Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Inhibition of protein synthesis enhances the lytic effects of tumor necrosis factor α and interferon γ in cell lines derived from gynecological malignancies

Summary Few clinical responses have occurred in preliminary studies using the cytokines tumor necrosis factor (TNF) or interferon (IFN) in cancer patients. This may be related to the observation that many malignant cell lines are resistant to lysis by these cytokinesin vitro. Resistance to lysis by TNF or IFN in many cells is controlled by a protein-synthesis-dependent mechanism, such that when protein synthesis is inhibited cells become sensitive to lysis by these cytokines. Because there is some evidence that TNF and IFN act through different lytic mechanisms and are opposed by different resistance mechanisms, we treated a panel of eight cell lines, five derived from human cervical carcinomas (ME-180, MS751, SiHa, HT-3, and C-33A) and three derived from ovarian carcinomas (Caov-3, SK-OV-3, and NIH: OVCAR-3) with both TNF and IFN to determine whether such combination treatment might maximizein vitro cell lysis. Our results showed that pretreatment with IFN followed by exposure to TNF in the presence of protein synthesis inhibitors increased lysis of seven of the eight cell lines above that seen with either TNF or IFN and inhibitors of protein synthesis. Only the cell line C-33A was resistant to lysis by TNF and IFN, when exposed to these agents both alone and in combination with protein synthesis inhibitors. Clinically, combining the cytokines TNF and IFN with protein synthesis inhibitors may maximize thein vivo lytic effects of these cytokines.Supported by American Cancer Society Career Development Award 90-221     

Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

A structural model for elongation factor 1 (EF-1) and phosphorylation by protein kinase CKII

EF-1a binds aminoacyl-tRNA to the ribosome with the hydrolysis of GTP; the complex facilitates the exchange of GDP for GTP to initiate another round of elongation. To examine the subunit structure of EF-1 and phosphorylation by protein kinase CKII, recombinant , , and subunits from rabbit were expressed in E. coli and the subunits were reconstituted into partial and complete complexes and analyzed by gel filtration. To determine the availability of the and subunits for phosphorylation by CKII, the subunits and the reconstituted complexes were examined as substrates for CKII. Formation of the nucleotide exchange complex increased the rate of phosphorylation of the subunit and reduced the Km, while addition of to or the complex inhibited phosphorylation by CKII. However, a had little effect on phosphorylation of . Thus, the and subunits in EF-1 were differentially phosphorylated by CKII, in that phosphorylation of was altered by association with other subunits, while the site on was always available for phosphorylation by CKII. From the availability of the subunits for phosphorylation by CKII and the composition of the reconstituted partial and complete complexes, a model for the subunit structure of EF-1 consisting of (22)2 is proposed and discussed.     

Forskolin Modulates Acetylcholine Receptor Gating by Interacting with the Small Extracellular Loop Between the M2 and M3 Transmembrane Domains

1. Forskolin acts as an allosteric modulator of muscle-type nicotinic acetylcholine receptors. Receptors from mouse muscle and Torpedo electroplax demonstrate differential sensitivity to inhibition by forskolin. Previous work from this laboratory suggested that the subunit is responsible for this differential sensitivity.2. We have used a series of mouse/Torpedo species-chimeric subunits to further define the site of forskolin interaction with the subunit. Analysis of the patterns of forskolin inhibition of receptors containing mouse/Torpedo chimeric subunits along with the mouse , , and subunits suggests that forskolin interacts with the small extracellular domain that links the M2 and M3 transmembrane domains (the M2–M3 linker).3. We suggest that the M2–M3 linker domain plays an important role in the transduction of ligand binding to the conformational changes that result in channel opening.     

Conservation status of parrot populations in an Atlantic rainforest area of southeastern Brazil

A census of four species of syntopic parrots was carried out using distance sampling methods on São Sebastião island, SE Brazil. Most of the 33593 ha island is covered by mature and secondary Atlantic rainforest. Almost 80% of these forests are within the Ilhabela Park. Although the species counted have marked differences in size and weight, density (individuals/km²) and estimated population size in 23500 ha of well-preserved forests were similar: Amazona farinosa (13.82±5.94; 3247±1395), Pionus maximiliani (15.79±7.04; 3712±1654), Brotogeris tirica (15.05±4.87; 3537±1143) and Pyrrhura frontalis (13.06±5.53; 3068±1298). Encounter rates of Forpus crassirostris and Pionopsitta pileata were very low, which suggests that there is only a small population of these species on the island. The São Sebastião forests still support healthy populations of parrots. Although woodpecker population estimates on the island are large enough to provide nesting sites for parrots, competition for holes with other secondary cavity nesters such as toucans, flycatchers and tytiras, and the selective cutting of dead trees for canoe construction, which is a common practice on the island, may limit hole availability for parrots.     

