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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Dihydrotestosterone propionate effects on dominance and sexual behaviors in gonadectomized male and female rhesus monkeys.

One triad of male and two triads of female gonadectomized rhesus monkeys were observed as social groups assembled for repeated hour-long sessions. Social relationships were measured in terms of aggressive behavior between the members of each group in order to determine the dominance hierarchical order. Sexual performance was assessed for each male, before and after castration, in tests with an estrogen-stimulated ovariectomized female. Similar measures were made when the same female was periodically introduced to the all-male triad. When dihydrotestosterone propionate (DHTP) was administered for a period of 6 weeks to the middle-ranking member of each group, social status changes occurred in two groups, one male and one female, resulting in the elevation of the treated monkeys to the highest rank in the dominance hierarchy. In the other female group, aggressive behavior was increased with DHTP treatment of the middle-ranking female. Somatic effects, particularly a gain in body weight, occurred in all treated animals. Yawning behavior also increased significantly in those animals receiving DHTP. The latter two effects returned toward pretreatment levels following the cessation of hormone injection; however, changes in dominance hierarchy persisted to the end of the experiment, 6 weeks following the last DHTP treatment.     

Two distinct adhesion mechanisms in embryonic neural retina cells. II. An immunological analysis

Antibodies were raised against neural retina cells prepared by dissociation in EGTA alone (E cells, Ca²⁺-independent aggregation), in trypsin + Ca²⁺ (TC cells, Ca²⁺-dependent aggregation), or in trypsin + EGTA (TE cells, nonadhesive). Anti-E-cell Fab selectively inhibited Ca²⁺-independent aggregation, anti-TC-cell Fab selectively inhibited Ca²⁺-dependent aggregation, and anti-TE-cell Fab inhibited neither. Fab from a fourth preparation, also raised against E cells, inhibited both Ca²⁺-independent and Ca²⁺-dependent aggregation but was separated by immunoadsorption into two fractions, one specific for each mode of aggregation. In cells which utilize both modes simultaneously (LTC cells), each was inhibited exclusively by the appropriate Fab. The immunological data presented here demonstrate the existence in the same cells of two distinct and functionally independent adhesion mechanisms, each responsible for one of the two modes of aggregation. The differing adhesive properties of retinal cells prepared by different procedures are explained by the presence, absence, or degree of activity of these two mechanisms, qualities regulated by the concentrations of trypsin and Ca²⁺ used in the tissue dissociation.     

Two distinct adhesion mechanisms in embryonic neural retina cells. I. A kinetic analysis

The reaggregation kinetics of embryonic chick neural retina cells prepared using several different dissociation procedures were monitored through decreases in the small-angle light scattering of aggregating samples. Two distinct modes of aggregation were revealed, one Ca²⁺ independent, the other Ca²⁺ dependent, suggesting the existence of two separate adhesion mechanisms. By varying the concentrations of Ca²⁺ and trypsin in the dissociation medium, we obtained cells which exhibited both, either, or neither mode of aggregation. The Ca²⁺-independent adhesiveness is active in the absence of proteolysis, is resistant to low levels of trypsin (0.001%), but is readily inactivated at higher trypsin concentrations in either the presence or absence of Ca²⁺. It is relatively temperature independent. By contrast, the Ca²⁺-dependent adhesiveness is not detected before exposure of the cells to proteolysis. It is expressed after tryptic proteolysis in the presence of Ca²⁺ and is then highly temperature dependent. It is resistant to further digestion by trypsin in the continued presence of Ca²⁺ but is lost when Ca²⁺ is subsequently removed, apparently through the expression of tryptic cleavage incurred earlier. We suggest that its increased activity may result at least in part from the clustering of surface components into adhesive patches. A provisional model is presented correlating these data.     

The application of PEI-cellulose thin-layer chromatography for the resolution of large oligonucleotide fragments of transfer ribonucleic acids.

Large oligonucleotide fragments from tRNA were separated on PEI-cellulose tle using stepwise gradients of increased concentrations of LiCl (containing 0.3 m Tris-HCl and 7.5 m urea at pH 7.9) or Li-formate (containing 7.5 m urea at pH 3.5). These large oligonucleotides, obtained by cleavage of tRNA with nuclease S₁, aniline-NaOH, or partial ribonuclease T₁ digestion and separated on PEI-cellulose, were analyzed by three different methods. The first method entailed elution and total base analysis by the tritium-postlabeling technique; the second involved complete ribonuclease T₁ digestion in situ, contact transfer to another PEI-cellulose tle plate, and two-dimensional tle fingerprinting; the third employed complete digestion in situ with ribonuclease T₁ and bacterial alkaline phosphatase, followed by the elution, periodate oxidation, introduction of a tritium into 3′-terminus, and subsequent two-dimensional PEI-cellulose fingerprinting. These techniques can aid in the determination of the complete nucleotide sequence of tRNA when only small quantities of pure tRNAs (less than 10 A₂₆₀ units) are available or when the tRNAs are not amenable to in vivo radioactive labeling.     

Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling

The eye lens contains a structural protein, alpha crystallin, composed of two homologous primary gene products alpha A2 and alpha B2. In certain rodents, still another alpha crystallin polypeptide, alpha AIns, occurs, which is identical to alpha A2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete alpha A crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the alpha A gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5' non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the alpha AIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3' non-coding region. The second exon is bordered by a 3' splice junction (A X G/G X C), which deviates from the consensus for donor splice sites (A X G/G X T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.