Purification of Neuronal Cell Surface Proteins and Generation of Epitope-Specific Monoclonal Antibodies Against Cell Adhesion Molecules

To establish a procedure for the purification of a broad spectrum of cell surface proteins, three separate methods based on different principles were compared with the aid of four marker proteins. Membrane preparation by sedimentation-flotation centrifugation, temperature-induced phase separation with Triton X-114, and lectin affinity chromatography were used separately as well as in combination. The two-step procedure of membrane preparation and lectin affinity chromatography provided by far the best enrichment of cell surface marker proteins. This result was further substantiated by screening greater than 6,600 hybridoma cultures that originated from mice that had been immunized with protein fractions obtained by different purification protocols. In addition, it was found that solubilized glycoproteins used as immunogens led to many more cell surface-specific monoclonal antibodies than glycoproteins immobilized on lectin-agarose beads. Three monoclonal antibodies that recognize distinct epitopes of cell adhesion molecules (CAMs) were isolated. Monoclonal antibody C4 bound to a detergent-labile epitope of G4 (neuron-glia CAM). Monoclonal antibody D1 recognized specifically nonreduced neural CAM (N-CAM) with intact disulfide bridges, and monoclonal antibody D3 recognized only the 180-kilodalton isoform of N-CAM. Because of these specificities, these monoclonal antibodies promise to be useful tools for the elucidation of the structural organization of adhesion molecules.     

Estimating recent growth in the cuttlefish Sepia officinalis: are nucleic acid-based indicators for growth and condition the method of choice?

A laboratory calibration study was undertaken with juvenile Sepia officinalis (80-85 g initial wet weight) to investigate the effects of different food rations and different starving intervals on RNA/dry weight (DW) ratios and RNA/DNA ratios in cephalopod mantle muscle at two different temperatures. The digestive gland index was also used as an additional indicator of recent growth. High food rations and low temperature went along with high RNA/DW ratios and high RNA/DNA ratios. Starving resulted in a linear decline in growth performance and a concomitant decrease in RNA/DW and RNA/DNA ratio, with RNA/DNA ratios representing the growth data better. RNA/DNA ratios decreased faster at higher temperatures. A fluorimetric assay for nucleic acid analysis was optimized for cephalopod mantle tissues and yielded reproducible RNA/DNA ratios with a relative variance below 10%. Thus, it may be possible to use this estimator of recently encountered feeding regime for the evaluation of mortality rates of early teuthid paralarvae to eventually support stock management. Also, log relative digestive gland weight showed a strong relationship with starving time, but, surprisingly, not with temperature. Data from the two temperatures analyzed could be combined to form a common regression line of relative digestive gland index with starving time. This indicator for recent growth might be especially suitable for large specimens with a well-developed digestive gland.     

Rad52 recruitment is DNA replication independent and regulated by Cdc28 and the Mec1 kinase

Recruitment of the homologous recombination machinery to sites of double‐strand breaks is a cell cycle‐regulated event requiring entry into S phase and CDK1 activity. Here, we demonstrate that the central recombination protein, Rad52, forms foci independent of DNA replication, and its recruitment requires B‐type cyclin/CDK1 activity. Induction of the intra‐S‐phase checkpoint by hydroxyurea (HU) inhibits Rad52 focus formation in response to ionizing radiation. This inhibition is dependent upon Mec1/Tel1 kinase activity, as HU‐treated cells form Rad52 foci in the presence of the PI3 kinase inhibitor caffeine. These Rad52 foci colocalize with foci formed by the replication clamp PCNA. These results indicate that Mec1 activity inhibits the recruitment of Rad52 to both sites of DNA damage and stalled replication forks during the intra‐S‐phase checkpoint. We propose that B‐type cyclins promote the recruitment of Rad52 to sites of DNA damage, whereas Mec1 inhibits spurious recombination at stalled replication forks.     