Adoptions expérimentales de larves entre des fourmis de genres différents (II):Myrmica laevinodis Nylander etAnergates atratulus Schenck

Résumé Nous avons fait élever des larves d'Anergates atratulus par des ouvrières deMyrmica laevinodis à 22°C. Pour y parvenir, il n'est pas utile de faire hivernerensemble les larves d'Anergates et les ouvrières deMyrmica. La présence de larves autochtones n'empêche pas lesMyrmica d'élever des larves d'Anergates. Dans toutes les expériences lesMyrmica ont été soumises au fridavant de recevoir des larves d'Anergates. Aucune reine deMyrmica n'a été utilisée dans ces expériences.Sur les 64 larves d'Anergates que nous avons utilisées, 38 se sont transformées en imagos. C'est au début de l'adoption et au moment des métamorphoses que périrent la plupart des 26Anergates perdus. Les femelles vécurent en général 2 ou 3 jours et cherchèrent très tôt à quitter le nid natal. Les mâles vécurent 2 à 3 semaines.

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Summary Larvae ofAnergates atratulus were experimentally reared by workers ofMyrmica laevinodis, at 22°C. An overwintering of both larvae ofAnergates and workers ofMyrmica is not necessary for the success of that experiment. The presence of larvae ofMyrmica does not keep theMyrmica from rearing larvae ofAnergates. The workers ofMyrmica have been cooled, in all the experiments, before receiving larvae ofAnergates. No queen ofMyrmica have been used in that experiments.38 of the 64 larvae ofAnergates used became imagos. Most of the 26 lostAnergates died at the beginning of the adoption and during the metamorphosis. The females lived generally 2 or 3 days and tried, very early, to leave their native nest. The males lived 2 or 3 weeks.

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Anergates atratulus Myrmica laevinodis, 22 . bmecme Anergates Myrmica. Myrmica Anergates. Myrmica Anergates. Myrmica . 64 Anergates , 38 . 26 Anergates 2 3 . 2 3 .

     

Slowly adapting stretch-receptor organs: Periodic stimulation with and without perturbations

