Preparation and properties of concanavalin A-binding glycopeptides derived from rat brain glycoproteins

Mannose-rich glycopeptides derived from brain glycoproteins were recovered by affinity chromatography on Concanavalin A-Sepharose. These glycopeptides, which adsorb to the lectin and are eluted with α-methylmannoside, constitute about 25–30% of the total glycopeptide material recovered from rat brain glycoproteins. They contain predominately mannose and N-acetylglucosamine (mannose/N-acetylglucosamine = 3), as well as small amounts of galactose and fucose. Approx. 65% of the Concanavalin A-binding glycopeptide carbohydrate was recovered after treatment with leucine aminopeptidase, gel filtration on Biogel P-4, and ion-exchange chromatography on coupled Dowex 50-hydrogen and Dowex 1-chrolide columns. The purified glycopeptide fraction contained six mannose and two N-acetylglucosamine residues per aspartic acid and possessed an apparent molecular weight of about 2000 as assessed by gel filtration and amino acid analysis. Galactose and fucose were absent. Treatment of the purified glycopeptides with α-mannosidase drastically reduced their affinity for Concanavalin A, suggesting the presence of one or more terminal mannose residues.     

The properties of trehalase from the mosquito-parasitizing water mold, Lagenidum sp

The soluble trehalase from the phycomycete Lagenidium sp., a parasite of many species of mosquitoes, was purified by acid titration, acetone precipitation, and Sephadex G-200 chromatography to give a 170-fold increase in specific activity over the crude extract. The enzyme was specific for trehalose. A β-glucosidase was copurified with the trehalase, but did not interfere with its characterization. Lagendium trehalase had a K_m of 1.43 mm, and E_a of 11.4 kcal/mole, and a pH of optimum activity of 5.5–6.5, and a molecular weight of 72,000. It was denatured by 30 min incubation at temperatures above 50°C, severely inhibited by heavy metals, and competitively inhibited by sucrose. No other reported inhibitors, including mannitol and ATP, were effective. Suggested physiological roles for the enzyme include the breakdown of stored trehalose in the mycelium and zoospores, and the digestion of hemolymph trehalose in infected mosquito larvae.     

Effects of active immunization of ram lambs and bull calves against synthetic luteinizing hormone releasing hormone

Ram lambs and bull calves were immunized against LH-RH by injections given in weeks 0, 6, 12 and 28 (ram lambs, week 0 = 16 to 20 weeks of age) and weeks 0, 6, 12 and 18 (bull calves, week 0 = approximately 4 weeks of age). The testis size of LH-RH-immunized animals was significantly less than that of controls from week 13 onwards in ram lambs and from week 15 onwards in bull calves. When ram lambs were sampled in week 17 and bull calves in week 20, mean plasma gonadotrophin and testosterone concentrations were consistently lower in LH-RH-immunized animals than in controls. Single intravenous injection of synthetic LH-RH or an analogue of LH-RH in week 27 failed to induce LH or testosterone responses in LH-RH-immunized ram lambs. Motile semen samples could not be obtained from any of the LH-RH-immunized ram lambs in weeks 24, 25 and 26 or from 7 of 10 in week 72, but samples of moderate motility were obtained in week 72 from three rams in which LH-RH antibody titres had fallen. No attempt was made to obtain semen from bull calves. After castration there was no increase in plasma LH in LH-RH-immunized rams and only a small increase in LH-RH-immunized bull calves. Mean testis weight was significantly lower in LH-RH-immunized animals than in controls of both species. Thus the normal development of the reproductive system in ram lambs and bull calves was blocked by active immunization against LH-RH. Some evidence was obtained for natural reversal of the effects with time and falling antibody titres. These findings demonstrate the potential of LH-RH immunization as an alternative to castration.     

