blowfly. II. Temperature

The response of the salt receptor of the blowfly increased with increasing ambient temperature. At constant ambient temperature, the response increased as the relative humidity was raised. At low relative humidity, the temperature of the stimulating solution near its air interface was markedly below ambient temperature, due to evaporation. Warming and cooling the tip of the sensillum while recording from its side respectively raised and lowered the frequency of "spontaneous" action potentials of the receptor. The results indicate that the response of the salt receptor is dependent on the temperature of the stimulus. The Q10 is probably several times greater than one.     

Comparisons of the hybrids Hordeum chilensexH. vulgare,H. chilensexH. bulbosum,H. chilensexSecale cereale and the amphidiploid of H. chilensexH. vulgare

Summary Diploid hybrids between Hordeum chilense and three other species, namely H. vulgare, H. bulbosum and Secale cereale, are described together with the amphidiploid of H. chilensexH. vulgare. Both the diploid hybrid and the amphidiploid of H. chilensexH. vulgare were chromosomally unstable, H. chilensexH. bulbosum was less so, while H. chilensexS. cereale was stable. Differential amphiplasty was found in all combinations. No homoeologous pairing was found in the Hordeum hybrids but in H. chilensexS. cereale there was chromosome pairing both within the two genomes and between the genomes.     

Shope fibroma virus. II. Role of the virion-associated nucleases.

The effect of Shope fibroma virus (SFV) infection on host DNA synthesis was investigated. The cytocidal strain, SFV-I, inhibited the incorporation of [3H]thymidine into nuclear DNA very shortly (2 h) after infection, whereas the noncytocidal strain, SFV-W, did so later (10 h postinfection) and to a lesser extent. Furthermore, a two- to threefold stimulation of host DNA synthesis was recorded in SFV-W-infected cells 3 to 4 h after infection. Since virion-associated nucleases have been implicated in the shutoff of host synthesis, these and other enzymatic activities were measured in purified virion preparations. The SFV strains and vaccinia virus contained equivalent amounts of DNA-dependent RNA polymerase, ATPase, and protein kinase activities. However, in SFV-W the pH 4.5 exonuclease activity was lower than in SFV-I and vaccinia virus, and the level of pH 7.8 endonuclease was almost undetectable. To test whether the lack of endonucleolytic activity had some effect on the removal of the cross-links in the parental DNA that occurs after viral penetration, the fate of the virion SFV DNA was followed. The majority (80%) of the SFV-I and SFV-W DNA molecules extracted after viral adsorption sedimented in alkaline sucrose gradients as cross-linked. After 3 h of infection, 75% of the SFV-I DNA molecules lacked cross-links, whereas 78% of the SFV-W DNA still remained cross-linked. The same results were obtained when the presence of cross-links was tested in restriction fragments. Taken together, these results indicate that virion-associated nucleases are involved in the early shutoff of host DNA synthesis and in the elimination of cross-links from the parental viral DNA.     

Topography of the rhodopsin molecule. Identification of the domain phosphorylated.

Studies on the light-stimulated phosphorylation of rod outer segments by [gamma-32P]ATP showed that although nearly 1 mol of [32P]phosphate was incorporated/mol of total opsin, only a small fraction of the molecules were phosphorylated, and these contained at least 2-3 mol of phosphate/mol. Rod outer segments containing the phosphorylated opsin were incubated with 11-cis-retinal to generate phosphorylated rhodopsin and then digested with papain to produce a cleaved complex comprising three fragments, heavy (H), medium (M) and light (L). It was shown that L-fragment of apparent mol.wt. 6000 contained all the phosphorylation sites. This suggests that one specific domain of rhodopsin is susceptible to multiple phosphorylation.     

Glutathione reductase in the sea urchin egg. III. Activation of the complex form by proteinases.

