Synthesis and comparative conformational energetics of D-phenylalanylsarcosine and its cyclic dehydration product, (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione

Summary Synthetic protocols are presented both for D-PheSar and the corresponding cyclised diketopiperazine, prepared from N-t-butoxycarbonylprotected D-PheSar. Deprotection conditions could be manipulated to yield either D-Phenylalanylsarcosine or (R)-1-methyl-3-(phenylmethyl)-2,5-piperazinedione. Molecular modelling revealed several low energy conformers which contained a Z-peptide bond and which were readily amenable to cyclisation. Cyclisation was found by HPLC to be fastest in strongly acidic conditions.Abbreviation HBTU o-Benzotriazolyl-tetramethyluronium hexafluorophosphate     

AN ASSESSMENT OF CHANGES WITH TIME IN THE MARKING PATTERNS USED FOR PHOTOIDENTIFICATION OF INDIVIDUAL SPERM WHALES, PHYSETER MACROCEPHALUS

We observed changes with time in the patterns of characteristic fluke markings used to identify sperm whales. Changes were categorized as minor, moderate, or major based on their severity. These change types were found to occur at rates of 0.9%, 11.8%, and 1.3% per individual per year, respectively. Gain and loss rates for each of seven different mark types were also calculated. The highest estimated rate was the gain of small nicks at 0.08 per individual per year. Most individuals identified by us possess at least a few characteristic marks and, therefore, changes of the type observed in this study are unlikely to severely affect their recognizability. For all but one mark type, gain rates were higher than loss rates, indicating that individuals may be accumulating marks with age. Over long periods this could eventually make individuals unrecognizable, with the result that population sizes calculated from these data may be overestimated. As long as photoidentification studies are conducted sufficiently often, and these changes are as gradual as they appear to be, this problem should be minimal.     

Influence of arbuscular mycorrhizae on the metabolism of maize under drought stress

A greenhouse experiment was carried out to investigate the influence of the arbuscular mycorrhizal (AM) fungus (Glomus intraradices Schenck & Smith) on metabolic changes in tropical maize (Zea mays L.) under drought. Two cultivars, Tuxpeno sequia CO (drought sensitive) and C8 (drought resistant), were subjected for 3 weeks to water stress following tasselling (75–95 days after sowing). Fully expanded 7th or 8th leaves were sampled and assessed for levels of chlorophyll, sugars, proteins, and amino acids. Chlorophyll content was not altered either by water stress or the presence of mycorrhizae. Mycorrhizal plants (M+) had higher total and reducing sugars than nonmycorrhizal plants (M-) at the end of 3 weeks of the drought cycle. An increase in protein content was observed with drought stress in M + plants of the cultivar C0. Most of the amino acids showed a linear increase during the period of water stress in M+ and M- plants for both cultivars. Total amino acids increased by 40.6% and 43.7% in M- plants of C0 and C8, respectively. With the presence of AM fungus, amino acid levels increased by only 10.7% and 19.2% of leaf dry mass in C0 and C8, respectively. Alanine, asparagine, glutamine, and glycine accounted for 70% of the amino acid pool. Under drought, AM inoculation enabled the plants to retain considerable amounts of sugars and proteins, especially in the drought-sensitive cultivar C0. This may be of physiological importance in helping the plant to withstand moderate drought.     

Thermodynamics of heme-induced conformational changes in hemopexin: role of domain-domain interactions.

Hemopexin is a serum glycoprotein that binds heme with high affinity and delivers heme to the liver cells via receptor-mediated endocytosis. A hinge region connects the two non-disulfide-linked domains of hemopexin, a 35-kDa N-terminal domain (domain I) that binds heme, and a 25-kDa C-terminal domain (domain II). Although domain II does not bind heme, it assumes one structural state in apo-hemopexin and another in heme-hemopexin, and this change is important in facilitating the association of heme-hemopexin with its receptor. In order to elucidate the structure and function of hemopexin, it is important to understand how structural information is transmitted to domain II when domain I binds heme. Here we report a study of the protein-protein interactions between domain I and domain II using analytical ultracentrifugation and isothermal titration calorimetry. Sedimentation equilibrium analysis showed that domain I associates with domain II both in the presence and absence of heme with Kd values of 0.8 microM and 55 microM, respectively. The interaction between heme-domain I and domain II has a calorimetric enthalpy of +11 kcal/mol, a heat capacity (delta Cp) of -720 cal/mol.K, and a calculated entropy of +65 cal/mol.K. By varying the temperature of the centrifugation equilibrium runs, a van't Hoff plot with an apparent change in enthalpy (delta H) of -3.6 kcal/mol and change in entropy (delta S) of +8.1 cal/mol.K for the association of apo-domain I with domain II was obtained.(ABSTRACT TRUNCATED AT 250 WORDS)     

Determination of the glucosidase-stimulating proteins by competitive enzyme-linked immunoassay