Subcellular and anatomical distribution in rat brain of glycoproteins that contain mannose-rich heteropolysaccharide chains

Mannose-rich glycopeptides derived from brain glycoproteins were obtained by proteolysis of bovine brain tissue or subcellular fractions derived from rat brain tissue. The dialyzable mannose-rich glycopeptides were isolated by colum electrophoresis and gel flitration. These glycopeptides contained, on the average, six mannose and two N-acetylglucosamine residues with variable amounts of fucose and galactose. Over 50% of the mannose-rich glycopeptides of rat brain were localized in the microsomal and synaptosomal fractions; myelin and the soluble fraction contained lesser amounts. None was recovered from the mitochondria. The amount, per mg protein, of mannose-rich oligosaccharide chains in the myelin exceeded the concentration found in the microsomal and synaptosomal fractions. The concentration of mannose-rich glycopeptides derived from glycoproteins was 50% higher in white matter than in gray. On the other hand, the non-dialyzable and acidic sialoglycopeptides showed a three-fold enrichment in gray matter compared to white. The relatively lower ratio of sialoglycopeptides to mannose-rich glycopeptides observed in white matter (2.5) compared to gray matter (6.9) is reflected in the lower value for the ratio in myelin (1.1) compared to synpatosomes (2.1). Although glycoproteins that contain mannose-rich oligosaccharide chains are present in the nerve cell and its terminals, these glycoproteins appear to be relatively enriched in myelin and/or glial membranes.     

Radiation-induced nondisjunction and Robertsonian translocation in Drosophila

In Drosophila melanogaster, gametes formed by oocytes in which Robertsonian translocations were induced in an immature stage usually show chromosomal imbalance. It is estimated that fewer than 20% of the gametes bearing newly induced Robertsonian translocations “fusing” X and fourth chromosomes are of balanced constitution. In contrast, when the two acrocentric pairs, X and fourth chromosomes, are replaced by an X-4 Robertsonian translocation, treatment of immature oocytes of homozygotes produces some 5–6-fold fewer sex-chromosome trisomics than do females of normal karyotype. In the place of such trisomics (having separate sex chromosomes), there is a much smaller number of compound-X chromosomes formed and a number of compound-fourth chromosomes as well. However, the production of “XO” males is not appreciably smaller in the translocation homozygotes. A number of possible mechanisms to account for this are suggested. The findings are consistent with the expectations of the hypothesis that radiation-induced nondisjunction results from improper conjunctions of heterologues, brought about by chromatid interchange^{7–12, 16}.     

Release of lutropin (LH) and follitropin (FSH) in cattle after administration of a new gonadoliberin (GnRH) analogue in comparison with the gonadoliberin decapeptide.

A gonadoliberin (GnRH) analogue nonapeptide (Hoe 766) was administered intramuscularly in concentrations between 2.5 and 50 μg to m?ture cows in order to study the response of lutropin (LH) and follitropin (FSH). Results were compared with those from experiments of the GnRH decapeptide (Hoe 471). Plasma LH and FSH were radio-immunologically determined. Increasing doses of GnRH analogue up to 15–20 μg caused an approximately linear increase in total plasma LH and FSH until the response reached a plateau. With these amounts peak values were about 60 fold higher for LH and 3.5 fold higher for FSH than basal levels about 135 minutes after injection. Higher values lasted for more than 6 h for LH and about 5 h for FSH. The LH response was much greater and more prolonged than for FSH.Doses of the nonapeptide analogue 50 to 70 times lower than the GnRH decapeptide provoked about the same height and duration of LH and FSH response.     

Diffusion models for firing of a neuron with varying threshold

A stochastic model for the firing of a neuron with refractory properties is treated analytically. Refractory behavior is modeled by a threshold function θ(t) which is infinite immediately after the neuron fires, as well as during the absolute refractory period, and then decreases monotonically to the quiescent threshold level, θ_∞, during the relative refractory period. Using Wald's identity, input-output relations are derived analytically for the exponential threshold which has a time constant equal to the membrane time constant. A method for computing these relations for a general threshold is presented and is explicitly used for the general exponential threshold and the Hagiwara threshold, θ(t) = θ_∞e^α/t, where a is a constant.     

Effect of active immunisation of ewes against synthetic luteinising hormone releasing hormone.

