A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant

The nodulation characteristics of soybean (Glycine max) mutant nts382 are described. The mutant nodulated significantly more than the parent cultivar Bragg in the presence and absence of several combined nitrogen sources (KNO₃, urea, NH₄Cl, and NH₄NO₃). The number of nodules on the tap root and on lateral roots was increased in the mutant line. In the presence of KNO₃ and urea, nitrogenase activity was considerably higher in nts382 than in Bragg. Mutant plants were generally smaller than wild-type plants. Although nts382 is a supernodulator, inoculation with Rhizobium japonicum was necessary to induce nodule formation and both trial strains CB1809 (= USDA136) and USDA110 elicited the mutant phenotype. Segregation of M₃ progeny derived from a M₂ wild-type plant indicated that the mutant character is inherited as a Mendelian recessive. The mutant is discussed in the context of regulation of nodulation and of hypotheses that have been proposed to explain nitrate inhibition of nodulation.     

The topography of glycerophosphate acyltransferase in the transverse plane of the mitochondrial outer membrane

In low ionic media, mitochondrial glycerophosphate acyltransferase was inhibited virtually completely within 15 min by the nonspecific proteases, proteinase K and subtilisin. In high ionic media, the mitochondrial enzyme was either not inhibited or was marginally inhibited by these proteases. Chymotrypsin and trypsin, regardless of the ionic strength of the medium, did not inhibit the acyltransferase. Substantial inhibition by proteinase K and subtilisin was observed in the high ionic media when the incubation was continued for 30 or 45 min. Adenylate kinase, an intermembrane enzyme, was not inhibited under any of the above conditions. These results demonstrate a cytosolic exposure of the mitochondrial acyltransferase. In a low ionic environment, when the outer membrane integrity was damaged either by gradually decreasing the tonicity of the medium or by stepwise addition of Triton X-100, either chymotrypsin or trypsin caused virtually parallel inhibition of glycerophosphate acyltransferase and adenylate kinase. A more direct approach in establishing the existence of protease-susceptible sites on the inner side of the outer membrane was taken by observing the inhibition of mitochondrial glycerophosphate acyltransferase and adenylate kinase in trypsinloaded right-side-out outer membrane vesicles incubated in the presence of externally located soybean trypsin inhibitor. The above results, taken together, suggest that mitochondrial glycerophosphate acyltransferase spans the transverse plane of the outer membrane.     

Comparison of Three 18F-Labeled Butyrophenone Neuroleptic Drugs in the Baboon Using Positron Emission Tomography

The butyrophenone neuroleptics spiroperidol, benperidol, and haloperidol were radiolabeled with fluorine-18 and studied in baboon brain using positron emission transaxial tomography (PETT). Pretreatment of the baboon with a high pharmacological dose of (+)-butaclamol reduced the specifically bound component of radioactivity distribution in the striatum to approximately the radioactivity distribution found in the cerebellum. Comparative studies of brain distribution kinetics over a 4-h period indicated that either [18F]spiroperidol or [18F]benperidol may be suitable for specific labeling of neuroleptic receptors. In an 8-h study with [18F]spiroperidol, striatal radioactivity did not decline, suggesting that spiroperidol either has a very slow dissociation rate or that it binds irreversibly to these receptors in vivo. [18F]Haloperidol may not be suitable for in vivo PETT studies, because of a relatively high component of nonspecific distribution and a faster dissociation from the receptor. Analysis of 18F in plasma after injection of [18F]spiroperidol indicated rapid metabolism to polar and acidic metabolites, with only 40% of the total radioactivity being present as unchanged drug after 30 min. Analysis of the metabolic stability of the radioactively labeled compound in rat striatum indicated that greater than 95% of [18F]spiroperidol remains unchanged after 4 h.     

Catabolism of myo-Inositol to Precursors Utilized for De Novo Glycerolipid Biosynthesis

Systemic injection of [2-3H]myo-inositol into frogs resulted in the incorporation of more than half of the label into glycerolipid classes other than phosphoinositides in retinal rod outer segment membranes. Following methanolysis and differential extraction of isolated lipid classes, radioactivity was recovered primarily in the aqueous phase. After phospholipase C hydrolysis of the total membrane lipids, 97% of the radioactivity was extractable with organic solvents, and 70% of the label in lipids was in 1,2-diglycerides. These results indicate that the label was incorporated primarily into the glyceryl moiety of the membrane glycerolipids. Intraocular injection of frog eyes or in vitro incubation of frog retinas with [2-3H]myo-inositol resulted in the incorporation of radioactivity almost exclusively into phosphoinositides in rod outer segment membranes. Incubation of retinas with [U-14C]glucuronic acid did not result in the formation of labeled retinal lipids. These results suggest that myo-inositol can be catabolized systemically to precursors utilized for glycerolipid biosynthesis in the retina.     