Fluid replacement following dehydration reduces oxidative stress during recovery

To investigate the effects of hydration status on oxidative DNA damage and exercise performance, 10 subjects ran on a treadmill until exhaustion at 80% VO_2max during four different trials [control (C), 3% dehydration (D), 3% dehydration + water (W) or 3% dehydration + sports drink (S)]. Dehydration significantly decreased exercise time to exhaustion (D < C and S). Plasma MDA levels were significantly higher at pre-exercise in D than C. Plasma TAS was significantly lower at pre-exercise in C and S than in D, and was significantly lower in S than D at 60 min of recovery. Dehydration significantly increased oxidative DNA damage during exercise, but fluid replacement with water or sports drink alleviated it equally. These results suggest that (1) dehydration impairs exercise performance and increases DNA damage during exercise to exhaustion; and (2) fluid replacement prolongs exercise endurance and attenuates DNA damage.     

Macromolecular synthesis in organ cultures of neonatal rat lung

Organ cultures of newborn rat lungs synthesize and accumulate DNA, RNA, collagen and noncollagenous proteins almost at a linear rate for at least 5 days. During this period the synthesis of collagen consistently exceeds the synthesis of noncollagenous proteins in a pattern similar to neonatal lung growth in vivo. Although some morphological characteristics of lung architecture are distorted after culture, fundamental structural similarities to lungs growing in intact animals are retained. When these cultures are maintained in atmospheres rich in oxygen, increased collagen synthesis is observed, a response similar to that of lungs in intact animals exposed to high oxygen concentrations in vivo. Our studies suggest that lung organ cultures may be a suitable system for investigating the biochemical aspects of lung tissue-environmental interaction. These studies were supported in parts by NIH Grant HL-19668, a contract (68-03-2005) from the U.S. Environmental Protection Agency, and grants from the California Lung Association.     

Purification of prostaglandin E2-9-oxoreductase from human decidua vera

Prostaglandin E2-9- oxoreductase (PGE2-9-OR), the enzyme which converts prostaglandin E2 (PGE2) to prostaglandin F2 alpha (PGF2 alpha), has been detected in human decidua vera. A 105-fold purification was achieved when the centrifuged homogenate was fractionated sequentially by DEAE-Trisacryl, hydroxyapatite-agarose gel, ultrogel AcA 44 and Matrex gel blue A gel chromatographies. The following kinetic constants for PGE2-9-OR have been obtained. The equilibrium constant with respect to PGE2 is 83 microM, the Michaelis constant, Km, for PGE2 is 80 microM, for NADPH 1.6 microM. The maximal velocity for the forward reaction is V1 = .203 pmol/min. The enzyme was inhibited by progesterone, oestradiol-17 beta, cortisol and pharmaceutical drugs. An activating effect could be demonstrated with Ca2+ and oxytocin. The occurrence of PGE2-9-OR in the decidua vera suggests that this enzyme may be responsible for the transformation of PGE2 to PGF2 alpha in these tissues. This may be an important mechanism for the initiation and maintenance of uterine contractions.     

CHARACTER DISCRIMINATORY POWER,CHARACTER-SET CONGRUENCE,AND THE CLASSIFICATION OF INDIVIDUALS FROM HYBRID ZONES: AN EXAMPLE USING STONE CRABS (MENIPPE)

Many investigators categorize individuals from hybrid zones to facilitate comparisons among genotypic classes (e.g., parental, F₁, backcross) for comparative studies in which components of fitness or geographic variation are being analyzed. Frequently, multiple character sets representing genetically independent traits are used to classify these individuals and various methodologies are employed to combine the classifications obtained from the different character sets. We adapted the principles of total evidence and taxonomic congruence (two formalized approaches used by systematists in formulating phylogenetic hypotheses) to address the problem of discriminating hybridizing species and classifying individuals from hybrid zones. As our model, we used two morphological (coloration and morphometric) and two molecular (allozyme and mitochondrial DNA restriction-fragment-length polymorphism) character sets that differentiate two stone crab species (Menippe adina and M. mercenaria). Using principal-components analysis, we determined that combining character sets and eliminating characters or character sets that did not have large eigenvector coefficients for the principal component that best separated the two species yielded the highest level of discrimination between species and allowed us to classify a broad range of morpho-genotypes as hybrids. For the stone crabs, three diagnostic allozyme loci and five diagnostic coloration characters best separated the species. The two character sets were not completely congruent, but they agreed in their classification of 50% of the individuals from the hybrid zone and rarely strongly disagreed in their classifications. Classification discrepancies between the two character sets probably represent variation between traits in interspecific gene flow rather than intraspecific, ecologically mediated variation. Our results support the assertions of previous investigators who espoused the benefits associated with using multiple character sets to classify individuals from hybrid zones and demonstrate that, if character sets are reasonably congruent and numerically balanced, combining diagnostic characters from multiple character sets (a total-evidence approach) can enhance discriminatory power between species and facilitate the assignment of hybrid-zone individuals to genotypic classes. On the contrary, classifying hybrid-zone individuals using character sets separately (a taxonomic-congruence approach) provides the opportunity to compare levels of introgression between species and to assess reasons for discordance among the data sets.     

