Synthesis of 27-Oxo, 27-Hydroxymilbemycins A3 and A4 and Novel 27-Alkoxymilbemycins A3 and A4 from Milbemycins A3 and A4 and…

27-Oxomilbemycins A₃ and A₄ and 27-hydroxymilbemycins A₃ and A₄ were identified as metabolites in soil metabolism studies of milbemycins A₃ and A₄. Chemical derivation methods were developed to synthesize 27-oxomilbemycins A₃ and A₄ and 27-hydroxymilbemycins A₃ and A₄ from milbemycins A₃ and A₄. In addition, 27-alkoxymilbemycin derivatives were also synthesized from the same precursors. Some of the synthesized compounds displayed satisfactory acaricidal activity against the organophosphorus-sensitive two-spotted spider mite (Tetranychus urticae), but did not have superior activity to corresponding milbemycins A₃ and A₄.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

A comparison of enzymatic phosphorylation and phosphatidylation of β-l- and β-d-nucleosides

Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers.     

Oligomerization and toxicity of Aβ fusion proteins

This study has found that the Maltose binding protein Aβ42 fusion protein (MBP-Aβ42) forms soluble oligomers while the shorter MBP-Aβ16 fusion and control MBP did not. MBP-Aβ42, but neither MBP-Aβ16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aβ42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aβ42 characterization.     

Purification and Separation of A and B Forms of N-Acetyl-β-D-Hexosaminidase from Human Aorta

Human aorta has been shown to possess multiple forms of N-Acetyl-6-D-hexosaminidase (β-2-acetamido-2-deoxy-D-glucoside-acetamido-deoxyglucohydro-lase, EC 3.2, 1.30). The enzyme was separable, by gel electrophoresis, into 2 enzymatically active bands representing A and B forms. By gel electro-focussing, A and B forms were further subdivided into at least 5 and 8 bands, respectively. The B form consisted of 4 bands (B₁) and 4 bands (B₂), which were not inactivated at 50° for 3 hr. (at pH 4.4) in the presence of serum; whereas, the 5 bands found in A form were completely inactivated. All forms of the enzyme were active towards naphthol-AS-BI-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide (about one-eighth of the hydrolysis rate of the former), suggesting each single enzyme acts on both substrates. The N-acetyl-hexosaminidases of bull epididymis, by comparison, were also found to be active towards both substrates and to possess 13 bands having pis more alkaline than those of the B form of the human enzyme, By heat inactivation we found that the aortic enzyme consisted of 51% of A and 49% of B (B₁ + B₂ .). Neuraminidase had no effect on either form of the aortic preparation. Both forms were partially purified and separated by conventional methods. They required BSA for their maximal activity; the A form being more dependent BSA than the B form, With PNP-N-acetyl-β-D-glucosaminide and the corresponding galactosaminide, Km of 1.04 mH and 0.54 mM, respectively, for A form and of 1.74 and 1.48 mM, respectively, for B form were obtained. While the purified B form was stable and did not transform into other species, the purified A form gradually transformed into B form as well as into other new forms during storage at -20°.     

Copper and Zinc Mediated Oligomerisation of Aβ Peptides

The accumulation of senile plaques composed primarily of aggregated amyloid β-peptide (Aβ), is the major characteristic of Alzheimer’s disease. Many studies correlate plaque accumulation and the presence of metal ions, particularly copper and zinc. The metal binding sites of the amyloid Aβ peptide of Alzheimer’s disease are located in the N-terminal region of the full-length peptide. In this work, the interactions with metals of a model peptide comprising the first 16 amino acid residues of the amyloid Aβ peptide, Aβ(1–16), were studied. The effect of Cu²⁺ and Zn²⁺ binding to Aβ(1–16) on peptide structure and oligomerisation are reported. The results of ESI-MS, gel filtration chromatography and NMR spectroscopy demonstrated formation of oligomeric complexes of the peptide in the presence of the metal ions and revealed the stoichiometry of Cu²⁺ and Zn²⁺ binding to Aβ(1–16), with Cu²⁺ showing a higher affinity for binding the peptide than Zn²⁺.     

Uranyl photoprobing of nonbent A/T- and bent A-tracts. A difference of flexibility?

