Construction of an ordered overlapping library of bacteriophage P1 DNA in phage vector lambda D69

A library of bacteriophage P1 DNA was constructed in the phage vector lambda D69. The DNA of some 150 randomly chosen lambda-P1 hybrid phages containing P1 DNA fragments 5-10 kb in size was analyzed by restriction endonuclease digestion using enzymes EcoRI, BglII, and BamHI that cleave P1 DNA at known positions on the physical map of P1. Approximately one third of the phages contained P1 DNA inserts having two or more restriction sites for any one of these enzymes, thus allowing the location of the insert to be determined with respect to the physical map. Genetic tests allowed detection of lambda-P1 hybrid phages possessing inserts with functional P1 ban and CmR genes. A subset of 18 phages was analyzed in more detail; their P1 DNA inserts comprise an ordered collection of overlapping P1 DNA fragments that cover almost 98% of the P1 genome.     

Gas exchange and water balance of a mistletoe species and its mangrove hosts

Summary The gas exchange and water relations of the hemiparasite Pthirusa maritima and two its mangrove host species, Conocarpus erectus and Coccoloba uvifera, were studied in an intertidal zone of the Venezuelan coast. Carbon uptake and transpiration, leaf osmotic and total water potential, as well as nutrient content in the xylem sap and leaves of mistletoes and hosts were followed through the dry and wet season. In addition, carbon isotope ratios of leaf tissue were measured to further evaluate water use efficiency. Under similar light and humidity conditions, mistletoes had higher transpiration rates, lower leaf water potentials, and lower water use efficiencies than their hosts. Potassium content was much higher in mistletoes than in host leaves, but mineral nutrient content in the xylem sap of mistletoes was relatively low. The resistance of the liquid pathway from the soil to the leaf surface of mistletoes was larger than the total liquid flow resistance of host plants. Differences in the daily cycles of osmotic potential of the xylem sap also indicate the existence of a high resistance pathway along the vascular connection between the parasite pathway along the vascular connection between the parasite and its host. P. maritima mistletoes adjust to the different physiological characteristics of the host species which it parasitizes, thus ensuring an adequate water and carbon balance.     

Can a simple function for the dielectric response model electrostatic effects in globular proteins?

The relationship between the effective dielectric constant that models the electrostatic effect from a charged side chain in a protein was evaluated both experimentally and theoretically. Experimental values were obtained from the shifts in pKa that resulted from point mutations of side chains in subtilisin. Theoretical values were obtained from an iterative solution to Poisson's equation that considers the dielectric response of the protein and the solvent together with charge positions. There is no simple relationship between the effective dielectric constant and the distance from the charge responsible for the interactions. For some charge positions a linear but not a direct proportional relationship of the effective dielectric with distance of separation was observed. Thus, simple models such as a linear distance-dependence for the dielectric response are not suitable to evaluate electrostatic effects in proteins.     

A predicted three-dimensional structure for the human immunodeficiency virus binding domains of CD4 antigen

A predicted three-dimensional structure of the two N-terminal extracellular domains of human CD4 antigen, a cell surface glycoprotein, is reported. This region of CD4, particularly the first domain, has been identified as containing the binding region for the envelope gp120 protein of the human immunodeficiency virus. The model was predicted based on the sequence homology of each domain with the variable light chain of immunoglobulins. The framework beta-sheet regions were taken from the crystal coordinates of REI. For one region in the first domain of CD4 there was an ambiguity in the alignment with REI and two alternate models are presented. Loops connecting the framework were modelled from fragments selected from a database of main chain coordinates from all known protein structures. Residues identified as involved in binding gp120 have been located in several other studies within the first domain of CD4. Epitopes from eight monoclonal antibodies have been mapped onto residues in both domains. Competition of these antibodies with each other and with gp120 can be interpreted from the structural model.     

Analysis and prediction of the location of catalytic residues in enzymes

The catalytic residues of an enzyme are defined as the amino acids directly involved in chemical catalysis. They mainly act as a general acid--base, electrophilic or nucleophilic catalyst or they polarize and stabilize the transition state. An analysis of the structural features of 36 catalytic residues in 17 enzymes of known structure and with defined mechanism is reported. Residues that bind metal ions (Zn2+ and Cu2+) are considered separately. The features examined are: residue type, location in secondary structure, separation between the residues, accessibility to solvent, intra-protein electrostatic interactions, mobility as evaluated from crystallographic temperature factors, polarity of the environment and the sequence conservation between homologous enzymes of residues that were sequentially or spatially close to the catalytic residue. In general the environment of catalytic residues is similar to that of polar side chains that have low accessibility to solvent. Two algorithms have been developed to identify probable catalytic residues. Scanning an alignment of homologous enzyme sequences for peaks of sequence conservation identifies 13 out of the 16 catalytic residues with 50 residues overpredicted. When the conservation of the spatially close residues is used instead, a different set of 13 residues are identified with 47 residues overpredicted. A combination of the two algorithms identifies 11 residues with 36 residues overpredicted.     

