A comparison of techniques for isolation of the outer membrane proteins of Haemophilus influenzae type b

We compared several rapid techniques used for extraction of outer membrane proteins from gram-negative enteric bacteria to Haemophilus influenzae type b. After lysis of cells with a French press, the inner and outer membranes were separated by isopycnic centrifugation. Each membrane was identified by density, morphology, enzymatic activity, and susceptibility to solid-phase iodination of intact cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, we identified 10 polypeptides which were enriched in the outer membrane band compared to the inner membrane band. Using these proteins, we compared the polypeptide pattern of outer membranes with that obtained by (1) selective solubilization with sodium dodecyl-beta-D-maltoside, octyl-beta-D-glucopyranoside, Triton X-100, sodium, or cholamidopropyl dimethylaminopropanesulfonate; (2) extraction with chaotropic agents and heat; and (3) differential centrifugation of vesicles shed during transition from log growth phase to stationary growth phase. There were definable differences between the polypeptide pattern of membranes obtained with each rapid technique compared to the polypeptide pattern of isolated outer membranes. The polypeptide pattern of lithium extracts and the Triton X-100 insoluble fractions of total membranes most closely approximated the polypeptide pattern of isopycnically isolated outer membranes. Depending on the outer membrane protein sought, one of these rapid techniques can be utilized when a rapid method of outer membrane protein isolation is required.     

Insect Feeding Deterrents in Endophyte-Infected Tall Fescue

The presence of an endophytic fungus, Acremonium coenophialum, in tall fescue (Festuca arundinacea) deterred aphid feeding by Rhopalosiphum padi and Schizaphis graminum. Both species of aphid were unable to survive when confined to endophyte-infected tall fescue plants. Feeding deterrents and toxic factors to R. padi and Oncopeltus fasciatus, large milkweed bug, were primarily associated with a methanol extract obtained when endophyte-infected tall fescue seed was serially extracted with hexane, ethyl acetate, and methanol. The concentrations of pyrrolizidine alkaloids were determined to be 30 to 100 times greater in the methanol extract than in the hexane and ethyl acetate extracts.     

Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes

Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater than 50 A. When gel-filtered rabbit muscle actin is polymerized in the presence of band 4.9, actin bundles are generated that are similar in appearance to those induced by "vinculin" or fimbrin. The bundles appear brittle and when they are centrifuged small pieces of filaments break off and remain in the supernatant. At low band 4.9 to actin molar ratios (1:30), band 4.9 lowers the apparent steady-state low-shear falling ball viscosity by sequestering filaments into thin bundles; at higher ratios, the bundles become thicker and obstruct the ball's movement leading to an apparent increase in steady-state viscosity. Band 4.9 increases the length of the lag phase and decreases the rate of elongation during actin polymerization as measured by high-shear Ostwald viscometry or by the increase in the fluorescence of pyrene-labeled actin. Band 4.9 does not alter the critical actin monomer concentration. We hypothesize that band 4.9, together with actin, erythrocyte tropomyosin, and spectrin, forms structures in erythroid precursor cells analogous to those formed by fimbrin, actin, tropomyosin, and TW 260/240 in epithelial brush borders. During erythroid development and enucleation, the actin filaments may depolymerize up to the membrane, leaving a membrane skeleton with short stubs of actin bundled by band 4.9 and cross-linked by spectrin.     

Thermal ecology of Pieris butterflies (Lepidoptera: Pieridae): a new mechanism of behavioral thermoregulation

Summary I document a new mechanism for behavioral thermoregulation, not previously described in animals, called reflectance basking. This behavior, described here for Pieris butterflies, involves the use of the wings as solar reflectors that reflect solar radiation onto the body to increase body temperature. Results show that Pieris require thoracic (body) temperature. between 29° and 40° C in order to take off and fly, and achieve these elevated temperatures by basking. Diurnal patterns of population flight activity are closely correlated with patterns of body temperature during basking. Behavioral studies indicate that 1) Pieris orient to solar radiation, 2) they use thermoregulatory postures consistent with reflectance basking, and 3) they do not use the basking postures found in other Pierid butterflies (i.e., the Coliadinae). There are consistent differences in wing angles used in reflectance basking between Pieris in different subgenera. Results are discussed with respect to thermoregulation and wing color in other Pierid butterflies, and suggest that a re-evaluation of the functional significance of melanization in Pieris is needed.     

