Computed three-dimensional structures for theras-binding domain of theraf-p74 protein complexed withras-p21 and with its suppressor protein, rap-1A

The three-dimensional structures of theras-p21 protein and its protein inhibitor, rap-1A, have been computed bound to theras-binding domain, RBD (residues 55–131), of theraf-p74 protein, a critical target protein ofras-p21 in theras-induced mitogenic signal transduction pathway. The coordinates of RBD have been reconstructed from the stereoview of an X-ray crystal structure of this domain bound to rap-1A and have been subjected to energy minimization. The energy-minimized structures of bothras- p21 and rap-1A, obtained in previous studies, have been docked against RBD, using the stereo figure of the RBD-rap-1A complex, based on a six-step procedure. The final energy-minimized structure of rap-1A-RBD is identical to the X-ray crystal structure. Comparison of theras-p21- and rap-1A-RBD complexes reveals differences in the structures of effector domains ofras-p21 and rap-1a, including residues 32–47, a domain that directly interacts with RBD, 60–66, 96–110, involved in the interaction ofras-p21 withjun kinase (JNK) andjun protein, and 115–126, involved in the interaction of p21 with JNK. The structure of the RBD remained the same in both complexes with the exception of small deviations in its-2 binding loop (residues 63–71) and residues 89–91, also involved in binding to rap-1A. The results suggest that the binding of these two proteins to RBD may allow them to interact with other cellular target proteins such as JNK andjun.     

Fatty acid double bond orientation alters interaction with L-cell fibroblasts

Relatively little is known of fatty acid specificity in cellular fatty acid uptake. In this study L-cells, a fibroblastic cell line with very low levels of endogenous cytosolic fatty acid binding protein, were used to examine the role of cis and trans unsaturation on fatty acid uptake. The fluorescent fatty acids, trans-parinaric acid and cis-parinaric acid, were used as analogs of straight-chain saturated, and kinked-chain unsaturated fatty acids, respectively, in order to evaluate the fatty acid specificity of the uptake system. Parinaric acid is poorly metabolizable; greater than 97% was unesterified while ³H-oleic acid was almost totally metabolized after 30 min uptake. Cis- and trans-parinaric acid uptake was saturable and dependent on the concentration of fatty acid. However, the initial rate and maximal amount of trans-parinaric acid taken up by the L-cells was greater than for cis-parinaric acid under the same conditions. The affinity of L-cell uptake for trans-parinaric acid (K_m = 0.12 uM) was 35-fold higher than that for cis-parinaric acid (K_m = 4.17 uM) . Based on competition studies with oleic and stearic acids, it was concluded that the cis- and trans-parinaric acid were taken up by the same L-cell fatty acid uptake system. The results suggest that the L-cell fatty acid uptake system has selectivity for straight chain rather than kinked chain unsaturated fatty acids.Abbreviations Cis-parinaric acid 9Z, 11E, 13E, 15Z-octatetraenoic acid - trans-parinaric acid 9E, I IE, 13E, 15E-octatetraenoic acid - EGTA ethylene glycol-bis(beta-amlno-ethyl ether) N,N,N,N-tetratacetic acid - BSA bovine serum albumin - PBS phosphate buffered saline     

Heterospecific pollen transfer between sympatric species in a midsuccessional old-field community

The cumulative (season-long) incidence of heterospecific pollen transfer (HPT) was examined using nine sympatric species in a midsuccessional old field. Inflorescences were collected weekly during the flowering season, and the proportion of foreign pollen/stigma was recorded. Flowering phenologies of sympatric species and ovule and seed counts of study species were also recorded. Heterospecific pollen was detected on some stigmas of each species. Medicago sativa (Fabaceae) received the most foreign pollen; in some cases, all of the grains on a stigma were heterospecific. Lotus corniculatus (Fabaceae) received the least amount of foreign pollen; the incidence of heterospecific pollen was near zero in most cases. The mean and range of foreign pollen received varied by as much as an order of magnitude between species. The six species with zygomorphic flowers, all Fabaceae, received more heterospecific pollen than the three species with actinomorphic flowers, Potentilla recta and P. simplex (Rosaceae) and Ranunculus acris (Ranunculaceae). This probably reflects a bias because our data were analyzed on a cumulative basis and the Fabaceae had longer flowering phenologies. HPT was not correlated with the species' relative abundance within the community. Proportion of foreign pollen received varied temporally within species, and this variation generally was not related to phenology of any sympatric taxa or the species' own phenology. Pollen grain diameter was positively related to levels of foreign pollen received by species. This might be caused by poor adhesion of large pollen grains to small stigmatic papillae or if generalist pollinators carrying large amounts of heterospecific pollen visit the large-grained species and specialists with little foreign pollen visit the small-grained species. The large proportions of heterospecific pollen on stigmas of many species indicate that HPT occurs frequently in the community we studied and the implications may include reduced seed set because of occlusion by foreign grains. As yet, however, it is unclear how important a factor HPT is in mediating pollen limitation of reproductive success.     