The length of isolated slowly adapting stretch-receptor organs (SAO) from crayfish was submitted to approximately sinusoidal modulations of 0.030–0.800 mm at frequencies of about 0.2, 1.0 or 3.0 cps. Sines were imposed either by themselves (clean) or mixed with (perturbed by) fast irregular length fluctuations or jitter. The amplitude of the latter remained within a specified fraction of the modulation, usually two-thirds (or x0.66), though from one twentieth to twice (or x0.05–2.00) were explored. The afferent impulse discharge was recorded: rate over bins of about 1/10 of the period was plotted as function of ongoing time. Within stationary epochs, average cycles were analyzed primarily in terms of lengths and discharge intensities. Each point in the displays corresponded to a particular bin along the average cycle, the abscissa and the ordinate being the length and the corresponding discharge rate, respectively; points were numbered in the order of their generation. Similar displays were constructed for rate vs velocity, rate vs acceleration, and rate-change vs velocity. Experiments without jitter. The non-perturbed state. With 0.2 and 1.0 cps, the rate vs length display had a clockwise loop with a flat extension to the left. The SAO at its shortest did not discharge and remained silent for some time. As length augmented, it eventually started firing, reaching maximum rate while being stretched (lead) or at maximum length. While length decayed, the organ fired less, slowing down, stopping at a length greater than where it had started, and then remaining silent until the end of the cycle. With 3.0 cps, the display had a counterclockwise loop with a flat extension on the left. The SAO did not discharge at its shortest, and fired more during relaxation than during stretching, peaking when relaxing (lag) and stopping at a length greater than where it had started. Artificial introduction of time separations between length and rate converted displays into acceptably straight lines only in a few, usually 0.2 cps, cases. The rate vs length relation exhibited consistent departures from monotonic increase and from linearity: departures were large fractions of the overall swings involving flat extensions, leads, lags, saturation effects, asymmetric rate sensitivities, and locking (which was frequent with 1.0 or 3.0 cps and could conceivably be more common with faster modulations). Other experimental paradigms have demonstrated multivaiued steady-state-rate-length relations. The variability from cycle to cycle of the rate in a particular bin was high or low for bins with low or high rates, respectively. Rate vs velocity plots were counter-clockwise loops with a base upon the abscissae, higher rates when velocity was decaying than when augmenting, and maxima at non-negative velocities. Rate-change vs velocity plots showed shortening associated with either discharge, decreases or small changes, and lengthenings associated with discharge increases, greater when augmenting if at 0.2 cps or when decaying if at 3.0 cps. Rate vs stimulus acceleration plots were, for the three tested frequencies, counterclockwise loops with a base upon the abscissae and higher rates when acceleration was decaying than when augmenting, suggesting a particular role in sensing accelerations. The sensitivity to length was evaluated by comparing the rate-swings at each frequency using identical bin-widths and depths. This procedure is considered to be most meaningful physiologically. Sensitivity was contingent upon the bin-width: with small bins (30 and 120 ms) it was highest for 3.0 cps, with intermediate bins (160 ms) it was highest for 1.0 cps, and with larger bins it was practically uniform. Differences involved factors of under x1.7. Experiments with jitter. The perturbed state. Jitter changed radically the rate vs length displays, converting all of them into clockwise folium-like loops, which implied a shift from lag to lead at 3.0 cps. Introduction of appropriate lags brought points acceptably close to straight, positively sloped segments: this occurred often at 0.2 cps, sometimes at 1.0 cps, and exceptionally at 3.0 cps. Jitter altered the variability profile along the cycle, decreasing peak variability and not affecting or increasing the minimum; variability along the cycle was thus reduced and uniformed and identification of stimuli on the basis of discharges was improved. Jitter amplitude was an important issue. The larger ones, about the modulation size, increased the lowest bin-rates, particularly at 0.2 and 1.0 cps, and increased the peaks at 0.2 cps, decreasing them at 1.0 and 3.0 cps. The smaller jitter, about 1/10 of the modulation, affected the lowest rates little, but increased the peak rate at 0.2 cps, decreasing it at 1.0 and 3.0 cps; display shapes changed little. A qualitative model of the SAO involves subsystems with linear features, as well as rectification, stiction-like and saturation-like properties. Depending upon back-ground length, modulation depth, jitter and several other issues, the organ can perform frankly nonlinearly, piece-wise linearly or linearly. Within each context, a particular stimulus is of interest and can be referred to as the signal; others are not, and can be referred to as noise, acting as perturbations. Small, erratic perturbations influence strongly transduction of large steps or sines, simplifying and making it more proportionate. Large regular perturbations act upon small signals too, but their influence has been explored less extensively.Supported by grants from NIH and NSF     

The addition of Dasypyrum villosum (L.) Candargy chromosomes to durum wheat (Triticum durum Desf.)

Summary Six monosomic addition lines were produced in which different Dasypyrum villosum (L.) Candargy chromosomes were added to the chromosome complement of Triticum durum Desf. cv. Creso. Each added alien chromosome was found to have a specific effect on plant morphology and fertility. Transmission rate varied widely (from 7.5 to 27.7%) among the six univalent chromosomes. Different monotelosomic addition plants derived by a relatively high frequency of chromosome misdivision were isolated. The addition lines should be useful for studying Dasypyrum chromosome homoeology and the introduction of alien variation into durum and common wheats.Research supported by a grant from the Italian Research Council for Finalized Project IPRA. Sub-project Plant Breeding, Paper No. 1095     

Zur Dynamik feiner Pseudopodien von Hochmoor-Amöben

Zusammenfassung Es werden verschiedene Vorgänge an sehr zarten Pseudopodien beschrieben und mit Hilfe der an submikroskopischen Fibrillen (schraubig gebauten Mikrofibrillen und Mikroröhrchen der Elektronenmikroskopie) auftretenden Torsionsspannungen und Rotationen gedeutet.

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On the dynamics of slender pseudopodia of bog amebae

Summary Different processes which occur on very slender pseudopods are described. They are interpreted with the help of torsional forces and revolutions of the submicroscopic fibrils (helical microfibrils and microtubules in electron microscopy).

     

Clonal propagation of Callistemon by tissue culture techniques

Callus development in Callistemon viminalis was readily achieved when axillary buds derived from nodal tissue were placed in a medium containing macro- and micro-nutrients, sucrose (0.06 M), inositol (300 M), nicotinic acid (20 M), pyridoxine hydrochloride (3 M), thiamine hydrochloride (2 M), riboflavin (10 M), cytokinins (5 M) and auxins (0.1 M). The presence of benzylaminopurine (5 M) and p-chlorophenoxyacetic acid (0.1 M) promoted the most vigorous callus development and sprout formation. Rooting of nodal material was rare but occurred readily following the transference of sprouts developed on callus to a basal medium containing sucrose and salts. Root initiation was stimulated, however, by the presence of auxins. Chlorophenoxyacetic acid while stimulating root initiation repressed root growth. Indole butyric acid stimulated both root initiation and shoot growth at concentrations of 0.005 to 0.1 M. The treatment of choice for rooting and shoot growth was the addition of indole butyric acid at a concentration of 0.01 M.     