In this study, we have systematically compared the uranyl photocleavage of a range of bent A-tracts and nonbent TA-tracts as well as interrupted A-tracts. We demonstrate that uranyl photocleavage of A-tracts and TA-tracts is almost identical, indicating a very similar minor groove conformation. Furthermore, a 10 base pair A-tract is divided into two independent tracts by an intervening TA or GC step. Uranyl probing also clearly distinguishes the bent A4T4 and the nonbent T4A4 sequences as adopting different structures, and our interpretation of the data is consistent with a structure for the bent A4T4 sequence that resembles a continuous A-tract, whereas the nonbent T4A4 sequences are closer to two independent and opposite A-tracts that cancel each other in terms of macroscopic bending. Finally, we also note that even single TA and TAT steps are highly sensitive to uranyl photocleavage and propose that in addition to average minor groove width, uranyl also senses DNA helix flexibility/deformability. Thus, the structural difference of TA-tracts and A-tracts may to a large extent reflect a difference in flexibility, and DNA curvature may consequently require a rigid narrow minor groove conformation that creates distinct A-tract-B-DNA junctions as the predominant cause of the bending.     

A critical evaluation of the predicted and X-ray structures of α-Lactalbumin

The rapidly increasing availability of protein amino-acid sequences, many of which have been determined from the corresponding gene sequences, has intensified interest in the prediction of related protein structures when the three-dimensional structure of another member of the family is known. The study of bovine α-Lactalbumin provides a classic example in which the three-dimensional structure was predicted, first by Browneet al. (1969) and later by Warmeet al. (1974), from the three-dimensional structure of hen-egg-white lysozyme (Blakeet al., 1965), taking into account the striking relationship between the amino acid sequences of the two proteins. A comprehensive comparison of these models with the structure of baboon α-Lactalbumin derived from X-ray crystallography (Acharyaet al., 1989) is presented. The models mostly compare well with the experimentally determined structure except in the flexible C-terminal region of the molecule (rms deviation on C^α of residues 1–95, 1.1 Å).     

A comparative study of hydrolysis and transglycosylation activities of fungal β-glucosidases

β-glucosidases (BGs) from Aspergillus fumigatus, Aspergillus niger, Aspergillus oryzae, Magnaporthe grisea, Neurospora crassa, and Penicillium brasilianum were purified to homogeneity, and investigated for their (simultaneous) hydrolytic and transglycosylation activity in samples with high concentrations of either cellobiose or glucose. The rate of the hydrolytic process (which converts one cellobiose to two glucose molecules) shows a maximum around 10–15 mM cellobiose and decreases with further increase in the concentration of substrate. At the highest investigated concentration (100 mM cellobiose), the hydrolytic activity for the different enzymes ranged from 10% to 55% of the maximum value. This decline in hydrolysis was essentially compensated by increased transglycosylation (which converts two cellobiose to one glucose and one trisaccharide). Hence, it was concluded that the hydrolytic slowdown at high substrate concentrations solely relies on an increased flow through the transglycosylation pathway and not an inhibition that delays the catalytic cycle. Transglycosylation was also detected at high product (glucose) concentrations, but in this case, it was not a major cause for the slowdown in hydrolysis. The experimental data was modeled to obtain kinetic parameters for both hydrolysis and transglycosylation. These parameters were subsequently used in calculations that quantified the negative effects on BG activity of respectively transglycosylation and product inhibition. The kinetic parameters and the mathematical method presented here allow estimation of these effects, and we suggest that this may be useful for the evaluation of BGs for industrial use.     

A study of the biochemistry and cytochemical localization of β-glycerophosphatase activity in root tips of maize and pea

Summary A critical study has been made of the standard lead phosphate precipitation technique for the localization of-glycerophosphatase activity in young root tips. The effects of fixatives on enzyme activity and on the loss of activity during fixation and incubation have been determined. Cytochemical studies showed that the most prominent sites of-glycerophosphatase activity were at particulate sites in the cytoplasm and in the nucleus. Good correlation was obtained between frozen section and electron microscope studies although a number of problems were encountered. These particularly concerned the penetration of the staining medium, the loss of activity during incubation and the use of the acetic acid rinse. Recommendations are made for the reliable localization of the enzyme. The use of controls in this method were studied and their validity discussed.The study was supported in part by a National Science Foundation Grant GB 12371 to Dr. J.Cronshaw.     

Comparison of the properties of concanavalin A and anti-α-d-glucose antibodies

Concanavalin A and anti-α-d-glucose antibodies form precipitin complexes with antigens havingα-d-glucose as terminal units. The sedimentation rates, molecular weights, gel electrophoretic mobilities, isoelectric points, and immunoglobulin type of Con A andα-Ab have been determined. The interactions of the compounds with antigens in the presence of potential inhibitors have been compared. The data show that the interaction of Con A with glucose units occurs with hydrogen bonding at hydroxyl groups at C1, 3,4, and 6 and van der Waals bonding at the pyranose ring oxygen. In theα-Ab complex with glucose units, in addition to the above bond types, a hydrogen bond at the hydroxyl at C2 occurs and this bond is essential for interaction.     

