Analysis of stimulatory and inhibitory amino acids for development of hamster one-cell embryos in vitro

Hamster embryo development to the blastocyst stage in vitro can be modulated by amino acids. This series of experiments employed both empirically and statistically designed approaches to elucidate which of 20 amino acids inhibit or stimulate development and to devise a complement of amino acids that best supports in vitro development of hamster 1-cell embryos. Development and/or mean cell number were significantly inhibited by the presence of leucine, tyrosine, valine, isoleucine, phenylalanine, arginine, methionine, or cysteine (at 0.5 mM) and isoleucine, phenylalanine, or tryptophan (at 0.05 mM). Three amino acids—glutamine, taurine, and glycine—were stimulatory and in combination improved development; the culture medium containing these amino acids was designated Hamster Embryo Culture Medium-5. Moreover, addition of another eight amino acids—asparagine, aspartic acid, serine, glutamic acid, histidine, lysine, proline and cysteine (medium designated HECM-6)—had a significant stimulatory effect on development over previously formulated culture media for hamster embryos. These results demonstrated that amino acids, alone and in combination, can markedly stimulate or inhibit hamster embryo development in vitro up to the blastocyst stage. Embryo transfer experiments showed that HECM-5 and ?6 (chemically defined, protein-free culture media) supported normal preimplantation embryo development in vitro. This study also indicates that empirically designed embryo culture media formulations can be as effective as those obtained by application of statistical methodologies. © 1995 wiley-Liss, Inc.     

Kinetics of the Competitive Degradation of Deoxyribose and Other Molecules by Hydroxyl Radicals Produced by the Fenton Reaction in the Presence of Ascorbic Acid

The competition method in which the Fenton reaction is employed as an OH radical generator and deoxyribose as a detecting molecule, has been used to determine the rate constants for reactions of the OH radical with its scavengers. Nonlinear competition plots were obtained for those scavengers which reacted with the Fenton reagents (Fe²⁺ or H₂O₂). Ascorbic acid is believed to overcome this problem. We have investigated the kinetics of deoxyribose degradation by -OH radicals generated by the Fenton reaction in the presence of ascorbic acid, and observed that the inclusion of ascorbic acid in the Fenton system greatly increased the rate of OH radical generation. As a result, the interaction between some scavengers and the Fenton reagents became negligeable and linear competition plots of A7A vs scavenger concentrations were obtained. The effects of experimental conditions such as, the concentrations of ascorbic acid, deoxyribose, H₂O₂ and Fe²⁺-EDTA, the EDTA/Fe²⁺ ratio as well as the incubation time, on the deoxyribose degradation and the determination of the rate constant for mercaptoethanol chosen as a reference compound were studied. The small standard error, (6.76± 0.21) ±' 10⁹M^-1s^-1 observed for the rate constant values for mercaptoethanol determined under 13 different experimental conditions, indicates the latter did not influence the rate constant determination. This is in fact assured by introducing a term, k_x, into the kinetic equation. This term represents the rate of-OH reactions with other reagents such as ascorbic acid, Fe²⁺-EDTA, H₂O₂ etc. The agreement of the rate constants obtained in this work with that determined by pulse radiolysis techniques for cysteine, thiourea and many other scavengers, suggests that this simple competition method is applicable to a wide range of compounds, including those which react with the Fenton reagents and those whose solubility in water is low.     

Ultrafast protein determinations using microwave enhancement

We present here microwave-based modifications of standard protein assays that dramatically reduce the time required to determine protein concentrations. Typical protein determinations involve incubation times ranging from 15–60 min. Microwave irradiation of specimens reduces this time requirement to 10–20 s without compromising accuracy or reliability. The remarkable speed with which protein determinations may be carried out using microwave enhancement greatly simplifies general laboratory procedures that depend on the estimation of protein concentrations. An erratum to this article is available at .     

Germination ofMusa velutina seeds: Comparison ofin vivo andin vitro systems

Summary In the genusMusa, germination is extremely variable and relatively difficult. Even more difficulties are faced when producing hybrids. The seed yield of hybrids in breeding programs is usually low and often, to ensure the viability and survival of seeds, it is necessary to attempt to germinate a large excess of these seeds. In this context,in vitro embryo culture might be an invaluable tool for obtaining desirable hybrid plants in a short time. Seeds ofMusa velutina were sown in seed trays in a peat-based mixture. Thein vivo seed germination reached 78% but only after 9 mo. Because of this delayed and intermittent germination, embryos were excised from seeds and inoculated onto half-strength Murashige and Skoog (1962) medium, with or without supplementation with various concentrations of gibberellic acid. Light and dark conditions were also used to test their effect on embryo germination. After 2 wk, 82% of embryos germinated in the dark on medium containing 0.1 μM gibberellic acid. Addition of gibberellic acid increased the shoot length and root number over the gibberellic acid-free treatment. Similarly, dark conditions gave a significant increase over light conditions for all the parameters except root number where light or dark conditions did not make any difference. Thus, the present study highlights the importance of various components of thein vitro culture ofMusa embryos and the advantage over direct use of greenhouse-sown seeds both in terms of the time taken to germinate and the final percentage.     

Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

Stress-elicited changes in amino acid levels of hemolymph of Aplysia californica

The effects of stress produced by noxious cutaneous stimulation on the concentration of 18 amino acids in the hemolymph of Aplysia californica were examined. The concentration of alanine and glutamate increased in response to stress and remained elevated for at least 6 h. The total amino acid concentration in the hemolymph did not change significantly in response to stress. It is concluded that stress evokes an increase in circulating alanine and glutamate levels. These factors may then act in a paracrine, endocrine, or neuroendocrine manner to modify stress-associated behaviors.Abbreviations AA amino acids(s) - bw body weight - SEM standard error of the mean - SW sea water     

Characterization of somatostatin receptor subtypes controlling rat gastric acid and pancreatic amylase release

An examination of the binding characteristics of a large number of somatostatin analogues with respect to the five known somatostatin receptor subtypes has recently resulted in the discovery of several peptides with some selectivity for types 2, 3, and 4 and little affinity for type 1 or 5 receptor. A panel of these peptides has thus far implicated type 2 receptors in the inhibition of release of pituitary growth hormone and type 4 receptors in inhibiting pancreatic insulin release. In the present article, we have examined the inhibitory effects of the same group of peptides on in vivo rat gastric acid and pancreatic amylase release and binding to rat pancreatic acinar cells. The type 2-selective ligand NC-8–12 was a potent inhibitor of gastric acid release (EC₅₀s in the 1.5 nM region) whereas the type 4-selective ligand, DC-23–99, elicited little response. However, some involvement of type 3 receptors could not be ruled out because the type 3-selective analoueg, DC-25–20, exhibited inhibitory effects at higher dose levels (EC₅₀ > 10 nM). Conversely, the type 4 analogue was a potent inhibitor of amylase release (EC₅₀ 1.1 nM) whereas the type 3 analogue had no significant effects at doses tested. DC-23–99 also bound with high affinity to rat acinar cells (EC₅₀ 3.8 nM), whereas DC-25-20 exhibited more than 10-fold less affinity. Thus, these two major biological functions of somatostatin appear to be controlled by different receptors and, furthermore, effects on both endocrine and exocrine pancreas appear to be type 4 receptor mediated.     

Developmental changes in the brain-stem serotonergic nuclei of teleost fish and neural plasticity

Summary 1. During early ontogeny, the serotonergic neurons in the brain stem of the three-spined stickleback shows a temporal and spatial developmental pattern that closely resembles that of amniotes.2. However, in the adult fish, only the midline nuclei of the rostral group (dorsal and median raphe nuclei) and the dorsal lateral tegmental nucleus are consistently serotonin-immunoreactive (5-HTir), whereas the groups of the upper and lower rhombencephalon (raphe pontis, raphe magnus, and raphe pallidus/obscurus nuclei) are variable and, when present, contain relatively small numbers of 5-HTir neurons.3. Using specific antisera against tryptophan 5-hydroxylase and aromaticl-amino acid decarboxylase, we have shown that the lateral B9 group and the groups of the upper and lower rhombencephalon are consistently present in adult sticklebacks. The results are discussed in relation to other known instances of neurotransmitter plasticity or transient neurotransmitter expression in teleost fish.4. While there are several instances of transient expression of neurotransmitter markers by discrete neuronal populations, there is so far no evidence of changes from one neurotransmitter phenotype to another in the brain of teleost fish. However, there are indications of plasticity of expression of catecholamines and indoleamines, and their respective synthesizing enzymes, as reflected in age-dependent changes and variation between individuals of different physiological status.5. As the brain grows continuously in teleost fish, and new neurons are added from proliferative regions, synaptic connections may be expected to undergo remodeling in all brain regions throughout life. Thus, the teleostean brain may be considered a suitable model for experimental studies of different aspects of neural plasticity.     

Homology between the seed cytolysin enterolobin and bacterial aerolysins

Enterolobin, a 55-kDa cytolytic, inflammatory, and insecticidal protein isolated from seeds of the Brazilian treeEnterolobium contortisiliquum (Leguminosae-Mimosoideae) has been further purified and partially sequenced by using both manual and automated methods. A computational search of enterolobin partial amino acid sequence against the PIR database revealed possible sequence similarities with aerolysins, cytolytic proteins fromAeromonas species. An alignment of enterolobin partial sequence to the amino acid sequences ofA. hydrophila andA. sobria aerolysins showed several similar regions with many residue identites. The seed protein enterolobin and the bacterial aerolysins may be homologous proteins despite the distant phylogenetic relationship.     

Immunoreactivity between a monoclonal lupus autoantibody and the arginine/aspartic acid repeats within the U1-snRNP 70K autoantigen is conformationally restricted

Immunoreactivity of the arginine/aspartic acid (RD) repeats of the 70K protein of U1 small nuclear ribonucleoprotein (snRNP) was determined to be conformationally dependent. The monoclonal autoantibody 2.73, isolated from a lupus-prone MRL/n mouse model, is reactive with the RD repeat regions of U1 snRNP 70K protein. Immunochemical analysis of the antigenic determinants with use of chemically synthesized peptides characterized the 2.73 epitope as the RD repeat [Pelsue, S.,et al. (1993)Autoimmunity,15, 231–236] Analysis by circular dichroism (CD) and nuclear magnetic resonance spectroscopy indicates conformational preferences in the immunoreactive peptides. Computer analyses of CD spectra obtained on the RD-containing peptides predict-turns and-sheet to be the preferred conformations of the RD repeats. This structure was also predicted by the Chou-Fasman algorithm. The RD repeat is believed to be a conserved structural motif; however, the biological function is still unclear. Immunological and biochemical analysis of autoimmune antibodies and their respective antigenic determinants has helped to characterize the possible mechanisms that lead to autoimmune diseases. This is the first report of a conformationally dependent, linear epitope of an autoantibody.