Diglycosyl diacylglycerol of Mycobacterium tuberculosis.

A diglycosyl diacylglycerol was isolated from Mycobacterium tuberculosis, and its structure was established by a combination of methylation analysis, 1H nuclear magnetic resonance, and fast atom bombardment-mass spectrometry. It is a 1,2-diacyl-[beta-D-glucopyranosyl(1"----6')-beta-D-glucopyranosyl(1'---- 3)]- sn-glycerol and exists in at least five molecular species differing in fatty acyl substituents. The major constituent fatty acids were identified as iso- and anteisopentadecanoate, iso- and n-hexadecanoate, and iso- and anteisoheptadecanoate. Although glycosyl diacylglycerols are common membrane components of gram-positive bacteria, this report represents the first substantial evidence for the presence of a glycosyl diacylglycerol within a member of the Mycobacterium genus. Although the glycolipid is not a major component of M. tuberculosis, it reacts readily in enzyme-linked immunosorbent assay against rabbit antibodies raised against whole bacteria and thus may be useful for the serodiagnosis of tuberculosis.     

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.     

A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant

The nodulation characteristics of soybean (Glycine max) mutant nts382 are described. The mutant nodulated significantly more than the parent cultivar Bragg in the presence and absence of several combined nitrogen sources (KNO₃, urea, NH₄Cl, and NH₄NO₃). The number of nodules on the tap root and on lateral roots was increased in the mutant line. In the presence of KNO₃ and urea, nitrogenase activity was considerably higher in nts382 than in Bragg. Mutant plants were generally smaller than wild-type plants. Although nts382 is a supernodulator, inoculation with Rhizobium japonicum was necessary to induce nodule formation and both trial strains CB1809 (= USDA136) and USDA110 elicited the mutant phenotype. Segregation of M₃ progeny derived from a M₂ wild-type plant indicated that the mutant character is inherited as a Mendelian recessive. The mutant is discussed in the context of regulation of nodulation and of hypotheses that have been proposed to explain nitrate inhibition of nodulation.     

Saccharomyces cerevisiae CYC1 mRNA 5''-end positioning: analysis by in vitro mutagenesis, using synthetic duplexes with random mismatch base pairs.

Expression of the Saccharomyces cerevisiae CYC1 gene produces mRNA with more than 20 different 5' ends. A derivative of the CYC1 gene (CYC1-157) was constructed with a deletion of a portion of the CYC1 5'-noncoding region, which includes the sites at which many of the CYC1 mRNAs 5' ends map. A 54-mer double-stranded oligonucleotide homologous with the deleted sequence of CYC1-157 and which included a low level of random base pair mismatches (an average of two mismatches per duplex) was used to construct mutants of the CYC1 gene and examine the role of the DNA sequence at and immediately adjacent to the mRNA 5' ends in specifying their locations. The effect of these mutations on the site selection of mRNA 5' ends was examined by primer extension. Results indicate that there is a strong preference for 5' ends which align with an A residue (T in the template DNA strand) preceded by a short tract of pyrimidine residues.     

Quantitative methods for determining the points of O-(carboxymethyl) substitution in O-(carboxymethyl)-guar

Two methods are described for locating the O-(carboxymethyl) groups in O-(carboxymethyl)guar. In Method I, O-(carboxymethyl)guar was depolymerized by methanolysis, the O-(carboxymethyl) groups were reduced, and the mixture of methyl glycosides and O-(2-hydroxyethyl)-substituted methyl glycosides was converted into a mixture of per-O-acetylated alditols and partially O-(2-acetoxyethyl)ated, partially O-acetylated alditols. Analysis of these alditols by gas-liquid chromatography-mass spectrometry allowed the positions of substitution of the O-(carboxymethyl) groups on the galactosyl groups and mannosyl residues to be determined. However, this method did not distinguish between O-(carboxymethyl) substitution on 4-linked and 4,6-linked mannosyl residues. This limitation was overcome by the more-detailed analysis provided by Method II, in which O-(carboxymethyl)guar was carboxyl-reduced, the product methylated, the glycosyl residues hydrolyzed, the sugars reduced, and the alditols acetylated to yield a mixture of partially O-acetylated, partially O-methylated alditols and partially O-acetylated, partially O-(2-methoxyethyl)ated, partially O-methylated alditols. These derivatives, when separated and quantitated by g.l.c., and identified by g.l.c.-m.s., gave a quantitative measure of every type of carboxymethyl substitution in guar.     