Role of surfactant and pH on dissolution properties of fenofibrate and glipizide—A technical note

Depending on the dose size and solubility characteristics of low solubility drugs, a meaningful and discriminatory power of dissolution rate testing can be demonstrated. Saturation solubility of fenofibrate and glipizide in different media were determined. Solubility of fenofibrate increased directly with SLS concentration. For a 54-mg fenofibrate tablet, SLS at 0.025 M level is required for a discriminative dissolution test, while for 160-mg tablet, dissolution condition and levels of SLS should be optimized; higher concentrations may be effective (ie, 0.052 M, ∼1.5%). A pH 6.8 phosphate buffer medium is appropriate for glipizide 10-mg tablet dissolution study, when formulation ingredients include excipients with surface activity (eg, HPMC).     

Self-Assembly of Aβ40, Aβ42 and Aβ43 Peptides in Aqueous Mixtures of Fluorinated Alcohols

Fluorinated alcohols such as hexafluoroisopropanol (HFIP) and trifluoroethanol (TFE) have the ability to promote α-helix and β-hairpin structure in proteins and peptides. HFIP has been used extensively to dissolve various amyloidogenic proteins and peptides including Aβ, in order to ensure their monomeric status. In this paper, we have investigated the self-assembly of Aβ40, Aβ42, and Aβ43 in aqueous mixtures of fluorinated alcohols from freshly dissolved stock solutions in HFIP. We have observed that formation of fibrillar and non-fibrillar structures are dependent on the solvent composition. Peptides form fibrils with ease when reconstituted in deionized water from freshly dissolved HFIP stocks. In aqueous mixtures of fluorinated alcohols, either predominant fibrillar structures or clustered aggregates were observed. Aqueous mixtures of 20% HFIP are more favourable for Aβ fibril formation as compared to 20% TFE. When Aβ40, Aβ42, and Aβ43 stocks in HFIP are diluted in 50% aqueous mixtures in phosphate buffer or deionized water followed by slow evaporation of HFIP, Aβ peptides form fibrils in phosphate buffer and deionized water. The clustered structures could be off-pathway aggregates. Aβ40, Aβ42, and Aβ43 showed significant α-helical content in freshly dissolved HFIP stocks. The α-helical conformational intermediate in Aβ40, Aβ42, and Aβ43 could favour the formation of both fibrillar and non-fibrillar aggregates depending on solvent conditions and rate of α-helical to β-sheet transition.     

Quantitative correlation between the residual activity of β-hexosaminidase A and arylsulfatase A and the severity of the resulting lysosomal storage disease

Summary A previously suggested model for the correlation between residual activity of a lysosomal enzyme and the turnover rate of its substrate(s) has been extended to a discussion of substrate accumulation rates in individual cells and whole organs. With these considerations, much of the observed variability in age of onset and clinical phenotype, as well as the phenomenon of pseudodeficiency, can be understood as the consequences of small differences in the residual activity of the affected enzyme. In order to experimentally verify the basic assumptions on which this model rests, studies were performed in cell culture. The radiolabeled substrates ganglioside G_M2 and sulfatide were added to cultures of skin fibroblasts with different activities of -hexosaminidase A or arylsulfatase A, respectively, and their uptake and turnover measured. In both series of experiments, the correlation between residual enzyme activity and the turnover rate of the substrate was essentially as predicted: degradation increased steeply with residual activity, to reach the control level at a residual activity of approximately 10–15% of normal. All cells with an activity above this critical threshold had a normal turnover. Comparison of the results of these feeding studies with the clinical status of the donor of each cell line basically confirmed our notions but also revealed the limitations of the cell culture approach.     