The G-200 flow-through fraction of the extract of sea urchin eggs contained a complex form of glutathione reductase (GR) [EC 1.6.4.2]. The complex was unstable and gradually dissociated with ain increase in GR activity. The activation was facilitated by high concentrations of EDTA, KCI or (NH4)2SO4. The rate of activation by salts was apparently dependent on the ionic strength. The complex form was also activated rather quickly by treatment with proteinases such as trypsin [EC 3.4.21.4], alpha-chymotrypsin [EC 3.4.21.1] or subtilisin [EC 3.4.21.14]. Trypsin caused the complex to release the free form of GR. Even after trypsin treatment, little change was observed in the dependence of the GR activity on GSSG or NADPH concentration. The GR activity of the complex form was not inhibited at all by 0.2 mM N-ethylmaleimide (NEM) in the presence of GSSG, but was reduced to 3% in the presence of NADPH. When excess NEM was sequestered with GSH, the NEM-treated complex form was strikingly activated by trypsin, while no activation was detected with the free form of enzyme pretreated with NEM. These results suggest that the active site of GR in the complex form is largely masked by a polypeptide moiety of theinhbitiory component.     

Studies on the product of antigenic recognition. I. Formation of the product in recognition of alloantigens.

A product of antigenic recognition (PAR) was produced whenever receptors for alloantigens from T lymphocytes or a principle present in T-cell dependent alloantisera interacted with alloantigen. With two forms of the PAR assay (direct and indirect) the mechanisms underlying these interactions have been analyzed. For the interaction of T-lymphocyte receptors with alloantigen measured with direct PAR assays, the following conclusion emerged: upon confrontation with alloantigen, receptors (if not already present in secreted form) had first to be released from T-cell membranes. Shed T-cell receptors interacted with alloantigens by solubilizing them. Both processes could be prevented by fixing cells with formaldehyde. Release of T-cell receptors was temperature-dependent, solubilization of alloantigens was not. Because in mixed cell cultures receptors had first to be shed, this process was considerably slower and, in concordance with temperature dependence of receptor release, took place only at 37 degrees C. Titration of T lymphocytes with 'bound' receptors by the direct PAR test revealed that in the presence of excess alloantigen 10(2) T cells sufficed to give measurable responses. Supernates of parental strain lymphocytes containing numerous T-cell receptor specificities could be depleted of one of them. Alloantisera raised in presence of T helper cells ('T alloantisera') contained a principle capable of recognizing alloantigens, alloantisera incited in the absence of T helpers ('B alloantisera') did not. The recognizing principle appeared to be IgG. Like T-cell receptors, it was capable of solubilizing alloantigens form target cell membranes. B alloantisera lacked this capacity and their alloantigen-recognizing moiety was found to be monomeric IgM. The mode of interaction of this IgM with alloantigen most likely consisted in fixation to and shielding of antigen.     

Cytophilic binding of IgE to the macrophage. I. Binding characteristics of IgE on the surface of macrophages in the rat.

Involvement of IgE antibody in macrophage cytotoxicity against Schistosoma mansoni schistosomula suggests a cytophilic interaction of this class of antibody with the membrane of macrophages. Peroxidase-labeled monoclonal IgE protein was used to investigate IgE-macrophage interaction in the rat. Benzidine-aggregated rat IgE bound to the surface of peritoneal macrophages under experimental conditions, preventing endocytosis of the labeled aggregates. Binding was inhibited by preincubation with unlabeled IgE (aggregated). When unaggregated IgE was used, labeling of the macrophage surface, even when endocytosis was inhibited, was also observed at 37 °C but not at 4 °C. This result indicated different binding characteristics than reported for cytophilic IgG. Radiolabeled monoclonal IgE (deaggregated by ultracentrifugation after labeling) bound to peritoneal rat macrophages at 37 °C with a maximum between 10 and 20 min and a progressive shedding thereafter, whereas no change in bound radioactivity was observed at 4 °C or after preincubation with unlabeled IgE. Radiolabeled bovine serum albumin as a control did not interact significantly with the macrophages at both temperatures in these experimental conditions. The use of ?-mono-specific rabbit anti-rat IgE allowed the identification of IgE on the surface of peritoneal macrophages from rats infected with S. mansoni.