A method for incorporating macromolecules into adherent cells

We describe a simple method for loading exogenous macromolecules into the cytoplasm of mammalian cells adherent to tissue culture dishes. Culture medium was replaced with a thin layer of fluorescently labeled macromolecules, the cells were harvested from the substrate by scraping with a rubber policeman, transferred immediately to ice cold media, washed, and then replated for culture. We refer to the method as "scrape-loading." Viability of cells was 50-60% immediately after scrape-loading and was 90% for those cells remaining after 24 h of culture. About 40% of adherent, well-spread fibroblasts contained fluorescent molecules 18 h after scrape-loading of labeled dextrans, ovalbumin, or immunoglobulin-G. On average, 10(7) dextran molecules (70,000-mol wt) were incorporated into each fibroblast by scrape-loading in 10 mg/ml dextran. The extent of loading depended on the concentration and molecular weight of the dextrans used. A fluorescent analog of actin could also be loaded into fibroblasts where it labeled stress fibers. HeLa cells, a macrophage-like cell line, 1774A.1, and human neutrophils were all successfully loaded with dextran by scraping. The method of scrape-loading should be applicable to a broad range of adherent cell types, and useful for loading of diverse kinds of macromolecules.     

Acidification of phagosomes is initiated before lysosomal enzyme activity is detected

We have measured changes of pH in a protein's microenvironment consequent on its binding to the cell surface and incorporation into pinosomes. Changes of pH were measured from single, living cells and selected regions of cells by the fluorescence ratio technique using a photon-counting microspectrofluorimeter. The chemotactic agent and pinocytosis inducer, ribonuclease, labeled with fluorescein (FTC- RNase), adsorbed to the surface of Amoeba proteus, and was pinocytosed by cells in culture media at pH 7.0. The FTC-RNase entered an apparently acidic microenvironment, pH approximately 6.1, upon binding to the surface of amoebae. Once enclosed within pinosomes, this protein's microenvironment became steadily more acidic, reaching a minimum of pH approximately 5.6 in less than 10 min. FTC-RNase pinocytosed by the giant amoeba, Chaos carolinensis, entered pinosomes whose pH was correlated with their cytoplasmic location during the initial 30-40 min after pinocytosis. The majority of pinosomes containing FTC-RNase clustered in the tail ectoplasm of C. carolinensis during this interval and had a pH of approximately 6.5; those released into endoplasm and carried into the tip of cells had a pH below 5.0. As pinosomes became distributed at random in C. carolinensis (1-2 h after initial pinocytosis), differences in pH between tip and tail pinosomes vanished. We have also measured the pH within single phagosomes of A. proteus. Phagosomal pH dropped steadily to approximately 5.4 within 5 min after particle ingestion in 70% of the cells measured, and reached this level of acidity within 10 min in 90% of the cells measured. By contrast, stain for the lysosomal enzyme, acid phosphatase, was evident within only 20% of 5-min-old phagosomes visualized by light microscopy, and within only 40% of 10-min-old phagosomes. A microfluorimetric assay was used to simultaneously record changes in pH, and the initial deposition of lysosomal esterases, within phagosomes of single, living Amoeba proteus. Near complete acidification of the phagosome was recorded from some cells before phagosomal fusion was evident by this microfluorimetric assay. From other cells, however, continued acidification of phagosomes was recorded after lysosomal fusion was initiated. We conclude that acidification of phagosomes by A. proteus is initiated but not necessarily completed prior to phagosome-lysosome formation, and that the two events are closely linked in time. Initial acidification of endosomes is a property intrinsic to the plasma membrane which envelops particles at the cell surface, rather than the result of lysosomal fusion with phagosomes.     

Blood volume determinations in sheep before and after splenectomy

Using 51Cr labelled RBCs, total blood volume, red cell volume and plasma volume were measured in fifteen adult, female, domestic sheep both before and after splenectomy. Eight of the fifteen animals studied were anemic. Statistical analyses revealed no significant differences in blood volume parameters whether animals were grouped together or separated into normal and anemic groups. We observed: (a) splenectomy produced modest reductions in blood volume parameters in 12 of 15 animals, (b) preoperative variability in blood volume parameters caused by release of sequestered RBCs from the spleen was eliminated after splenectomy, and (c) equilibration of 51Cr required at least 30 minutes in intact animals, but only 10 minutes in splenectomized animals. After volume parameters were normalized to body weight, they were found to agree closely with values reported previously. This study demonstrates the dynamic function of the sheep spleen in the regulation of blood volume.     

Chromosomal Assignment of Human Nuclear Envelope Protein Genes LMNA, LMNB1, and LBR by Fluorescencein SituHybridization

We have used fluorescencein situhybridization to establish precise chromosomal localizations for three human genes encoding four different nuclear envelope proteins. Lamin A/C (LMN1, HGMW-approved symbol LMNA) mapped to 1q21.2–q21.3, with a most probable gene assignment to 1q21.3; lamin B receptor (LBR) was localized to 1q42.1; and lamin B1 (LMNB1) was mapped to the interface of bands 5q23.3–q31.1. Assignments were determined by direct placement of signals relative to high-resolution DAPI or G-bands. Comparison of these results of band positions predicted from fractional length measurements to signal placement indicated that more accurate predictions are made using Francke idiograms and that measurement strategy avoids variance due to polymorphic chromosome segments.