Utilization of amino acids by Frankia sp. strain CpI1

The utilization of some amino acids, added at 1 mM and 10 mM concentrations, as the sole combined nitrogen sources by Frankia sp. strain CpI1, has been investigated. Glutamine, like NH ₄ ⁺ , provided rapid growth without N₂ fixation. Histidine at 1 mM yielded poor N₂-fixing activity but better cell growth than N₂. Aspartate, glutamate, alanine, proline, each at 1 mM concentration, supported similar levels of N₂ fixation and growth. Growth on 10 mM glutamate, proline, or histidine resulted in poor N₂-fixing activity and poor cell growth. Cells grown on 10 mM alanine had about half the N₂-fixing activity of cells grown on N₂ but growth was good. Aspartate at 10 mM concentration, however, stimulated N₂-fixing activity dramatically and promoted faster growth. Enzyme analysis suggested that asparate is catabolized by glutamate-oxaloacetate transaminase (GOT), since GOT specific activity was induced, and aspartase activity was not detected, in cells grown on aspartate as the sole combined nitrogen source. Thinlayer chromatography (TLC) of metabolites extracted from N₂-grown cells fed with [¹⁴C]-aspartate showed that label was rapidly accumulated mainly on aspartate and/or glutamate, depending on the cells' physiological state, without detectable labeling on fumarate or oxaloacetate (OAA). These findings provide evidence that aspartate is catabolized by GOT to OAA which, in turn, is rapidly converted to -ketoglutarate through the TCA cycle and then to glutamate by GOT or by glutamate synthase (GOGAT). The stimulation of N₂ fixation and growth by aspartate is probably caused by an increased intracellular glutamate pool.     

Evidence of abortive recombination in ruv mutants of Escherichia coli K12

Summary Genetic recombination in Escherichia coli was investigated by measuring the effect of mutations in ruv and rec genes on F-prime transfer and mobilization of nonconjugative plasmids. Mutation of ruv was found to reduce the recovery of F-prime transconjugants in crosses with recB recC sbcA strains by about 30-fold and with recB recC sbcB sbcC strains by more than 300-fold. Conjugative plasmids lacking any significant homology with the chromosome were transferred normally to these ruv mutants. Mobilization of the plasmid cloning vectors pHSG415, pBR322, pACYC184 and pUC18 were reduced by 20- to 100-fold in crosses with ruv rec ⁺ sbc ⁺ strains, depending on the plasmid used. Recombinant plasmids carrying ruv ⁺ were transferred efficiently. With both F-prime transfer and F-prime cointegrate mobilization, the effect of ruv was suppressed by inactivating recA. It is proposed that the failure to recover transconjugants in ruv recA ⁺strains is due to abortive recombination and that the ruv genes define activities which function late in recombination to help convert recombination intermediates into viable products.     

Macroalgal farming in the sea: water motion and nitrate uptake

A better understanding of water motion effects on nutrient uptake by marine crop plants should make it possible to farm the sea more effectively. Farms in China, Japan and the Philippines now grow plants on slack lines or nets that move with passing waves and currents. Nutrient uptake rates are increased onLaminaria farms in China by adding nitrogen-containing fertilizer. In contrast, forests of the giant kelp,Macrocystis grow in California at low nutrient levels without fertilization. The giant kelp, compared as a structure with the slack Chinese farms, has float-supported, spring-like stipes that stretch and recoil as waves pass. This motion seems likely to enhance flow over the thallus surface. In thus study we modified flow around kelp blades in a water tunnel in the laboratory by changing orifice plates, and flow around Chinese-style long-line farms in the sea by tightening them under various sea conditions. Our measurements suggest that if marine farms were designed and operated to increase water movement over the plants being grown, their rates of nutrient uptake, and growth would increase.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

DNA Sequence Determinants of λ Repressor Binding in Vivo

The critical operator determinants for λ repressor recognition have been defined by analyzing the binding of wild-type repressor to a set of mutant operators in vivo. Base pair substitutions at six positions within the λ operator half-site impair binding severely, and define these base pairs as critical for operator function. One mutant operator binds repressor better than the consensus operator, and is a superoperator. The model proposed by M. Lewis in 1983 for the binding of λ repressor to its operator accurately predicts the observed operator requirements for binding in vivo, with several minor exceptions. The order of affinities of the six natural λ operators has also been determined.     

Isolation and characterization of OmpC porin mutants with altered pore properties.

The LamB protein is normally required for the uptake of maltodextrins. Starting with a LamB- OmpF- strain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex+ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB- OmpF- strains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of [14C]maltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain beta-lactam antibiotics and sodium dodecyl sulfate, but did not exhibit an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.     

Transfer of phospholipids by a protein fraction obtained from canine pulmonary lavage

Surfactant phospholipid exists in multicompartment pools within the subphase of the lung. Movement among these pools and back into type II alveolar cells may be catalyzed by a phospholipid transfer protein resident in the subphase. We demonstrate here that a protein fraction obtained from canine lung lavage catalyzes the intermembrane transfer of all the major surfactant phospholipids. The protein is probably not derived from serum and is unrelated to surfactant proteins that have already been described.     

