Determination of choline in erythrocytes using high-resolution proton nuclear magnetic resonance spectroscopy: Comparison with a choline oxidase method

Detailed operating conditions are reported for the determination of choline in human erythrocytes using proton nuclear magnetic resonance spectroscopy in conjunction with the inversion-recovery spin-echo pulse sequence. The results of the NMR method were in excellent agreement with those obtained using an enzymatic (choline oxidase) assay; however, they were approximately three times higher than those reported using gas chromatography/mass spectrometry techniques. The differences may be partly due to the method of preparing or sampling cells since there is a distribution of choline in cells of different ages. However, choline levels were not affected by the methods used in the present study for storing or preparing cells.     

Comparison of gravimetry and planimetry in determining dry matter budgets for three species of phytophagous lepidopteran larvae

Gravimetric and a combination areal-gravimetric methods for determining dry matter budgets for leaf eating Lepidoptera were compared. The gravimetric method is based on dry weight/live weight ratios of the leaves fed to the larvae. In the areal-gravimetric method, the quantity of food offered to the larvae is determined from the area of leaf tracings and the dry matter content per unit area of the leaves. The areal-gravimetric method permits the use of larger leaf sprays and an open, gauze enclosed rearing chamber. There were no consistent differences in budget factors (growth, ingestion or egestion), nor were there any differences in the observed variability of the data attributable to the method used. However, the expected variability based on instrument precision for the gravimetric method is less than for the areal-gravimetric method. Experimental factors inherent in the gravimetric method introduce variability to the measurements that are not present in the areal method. Thirty to 60% of the variability in budget factors was attributed to intrinsic properties of the larvae, even though the larvae were taken from the same egg masses.

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Résumé Les budgets en matière sèche consommée par des lépidoptères ont été comparés par les méthodes gravimétrique et planimétrique. La méthode gravimétrique est basée sur le rapport poids sec/poids frais de feuilles consommées par les chenilles. Avec la méthode planimétrique, la quantité d'aliment proposée aux chenilles est déterminée par les tracés de la surface des feuilles et le contenu de matière sèche par unité de surface des feuilles. La méthode de planimétrie permet l'utilisation de plus grands rameaux de feuilles et de cages d'élevage extérieures en gaze. Il n'y avait pas de différence appréciable dans les éléments du budget (croissance, ingestion et déjection), ni aucune différence dans la variabilité observée des données attribuable à la méthode utilisée. Cependant, la variabilité attendue d'après la précision des mesures avec la méthode gravimétrique est inférieure à celle de la méthode planimétrique. est inférieure à celle de la méthode planimétrique. Des éléments expérimentaux, inhérents à la méthode gravimétrique, introduisent une variabilité dans les mesures que l'on n'a pas avec la méthode planimétrique. 30–60% de la variabilité dans la consommation ont été attribués à des paramètres internes à la chenille, même quand elles provenaient toutes de la même ooplaque.

     

Rapid methods for the characterization of unilamellar phospholipid vesicles. Application to studies on fatty acid donor and acceptor properties of membranes and fatty acid binding proteins

Vesicles having diameters from 20 to 200 nm were prepared from egg-yolk phosphatidylcholine (PC) and were separated as well as analyzed by methods that can be carried out with standard laboratory equipment. Gel-chromatography on Sephacryl S 1000 was adapted for expeditious size analysis of vesicles as well as for isolation of vesicle populations having a narrow range of diameters. The internal volume of vesicles was derived from enzymic tests for PC and for glucose encapsulated. Size analysis and enzymic determinations provided a convenient check for the lamellarity of membranes produced.Fatty acids and fatty acid binding proteins (FABPs) must interact in vivo in the presence of cellular membranes. As a model, interactions between unilamellar vesicles, anthroyloxypalmitic acid (A16:0) and FABPs were studied with the aid of gel-chromatographic methods elaborated and of fluorescence spectroscopy. FABP from bovine heart donated A16:0 to membranes, whereas FABP from bovine liver removed this fatty acid from vesicle membranes. The results revealed characteristic differences between cardiac and hepatic FABPs with regard to binding a fatty acid.     