Chloroplasts in the epidermis of Sarracenia (the American pitcher plant) and their possible role in carnivory - An immunocytochemical approach

A photosynthetic apparatus is present in the epidermis of the bottom zone of the pitcher of Sarracenia purpurea L. ssp. purpurea. This has been demonstrated using conventional light and electron microscopy, as well as fluorescent and immunohistochemical techniques. Red intrinsic fluorescence by these chloroplasts indicates photochemical activity. Antibodies against the coupling factor of chloroplast ATPase and against the subunits of ribulose-bis-phosphate-carboxylase were bound to the epidermal chloroplasts. This has been visualized using a ferritin-isothiocyanate labeled second antibody. These results unequivocally prove the existence of the two main proteins which are associated with the photophosphorylation (membrane protein) and carbon dioxide fixation (stromal protein). The possible implication of this system to interrelationships between the carnivorous plant and aquatic insects inhabiting its leaves is discussed.     

Effects of Neuronal Activity on Inositol Phospholipid Metabolism in the Rat Autonomic Nervous System

The effect of nerve stimulation on inositol phospholipid hydrolysis in autonomic tissue was assessed by direct measurement of [3H]inositol phosphate production in ganglia that had been preincubated with [3H]inositol. Within minutes, stimulation of the preganglionic nerve increased the [3H]inositol phosphate content of the superior cervical sympathetic ganglion indicating increased hydrolysis of inositol phospholipids. This effect was blocked in a low Ca2+, high Mg2+ medium. It was also greatly reduced when nicotinic and muscarinic antagonists were present together in normal medium. However, neither the nicotinic antagonist nor the muscarinic antagonist alone appeared to be as effective as both in combination. In other experiments, stimulation of the vagus nerve caused dramatic increases in [3H]inositol phosphate in the nodose ganglion but did not increase [3H]inositol phosphate in the nerve itself. This effect was insensitive to the cholinergic antagonists. Thus, neuronal activity increased inositol phospholipid hydrolysis in a sympathetic ganglion rich in synapses, as well as in a sensory ganglion that contains few synapses. In the sympathetic ganglion, synaptic stimulation activated inositol phospholipid hydrolysis and this was primarily due to cholinergic transmission; both nicotinic and muscarinic pathways appeared to be involved.     

Retention of ingested latex particles in Peyer's patches of germfree and conventional mice

Conventional and germfree mice ingested a suspension of 2-micron latex particles in drinking water for a 15-day period. Number and distribution of intestinal Peyer's patches did not differ significantly in the two types of mice. Cleared Peyer's patches were compared with regard to size and particle content. The location of particles within Peyer's patch follicles of germfree mice was similar to that of conventional mice, but the latter had significantly larger follicles and greater accumulations of latex particles. Latex concentration varied with patch location. Proximal patches contained the majority of particles in germfree mice, whereas particles were most abundant in distal patches of conventional mice. The results show that particle uptake into Peyer's patches takes place even in the complete absence of bacteria in the gut.     

Formation of prostaglandins by ovarian carcinomas

Tissue contents of prostaglandins (PG) PGE2, PGE2a and 6-keto-PGF1a (degradation product of PGI2) were determined in specimens of advanced human ovarian cancer (n = 11). The PG levels (ng/mg tissue protein) varied widley: PGE2 17-515; PGF2a 2-43 and 6-keto-PGF1a 5-105. Tumors of patients without response to chemotherapy contained more PGE2, PGF2a and 6-keto-PGF1a than did tumors responding to chemotherapy. PG production was investigated in two ovarian carcinoma-derived cell lines. The ability of these cells to synthesize PG varied depending on the cell density. An increase of cell number was associated with a decrease of PG yield. PG formation was inhibited by indomethacin in a concentration-dependent manner. The present study suggests that ovarian carcinoma cells form PG in vivo and vitro.     

Effect of the ovulatory cycle on oviductal sperm storage in the domestic fowl

Anovulatory domestic hens (pregnant mare serum-treated) and normally cyclic domestic hens were artificially inseminated with 0.034 ml of pooled semen. A subsequent microscopic assessment of the uterovaginal sperm storage glands on days 1, 3, 6, 9 and 12 post-insemination indicated that the sperm glands emptied over time in the normally cyclic hens, but not in the anovulatory hens. The data suggest that events associated with ovulation and/or oviposition are important to the sperm gland emptying process.     

The nucleotide sequence of tobacco rattle virus RNA-2 (CAM strain).

The nucleotide sequence of the smaller genomic strand (RNA-2) of the bipartite tobacco rattle virus (CAM strain) has been determined. RNA-2 is capped at the 5' terminus and contains 1799 nucleotide residues. There is a single 223 codon long open reading frame extending from nucleotide 574 to 1242 which designates a protein of Mr 23,654. The derived amino acid composition, in percent, matches that previously determined for the virus capsid protein. The long open reading frame is flanked by 5' and 3' untranslated regions of 573 and 554 nucleotides, respectively. The 5' leader sequence contains two different sets of direct repeats, one of 119 nucleotides and the other of 76. It also contains 13 apparently unused AUG codons, four of which lie in the same frame as the capsid protein cistron. The 3' terminal sequence of RNA-2 is identical to that of the larger genomic strand (RNA-1) for 459 nucleotides.