At the start of the breeding season 13 intact and four ovariectomised ewes were immunised against LH-RH which was rendered immunogenic by conjugation to bovine serum albumin using carbodiimide. The immunogen was emulsified with Freund's complete adjuvant prior to multi-site intradermal injection into a shaved area on the back of each animal. All the ewes were boosted using an identical procedure six and twelve weeks later. LH-RH antibody titres were monitored from weekly blood samples. Oestrous cycles were shown to stop in all but one of the intact ewes after anti-LH-RH titres had developed, but before the seasonal anoestrus. Laparoscopy of the ewes at this time showed that the ovaries and uteri were in various stages of regression. Plasma gonadotrophin levels of ovariectomised ewes fell significantly after immunisation and in intact immunised ewes ovariectomy failed to result in any increase in plasma gonadotrophins. Injection of 150μg synthetic Lh-RH or 6μg of an immunologically distinct analogue of LH-RH failed to induce LH or FSH responses approaching those previously demonstrated with identical doses in non-immunised anoestrous ewes. These results suggest that immunisation against LH-RH could provide an alternative to ovariectomy for the suppression of unwanted oestrous symptoms and ovulation but that reversal of the effects of immunisation might be difficult to achieve routinely.     

The effects of injecting [D-Ser(But)6] Des Gly-NH2(10) luteinizing hormone releasing hormone ethylamide in early, mid and late seasonal anoestrus on gonadotrophin secretion and luteal function in the ewe

The ability of the luteinizing hormone releasing hormone (LH-RH) analogue [D-Ser(Bu(t))(6)] Des-Gly-NH(2)(10) LH-RH ethylamide to stimulate the secretion of luteinizing hormone (LH) and follicle stimulating hormone (FSH) and to induce ovulation and luteal function in seasonally anoestrous ewes was investigated by injecting the analogue at three stages of the anoestrus (day 118, day 182 and day 235 of the year). After injection on day 118, eight of nine ewes ovulated and all of the former secreted progesterone during the subsequent 20 days. After injection on day 182, six of the nine ewes ovulated, of which none showed luteal function. Only two of the nine ewes were not already secreting progesterone on day 235. Both of these responded to the analogue by secreting normal luteal levels of progesterone. The mean LH peak heights in response to injection at the three stages showed no significant differences from one another. The mean FSH peak heightafter injection on day 182 was significantly lower than the mean FSH peak height associated with the other two challenges (P < 0.05). On day 116 of the following year, 20 ewes were treated with the analogue as before. The high progesterone levels confirmed the results of the day 118 challenge in the previous year. However, none of the ewes conceived when inseminated artificially 24 and 36 hours after analogue treatment.     

Yeast episome: oligomycin resistance associated with a small covalently closed non-mitochondrial circular DNA

We have isolated a single step spontaneous mutant of S. cerevisiae resistant simultaneously to oligomycin, venturicidin, chloramphenicol, cycloheximide and triethyltin. This multiple drug resistance results from the interaction of two genetic factors showing both chromosomal location and episomal characteristics. One factor (π) confers oligomycin resistance, the other (τ) confers the other resistances. π can be lost spontaneously while τ can be completely eliminated with ethidium bromide. All π⁺ strains, whether grande or petite, τ⁺ or τ^?, carry a covalently closed circular DNA while π^? strains are devoid of it. We hypothesise that this circular DNA may play an informational role in the biogenesis and/or function of membranes.     

Cellular anamnesis in earthworms

The quantitative response of coelomic cells associated with first- and second-set Eisenia xenografts transplanted to Lumbricus hosts at 20 ° C was compared with autografts and nonspecific wounds. Coelomocyte numbers were significantly lower in response to first than second-set xenografts. Coelomocytes also increased in association with autografts and nonspecific wounds, but the reaction is short lived, and essential for early wound healing and repair. Such nonspecific increases are different from subsequent specific immunologic longer-lasting coelomocyte responses. First-set xenografts induced a relatively slow increase in coelomocytes, which declined after 3–4 days postgrafting. By contrast, second-set xenografts caused an accelerated rise in coelomocytes, usually 20 to 30% greater than the maximum coelomocyte response induced by first-set xenografts. The mean survival time for first-set xenografts (non-self) was 17 ± 1 days, but repeat second-sets were rejected in an accelerated time of 6 ± 1 days. Autografts (self) are never destroyed. After priming with a first-set xenograft, this heightened coelomocyte reaction, to a second-set xenograft, was interpreted as an anamnestic response. The memory response is measurable in two ways: grossly as accelerated rejection of repeat xenografts, and at the cellular level, heightened coelomocyte numbers. Specific cellular immunity is demonstrable phylogenetically at the level of annelid worms.     

