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.     

AORTOGRAPHY—A Discussion of Technique and Complications

Aortography is valuable in the demonstration of vascular blocks, in determining the exact location of the disease and as a guide to surgeons if operation is done. Since complications may develop, indications for the procedure should be carefully considered.One death and 15 nonfatal complications occurred in 594 cases in which the authors made angiographic examinations.A technique is described which takes a minimum of time.     

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°.     

HYPOTHALAMIC DYSFUNCTION—A Review of Experimental and Clinical Observations of Cardiac and Renal Aspects

The nuclear cell masses of the hypothalamus act as autonomic regulators for visceromotor function. Through the correlation of impulses arising in or about the hypothalamus with the changes in cellular chemistry, there is provided, by mediation of the endocrines, a balanced control of water metabolism, renal function and cardiac action.Derangement of hypothalamic regulation causes specific clinical syndromes described by the general term “diencephalohypophyseal dystrophy.” Cardiac abnormalities attributable to hypothalamic dysfunction include alterations in rate and various arrhythmias. Alteration in renal function includes hematuria, polyuria or relative anuria, and specific effects on electrolyte and nitrogen output.     

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.     

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).     

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.     

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.     

A Detailed Family History of Myocardial Infarction and Risk of Myocardial Infarction – A Nationwide Cohort Study

BackgroundFamily history of myocardial infarction (MI) is an independent risk factor for MI. Several genetic variants are associated with increased risk of MI and family history of MI in a first-degree relative doubles MI risk. However, although family history of MI is not a simple dichotomous risk factor, the impact of specific, detailed family histories has not received much attention, despite its high clinical relevance. We examined risk of MI by MIs in first- and second-degree relatives and by number and age of affected relatives.ConclusionA detailed family history, particularly number of affected first- and second-degree relatives, contributes meaningfully to risk assessment, especially in middle-aged persons. Future studies should test for potential improvement of risk algorithm prediction using detailed family histories.     

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.