Effect of plant genotype and nitrogen fertilizer on symbiotic nitrogen fixation by soybean cultivars

Summary Isotopic as well as non-isotopic methods were used to assess symbiotic nitrogen fixation within eight soybean [Glycine max (L.) Merr.] cultivars grown at 20 and 100 kg N/ha levels of nitrogen fertilizer under field conditions.The¹⁵N methodology revealed large differences between soybean cultivars in their abilities to support nitrogen fixation. In almost all cases, the application of 100 kg N/ha resulted in lower N₂ fixed in soybean than at 20 kg N/ha in the first year of the study. However, N₂ fixed in one cultivar, Dunadja, was not significantly affected by the higher rate of N fertilizer application. These results were confirmed by measurements of acetylene reduction activity, nodule dry weight and N₂ fixed as measured by the difference method. Further proof of differences in N₂ fixed within soybean cultivars and the ability of Dunadja to fix similar amounts of N₂ at 20 and 100 kg N/ha was obtained during a second year experiment. Dunadja yield was affected by N fertilizer and produced larger yield at 100 kg N/ha than at 20 kg N/ha. This type of cultivar could be particularly useful in situations where soil N levels are high or where there is need to apply high amounts of N fertilizer.The present study reveals the great variability between legume germplasms in the ability to fix N₂ at different inorganic N levels, and also the potential that exists in breeding for nitrogen fixation associative traits. The¹⁵N methodology offers a unique tool to evaluate germplasms directly in the field for their N₂ fixation abilities at different N fertilizer levels.     

Analysis of penicillin-binding sites with a micro method — The binding site of PBP 5 from Escherichia coli

Abstract A micro method for the isolation and characterization of the penicillin-binding sites in penicillin-binding proteins (PBPs) was developed. Only 10 nmol of a pure PBP are required for the whole procedure which is based on high-pressure liquid chromatography (HPLC). We showed that serine 44 in PBP 5 from Escherichia coli binds penicillin covalently.     

A spectrophotometric method for the determination of the kinetic parameters of NH+4 uptake by yeast cells

Abstract The kinetic parameters of NH⁺₄-uptake in yeast cells were determined by a method that is based on the following changes in the external NH⁺₄ concentration in cell suspensions by using NADH-dependent glutamate formation from NH⁺₄ and 2-oxoglutarate. The kinetics of the observed NADH oxidation were analyzed by computer and enabled an estimation of V _max and K _m of the NH⁺₄-uptake system of the cells.     

Assessment of a model for intron RNA secondary structure relevant to RNA self-splicing--a review

A widespread class of introns is characterized by a particular RNA secondary structure, based upon four conserved nucleotide sequences. Among such "class I" introns are found the majority of introns in fungal mitochondrial genes and the self-splicing intron of the large ribosomal RNA of several species of Tetrahymena. A model of the RNA secondary structure, which must underlie the self-splicing activity, is here evaluated in the light of data on 16 further introns. The main body or "core structure" of the intron always consists of the base-paired regions P3 to P9 with the associated single-stranded loops, with P2 present also in most cases. Two minority sub-classes of core structure occur, one of which is typical of introns in fungal ribosomal RNA. Introns in which the core structure is close to the 5' splice site all have an internal guide sequence (IGS) which can pair with exon sequences adjacent to the 5' and 3' splice sites to align them precisely, as proposed by Davies et al. [Nature 300 (1982) 719-724]. In these cases, the internal guide model allows us to predict correctly the exact location of splice sites. All other introns probably use other mechanisms of alignment. This analysis provides strong support for the RNA splicing model which we have developed.     

Cytological observations on the ventromedial hypothalamic nucleus

Two sorts of neurons are recognized in Golgi impregnations of the rat ventromedial hypothalamic nucleus (HVM). The two cell types, category I and II neurons, are differentiated on the basis of their somatic, dendritic, and axonal characteristics. Category I neurons form most of the neuronal population and are located throughout HVM. The small number of category II neurons that have been studied occur in lateral HVM. Two varieties of neuronal profile, "common" and "uncommon cells", are seen in thin sections of HVM. The "uncommon cells", in comparison with the "common ones", appear to have a larger soma, a more electron-dense cytoplasmic matrix, an abundance of Nissl bodies, and a population of dense-cored vesicles (100--130 nm in diameter). Some of the somata and proximal dendrites of "common", but not "uncommon" cells, are wrapped in multiple layers of astrocytic processes. Although the correlation is tentative, it is argued that category I neurons correspond to "common cells" and category II, to "uncommon cells". One possible implication of this correspondence is discussed regarding neuronal alteration in response to change in the endocrinological environment of the brain.