Analgesic activity of substance P following intracerebral administration in rats

Substance P was found to be a potent, long-lasting analgesic in the tail flick test in rats following intracerebral administration, via chronically indwelling cannulae, into the midbrain periaqueductal gray. Substance P was approximately five times as potent as morphine sulfate on a weight basis; however, it was 25 times more potent than morphine on a molar basis. The analgesic activity produced by Substance P was significantly antagonized by pretreatment with naloxone, a narcotic antagonist. The analgesic activity of Substance P exhibited a rapid onset (1 min.), peaked by 3 minutes post infusion and its duration of activity was between 30 and 60 minutes. Thus, Substance P may be yet another endogenous analgesic peptide.     

In vitro induction of IgM synthesis and secretion in rabbit spleen cells by soluble Concanavalin A

Rabbit spleen cells are not activated by Concanavalin A (Con A) conjugated to Sepharose 4B but are stimulated by soluble Con A which induces DNA and protein synthesis. At optimal concentration (5 μg/ml) one notes an increased intracellular protein and IgM synthesis and then secretion. This increase in protein synthesis is seen at all phases of the culture. At the intracellular level, IgM is found in the form of 7S molecules and a significant proportion of polymers with a centrifugation constant smaller than 19S. Fully assembled 19S polymers are found in the fluid phase. These results are compatible with a model of cellular cooperation, the basis of which is not the presentation of the inducer (mitogen or antigen) by a cell type to another, but rather the secretion of mediators by one of the cell populations making other cells responsive to the inducer.     

The influence of caging conditions and hormone treatments on fighting in male and female hamsters

After gonadectomy, more individually caged female hamsters fought prior to the initiation of hormone treatments than did group-caged females. Daily injections of testosterone propionate (TP), estradiol benzoate (EB), or progesterone (Prog) had no influence on the number of individually caged females that fought. However, TP and EB were effective in increasing the number of group-caged females that fought. In contrast to females, both individually and group-caged males fought infrequently after castration. Daily injections of TP, EB, or Prog were effective in increasing the number of individually caged males that fought, while only TP and EB were effective in group caged males. Prog failed to increase the number of group-caged hamsters of either sex that fought.     

Origin of hydrogen required for fatty acid synthesis in isolated rat adipocytes.

Beef liver and beef spinal cord d-glycerate dehydrogenases have been shown to be extremely similar. No differences between the two enzymes could be shown by polyacrylamide electrophoresis, sodium dodecyl sulfate polyacrylamide electrophoresis, immunodiffusion, immunoelectrophoresis, or their response to certain inhibitors. Differences could be obtained, however, between the beef spinal cord enzyme and the hog spinal cord enzyme by immunodiffusion and immunoelectrophoresis.Only by the very sensitive technique of microcomplement fixation could a small but significant difference be shown between the beef liver and beef spinal cord enzymes. Like the beef liver and hog spinal cord enzymes, the beef spinal cord enzyme was not inhibited by high concentrations of serine or glycine. The enzyme was inhibited however by low concentrations of phosphohydroxypyruvate and by other phosphorylated compounds.     

Reaction of o-phthalaldehyde and thiols with primary amines: Fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles

The fluorescence properties of 1-alkyl(and aryl)thio-2-alkylisoindoles, formed by the reaction of o-phthalaldehyde (OPTA) and thiols with primary amines, are reported. Variations in thiol and amine substituents and solvent polarity have large effects on the isoindole fluorescence spectra. These parameters, in addition to 3-thiol substitution of the isoindoles, pH, and the use of phosphate vs borate aqueous buffers, were found to have dramatic effects on the corrected relative fluorescence intensity. Low concentrations and nonaqueous solvents apparently stabilized most adducts while aqueous solutions, especially at low pH, caused pseudo-first-order decomposition, probably via hydrolysis to the corresponding 2,3-dihydro-1H-isoindole-1-one. However, 3.3 × 10⁻⁸ solutions of the more intensely fluorescent adducts (total adduct 5 pmol) were readily detected if the fluorescence was determined shortly after adding the isoindole to pH 9.2 borate buffer. The adduct formed using ethanethiol and n-propylamine possessed spectral properties which were the most responsive to changes in solvent polarity and was the most stable under the various conditions employed. Finally, arguments are presented that these isoindoles are the products in several other fluorogenic assays using OPTA.     