A comparison of the effects of NN′-dicyclohexylcarbodi-imide, oligomycin A and aurovertin on energy-linked reactions in mitochondria and submitochondrial particles

1. The effects of dicyclohexylcarbodi-imide, oligomycin A and aurovertin on enzyme systems related to respiratory-chain phosphorylation were compared. Dicyclohexylcarbodi-imide and oligomycin A have very similar functional effects, giving 50% inhibition of ATP-utilizing and ATP-generating systems at concentrations below 0.8nmole/mg. of submitochondrial-particle protein. Aurovertin is a more potent inhibitor of ATP synthesis, giving 50% inhibition at 0.2nmole/mg. of protein. However, aurovertin is a less potent inhibitor of ATP-utilizing systems: the ATP-driven energy-linked nicotinamide nucleotide transhydrogenase is 50% inhibited at 3.0nmoles/mg. of protein and the ATP-driven reduction of NAD(+) by succinate is 50% inhibited at 0.95nmole/mg. of protein. 2. With EDTA-particles (prepared by subjecting mitochondria to ultrasonic radiation at pH9 in the presence of 2mm-EDTA) the maximum stimulation of the ATP-driven partial reactions is effected by similar concentrations of oligomycin A and dicylcohexylcarbodi-imide, but the latter is less effective. The stimulatory effects of suboptimum concentrations of dicyclohexylcarbodi-imide and oligomycin A are additive. Aurovertin does not stimulate these reactions or interfere with the stimulation by the other inhibitors. 3. Dicyclohexylcarbodi-imide and oligomycin A stimulate the aerobic energy-linked nicotinamide nucleotide transhydrogenase of EDTA-particles, but the optimum concentration is higher than that required for the ATP-driven partial reactions. Aurovertin has no effect on this reaction. 4. The site of action of dicyclohexylcarbodi-imide is in CF(0), the mitochondrial fraction that confers oligomycin sensitivity on F(1) mitochondrial adenosine triphosphatase.     

Biomimetic Leukocyte Adhesion： A Review of Microfluidic and Computational Approaches and Applications

Leukocyte rolling and adhesion are complex physiological processes that have received a great deal of attention over the past decade. Significant increases in the knowledge base related to how leukocytes adhere in shear flows have occurred as a result of the development of novel experimental and computational techniques. Micro- and nano-fabrication techniques have enabled the development of novel flow devices for studying leukocyte adhesion in simple and complex geometries. Improvements in computer technology have enabled simulations of complex flow processes to be developed. As a result of these advances in knowledge related to leukocyte adhesion, numerous novel devices have been developed that mimic the leukocyte rolling and adhesion process. Examples of these devices include cell separation and enrichment devices and targeted ultrasound contrast agents. Future advances related to leukocyte rolling and adhesion processes hold great promise for advancing our knowledge of disease processes as well as development of novel therapeutic devices.     

Energetics of ligand recognition and self-association of bovine β-lactoglobulin: differences between variants A and B

An understanding of the interplay between structure and energetics is crucial for the optimization of modern protein engineering techniques. In this context, the study of natural isoforms is a subject of major interest, as it provides the scenario for analyzing mutations that have endured during biological evolution. In this study, we performed a comparative analysis of the ligand-recognition and homodimerization energetics of bovine β-lactoglobulin variants A (βlgA) and B (βlgB). These variants differ by only two amino-acid substitutions: 64th (Asp(A) → Gly(B)), which is fully exposed to the solvent, and 118th (Val(A) → Ala(B)), immersed in the hydrophobic core of the protein. Calorimetric measurements revealed significant enthalpic and entropic differences between the isoforms in both binding processes. A structural comparison suggests that a variation in the conformation of the loop C-D, induced by mutation Asp/Gly, could be responsible for the differences in ligand-binding energetics. While recognition of lauric acid was entropically driven, recognition of sodium dodecyl sulfate was both entropically and enthalpically driven, confirming the key role of the ligand polar moiety. Because of a more favorable enthalpy, the dimerization equilibrium constant of βlgB was larger than that of βlgA at room temperature, while the two dimers became similarly stable at 35 °C. The isoforms exchanged the same number of structural water molecules and protons and shared similar stereochemistry at the dimer interface. MD simulations revealed that the subunits of both variants become more flexible upon dimer formation. It is hypothesized that a larger increase of βlgA mobility could account for the dimerization energetic differences observed.     

Macromolecular and small-molecule modulation of intracellular Aβ42 aggregation and associated toxicity

Aβ (amyloid β-peptide) has a central role in AD (Alzheimer's disease) where neuronal toxicity is linked to its extracellular and intracellular accumulation as oligomeric species. Searching for molecules that attenuate Aβ aggregation could uncover novel therapies for AD, but most studies in mammalian cells have inferred aggregation indirectly by assessing levels of secreted Aβ peptide. In the present study we establish a mammalian cell system for the direct visualization of Aβ formation by expression of an Aβ(42)-EGFP (enhanced green fluorescent protein) fusion protein in the human embryonic kidney cell line T-REx293, and use this to identify both macromolecules and small molecules that reduce aggregation and associated cell toxicity. Thus a molecular shield protein AavLEA1 [Aphelenchus avenae LEA (late embryogenesis abundant) protein 1], which limits aggregation of proteins with expanded poly(Q) repeats, is also effective against Aβ(42)-EGFP when co-expressed in T-REx293 cells. A screen of polysaccharide and small organic molecules from medicinal plants and fungi reveals one candidate in each category, PS5 (polysaccharide 5) and ganoderic acid DM respectively, with activity against Aβ. Both PS5 and ganoderic acid DM probably promote Aβ aggregate clearance indirectly through the proteasome. The model is therefore of value to study the effects of intracellular Aβ on cell physiology and to identify reagents that counteract those effects.     

