Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture

Summary Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.Abbreviations BAME bovine adrenal medullary endothelial cells - DMEM Dulbecco's modified essential medium - ECM extracellular matrix - EMEM Eagle's modified essential medium - FCS fetal calf serum - PBS phosphate-buffered saline - PC12 rat pheochromocytoma cells     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

Identification of variable region differences in Neisseria meningitidis class 3 protein sequences among five group B serotypes

Strains of Neisseria meningitidis express one of two porin proteins. These porins have been identified as the class 2 and class 3 proteins, and express serotype-specific epitopes. The gene for the class 3 protein was amplified by the polymerase chain reaction from the DNA of a serotype 4 strain as a 1025 bp fragment. The nucleotide sequence of this gene was determined and compared with two recently published sequences. On the basis of this comparison we have identified two major variable regions in the translated protein sequence, VR1 and VR2, that may be associated with serotype specificity. Three other variable regions were also identified. The sequences in the VR1 and VR2 regions from five additional group B N. meningitidis strains of serotypes 1, 4, 8, 12, and 15, all expressing class 3 proteins, were determined. The VR1 and VR2 regions were variable and were flanked by highly conserved regions among eight different class 3 sequences. These two variable regions of 15 and 9 amino acids are predicted to be in surface-exposed loops.     

Quantifying sink and source limitations on dry matter partitioning to fruit growth in peach trees

We describe an approach for determining the degree of sink and source limitations on peach ( Prunus persica L. Batsch) fruit growth during several growth periods. Source limitations on fruit growth may be due to either a shortfall in assimilate supply within the tree (supply limitation) or to a deficiency in the capacity of the translocation system to deliver assimilates in sufficient quantity to support the maximum fruit growth rate (transport/competition limitation). To ascertain the potential maximum rate of fruit growth, fruit thinning treatments were used. One month after bloom, the number of fruits per tree was adjusted to between 50 and 700 on an early and a late maturing peach cultivar (cvs Spring Lady and Cal Red, respectively). Rates of potential sink demand, potential source supply and actual fruit growth were estimated from sequential harvests of all fruits on 42 trees on two (Spring Lady) and three (Cal Red) dates. These values were used to estimate the proportion of potential growth achieved, and the supply and transport/competition limitations on fruit growth. The results indicated that source limitations were significant on trees with moderate to high fruit numbers. These source limitations were due to supply limitations during all harvest intervals and to transport/competition limitations during the early harvest intervals. Sink limitations occurred to the greatest extent during the mid-period of fruit growth on the later maturing cultivar.     

Maximum Vegetative Growth Potential and Seasonal Patterns of Resource Dynamics during Peach Growth

The maximum vegetative growth potential of two peach [Prunuspersica (L.) Batsch] cultivars that differ in the timing ofresource demand for reproductive growth was determined in termsof stem extension, stem and leaf dry weight accumulation, andtrunk radial increment on defruited trees. The maximum vegetativegrowth potentials were similar on the two cultivars indicatingthat the greater partitioning of dry weight to vegetative growthfrequently observed on early maturing cultivars compared tolate maturing cultivars is the result of a shorter period ofcompetition between reproductive and vegetative growth, ratherthan a genetic difference in vegetative growth potential. Onboth cultivars, stem extension and leaf dry weight accumulationceased in mid-summer, however stem dry weight accumulation andtrunk radial increment increase continued through the autumn. The presence of fruit did not have a detectable effect on thefinal stem length, stem dry weight or leaf dry weight on theearly maturing cultivar, but it reduced final stem length anddry weight by 43 and 56%, respectively on the late maturingcultivar. The presence of fruit did decrease stem length, stemdry weight and leaf dry weight on the early maturing cultivarfor 1 month prior to and 1 month after fruit harvest. Fruitdecreased final trunk radial increment by 42 and 77% on theearly and late maturing cultivars, respectively. These reductionsin vegetative growth indicate that resource partitioning tovegetative growth was reduced by competition with fruit growth. Comparison of stem relative extension rates and stem and leafrelative growth rates on fruited and defruited trees indicatedthat vegetative growth was resource-limited shortly after vegetativebud break on fruited trees of both cultivars. This period ofresource-limited vegetative growth corresponded to a periodof resource-limited fruit growth identified in an earlier study.During the period of resource-limited vegetative growth, assimilatesupply was low due to low leaf area index, and carbohydratedemand was relatively high due to high vegetative and reproductivegrowth potentials, creating resource-limited growth conditions.Copyright1995, 1999 Academic Press Maximum vegetative growth potential, carbon economy, partitioning, resource availability, resource limitation, source-limited growth, growth analysis, relative growth rate, peach, Prunus persica (L.) Batsch     

Maximum Fruit Growth Potential and Seasonal Patterns of Resource Dynamics During Peach Growth

