Analysis of extractables from one euphorbia

Chemical analyses have been made of the heptane extractable material ofEuphorbia lathyris, a plant which has been proposed as an “energy farm” candidate. The heptane extract is 4–5% of the dry plant weight and has a heat value of ≈18×10³ BTU/Ib. This reduced photosynthetic material consists almost entirely of polycyclic triterpenoids.     

Liquid fuels fromMesua ferrea L. seed oil

Mesua ferrea L. seed oil consists of triglycerides of linoleic, oleic, palmitic and stearic acids. These acids were pyrolyzed separately in the presence of different amounts of solid sodium carbonate. Pyrolysis experiments revealed that linoleic and oleic acids can be converted to hydrocarbons of a wide range of molecular weights by pyrolyzing them with even 1% by wt of sodium carbonate up to a temperature of 500°C, whereas palmitic and stearic acids can be converted to hydrocarbons only by pyrolyzing them with equivalent amounts or more of sodium carbonate up to a temperature of about 650°C. The fractions of boiling range 60–320°C of all of the pyrolytic oils were analyzed for their hydrocarbon types by the method of fluorescent indicator adsorption (FIA). The aromatic contents of the pyrolytic oils of linoleic and oleic acids were found to be much higher than those of palmitic and stearic acids. GS and GC-MS analyses of all the saturate fractions indicated mainly normal alkanes with a carbon number range of 6 to 17.     

Hepatic lipase activity influences high density lipoprotein subclass distribution in normotriglyceridemic men. Genetic and pharmacological evidence

Inhibin is a heterodimeric glycoprotein originally detected in gonadal tissues. One report described inhibin immunopositivity in 17 of 19 hepatocellular carcinomas (HCCs) and the hepatocytes of the surrounding nonneoplastic parenchyma. The reported immunohistochemical method, which used avidin-biotin complex, did not describe blocking endogenous biotin. Since liver tissue may contain high levels of biotin, endogenous biotin may result in false-positive immunostaining. We wondered whether this reported immunopositivity represented a false-positive result due to unblocked endogenous biotin. By using a similar antigen retrieval technique and the same specificity, titer, and clonal source of primary antibody as the aforementioned study, we performed immunostaining for inhibin with and without an endogenous biotin blocking step on 23 cases of HCC and the surrounding cirrhotic liver. In all cases, the HCC and the hepatocytes in the cirrhotic nodules were negative for inhibin when the endogenous biotin blocking step was used. When the blocking step was omitted, apparent immunostaining was noted in 20 of 23 HCCs and in the hepatocytes in all cases. Accordingly, HCC and the hepatocytes of the surrounding cirrhotic liver are immunohistochemically negative for inhibin. The previously reported immunopositivity of HCC and nontumoral hepatocytes for inhibin represents a false-positive result due to endogenous biotin.     

Poly (DL-lactide-co-glycolide) microspheres as carriers for peptide vaccines

Peptides carrying an immunodominant T-helper cell epitope delineated from the rabies virus nucleoprotein either alone or in combination with a linear B-cell epitope from the same protein were incorporated into three different formulations of poly(DL-lactide-co-glycolide) (PLG) which were distinct in their composition, and consequently in their peptide release rates. In vitro peptides incorporated into any of the PLG formulations stimulated a peptide-specific T-cell line. Upon subcutaneous immunization of mice, the PLG formulation that showed the fastest peptide release rate induced the best immune response. This immune response was in magnitude comparable or even superior to that induced by peptide emulsified in complete Freund's adjuvant.     

Conformational studies by NMR of the antimicrobial peptide, drosocin, and its non-glycosylated derivative: effects of glycosylation on solution conformation

Drosocin is a cationic 19 amino acid peptide secreted by Drosophila in response to septic injury. The sequence (GKPRPYSPRPTSHPRPIRV) contains six Pro and four Arg residues which are incorporated into three repeated triplet sequences Pro-Arg-Pro. The peptide is glycosylated at Thr11 and has potent antimicrobial activity. This activity is markedly reduced on deglycosylation, but a structural basis for this has not been previously established. In the current study, the solution conformations of drosocin and its non-glycosylated derivative were determined by NMR spectroscopy and structure calculations. The NMR and structure studies showed that the peptides have significant populations of essentially random coil conformations in aqueous solution. Addition of 50% trifluoroethanol causes the development of small populations of folded conformations, mainly in the form of turns. In particular, turn elements occur near residues 4-7, 10-13, 17, and 18. No substantial difference was detected in the predominantly random coil conformation of the glycosylated and non-glycosylated forms, but there are subtle differences in the small populations of folded conformers. In particular, the turn at residues 10-13 tends toward a more extended structure on glycosylation, while there is some tightening of the downstream turn at residues 17 and 18. There are a significant number of nuclear Overhauser enhancement contacts between the sugar moiety and the peptide near the glycosylation site, consistent with a close association between them. Despite this close association, the pKa of H13, which is proximate to the glycosylation site, was found to be unaffected by glycosylation.     

