N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Adenine photodimerization in deoxyadenylate sequences: elucidation of the mechanism through structural studies of a major d(ApA) photoproduct.

The mechanism of the photodimerization of adjacent adenine bases on the same strand of DNA has been elucidated by determining the structure of one of the two major photoproducts that are formed by UV irradiation of the deoxydinucleoside monophosphate d(ApA). The photoproduct, denoted d(ApA)*, corresponds to a species of adenine photodimer first described by P?rschke (P?rschke, D. (1973) J.Am.Chem.Soc. 95, 8440-8446). From a detailed examination of its chemical and spectroscopic properties, including comparisons with the model compound N-cyano-N1-(1-methylimidazol-5-yl)formamidine, it is deduced that d(ApA)* contains a deoxyadenosine unit covalently linked through its C(8) position to C(4) of an imidazole N(1) deoxyribonucleoside moiety bearing an N-cyanoformamidino substituent at C(5). On treatment with acid, d(ApA)* is degraded with high specificity to 8-(5-amino-imidazol-4-yl)adenine whose identity has been confirmed by independent chemical synthesis. It is concluded that the primary event in adenine photodimerization entails photoaddition of the N(7)-C(8) double bond of the 5'-adenine across the C(6) and C(5) positions of the 3'-adenine. The azetidine species thus generated acts as a common precursor to both types of d(ApA) photoproduct which are formed from it by competing modes of azetidine ring fission.     

Effects of nectar concentration on butterfly feeding: measured feeding rates for Thymelicus lineola (Lepidoptera: Hesperiidae) and a general feeding model for adult Lepidoptera

Summary Field observations of the adult European skipper, Thymelicus lineola (Ochs), feeding on concentrated nectars (40–65% sucrose) from a variety of flower species led us to question recent literature stating that butterflies feed primarily, and most effectively, on dilute nectars. Rate of sucrose solution intake, volume consumed and feeding duration were measured for males and females at 25 and 35°C under laboratory conditions. As sucrose concentration increased, the volume of solution ingested per meal first increased and then decreased gradually, while sucrose intake was highest at concentrations 40%. Females fed more than males at all concentrations >10% while temperature had no significant effect on meal size. Feeding duration increased with concentration, was shorter at 35 than at 25°C, and was longer for females than males.The rate of volume intake decreased as concentration incresed, but not nearly as rapidly as predicted by earlier models. Rates did not differ between the sexes but were faster at 35 than 25°C. This increase was contributed to equally by a reduction in viscosity and an increase in power output of the cibarial pump. The form of the relations was similar, with maximum rate of sucrose intake occurring at 40% sucrose.A new mathematical model was developed to describe the rate — concentration relation based on the Hagen-Poiseuille equation for laminar fluid flow through pipes. Our model differs from previous models principally in that the power output of the insect's cibarial pump remains relatively constant while the pressure drop created by the pump to induce suction is highly variable. This change results in a very different feeding rate — sucrose concentration function with the optimal rate of sucrose intake at a concentration of approximately 40%. The model indicates that the same relation should hold for a wide range of proboscis shape and size and type of suction pump, and should therefore be applicable to all other nectar feeders with sucking mouth parts. Independent verifications of the model were carried out by measuring the rate of uptake of sucrose solutions of the adult common armyworm, Pseudaletia unipuncta (Haw.), and of human subjects using a volumetric pipette, both of which gave an excellent fit.Nectar concentrations which correspond to optimal rates of sucrose intake should be highly preferred by insects with high feeding costs, those which are time-limited, or which are very vulnerable while feeding. High transport costs and severe water stress may shift preferences to higher and lower concentrations respectively.     