Release of macromolecular markers (enzymes) from liposomes treated with antibody and complement: An attempt at correlation with electron microscopic observations

Antibody-complement dependent damage to liposomal model membranes has been previously investigated by measuring the release of low molecular weight markers such as glucose. To determine whether larger solutes are also released under these conditions, experiments have been performed using immunologically sensitive liposomes that contained not only trapped glucose, but also enzymes (hexokinase, glucose-6-phosphate dehydrogenase, β-galactosidase) as macromolecular markers. The largest of these enzymes (β-galactosidase) has dimensions which closely approximate the diameter of the lesions detected by negative staining in natural membranes after immune lysis. Liposomes prepared with lecithin, and either actively sensitized with globoside or passively sensitized with alkali-treated lipopolysaccharide, released the enzymes in parallel with glucose upon incubation with the appropriate antiserum and native guinea pig serum as source of complement. Immune damage to sphingomyelin liposomes was characterized by a significantly lower loss of the enzymes in comparison to the percentage of glucose released; a comparable response was manifested by liposomes prepared from sheep erythrocyte lipids. Electron microscopic examination of negatively stained lecithin liposomes, which had released the macromolecular markers, failed to reveal the characteristics lesions; these findings are consistent with evidence obtained by other laboratories suggesting that the lesions may not correspond to functional holes. Lesions were, however, consistently observed in liposome preparations that had been treated with the polyene antibiotics, filipin; this antibiotic causes appreciable loss of both glucose and enzymes from either lecithin or sphingomyelin liposomes.     

Immunobiological properties of a concanavalin A derivative

A monovalent subunit of concanavalin A (Con A) was tested for mitogenic effects on murine splenic lymphocytes in vitro. In contrast to the effects of intact Con A, the monovalent derivative was not mitogenic at any concentration tested. Furthermore, prior exposure of splenic lymphocytes to monovalent Con A rendered the cells refractory to stimulation by the unmodified lectin.     

Mechanisms of inhibition of DNA replication by ultraviolet light in normal human and xeroderma pigmentosum fibroblasts

The inhibition of DNA replication in ultraviolet-irradiated human fibroblasts was characterized by quantitative analysis of radiation-induced alterations in the steady-state distribution of sizes of pulse-labeled, nascent DNA. Low, noncytotoxic fluences (<1 J/m², producing less than one pyrimidine dimer per replicon) rapidly produced an inhibition of DNA synthesis in half-replicon-size replication intermediates without noticeably affecting synthesis in multi-repliconsize intermediates. With time, the inhibition produced by low fluences spread progressively to include multi-replicon-size intermediates. The results indicate that ultraviolet radiation inhibits the initiation of DNA synthesis in replicons. Higher (>1 J/m², producing more than one dimer per replicon) cytotoxic fluences inhibited DNA synthesis in operating replicons presumably because the elongation of nascent strands was blocked where pyrimidine dimers were present in template strands. Xeroderma pigmentosum fibroblasts with deficiencies in DNA excision repair exhibited an inhibition of replicon initiation after low radiation fluences. indicating the effect was not solely dependent upon operation of the nucleotidyl excision repair pathway. Owing to their inability to remove pyrimidine dimers ahead of DNA growing points, the repair-deficient cells also were more sensitive than normal cells to the ultraviolet-induced inhibition of chain elongation. Xeroderma pigmentosum cells belonging to the variant class were even more sensitive to inhibition of chain elongation than the repair-deficient strains despite their ability to remove pyrimidine dimers. This analysis suggests that normal and repair-deficient human fibroblasts either are able to rapidly bypass certain dimers or these dimers are not recognized by the chain elongation machinery.