Physical properties of a single-motif erythrocyte spectrin peptide: a highly stable independently folding unit

Spectrin is a long flexible rod-like actin cross-linking protein mostly comprised of many tandem homologous 106-residue motifs. In this study, the conformational stability and physical properties of a single homologous motif peptide, alpha1, were evaluated and compared to intact spectrin monomers and alphabeta heterodimers. It is interesting that while spectrin dimers elongate by about 3-fold in low ionic strength buffers relative to their size in physiological buffers, the single-motif peptide does not show significant changes in secondary structure in 10 mM phosphate buffer compared with isotonic buffer. This single-motif peptide is monomeric in physiological buffer as demonstrated by equilibrium sedimentation studies, and its hydrodynamic radius determined by gel filtration and dynamic light scattering of about 2.2 nm is consistent with an elongated rod-like shape. Unfolding of the single-motif peptide in urea solutions was similar to unfolding of intact heterodimers. Differential scanning calorimetry analyses showed that this single motif undergoes a reversible two-state transition with a Tm of 53 degrees C and an enthalpy of 65 kcal/mol in physiological buffer. Thermal stability was unaffected by ionic strength changes, but was decreased below physiological pH. These data show that this 13 kDa spectrin motif is a monomeric, highly stable, triple-helical, independently folding protein building block with physical characteristics that define many of the structural properties of the 526 kDa spectrin heterodimer. In contrast, interactions between adjacent motifs are probably responsible for spectrin's molecular flexibility and elasticity.     

A monoclonal antibody to a multiphosphorylated, conformational epitope at the carboxy-terminus of p53

Mutations of the gene encoding the tumor suppressor protein p53 are the most common molecular alterations of cancer cells found in about half of all human tumors. Mutations which cluster in well-defined hot spots change the structure of the protein thus affecting its ability to bind to DNA. Post-translational modifications, primarily phosphorylation, might also influence how p53 binds to DNA or folds to its active tetrameric form. However, the lack of appropriate biochemical markers to characterize the status of phosphorylation in different cell types and in cells at different stages of tumor progression has prohibited such investigations. To generate a sensitive and phosphorylation-specific monoclonal antibody (mAb), we chemically synthesized the C-terminal 23 amino acid stretch of human p53 in a double-phosphorylated form. The peptide 371-393, carrying phosphate groups on Ser378 and Ser392, was co-synthesized with a turn-inducing spacer and peptide 31D, an immunodominant T-helper cell epitope in mice of the H-2k haplotype. After immunization and fusion of splenocytes with myeloma cells, a number of mAbs were obtained, from which mAb p53-18 emerged as a highly sensitive reagent. By enzyme-linked immunosorbent assay, p53-18, a mAb of the IgM isotype, recognized phosphorylated p53, expressed in insect cells infected with a recombinant baculovirus but not p53 expressed in Escherichia coli. Moreover, murine p53 from insect cells could be immune purified with mAb p53-18. Mass spectrometry following tryptic digestion of the purified protein and liquid chromatography of the fragments verified the presence of phosphate groups at both Ser375 and Ser389. From the corresponding human protein fragments, mAb p53-18 bound to the immunizing peptide phosphorylated on Ser378 and on Ser392, but failed to cross-react with the unphosphorylated peptide, or peptides phosphorylated individually on either Ser378 or Ser392. The binding to the unphosphorylated peptide could be restored, however, if the peptide conformation was stabilized to that of an alpha-helix. The immunogenic nature of the multiphosphorylated C-terminus of p53 is indicated by the finding that human sera, mostly from cancer patients, preferentially recognized the double-phosphorylated peptide over the monophosphorylated or unphosphorylated analogs. Antibody p53-18 appears to be a highly useful biochemical marker to detect low levels of p53 protein in different tissues, and to be a key tool to characterize the phosphorylation status of the C-terminus of p53 protein originated from various sources.     