Clinical Efficacy and Tolerability of Praziquantel for Intestinal and Urinary Schistosomiasis—A Meta-analysis of Comparative and Non-comparative Clinical Trials

Background

Extensive use of praziquantel for treatment and control of schistosomiasis requires a comprehensive understanding of efficacy and safety of various doses for different Schistosoma species.

Methodology/Principal Findings

A systematic review and meta-analysis of comparative and non-comparative trials of praziquantel at any dose for any Schistosoma species assessed within two months post-treatment. Of 273 studies identified, 55 were eligible (19,499 subjects treated with praziquantel, control treatment or placebo). Most studied were in school-aged children (64%), S. mansoni (58%), and the 40 mg/kg dose (56%); 68% of subjects were in Africa. Efficacy was assessed as cure rate (CR, n = 17,017) and egg reduction rate (ERR, n = 13,007); safety as adverse events (AE) incidence. The WHO-recommended dose of praziquantel 40 mg/kg achieved CRs of 94.7% (95%CI 92.2–98.0) for S. japonicum, 77.1% (68.4–85.1) for S. haematobium, 76.7% (95%CI 71.9–81.2) for S. mansoni, and 63.5% (95%CI 48.2–77.0) for mixed S. haematobium/S. mansoni infections. Using a random-effect meta-analysis regression model, a dose-effect for CR was found up to 40 mg/kg for S. mansoni and 30 mg/kg for S. haematobium. The mean ERR was 95% for S. japonicum, 94.1% for S. haematobium, and 86.3% for S. mansoni. No significant relationship between dose and ERR was detected. Tolerability was assessed in 40 studies (12,435 subjects). On average, 56.9% (95%CI 47.4–67.9) of the subjects receiving praziquantel 40 mg/kg experienced an AE. The incidence of AEs ranged from 2.3% for urticaria to 31.1% for abdominal pain.

Conclusions/Significance

The large number of subjects allows generalizable conclusions despite the inherent limitations of aggregated-data meta-analyses. The choice of praziquantel dose of 40 mg/kg is justified as a reasonable compromise for all species and ages, although in a proportion of sites efficacy may be lower than expected and age effects could not be fully explored.     

Production and characterization of mice transgenic for the A and B isoforms of human FcγRIII

Fcγ receptor (FcγR) engagement is pivotal for many effector functions of macrophages, polymorphonuclear neutrophils (PMN), and natural killer (NK) cells. Mice transgenic for the A and B isoforms of human (h) FcγRIII on macrophages, PMN, and NK cells were constructed to permit the study of mechanisms and potential in vivo strategies to utilize the cytotoxic effector and antigen-presenting functions of cells expressing the hFcγR. The present report characterizes the phenotypic and functional expression of hFcγRIII in transgenic mice derived by crossing hFcγRIIIA and hFcγRIIIB transgenic mice. Interleukin-2 (IL-2) induces hFcγRIII expression by myeloid cells and their precursors, and these transgenic receptors promote in vitro cytotoxicity and anti-hFcγRIII antibody internalization. Splenocytes from untreated and IL-2-treated hFcγRIIIA, hFcγRIIIB, and hFcγRIIIA/B mice exhibited enhanced in vitro cytotoxicity toward HER-2/neu-overexpressing SK-OV-3 human ovarian carcinoma cells when incubated with the murine bispecific mAb 2B1, which has specificity for HER-2/neu and hFcγRIII. These results indicate that hFcγRIII transgenes are expressed on relevant murine cellular subsets, exhibit inducible up-regulation patterns similar to those seen in humans, and code for functional proteins. hFcγRIII transgenic mice exhibiting specific cellular subset expression will permit the examination of strategies designed to enhance hFcγRIII-dependent immunological effector functions and will provide a model system in which to evaluate preclinically potential candidate molecules that recognize hFcγRIII for the immunotherapy of cancer. Received: 5 December 1998 / Accepted: 14 July 1999