Structural studies of the glycopeptides of B-chain of cinnamomin – a type II ribosome-inactivating protein by nuclear magnetic resonance

Cinnamomin is a plant type II ribosome-inactivating protein (RIP) isolated from the seeds of Cinnamomum camphora. It consists of two nonidentical polypeptide chains (A- and B-chain) held together through one disulfide linkage. Its A- and B-chain contain 0.3% and 3.9% sugars respectively. The B-chain of cinnamomin was digested by pronase E and then the liberated glycopeptides were separated from non-glycopeptides by gel filtration chromatography on a Bio-Gel P-4 column. Three crude glycopeptides were obtained by continuing chromatography over anion-exchange resin (AG1-X2) in the buffer of 2% pyridine-acetic acid (pH 8.3) with a polygradient elution system. Through further purification by the gel filtration chromatography and HPLC, three major glycopeptides, GP1, GP2 and GP3 were obtained. Mainly by two-dimensional Nuclear Magnetic Resonance (NMR) including TOCSY, DQF-COSY, NOESY, HMQC and HMBC, their primary structures were analyzed as: Man1,3Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4GlcNAc1-(Gly-)Asn-Asn-Thr(GP1), Man1,6(Man1,3)(Xyl1,2)Man1,4GlcNAc1,4(Fuc1,3)GlcNAc1-Asn-Ala-Thr(GP2),Man1,6(Man1,3)Man1,6(Man1,2 Man1,3)Man1,4GlcNAc1,4GlcNAc1-(Ala-)Asn-Gly-Thr(GP3).     

A comparison of male and female recombination frequency in wheat using RFLP maps of homoeologous group 6 and 7 chromosomes

A novel approach was used to compare male and female recombination rates in wheat. Doubled haploid lines were developed from an F₁ using two distinct approaches: the anther-culture technique and the Hordeum bulbosum system, from which sets of lines were developed from male and female meioses, respectively. The genotype of the lines was established at RFLP and isozyme markers polymorphic on chromosomes of homoeologous groups 6 and 7, and male and female linkage maps were calculated using this information. The markers in one segment of chromosome 6B exhibited disturbed segregation frequencies in the anther-culture population. The male and female maps differed significantly in recombination frequency between some markers on two chromosomes, and these were consistent in direction within chromosomes and inconsistent in direction between chromosomes. In two of the four chromosomes studied the male map was much longer than the female map. These results suggest that significant differences may exist in male and female recombination frequencies in bread wheat which are specific to certain chromosomal segments but are inconsistent in direction between chromosomes. Other factors, such as environmental influences, may also be important in creating differences.     

Coiled mechanoreceptors inAplysia revealed by sensorin immunofluorescence and confocal microscopy

Identified mechanosensory neurons ofAplysia are established model neurons for studies on learning and memory, and for examining responses to axonal injury. Although many characteristics of these sensory neurons have received intensive study, the nature of the peripheral mechanoreceptive endings remains unknown. Identification of a peptide, sensorin, specific inAplysia for mechanosensory neurons, led to the development of an antibody which proved useful in studying the peripheral morphology of these neurons. Immunostaining for sensorin in tail body wall revealed that sensorin is present in peripheral arborizations. Examination of sensorin-positive fibers in the periphery revealed that they terminate as coiled structures in the muscle layer of the body wall. These coiled structures (0.5 m diameter processes, 2–3 m across the coil, 60) m long) run parallel to muscle fibers and have a pitch of about one turn per 4 um. Sensorin immunostaining was particularly intense in varicosities, both along peripheral fibers and along the coiled structure. The localization of sensorin suggests that it may be released peripherally where it could have various paracrine and/or autocrine neuromodulatory actions.     