Biphasic kinetics of Zn2+ removal from Zn metallothionein by nitrilotriacetate are associated with differential reactivity of the two metal clusters

We investigated the kinetics of nitrilotriacetate (NTA) extraction of Zn2+ from Zn7-metallothionein (MT) and a metal-hybrid derivative, Zn4Ag6MT, in which the Zn2+ and Ag+ ions occupy sites in the C-terminal alpha and N-terminal beta domains of the protein, respectively. Biphasic kinetics were observed for Zn7MT under pseudo-first-order conditions. Rate constants were (5.2 +/- 0.6) x 10(-3) and (1.0 +/- 0.3) x 10(-4)s-1 in 20 mM phosphate, 100 mM KF, pH 7.5 at 23 degrees C. In contrast, Zn4Ag6MT showed a single kinetic step with a rate constant of (2.9 +/- 0.4) x 10(-3)s-1. These results indicate that the biphasic reactivity of Zn7MT stems from differential susceptibility of the metal in the two metal-thiolate clusters to removal by competing ligands, with Zn2+ in the more stable alpha-domain cluster reacting faster than that in the less stable beta-domain cluster. Such behavior suggests that the structures of the two domains of mammalian MT may have evolved to assure that Cu binding does not compromise the structural characteristics that allow Zn to be rapidly transferred from MT to essential cellular ligands.     

A conformation- and phosphorylation-dependent antibody recognizing the paired helical filaments of Alzheimer's disease

Hyperphosphorylated tau (PHF-tau) is the major constituent of paired helical filaments (PHFs) from Alzheimer's disease (AD) brains. This conclusion has been based largely on the creation and characterization of monoclonal antibodies raised against PHFs, which can be classified in three categories: (a) those recognizing unmodified primary sequences of tau, (b) those recognizing phosphorylation-dependent epitopes on tau, and (c) those recognizing conformation-dependent epitopes on tau. Recent studies have suggested that the antibodies recognizing primary sequence and phosphorylation-dependent epitopes on tau are unable to distinguish between normal adult biopsy tau and PHF-tau. We now present evidence for a new fourth class of monoclonal antibodies recognizing conformation-dependent phosphoepitopes on tau, typified by TG-3, a monoclonal antibody raised to PHFs from AD brain homogenates. Studies using a series of deletional tau mutants, site-directed tau mutants, and synthetic peptides enable the precise epitope mapping of TG-3. Additional studies demonstrate that TG-3 reacts with neonatal mouse tau and PHF-tau but does not recognize adult mouse tau or tau derived from normal human autopsy or biopsy tissue. Further investigation reveals that TG-3 recognizes a unique conformation of tau found almost exclusively in PHFs from AD brains.     

Enlarged scale chemical synthesis and range of activity of drosocin, an O-glycosylated antibacterial peptide of Drosophila

OBJECTIVE: Poor cardiorespiratory endurance is a common finding in neuromuscular disease (NMD), and the capacity of such patients to respond to aerobic training is unclear. This study was conducted to determine if a 12-week walking program results in increased aerobic capacity in slowly progressive NMD subjects, whether such a program is safely tolerated, and whether such patients can adhere to a self-monitored, home-based training program. DESIGN: Before-after trial. SETTING: Subjects' homes. PATIENTS: A cohort of 8 slowly progressive NMD subjects (4 men, 4 women) followed in the neuromuscular disease clinic participated (age, 36.6 +/- 8.0 yrs; ht, 170 +/- 11 cm; wt, 74.3 +/- 19.0 kg) (Mean +/- SD). INTERVENTION: Subjects walked 15 to 30 min 3 to 4 days a week at 50% to 60% of their heart rate reserve. MAIN OUTCOME MEASURES: Resting, submaximal, and peak heart rates, systolic and diastolic blood pressures, oxygen uptake, and peak power output. RESULTS: Graded exercise testing to volitional fatigue using a semirecumbent cycle ergometer before and after the training program found significant decreases in submaximal heart rate by 7 +/- 3 beats/min (Mean +/- SEM) (95% CI = -23 to 9) (p = .046) and submaximal systolic blood pressure by 11 +/- 4 mmHg (95% CI = -31 to 9) (p = .019), and nonsignificant increases in peak power output and VO2. CONCLUSIONS: These results suggest that moderate-intensity aerobic exercise training is well tolerated and may provide modest improvement in aerobic capacity in slowly progressive NMD subjects.