Cell surface changes in preimplantation mouse embryos during compaction investigated using FITC conjugated lectins after proteolytic enzyme treatment

Summary Fluorescein-conjugated lectins were used to examine the reappearance of glycoproteins on the surface of 8-cell mouse embryos after treatment with proteolytic enzymes. Embryos were decompacted in calcium free medium, treated with various proteases and the process of recompaction monitored. The most effective enzymes in delaying recompaction were subtilopeptidase A and proteinase K at 1 mg/ml; the initiation of recompaction was delayed by about 5 h and 90% recompaction by 14–18 h. Papain and -chymotrypsin were only effective in the absence of calcium. The reappearance of receptors for fluorescein-conjugated Con-A, MPA, RCA-I, FBP, BSL-II and DBA was examined photometrically at 0,8–10 and 17–18 h after proteinase K treatment. There was an increase in binding of MPA, RCA-I, FBP and BSL-II in control embryos during the period of the experiment, between approx. 61 and 80 h post coitum in which embryos passed from the 8-cell stage to the 16–32 cell stage. Con-A binding remained the same and that of DBA decreased. By the time that 50% of enzyme treated embryos had recompacted (8–10 h) binding of Con-A was similar to control embryos. Binding of FBP had almost reached control levels while that of BSL-II, DBA, RCA-I and MPA had reached 60–85% of control levels. When embryos were fully compact (17–18 h) Con-A, FBP and DBA were bound in equal or slightly greater amounts to enzyme treated as to control embryos, and receptors for BSL-II, MPA and RCA-I had recovered almost to control levels. The results clearly show that the recovery of glycoproteins on the surface of 8–16 cell embryos parallels recompaction, providing further evidence for the role of these molecules in compaction.     

Computer-linked HPLC system for feedback control of fermentation substrates

Summary A data acquisition/control microcomputer system was interfaced to a commercial HPLC data transmission module. Control of substrate (ethanol) levels for four 7.5 L fermenters containing 100 g/L wet weight of the yeastCandida norvegensis was accomplished by employing intermittent, automated HPLC monitoring and a BASIC-encoded proportional integral policy for controlling substrate feed rates. Ethanol levels were maintained at 0.25, 0.50, 0.75 and 1.00% w/v.     

The utility of photo-affinity labels as `mapping' reagents. A study of sub-populations of a specific rabbit antibody by using structurally related photo-affinity reagents

A specific rabbit antibody against the 4-azido-2-nitrophenyl determinant was photo-labelled by the homologous hapten ε-(4-azido-2-nitrophenyl)-l-lysine, and by the close structural isomer ε-(5-azido-2-nitrophenyl)-l-lysine. The extents of covalent labelling of the antibody-binding site were assessed by using radioactive haptens and exhaustive displacement dialysis, which leaves the unlabelled sites empty but largely intact. A single photolysis of hapten–antibody complex suffices to label those sites that are capable of being labelled. Although there is considerable overlap among sub-populations of antibody that will bind the two haptens non-covalently, sites that can be covalently labelled by one reagent cannot be labelled by the other.     

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast

Summary Host-cell reactivation of gamma-irradiated phage T1 in strains of E. coli K-12 has been compared with HCR of UV-irradiated phage in these same strains and with the radiation sensitivities of these strains (Fig. 1–4). The pattern of the HCR of gammairradiated phage in these strains is like that of the HCR of UV-irradiated phage. HCR in strains whose genotype is uvr ⁺rec^- is like that of the wild type; whereas, HCR is minimal in strains which are uvr ^-. It is suggested that some type of gamma-ray-induced base damage in phage DNA is repaired in uvr ⁺ strains.This work was supported by the United States Atomic Energy Commission Contract No. AT(11-1)-1686. — This is report No. COO-1686-6.Supported in part by the United States Public Health Service Training Grant No. 5T1 RH-80-02(67).     

The integument of the Queensland fruit fly,Dacus tryoni (Frogg.)