Analytical characterization and purification of plasma membrane from cultured hepatoma cells (HTC cells)

The plasma membrane of the hepatoma cell line, HTC cells, has been characterized and purified by cell fractionation techniques. In the absence of true 5′-nucleotidase in HTC cells, alkaline phosphodiesterase I has been used as a marker enzyme, following conclusions gained from differential and isopycnic centrifugation studies (Lopez Saura, P., Trouet A. and Tulkens P. (1978) Biochim. Biophys. Acta 543, 430–449). To confirm this localization, HTC cells were exposed to anti-plasma membrane IgG at 4°C and fractionated. Alkaline phosphodiesterase I and IgG showed super imposable distribution patterns in linear sucrose gradients. Alkaline phosphodiesterase I is, however, only poorly resolved from enzyme markers of other organelles, especially NADPH-cytochrome $\text{c}$ reductase (endoplasmic reticulum) and galactosyltransferase (Golgi complex). Maximal purification from the homogenate is only 13-fold, on a protein basis, even when using a microsomal fraction (67 and 13% of alkaline phosphodiesterase I and protein, respectively) as the starting material. Improved resolution can be obtained after the addition of small quantities of digitonin (equimolar with respect to the cholesterol content). Digitonin increases the buoyant density of alkaline phosphodiesterase I by approx. 0.05 g/cm³, whereas the buoyant densities of galactosyltransferase and NADPH-cytochrome $\text{c}$ reductase are increased only by 0.03 and 0.015 g/cm³, respectively. Accordingly, a procedure has been designed which yields a fraction containing 22.8% of alkaline phosphodiesterase I with a purification of 21-fold on a protein basis. The content of NADPH-cytochrome $\text{c}$ reductase and galactosyltransferase is 1.2 and 2.1%, respectively. Electron microscopy shows smooth surface membrane elements and vesicles, with only occasional other recognizable elements.     

Single copy DNA homology in sea stars

Summary The sequence homology in the single copy DNA of sea stars has been measured. Labeled single copy DNA fromPisaster ochraceus was reannealed with excess genomic DNA fromP. brevispinus, Evasterias troschelii, Pycnopodia helianthoides, Solaster stimpsoni, andDermasterias imbricata. Reassociation reactions were performed under two criteria of salt and temperature. The extent of reassociation and thermal denaturation characteristics of hybrid single copy DNA molecules follow classical taxonomic lines.P. brevispinus DNA contains essentially all of the sequences present inP. ochraceus single copy tracer whileEvasterias andPycnopodia DNAs contain 52% and 46% of such sequences respectively. Reciprocal reassociation reactions with labeledEvasterias single copy DNA confirm the amount and fidelity of the sequence homology. There is a small definite reaction of uncertain homology betweenP. ochraceus single copy DNA andSolaster orDermasterias DNA. SimilarlySolaster DNA contains sequences homologous to approximately 18% ofDermasterias unique DNA. The thermal denaturation temperatures of heteroduplexes indicate that the generaPisaster andEvasterias diverged shortly after the divergence of the subfamilies Pycnopodiinae and Asteriinae. The twoPisaster species diverged more recently, probably in the most recent quarter of the interval since the separation of the generaPisaster andEvasterias.