Maximum fruit growth potential, the growth attained by fruitswhen they are grown under optimal environmental conditions inthe presence of a non-limiting supply of resources, was estimatedfor two peach [Prunus persica (L.) Batsch] cultivars that differin the timing of resource demand for reproductive growth. Maximumpotential fruit growth was estimated on trees that were heavilythinned at bloom. On these trees, resource availability exceededresource demand for fruit growth. For both cultivars, the mean dry weights of fruits grown onunthinned trees were approximately half the mean dry weightsof fruits grown on trees that were heavily thinned at bloom,indicating that fruit growth was source-limited on unthinnedtrees. Comparison of the seasonal patterns of relative growthrate of fruits on unthinned and heavily thinned trees indicatedthe source-limited fruit growth occurred during distinct periodsof the growing season. On the early maturing cultivar, source-limitedfruit growth occurred from 300 degree-days after bloom untilharvest (4·5-10 weeks after bloom). On the late maturingcultivar, source-limited fruit growth occurred from 200-900and 1600-1900 degree-days (3·5-12 and 18-20 weeks) afterbloom. Although the final dry weight of fruits on the early maturingcultivar was only half that of fruits on the late maturing cultivar,the potential net sink strength of fruits was significantlyhigher on the early than the late maturing cultivar throughoutthe entire growth period of the early maturing cultivar. Resourceavailability for fruit growth was similar on the early and latematuring cultivars, indicating that selection for early maturingfruits has not changed the patterns of resource availabilityfor fruit growth.Copyright 1995, 1999 Academic Press Maximum fruit growth potential, carbon economy, partitioning, resource availability, resource limitation, source-limited growth, sink activity, sink strength, growth analysis, relative growth rate, Prunus persica (L.) Batsch, peach     

Metabolism of lignin related aromatic compounds by Aspergillus japonicus

Archives of Microbiology - Aspergillus japonicus metabolizes a wide variety of aromatic compounds, some of them resulting from lignin degradation. The efficient conversion of such compounds is...     

The natural cytotoxicity receptor 1 contribution to early clearance of Streptococcus pneumoniae and to natural killer-macrophage cross talk

Natural killer (NK) cells serve as a crucial first line of defense against tumors, viral and bacterial infections. We studied the involvement of a principal activating natural killer cell receptor, natural cytotoxicity receptor 1 (NCR1), in the innate immune response to S. pneumoniae infection. Our results demonstrate that the presence of the NCR1 receptor is imperative for the early clearance of S. pneumoniae. We tied the ends in vivo by showing that deficiency in NCR1 resulted in reduced lung NK cell activation and lung IFNγ production at the early stages of S. pneumoniae infection. NCR1 did not mediate direct recognition of S. pneumoniae. Therefore, we studied the involvement of lung macrophages and dendritic cells (DC) as the mediators of NK-expressed NCR1 involvement in response to S. pneumoniae. In vitro, wild type BM-derived macrophages and DC expressed ligands to NCR1 and co-incubation of S. pneumoniae-infected macrophages/DC with NCR1-deficient NK cells resulted in significantly lesser IFNγ levels compared to NCR1-expressing NK cells. In vivo, ablation of lung macrophages and DC was detrimental to the early clearance of S. pneumoniae. NCR1-expressing mice had more potent alveolar macrophages as compared to NCR1-deficient mice. This result correlated with the higher fraction of NCR1-ligand(high) lung macrophages, in NCR1-expressing mice, that had better phagocytic activity compared to NCR1-ligand(dull) macrophages. Overall, our results point to the essential contribution of NK-expressed NCR1 in early response to S. pneumoniae infection and to NCR1-mediated interaction of NK and S. pneumoniae infected-macrophages and -DC.     

A cytokine promoter/yellow fluorescent protein reporter transgene serves as an early activation marker of lymphocyte subsets

A mouse containing an IL-4 promoter linked to the yellow fluorescent protein (YFP) reporter transgene was created to follow aspects of lymphocyte development and function. Following stimulation with phorbol 12-myristate 13-acetate and ionomycin, anti-CD3/CD28, antigen-specific peptide, or allogeneic cells, both CD4 and CD8 T cells expressed the transgene within 24h in a manner that was consistent with cellular activation markers. Transgene induction was inhibited by cyclosporine and FK506, suggesting that its activation occurs in an NFAT-dependent manner. B lymphocytes were also able to express the transgene when stimulated with LPS. This induction was inhibited in part by rapamycin. The results suggest that this transgene can function as an indicator of lymphocyte activation. Because YFP is not toxic and requires no preparation of the cells to view the reporter gene, this system provides a unique tool to follow lymphocyte activation in a number of model systems, such as those involving transplantation, allergy, and vaccine development.     

Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

In Streptococcus pneumonia, phosphoenolpyruvate protein phosphotransferase (PtsA) is an intracellular protein of the monosaccharide phosphotransferase systems. Biochemical and immunostaining methods were applied to show that PtsA also localizes to the bacterial cell-wall. Thus, it was suspected that PtsA has functions other than its main cytoplasmic enzymatic role. Indeed, recombinant PtsA and anti-rPtsA antiserum were shown to inhibit adhesion of S. pneumoniae to cultured human lung adenocarcinoma A549 cells. Screening of a combinatorial peptide library expressed in a filamentous phage with rPtsA identified epitopes that were capable of inhibiting S. pneumoniae adhesion to A549 cells. The insert peptides in the phages were sequenced, and homologous sequences were found in human BMPER, multimerin1, protocadherin19, integrinβ4, epsin1 and collagen type VIIα1 proteins, all of which can be found in A549 cells except the latter. Six peptides, synthesized according to the homologous sequences in the human proteins, specifically bound rPtsA in the micromolar range and significantly inhibited pneumococcal adhesion in vitro to lung- and tracheal-derived cell lines. In addition, the tested peptides inhibited lung colonization after intranasal inoculation of mice with S. pneumoniae. Immunization with rPtsA protected the mice against a sublethal intranasal and a lethal intravenous pneumococcal challenge. In addition, mouse anti rPtsA antiserum reduced bacterial virulence in the intravenous inoculation mouse model. These findings showed that the surface-localized PtsA functions as an adhesin, PtsA binding peptides derived from its putative target molecules can be considered for future development of therapeutics, and rPtsA should be regarded as a candidate for vaccine development.