Localization of C4 genes within the HLA complex by molecular genotyping

Segregation of the complement component, C4, was analyzed in six families that each included an individual who inherited an HLA haplotype where a crossover event had occurred in the region between HLA-B and HLA-DR. Two cDNA clones corresponding to the C4 gene were utilized as probes in Southern blot analysis of DNA from members of each family. Restriction fragment length polymorphisms (RFLP) were observed and were assigned to haplotypes. In one family RFLP, hybridizing with the C4 probes, segregrated with HLA-B, and in four families RFLP segregated with HLA-DR; one family was not informative in this respect. These analyses have made it possible to localize the genes for C4 between HLA-B and HLA-DR by molecular genotyping and to characterize three different genomic configurations of C4 genes by limited restriction mapping.Abbreviations RFLP restriction fragment length polymorphisms - LCL lymphoblastoid cell lines     

Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis

By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.     

Paramagnetic 1H and 13C NMR studies on cobalt-substituted human carbonic anhydrase I carboxymethylated at active site histidine-200: molecular basis for the changes in catalytic properties induced by the modification

Using bromo[1-13C]acetate to modify N tau of His-200 of human carbonic anhydrase isozyme I leads to the introduction of a useful 13C NMR probe into the active site. To complement our previous diamagnetic NMR studies with this probe, we have now succeeded in directly observing the paramagnetically perturbed resonance of the carboxylate in the cobalt-substituted modified enzyme above pH 8. In the pH range 8-10, the resonance undergoes a pH-dependent slow-exchange process, with the more alkaline form having a much smaller pseudocontact shift and a narrower line width. Below pH 8, the resonance apparently undergoes a very large paramagnetic downfield shift that was estimated by extrapolation. An ionization of approximate pK of 6 appears to control this process. Paramagnetic spin-relaxation studies on the resonance under conditions where it was directly observed yielded distance measurements between the carboxylate carbon and the active site cobalt ion. In inhibitor complexes, this distance was in the range of 5-7 A. In the absence of inhibitors, the distance was approximately 3.0-3.2 A at pH 7.9, consistent with the coordination of the carboxylate to the metal. However, at pH 10, the distance was increased to 4.8 A. These distance determinations were aided by relaxation measurements of a paramagnetically shifted proton resonance at 60-65 ppm downfield assigned by others to a proton of a ligand histidine of metal and confirmed by us to be 5.2 +/- 0.1 A from the metal. Our findings provide a molecular basis for the observed changes in catalytic properties that accompany the carboxymethylation.     

Recycling of platelet phosphorylation and cytoskeletal assembly

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.     

Exchange assay for androgen receptors in the presence of molybdate

Several reports have shown that sodium molybdate stabilizes steroid hormone receptors. We have utilized these observations to develop an exchange assay for the androgen receptor at elevated temperatures. Exchange was found to be complete after 30 min at 30 degrees C. Receptor degradation was negligible during this treatment. Scatchard analysis indicated that the dissociation constant of the androgen receptor was similar both in the absence (Kd = 3.9 nM) and presence (Kd = 2.9 nM) of molybdate. Steroid specificity of the androgen receptor was unaltered by this treatment. The exchange procedure was reproducible, with an interassay variation of 2.45% and intraassay variation less than 10.0%. Using this assay, highest concentrations of androgen binding were measured in androgen target tissues of the rat (Dunning R3327 tumor, prostate and seminal vesicle; 23.37, 20.20 and 19.84 fmol/mg protein respectively). Lower concentrations were observed in other tissues (lung, brain, heart, spleen, liver and kidney; 9.06, 5.63, 3.50, 2.42, 2.33 and 1.36 fmol/mg protein respectively). These results demonstrate that molybdate stabilization of the androgen receptor allows efficient steroid exchange without significant alteration of the receptor's steroid binding properties. Furthermore, this exchange assay can be used to obtain a reasonable measurement of receptor concentrations in different androgen target tissues.     

Visual fields of orb web and single line web spiders of the family Uloboridae (Arachnida,Araneida)

Summary In the family Uloboridae, web reduction is associated with changes in web monitoring posture and prosomal features. A spider must extend its first pair of legs directly forward to monitor the signal line of a reduced web. This posture is facilitated by shifts in prosomal musculature that cause reduced web uloborids to have a narrower anterior prosoma, a reduced or absent anterior eye row, and prominent posterior lateral eye tubercles. The eye tubercles and larger posterior eyes of these uloborids suggest that web reduction may also be accompanied by ocular changes that compensate for reduction of the anterior eyes by expanding the visual fields of the posterior eyes. A comparison of the visual fields of the eight-eyed, orb web species Octonoba octonaria and a four-eyed, reduced web Miagrammopes species was made to determine if this is true. Physical and optical measurements determined the visual angles of each species' eyes and the pattern of each species' visual surveillance. Despite loss of the anterior four eyes, the Miagrammopes species has a visual coverage similar to that of O. octonaria. This is due to (1) an increase in the visual field of each of the four remaining Miagrammopes eyes, accruing from an extension of the retina and an increase in the lens' rear radius of curvature, and (2) a ventral shift of each visual axis, associated with the development of an eye tubercle and an asymmetrical expansion of the retina. Miagrammopes monitor their simple webs from twigs or moss where they are vulnerable to predation. Therefore, maintenance of visual cover may enable them to detect predators in time to assume or maintain their characteristic, cryptic posture. It may also allow them to observe approaching prey and permit them to adjust web tension or prepare to jerk their webs when prey strikes.