Interaction of Rhizobium sp. with Toxin-Producing Fungus in Culture Medium and in a Tropical Soil

Experiments were conducted to determine the influence of a toxin-producing fungus on a rhizobial population in yeast-mannitol medium and in a tropical soil. The fungus, which was isolated from a highly weathered soil (Tropeptic Eutrustox), was identified as a Metarhizum sp. The density of rhizobial populations established in yeast-mannitol medium in the absence of the fungus was 10⁵ times higher than that established in its presence. However, the fungus did not exert similar antagonistic influence on the rhizobial population incubated with it in the sterilized test soil. Rhizobial growth activity in yeast-mannitol medium was also insensitive to the presence of the fungus when the medium was amended with 1% (wt/vol) kaolinite or montmorillonite. The results suggest that clay minerals may be responsible for protecting rhizobia against toxin-producing fungi in soil.     

The Relationship Between Arachidonic Acid Release and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

Increased arachidonic acid release occurred during activation of catecholamine secretion from cultured bovine adrenal medullary chromaffin cells. The nicotinic agonist 1,1-dimethyl-4- phenylpiperazinium (DMPP) caused an increased release of preincubated [3H]arachidonic acid over a time course which corresponded to the stimulation of catecholamine secretion. Like catecholamine secretion, the DMPP-induced [3H]arachidonic acid release was calcium-dependent and was blocked by the nicotinic antagonist mecamylamine. Depolarization by elevated K+, which induced catecholamine secretion, also stimulated arachidonic acid release. Because arachidonic acid release from cells probably results from phospholipase A2 activity, our findings indicate that phospholipase A2 may be activated in chromaffin cells during secretion.     

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K⁺ stimulated ⁴⁵Ca²⁺ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca²⁺ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca²⁺ uptake occurred within 15 s of stimulation by carbachol or elevated K⁺ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K⁺ was similar to that of Ca²⁺ uptake. There was a close correlation between Ca²⁺ uptake and catecholamine secretion at various concentrations of Ca²⁺. The concentration dependencies for inhibition of both processes by Mg²⁺ or Cd²⁺ were similar. Ca²⁺ uptake saturated with increasing Ca²⁺ concentrations, with an apparent K_m for both carbachol-induced and elevated K⁺-induced Ca²⁺ uptake of approximately 2 mM. The Ca²⁺ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca²⁺ entry and a presumed increase in cytosolic Ca²⁺ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca²⁺- dependent processes associated with catecholamine secretion. Ca²⁺ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca²⁺ channels.     

Progestins Can Have a Membrane-Mediated Action in Rat Midbrain for Facilitation of Sexual Receptivity

In rats, progesterone (P) facilitates sexual receptivity by interacting with intracellular progestin receptors in the ventromedial hypothalamus (VMH). This experiment concerns whether P can also facilitate receptivity in rats by acting extragenomically within the ventral tegmental area (VTA). Ovariectomized rats (n= 10) with bilateral guide cannulas over the VMH and VTA were primed with 2 μg subcutaneous estradiol benzoate 44 hr prior to testing. After a pretest for sexual receptivity, animals received implants to the VMH of P, P conjugated to bovine serum albumin (P:BSA), or cholesterol control (CHOL), and were retested. Two hours later, animals were again tested for receptivity, and P, P:BSA, the P metabolite 5α-pregnan-3α-ol-20-one (3α,5α-THP), or CHOL implants were applied to the VTA. Subjects were retested immediately, 30, 90, and 150 min later. Animals that received P in the VMH and had P, P:BSA, or 3α,5α-THP applied to the VTA exhibited facilitated receptivity at all time points compared with all other combination implants. That P:BSA and P were equally effective when applied to the VTA, but not the VMH, suggests that in the VTA P's membrane-mediated actions are sufficient to facilitate receptivity, whereas in the VMH they are not. Since the steroid (P) and its metabolite (3α,5α-THP) are similarly effective when applied to the VTA, given P application to the VMH earlier, P's effects in the VTA may be subsequent to metabolism and/or actions at GABA receptors. Overall, these data suggest that in rats P can act at the membrane of neurons within the VTA to modulate lordosis and that these effects may be subsequent to P's metabolism and/or actions at GABA receptors.     

Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age

The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning³¹P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.     

After the Revolution: Post-Neolithic Subsistence in Northern Mesopotamia

Over the past two decades there has been a proliferation of studies that reveal a far greater diversity in the pathways to food production worldwide than would have been predicted by traditional models of the Neolithic Revolution. It would now appear that, while there are certain universal elements in the origins of food production, each instance of the incorporation of domestic resources into the subsistence economy is shaped by highly localized social and natural forces. It is perhaps not surprising, then, to find that the times after the Neolithic Revolution do not conform to earlier uniformitarian conceptions of the origin of food production as either a technological blessing or an environmental blight that locked people into an economy based solely on domestic resources. This examination of subsistence at Umm Qseir, one small post-Neolithic community in the Khabur Draïnage of Northern Mesopotamia, highlights the remarkable degree of flexibility and individualized response in post-Neolithic economies in the Near East, especially in marginal areas. The incorporation of both domestic and wild resources into the economy of inhabitants of this site is further shown not to be just an isolated curiosity. Rather, Umm Qseir joins a number of other post-Neolithic communities situated in marginal areas, persisting several millennia after the first introduction of domestic crops and animals, that also fashioned highly individualized subsistence economies based on both wild and damestic resources. In the Khabur Drainage, the blending of domestic and wild resources in the subsistence of people situated in more arid areas is replaced by a more "conventional" agropastoral economy only with development of urbanism and the imposition of an agricultural economy of scale on this fragile environment.     