Nerve growth factor (NGF) regulates adult rat cultured dorsal root ganglion neuron responses to the excitotoxin capsaicin

An overlap between subpopulations of nerve growth factor (NGF)-responsive and capsaicin-sensitive dorsal root ganglion (DRG) sensory neurons has been suggested from a number of in vivo studies. To examine this apparent link in more detail, we compared the effects of capsaicin on adult rat DRG neurons cultured in the presence or absence of NGF. Capsaicin sensitivity was assessed histochemically by a cobalt staining method, by measuring capsaicin-induced 45Ca2+ uptake, and by electrophysiological recording of capsaicin-evoked membrane currents. When cultured with NGF, approximately 50% of these adult DRG neurons were capsaicin-sensitive, whereas adult sympathetic neurons or ganglionic nonneuronal cells were insensitive. DRG cultures grown in the absence of NGF, however, were essentially unresponsive to capsaicin. Capsaicin sensitivity could be regained fully within 4-6 days of replacement of NGF. These results indicate that, at least in vitro, NGF can modify the capsaicin sensitivity of adult DRG neurons.     

Effect of serotonin on murine macrophages: suppression of Ia expression by serotonin and its reversal by 5-HT2 serotonergic receptor antagonists

Serotonin (5-HT), a mediator released from platelets at sites of inflammation, suppressed IFN-gamma-induced Ia expression in mouse bone marrow macrophages maintained in vitro. (Mean percent suppression = 63.9% +/- 9.2, n = 40.) This suppression was not toxic or endotoxin-related, was concentration-dependent, and occurred at the physiologic concentrations of 5-HT present at inflammatory sites. The concentration of 5-HT producing the half-maximal effect was 2.5 to 5.5 X 10(-8) M. Related compounds, dopamine, histamine, and tryptamine, were much less potent in suppressing IFN-gamma-induced Ia, with maximally suppressing concentrations more than 100-fold higher than the maximally suppressing 5-HT concentration. L-5-hydroxytryptophan (5-HTP), the most potent analog tested, was 10-fold less potent than 5-HT in suppressing Ia expression. The concentration of 5-HTP producing the half-maximal effect = 4 X 10(-7) M. 5-HT suppression of IFN-gamma-induced Ia expression was antagonized by the 5-HT2 type receptor antagonists spiperone, ketanserin, and LY53857. Concentrations of these agents resulting in 50% inhibition of the serotonin effect were 1.5 X 10(-8) M, 7.5 X 10(-8) M, and 4.5 X 10(-12) M, respectively. 5-HT was most effective in suppressing IFN-gamma-induced Ia when added early in culture simultaneously with IFN-gamma. These data provide functional evidence that 5-HT suppression of IFN-gamma-induced Ia expression is mediated through a 5-HT receptor with some characteristics of the 5-HT2 type. 5-HT may play a physiologic role at sites of inflammation as a modulator of the effects of IFN-gamma on macrophage function.     

Physiological and isotopic aspects of photosynthesis in peperomia

Physiological and isotopic aspects of several Peperomia species were investigated. All but one species had C₃-like stomatal behavior, in that stomata were open during the day and closed during the night. In these species, most atmospheric CO₂ uptake occurred during the day. Concurrent with this stomatal behavior, there were Crassulacean acid metabolism-like acid fluctuations in most species. Carbon and hydrogen isotope ratios of cellulose nitrate from Peperomia reflect their physiological behavior. The δ¹³C values of cellulose nitrate from Peperomia species were similar to values observed in C₃ plants and consistent with the daytime uptake of exogeneous CO₂ via the C₃ photosynthetic pathway. The δD values of cellulose nitrate from Peperomia species approach those of Crassulacean acid metabolism plants. These elevated δD values are caused by fractionations occurring during biochemical reactions and not as a consequence of water relations.     

Variable Photosynthetic Metabolism in Leaves and Stems of Cissus quadrangularis L

By measuring titratable acidity, gas exchange parameters, mesophyll succulence, and ¹³C/¹²C ratios, we have shown that Cissus quadrangularis L. has C₃-like leaves and stems with Crassulacean acid metabolism (CAM). In addition, the nonsucculent leaves show the diurnal fluctuations in organic acids termed recycling despite the fact that all CO₂ uptake and stomatal opening occurs during the day. Young succulent stems have more C₃ photosynthesis than older stems, but both have characteristics of CAM. The genus Cissus will be a fruitful group to study the physiology, ecology, and evolution of C₃ and CAM since species occur that exhibit characteristics of both photosynthetic pathways.