Discovery of very late antigen-4 (VLA-4, alpha4beta1 integrin) allosteric antagonists

Integrins are cell adhesion receptors that mediate cell-to-cell, or cell-to-extracellular matrix adhesion. They represent an attractive target for treatment of multiple diseases. Two classes of small molecule integrin inhibitors have been developed. Competitive antagonists bind directly to the integrin ligand binding pocket and thus disrupt the ligand-receptor interaction. Allosteric antagonists have been developed primarily for α(L)β(2)- integrin (LFA-1, lymphocyte function-associated antigen-1). Here we present the results of screening the Prestwick Chemical Library using a recently developed assay for the detection of α(4)β(1)-integrin allosteric antagonists. Secondary assays confirmed that the compounds identified: 1) do not behave like competitive (direct) antagonists; 2) decrease ligand binding affinity for VLA-4 ～2 orders of magnitude; 3) exhibit antagonistic properties at low temperature. In a cell based adhesion assay in vitro, the compounds rapidly disrupted cellular aggregates. In accord with reports that VLA-4 antagonists in vivo induce mobilization of hematopoietic progenitors into the peripheral blood, we found that administration of one of the compounds significantly increased the number of colony-forming units in mice. This effect was comparable to AMD3100, a well known progenitor mobilizing agent. Because all the identified compounds are structurally related, previously used, or currently marketed drugs, this result opens a range of therapeutic possibilities for VLA-4-related pathologies.     

A simple method for DNA purification from peripheral blood

A new, simple, and inexpensive method for the rapid isolation of DNA from whole blood is described. Cell nuclei are prepared by lysis of cytoplasmic membranes and DNA within the nuclear pellet is dispersed with guanidine isothiocyanate and precipitated with isopropanol. DNA prepared in this way restricts completely and results in low backgrounds of nonspecific hybridization after Southern analysis. The yields of DNA are similar to those obtained by more tedious traditional procedures. Numerous genomic DNA samples can be prepared from whole blood in 2 h, thus facilitating gene linkage or other molecular studies in which large numbers of individuals are required.     

Biomass and nutrient allocation of sawgrass and cattail along a nutrient gradient in the Florida Everglades

Biomass and nutrient allocation in sawgrass (Cladium jamaicense Crantz) and cattail (Typha domingensis Pers.) were examined along a nutrient gradient in the Florida Everglades in 1994. This north to south nutrient gradient, created by discharging nutrient-rich agricultural runoff into the northern region of Water Conservatio ea 2A, was represented by three areas (impacted, transitional and reference). Contrasting changes of plant density and size along the gradient were found for communities of both species. For the sawgrass community, more small plants were found in ref ce areas, whereas few large plants were found in impacted areas. In contrast, for the cattail community, bigger plants were found in reference areas, and smaller plants were found in impacted areas. Both species allocated approximately 60% of their total biomass to leaves and 40% to belowground tissues. However, sawgrass biomass allocation to leaves, roots, shoot bases and rhizomes (65%, 19%, 11%, and 5%, respectively) was similar among the three areas. In contrast, cattail plants growing in referen reas showed higher root allocation (27.3%), but lower leaf allocation (51.1%) than those growing in impacted areas (14.6% and 65.8% for root and leaf allocation, respectively). Cattail had higher phosphorus concentrations than sawgrass in tissues associated with growth functions (leaves, roots, and rhizomes). In contrast, sawgrass had higher phosphorus and nitrogen concentrations than cattail in tissues primarily associated with resource storage (shoot bases). From impacted to reference areas, for sawgrass, there was a decrease of leaf TP from 605 to 248 (mg/kg), root TP from 698 to 181 (mg/kg), rhizome TP from 1,139 to 142 (mg/kg), and shoot base TP from 5,412 to 400 to (mg/kg). For cattail, leaf TP decreased from 1,175 to 556 (mg/kg), root TP de sed from 1,100 to 798 (mg/kg), rhizome TP decreased from 1390 to 380 (mg/kg), and shoot base TP decreased from 2,990 to 433 (mg/kg). N/P ratios of sawgrass in reference areas were 27, 63, 38, and 50 for leaves, roots, rhizomes, and shoot bases, respectively, whereas in impacted areas they were 11, 21, 6, and 2, respectively. The greatest TP storage was found in impacted areas. Differences in seed output, seed number, and mean seed weight were found for both species as well. Each cattail flower stalk duced approximately 105 tiny seeds (0.048 ± 0.001 mg) while each sawgrass flower stalk produced about 103 large seeds (3.13 ± 0.005 mg). These results suggest that phosphorus is a limiting resource in the Everglades and that the two species have different life history strategies. These data provide an ecological basis for making informed management and planning decisions to protect and restore the Everglades.     