A procedure for the immunoassay of cohydrolase sphingolipid-I in mouse tissue is described. This cohydrolase (actually a mixture of at least four related proteins) stimulates or activates the beta-glucosidase which hydrolyzes ceramide glucoside, a widely occurring glycosphingolipid. The method involves extraction of cohydrolase from tissue homogenate with a salt-buffer solution, removal of proteins by adjustment to pH 6, further removal of proteins by heating, and removal of interfering materials with a small size exclusion column. Antibodies were raised to bovine cohydrolase in rabbits and purified with an affinity column made from cohydrolase. The immunoassay involves binding of antibody by the cohydrolase sample (20-200 pg) in competition with cohydrolase that has been chemically linked to horseradish peroxidase. The mixture is treated with particle-linked second antibody and centrifuged; the pellet is then assayed fluorometrically for peroxidase content. Initial application of the method showed that cohydrolase was present in all mouse tissues studied and that its concentration paralleled that of glucocerebrosidase relatively closely. Changes with age (14 and 92 days) occurred in a similar fashion for the two substances.     

Dynamics of the cytoskeleton inAmoeba proteus: II. Influence of different agents on the spatial organization of microinjected fluorescein-labeled actin

Summary Iodoacetamido-fluorescein-(IAF)-labeled actin was microinjected into normal locomotingAmoeba proteus. Thereafter (30–60 minutes) changes in the cytoplasmic fluorescence distribution pattern and contractile activity were induced by internal and external chemical stimulation. Different agents such as phalloidin, procaine, 2.4-dinitrophenol (DNP), puromycin, ouabain and n-ethyl maleimide (NEM) interfere with the excitation-contraction mechanism involved in ordered pseudopodium formation during ameboid movement and cause various morphogenetic reactions based on actin polymerization-depolymerization cycles. Most frequent changes are (a) local condensation of IAF-actin and formation of a continuous IAF-actin layer at the cytoplasmic surface of the cell membrane and around the pulsating vacuole, (b) immobilization and hyalo-granuloplasm separation by combined contraction and detachment of the IAF-actin layer from the cell membrane, (c) organized and disorganized formation of pseudopodia by local contraction and disintegration of the IAF-actin layer, and (d) alterations in the rheological properties of the protoplasmic matrix by changes in the molecular state of soluble actin not incorporated into the cytoskeleton. The experimental approaches to the function of the actomyosin system in large amebas attainable by the method ofin vivo molecular cytochemistry are discussed in detail with respect to the participation of the cytoskeleton in motive force generation for cytoplasmic streaming and ameboid movement.     

Problems in Fitting a Cosine Curve: Short Communications

In fitting of cosine curves latent experimental inequalities due to a serial effect have to be excluded. Though cosinor analysis may be sufficient then, inclusion of biological time, i.e. not fitting values to time but to a function of time, will lead to further improvement.     

Use of proton Nuclear Overhauser Effects for the determination of the conformations of amino acid residues in oligopeptides

The use of the Nuclear Overhauser Effect to determine backbone and side-chain conformations of oligopeptides is discussed. The distance between the H^α proton of a given residue and the amide proton of the following residue depends only on the dihedral angle ψ. A calibration curve is given for the determination of ψ from the Nuclear Overhauser Effect involving these protons. In amino acids with branched side chains, e.g., threonine, isoleucine, and valine, the Nuclear Overhauser Effect involving the H^β proton and the amide proton in either the same or the following residue gives limited information about both χ¹ and either or ψ. The Nuclear Overhauser Effect involving the H^α and H^γ protons in leucine gives information about χ¹ and χ².     

Effect of L-cystine on macromolecular changes during spore and parasporal crystal formation inBacillus thuringiensis var.thuringiensis

High concentration of L-cystine (0.25%) when present in a glucose-mineral salt medium inhibited sporulation-specific events like protease production, calcium uptake and dipicolinic acid synthesis inBacillus thuringiensis var.thuringiensis. In addition, the enzymes of the Krebs cycle from aconitase onwards were completely inhibited by a high concentration of cystine. At a low concentration of cystine (0.05%), none of the above mentioned macromolecular changes were affected. Lipid synthesis monitored by [1,2¹⁴ C]-acetate incorporation into lipid as well as into whole cells was completely inhibited.     

Ultrastructural changes in the urophysis of Mollienesia sphenops following adaptation to seawater

Summary The urophysis or neurohemal contact site of the caudal neurosecretory system of Mollienesia sphenops, the black molly, was studied in animals adapted to an artificial seawater environment. This species of fish was chosen for these studies because of its known ability to osmoregulate and its adaptability to the laboratory aquarium. The urophysis of freshwater acclimated mollys contained an abundance of neurosecretory granules. However, in fish subjected to a seawater environment for one week the number of neurosecretory granules was significantly decreased. In addition, there was an increase in blood cell infiltration of the urophysis.Supported by PHS 5429-16-19 (5-23311)The Author wishes to thank Drs. W. Young and J. Wells for their careful reading of this report and W. Boldosser for technical assistance