An Experimental Test of the Rhizopine Concept in Rhizobium meliloti

In some Rhizobium-legume symbioses, compounds known as rhizopines are synthesized by bacteroids and subsequently catabolized by free-living cells of the producing strain. It has been suggested than rhizopines act as proprietary growth substrates and enhance the competitive ability of the producing strain in its interactions with the diverse microbial community found within the rhizosphere. Wild-type, rhizopine-producing Rhizobium meliloti L5-30 and mutant L5-30 strains deficient for either rhizopine synthesis or catabolism were inoculated onto lucerne host plants in competition experiments. These experiments demonstrated that no apparent advantage resulted from the ability to synthesize a rhizopine, whereas the ability to catabolize rhizopine provided a clear advantage when an organism was in competition with a strain without this ability. The results suggest that when an organism is in competition with a catabolism-deficient mutant, the ability to catabolize rhizopine results in enhanced rates of nodulation. The results of the experiments were not consistent with the hypothesis that the sole role of rhizopines is to act as proprietary growth substrates for the free-living population of the producing strain.     

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation.     

In situ treatment of Hamilton Harbour sediment

To enhance the biodegradation of organic contaminants, approximately 18.5 tonnes of oxidant (calcium nitrate) and 5 tonnes of nutrients were injected into sediments of the Dofasco Boatslip, Hamilton Harbour. In the laboratory 78% and 68% of the oil (TPHs) and polynuclear aromatic hydrocarbons (PAHs), respectively biodegraded in 197 days. In the 1992 treatment in the Ddofasco Boatslip, biodegradation of organic contaminants varied from 79% for low molecular weight compounds (BTXs), to 25/15 of the 16 priority pollutant PAHs. At first biodegraduation of large molecular weight PAHs resulted in the production of naphthalene (from 280 g/g to 549 g/g). In the 1993 treatments, 94% of the naphthalene, and 57% of the TPHs biodegraded. The in situ biotreatment of organic contamination takes time but for some sites the significantly lower cost relative to dredging and confinement makes in situ treatment a viable alternative.     

The dual-specificity CLK kinase induces neuronal differentiation of PC12 cells.

CLK is a dual-specificity protein kinase capable of phosphorylating serine, threonine, and tyrosine residues. We have investigated the action of CLK by establishing stable PC12 cell lines capable of inducibly expressing CLK. Expression of CLK in stably transfected PC12 cells mimicked a number of nerve growth factor (NGF)-dependent events, including the morphological differentiation of these cells and the elaboration of neurites. Moreover, CLK expression enhanced the rate of NGF-mediated neurite outgrowth of these cells, indicating that CLK expression and NGF treatment activate similar signal transduction pathways. CLK expression, unlike NGF, was not able to promote PC12 cell survival in serum-free media, demonstrating that CLK only partially recapitulated the actions of NGF on these cells and that the biochemical pathways necessary for morphological differentiation can be stimulated without also stimulating those necessary for survival. Induction of CLK expression also resulted in the selective activation of protein kinases that are components of growth factor-stimulated signal transduction cascades, including ERK1, ERK2, pp90RSK, and S6PKII. Induction of CLK expression, however, did not stimulate pp70S6K or Fos kinase, two NGF-sensitive protein kinases. These data indicate that CLK action mediates the morphological differentiation of these cells through its capacity to independently stimulate signal transduction pathways normally employed by NGF.     

Lectin-induced deciduoma formation in the pseudopregnant rat

Decidual cell induction in the pseudopregnant rat was examined in this study using the lectin concanavalin A (ConA). The histochemical binding of the lectin to the uterine cell surface at the time of deciduomatic induction was also studied. ConA was found to induce significant deciduomata (decidual-like tissue) in the uterine horn when injected intraluminally on day 5 of pseudopregnancy (PSP). ConA-induced deciduomata appeared as a series of discrete nodules in the uterine horn, reminiscent of the anatomical appearance of normal embryo implantation sites. Deciduoma induction by ConA was greatly reduced by pre-absorption of the lectin with its competitive sugar. Lectin histochemistry revealed binding of ConA to the cell surface on day 5 of PSP. Pre-absorption of the lectin with its competitive sugar also significantly reduced surface binding of the lectin, and this finding may be correlated with the greatly reduced ability of the pre-absorbed lectin to induce deciduomata. Possible mechanisms for the induction of deciduomata by lectins are considered.     

Genetic Analysis of Hispanic Individuals with Cystic Fibrosis

We have performed molecular genetic analyses of Hispanic individuals with cystic fibrosis (CF) in the southwestern United States. Of 129 CF chromosomes analyzed, only 46% (59/129) carry ΔF508. The G542X mutation was found on 5% (7/129) of CF chromosomes. The 3849+10kbC→T mutation, detected primarily in Ashkenazi Jews, was present on 2% (3/129). R1162X and R334W, mutations identified in Spain and Italy, each occurred on 1.6% (2/129) of CF chromosomes. W1282X and R553X were each detected once. G551D and N1303K were not found. Overall, screening for 22 or more mutations resulted in detection of only 58% of CF transmembrane conductance regulator gene mutations among Hispanic individuals. Analysis of KM19/XV2c haplotypes revealed an unusual distribution. Although the majority of ΔF508 mutations are on chromosomes of B haplotypes, the other CF mutations are on A and C haplotypes at higher-than-expected frequencies. These genetic analyses demonstrate significant differences between Hispanic individuals with CF and those of the general North American population. Assessment of carrier/affected risk in Hispanic CF individuals cannot, therefore, be based on the mutation frequencies found through studies of the general population but must be adjusted to better reflect the genetic makeup of this ethnic group. Further studies are necessary to identify the causative mutation(s) in this population and to better delineate genotype/phenotype correlations. These will enable counselors to provide more accurate genetic counseling.