Small-volume rapid-mix device for subsecond kinetic analysis in flow cytometry.

BACKGROUND: Rapid-mix flow cytometry has emerged as a powerful tool for mechanistic analysis of ligand binding, cell response, and molecular assembly. Although progress has come from improving sample delivery capabilities, little attention has been paid to the volumetric requirements associated with precious biological reagents. METHODS: By using programmable syringes, valves, and other fluidic components, we created a modular, precisely regulated rapid-mix device for the delivery of small-volume samples to the flow cytometer. The device was tested using a bead-based assay in which the binding kinetics between native biotin and fluorescein biotin-bearing beads were characterized. RESULTS: Bead suspensions and reagents paired in 35- to 45-microl aliquots were efficiently mixed by the device and delivered to the flow cytometer. Kinetic data associated with the fluorescein biotin beads were analyzed and used to calibrate the performance characteristics of the device in terms of sample delivery and mixing efficiency. CONCLUSION: The rapid-mix device is capable of detecting subsecond kinetics of biological reactions using microliter volume of samples. Dimensions of the device have been minimized, and the quantitative aspects of sample delivery and analysis have been optimized. Further, the modular design has been optimized for adaptation to a variety of experimental protocols.     

The fate of an N-formylated chemotactic peptide in stimulated human granulocytes. Subcellular fractionation studies

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.     

Regulation of ligand-receptor dynamics by guanine nucleotides. Real-time analysis of interconverting states for the neutrophil formyl peptide receptor

Intact neutrophils exhibit interconverting active and inactive receptor states with half-times for dissociation of 10 s and 2 min, respectively. We examined the effect of guanine nucleotides on ligand-receptor dynamics at 37 degrees C in neutrophils permeabilized with digitonin using continuous fluorometric measurements. The permeabilized cells exhibit a single class of slowly dissociating receptors with a half-time similar to the inactive state. The slowly dissociating state is lengthened in the presence of 10 mM by Mg2+ about two-fold but is relatively insensitive to substitutions of Na+ or K+. When guanine nucleotide is added the receptors dissociate uniformly with a half-time similar to the active state but are sensitive to the substitution of Na+ or K+ (K+ or K+/Mg2+ approximately 10 s; Na+ or Na+/Mg2+ approximately 4 s). When receptors in permeabilized cells are ADP-ribosylated with pertussis toxin the rapidly dissociating state is detected. In the presence of nonsaturating nucleotide or incomplete ribosylation, complex rates of ligand dissociation intermediate between the active and inactive forms are observed. Micromolar concentrations of Ca2+ block the effect of guanine nucleotide on the receptor. The relationships between ligand-receptor dynamics in intact neutrophils and interconverting states regulated by guanine nucleotides and ions in permeabilized cells are discussed.     

The odorant-sensitive adenylate cyclase of olfactory receptor cells. Differential stimulation by distinct classes of odorants

We have characterized odorant-stimulated adenylate cyclase activity in isolated chemosensory cilia prepared from frog and rat olfactory epithelium. Cilia from both species exhibit high levels of adenylate cyclase activity. Basal activity is stimulated approximately 2-fold by GTP and approximately 5-fold by guanosine 5'-(3-O-thio)triphosphate and forskolin. Odorants augment enzyme activity 30-65% above the basal level in a tissue-specific and GTP-dependent manner. Calcium reduces GTP-stimulated activity with a 50% effective concentration at 10 microM. Odorants vary in their influence upon olfactory adenylate cyclase activity. Most fruity, floral, minty, and herbaceous odorants stimulate the enzyme. 3,7-Dimethyl-2,6-octadienenitrile (citralva), menthone, D-carvone, L-carvone, and 2-isobutyl-3-methoxypyrazine display similar potencies in activating the adenylate cyclase upto concentrations of 100 microM. Putrid odorants, such as isovaleric acid, triethylamine, pyridine, thiazole, and methoxypyrazine, and odorous chemical solvents, do not stimulate enzyme activity. In homologous series of pyrazine, thiazole, and pyridine odorants, compounds with the longest hydrocarbon side chains are best able to enhance enzyme activity. The failure of certain odorants to affect adenylate cyclase activity suggests that additional transduction mechanisms besides the formation of cAMP are involved in olfaction.