Thyroid hormone regulation of Na,K-ATPase subunit-mRNA expression in neonatal rat myocardium

Summary Regulation of Na,K-ATPase mRNA isoform and mRNA expression by thyroid hormone (T₃) in neonatal rat myocardium was examined. In euthyroid neonates between ages of 2 and 5 days, mRNA₁, mRNA₃, and mRNA₁ abundances were nearly constant while mRNA₂ was undetectable. During the interval between postnatal days 5 and 15, mRNA₃ decreased to negligible levels and mRNA₂ became expressed and increased in abundance to account for 20% of the mRNA pool by the 15^th postnatal day. To examine the effect of T₃ on this developmental program, neonates were injected with 75 g T₃/100 g body weight or diluent alone on the second and third postnatal days and myocardial Na,K-ATPase subunit-mRNA abundances were determined on the third and fourth postnatal days. Because T₃ treatment increased the RNA/DNA ratios of myocardial tissue, the subunit-mRNA abundances were normalized per unit DNA. Following 24 and 48 hr of T₃ treatment, the abundances of mRNA₁, mRNA₃, and mRNA₁ increased, while mRNA₂ continued to remain undetectable during the 2-day interval between the second to fourth postnatal days. It is concluded that T₃ augments the abundance of Na,K-ATPase subunit mRNAs that are already being expressed in the neonatal rat myocardium. The results further suggest that T₃ does not act as a molecular switch in the developmental expression of the mRNA isoforms in rat myocardium during the first four postnatal days.     

DNA homology and adsorption specificity of Pseudomonas aeruginosa virulent bacteriophages

Summary The DNA homology and adsorption specificity of newly isolated virulent bacteriophages of P. aeruginosa have been studied. On the basis of this analysis all phages were divided into four groups: k, m, mnP78-like and mnF82-like bacteriophages. DNA's of k as well as m phages were shown to possess different restriction patterns although they have an extensive homology. Unlike other groups, k phages were characterized by the presence of T4 DNA ligase-repaired, single-chain breaks.Abbreviations kbp kilobase pairs - EM electron microscopy     

The effect of ferrous sulphate on the yield and manganese uptake of oats on sandy soil fertilized with pyrolusite

Summary In pot experiments with oats on a sandy soil deficient in managanese and with an extremely low content of reducible managanese, the effect of 0.125 g reducible managanese in two pyrolusite fertilizers (-managanese dioxides) made in 1938 and 1955, respectively, was compared with the effect of 0.125 g water-soluble managanese as managanese sulphate.On soil to which no ferrous sulphate was added, the response to manganese added as Pyrolusite 1938 and Pyrolusite 1955 was of the same magnitude, but slightly less than the response to managanese added as managanese sulphate. The difference between the effect of pyrolusite and that of managanese sulphate was not, however, statistically significant.On soil to which ferrous sulphate had been added, a response was obtained to Pyrolusite 1938 twice as high, and to Pyrolusite 1955 three times as high as the response to manganese sulphate. These results support the suggestion² that the manganese effect of ferrous sulphate is due to its ability to reduce higher managanese oxides in the soil.Since soil dressed with ferrous sulphate gave a response to added Pyrolusite 1955 which was about 50 per cent higher than the response to Pyrolusite 1938, and since, furthermore, the same amount of reducible manganese and the same number of manganese atoms on the surface of the two fertilizers were involved, it is probable that the difference between the effects of the two pyrolusite fertilizers found under these circumstances is due to difference in their crystallinity.     

Orientation of the porphyrin ring in artificial chlorophyll membranes

Summary A sensitive photometric method is described by which the dichroism of lipid bilayer membranes in aqueous phase can be measured. The method is applied to black films with incorporated chlorophylla andb. With chlorophylla a relatively large dichroism is found in the Soret band and a much weaker dichroism in the red band. From the experimental data, the angles _B and _R between the blue and red transition moments and the membrane can be obtained. _B and _R are then used to calculate the angle of the porphyrin ring with respect to the membrane surface. For chlorophylla and three different lipids, values of between 44 and 49° are found.     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

Lipid profile of rat myelin subfractions

Myelin from adult rat brains was separated on a discontinuous sucrose gradient into three subfractions. Analysis of light, heavy and membrane fraction lipid classes was performed by HPTLC and densitometry while fatty acid composition was determinated by GLC. The more interesting results observed are: i) the membrane fraction resembles in its lipid and fatty acid composition other cell membranes (particulary oligodentrocytes); ii) light and heavy myelin are quite similar between them but the former has a higher content of sphingomyelin, a lower hydroxy/nohhydroxy cerebrosides ratio and a lower content of monoenoic fatty acids than the heavy subfraction. The results obtained could explain the different structures observed in each myelin subfraction since fatty acid composition, hydroxy fatty acids, sphingomyelin and cholesterol play a key role in the stability and structure of membranes.