Summary Testes of the Japanese freshwater turtle Clemmys japonica Temmnick et Schlegel were fixed in 3% potassium permanganate buffered to pH 7.2 with Veronal-acetate buffer, and thin sections of the tissue, embedded in epoxy Epon resin, were studied under the electron or light microscope.At the early stage of differentiation of the spermatid, the cytoplasm contains a few mitochondria provided with cristae which are oriented transversely or longitudinally. As the differentiation of spermatids proceeds, the mitochondrion has been modified into a cupshaped body with a wall consisting of several concentric layers. Such body has been referred to the mitochondrial lamellar body. The formation of such a body is mainly attributed to the mitochondrial cristae, and subsequently to the membrane system of the endoplasmic reticulum. In a more advanced stage of differentiation, the mitochondrial lamellar bodies appear wrapped around a bundle of tail filaments, and seem to present a very wide surface available for the localization of organized enzyme systems to facilitate the motion of spermatozoa.Prior to the formation of the mitochondrial lamellar bodies, the Golgi apparatus has been reorganized into a peculiar body with a floral appearance, consisting of numerous tubular elements, and revealing to be positive in PAS-reaction. The body has been designated as the tubular body which has never been demonstrated in any spermatogenic cells through animal kingdom.One to three tubules oval in cross section, approximately 430 × 700 Å in diameter, have been found in the nucleoplasm along the longitudinal axis of a greatly elongated, cone-shaped nucleus of the spermatid. The tubules open on the apex surface of the nucleus, but they are not encountered in the acrosome. A possible physiological significance of the tubules has been discussed in view of the function of the acrosomal tubules in the decapod and other species spermatozoa as well as on the basis of the metabolism of nucleus.This study was supported by Grant GM-8327 from the United States Public Health Service.We wish to express our gratitude to Dr. B. A. Afzelius, Wenner-Gren Institute, University of Stockholm, for his valuable suggestion to the present work.     

Identification of a common molecular basis for combined 17α-hydroxylase/17,20-lyase deficiency in two Mennonite families

Summary During the course of studies to characterize mutations of the CYP17 gene that cause the 17-hydroxylase-deficient form of congenital adrenal hyperplasia we have discovered two ostensibly unrelated Mennonite families in which affected individuals are homozygous for the same mutation. The defect is a four-base duplication in exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 animo acids of cytochrome P450₁₇.     

ATP-Evoked Ca2+ Mobilisation and Prostanoid Release from Astrocytes: P2-Purinergic Receptors Linked to Phosphoinositide Hydrolysis

Astrocyte cultures prelabelled with either [3H]inositol or 45Ca2+ were exposed to ATP and its hydrolysis products. ATP and ADP, but not AMP and adenosine, produced increases in the accumulation of intracellular 3H-labelled inositol phosphates (IP), efflux of 45Ca2+, and release of thromboxane A2 (TXA2). Whereas ATP-stimulated 3H-IP accumulation was unaffected, its ability to promote TXA2 release was markedly reduced by mepacrine, an inhibitor of phospholipase A2 (PLA2). ATP-evoked 3H-IP production was also spared following treatment with the cyclooxygenase inhibitor, indomethacin. We conclude that ATP-induced phosphoinositide (PPI) breakdown and 45 Ca2+ mobilisation occurred in parallel with, if not preceded, the release of TXA2. Following depletion of intracellular Ca2+ with a brief preexposure to ATP in the absence of extracellular Ca2+, the release of TXA2 in response to a subsequent ATP challenge was greatly reduced when compared with control. These results suggest that mobilisation of cytosolic Ca2+ may be the stimulus for PLA2 activation and, thus, TXA2 release. Stimulation of alpha 1-adrenoceptors also caused PPI breakdown and 45 Ca2+ efflux but not TXA2 release. The effects of ATP and noradrenaline (NA) on 3H-IP accumulation were additive, but their combined ability to increase 45Ca2+ efflux was not. Interestingly, in the presence of NA, ATP-stimulated TXA2 release was reduced. Our data provide evidence that functional P2-purinergic receptors are present on astrocytes and that ATP is the first physiologically relevant stimulus found to initiate prostanoid release from these cells.