Rapid cessation of estrous cyclicity and depressed castration response in short photoperiod-treated, inbred LSH/SsLaK hamsters

This study examined the effects of transfer from long photoperiod (LP) to short photoperiod (SP) on the cessation of ovarian cyclicity and the castration response in inbred LSH/SsLak golden Syrian hamsters. Forty-six 8 to 10-wk-old female hamsters were acclimatized in LP (14L:10D; lights on at 0600 h) during which time animals were monitored for regular ovarian cyclicity. Twenty-six animals were transferred to SP (8L:16D; lights on at 0600 h) and examined daily for vaginal discharges. One day after the day of the first missed ovulation, individual SP-exposed animals were bilaterally ovariectomized; concomitantly, an LP control animal in diestrus I underwent the same procedure. Thirty days after ovariectomy, the hamsters were fitted with intra-atrial silastic cannulae. On the following two postoperative days, 0.6 ml blood samples were collected at 0700, 1200, 1400, and 1600 h for SP animals and at 0700, 1400, 1600 and 1800 h for LP controls. On the third day, the animals were decapitated and sera and pituitaries saved for determination of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) by radioimmunoassay (RIA). All SP-exposed animals displayed their last estrous discharge 14-34 days after transfer to SP (mean = 23.0 +/- 0.8 days). Their ovaries were characterized by the absence of corpora lutea, the presence of large atretic antral follicles, few growing follicles, and interstitium that was stimulated to varying degrees. Total and adjusted pituitary weights were decreased by SP exposure (p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA sequence evidence for polymorphic forms of human serum amyloid A (SAA)

Serum amyloid A (SAA) is an acute-phase reactant and precursor to amyloid A protein, the major constituent of the fibril deposits of reactive amyloidosis. The factors determining whether the 104-amino acid SAA molecule is converted into the 76-amino acid amyloid A protein and deposited as fibrils are not known. As an initial step toward investigating the possibility that a particular primary structure of SAA is involved in amyloid formation, we have cloned and determined the nucleotide sequence of human SAA-specific cDNAs. The first clone, selected using an oligonucleotide probe, was shown to encode the signal peptide and amino-terminal region of SAA. The cDNA of this clone served as probe in the selection of two distinct, full-length SAA cDNAs, initially differentiated by the presence (pSAA21) or absence (pSAA82) of a PstI site in the coding sequence. The complete nucleotide sequence of pSAA82 cDNA was determined. Since there appear to be multiple human SAA alleles, it is conceivable that their differential expression is important to amyloid formation.     

Apolipoprotein A-IV. A determinant for binding and uptake of high density lipoproteins by rat hepatocytes

To identify the role of a specific apoprotein other than apoE which might be responsible for the receptor-mediated uptake of high density lipoprotein (HDL) by rat hepatocytes, 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) was combined with rat apoE, apoA-I, or apoA-IV to form apoprotein-phospholipid complexes and the complexes were tested for their binding and uptake by primary rat hepatocytes. Apoprotein-POPC complexes were labeled with the specific fluorescent probe, 1,1-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine to monitor their uptake by cultured rat hepatocytes at 37 degrees C using digital fluorescence imaging microscopy or were labeled with 125I to study their binding to hepatocytes at 4 degrees C. POPC, either alone or with apoA-I, was not internalized by rat hepatocytes while complexes containing apoE or apoA-IV were taken up by the cells. Specific binding at 4 degrees C was demonstrated for apoE-free HDL, apoA-IV X POPC, and apoE X POPC but not for apoA-I X POPC. The binding of apoE-free HDL was inhibited by apoA-IV X POPC, apoE-free HDL, and apoA-IV + apoA-I X POPC but not by apoA-I X POPC. Binding of apoA-IV X POPC was inhibited by apoE-free HDL, apoA-IV X POPC, and apoA-IV + apoA-I X POPC, but not by apoE X POPC or apoE-enriched HDL. These data indicate that apoA-IV is a ligand responsible for the rat HDL binding to primary rat hepatocytes and that apoA-IV binds to a receptor site distinct from apoE-dependent receptors such as the apoB,E or chylomicron-remnant receptor.     

Fluorometric analysis of transferable membrane pores

When pore-forming factors insert into the hyperpolarized membranes of lipid vesicles, ion gradients are rapidly equilibrated, effecting complete depolarization. This process can be conveniently followed with a potentiometric cyanine dye. The generality of the method is demonstrated by applications to three diverse materials. The well-studied gramicidin channel is used to demonstrate that the method is sensitive down to concentrations of 10(-12)M. An extract from the shark repellent skin secretion of the Red Sea flatfish displays activity in the assay and is used to demonstrate the potential of the method to elucidate some of the characteristics of the pore, including its molecularity. That membrane-active factors can be detected and assayed in crude preparations is demonstrated with an impure extract of "amoebapore" from Entamoeba histolytica. In addition, variation of the buffer composition surrounding the vesicles can provide information about the ion selectivity of the pore under investigation.