Membrane peroxidation: Inhibiting effects of water- soluble antioxidants on phospholipids of different charge types

Quantitative kinetic methods of autoxidation are used to determine the antioxidant activities of two water-soluble antioxidants of the chromanol type, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 6-hydroxy-2,5,7,8- tetramethyl-2-N,N,N-trimethylethanaminium methylbenzene-sulfonate (MDL 73404), during free radical peroxidation of phospholipid membranes of different charge types. The stoichiometric factor (n) for peroxyl radical trapping for both Trolox and MDL 73404 was found to be 2. Trolox was found to partition partially, approximately 20%, into the lipid phase of liposomes. The antioxidant activity of Trolox during peroxidation of membranes determined by measurements of the absolute rate constant for inhibition of oxygen uptake,k_inh, was found to vary with the membrane surface charge that is controlled by variation in pH. When peroxidation is initiated in the lipid phase by azo-bis-2,4-dimethylvaleronitrile (ADVN), using a typical zwitterionic liposome, dilinoleoylphosphatidyl choline (DLPC), the k_inh was found to be 2.98 × 10³ M⁻¹s⁻¹. The k_inh of Trolox increased approximately 2-fold for membranes that have positive surface, including DLPC at pH 4, DLPC containing stearylamine at pH 7, and for a membrane of dimyristoylphosphatidic acid containing linoleic acid (DMPA/LA). Conversely, Trolox does not inhibit peroxidation of negatively charged dilinoleoylphosphatidyl glycerol (DLPG) at pH 7–11. Studies made of the positively charged MDL 73404 show that its antioxidant activity using DLPC and DLPG is pH dependent. Trolox inhibits the peroxidations of DLPC initiated in the aqueous phase by azo-bis(2-amidinopropane·HCl)(ABAP) at pH 4 or 7. However, Trolox does not inhibit the peroxidation of DLPG at pH 7. The different antioxidant activities of Trolox and MDL 73404 are rationalized in terms of a peroxyl-radical diffusion model and specific charge interactions between antioxidants and membrane surface.     

Beta-D-Allose inhibits fruiting body formation and sporulation in Myxococcus xanthus

Myxococcus xanthus, a gram-negative soil bacterium, responds to amino acid starvation by entering a process of multicellular development which culminates in the assembly of spore-filled fruiting bodies. Previous studies utilizing developmental inhibitors (such as methionine, lysine, or threonine) have revealed important clues about the mechanisms involved in fruiting body formation. We used Biolog phenotype microarrays to screen 384 chemicals for complete inhibition of fruiting body development in M. xanthus. Here, we report the identification of a novel inhibitor of fruiting body formation and sporulation, beta-d-allose. beta-d-Allose, a rare sugar, is a member of the aldohexose family and a C3 epimer of glucose. Our studies show that beta-d-allose does not affect cell growth, viability, agglutination, or motility. However, beta-galactosidase reporters demonstrate that genes activated between 4 and 14 h of development show significantly lower expression levels in the presence of beta-d-allose. Furthermore, inhibition of fruiting body formation occurs only when beta-d-allose is added to submerged cultures before 12 h of development. In competition studies, high concentrations of galactose and xylose antagonize the nonfruiting response to beta-d-allose, while glucose is capable of partial antagonism. Finally, a magellan-4 transposon mutagenesis screen identified glcK, a putative glucokinase gene, required for beta-d-allose-mediated inhibition of fruiting body formation. Subsequent glucokinase activity assays of the glcK mutant further supported the role of this protein in glucose phosphorylation.     

Sequential roles for myosin-X in BMP6-dependent filopodial extension, migration, and activation of BMP receptors

Endothelial cell migration is an important step during angiogenesis, and its dysregulation contributes to aberrant neovascularization. The bone morphogenetic proteins (BMPs) are potent stimulators of cell migration and angiogenesis. Using microarray analyses, we find that myosin-X (Myo10) is a BMP target gene. In endothelial cells, BMP6-induced Myo10 localizes in filopodia, and BMP-dependent filopodial assembly decreases when Myo10 expression is reduced. Likewise, cellular alignment and directional migration induced by BMP6 are Myo10 dependent. Surprisingly, we find that Myo10 and BMP6 receptor ALK6 colocalize in a BMP6-dependent fashion. ALK6 translocates into filopodia after BMP6 stimulation, and both ALK6 and Myo10 possess intrafilopodial motility. Additionally, Myo10 is required for BMP6-dependent Smad activation, indicating that in addition to its function in filopodial assembly, Myo10 also participates in a requisite amplification loop for BMP signaling. Our data indicate that Myo10 is required to guide endothelial migration toward BMP6 gradients via the regulation of filopodial function and amplification of BMP signals.