Possible Mechanism of Action of β-Lactam-Enhancing Factor on Methicillin-Resistant Staphylococcus aureus

We have recently found a factor (Factor T) in aged mixtures of tungstate and phosphate which greatly enhances the antibacterial effects of β-lactams on methicillin-resistant strains of staphylococcal species such as methicillin-resistant Staphylococcus aureus (MRSA), but shows only weak effects on methicillin-susceptible S. aureus and bacterial strains other than staphylococci. Factor T alone did not strongly inhibit cell metabolism and bacterial growth unless an excess amount was added. When Factor T was added to the culture medium beforehand, the growth of MRSA cells was rapidly suppressed just after addition of oxacillin (MPIPC). However, the growth of the cells was inhibited gradually when these two reagents were added in reverse order. For full expression of the enhancing effect, it seemed necessary for cells of MRSA strains to be incubated with Factor T for at least 2–3 hr. When the cells were washed after being sensitized by incubating them for 5 hr with Factor T, it took approximately 1 hr for the cells to recover their resistance to MPIPC. Factor T reduced the amount of penicillin-binding protein-2′ (PBP 2′), and thus sensitized the MRSA strains to β-lactams.     

Effects of tungstate on the growth of Desulfovibrio gigas NCIMB 9332 and other sulfate-reducing bacteria with ethanol as a substrate

Growth of Desulfovibrio gigas NCIMB 9332 in mineral, vitamin-supplemented media with ethanol as substrate was strongly stimulated by the addition of tungstate (optimal level approximately 10^-7 M). At suboptimal tungstate concentrations, up to 1.0 mM acetaldehyde was detected in the culture supernatant and growth was slow. Omission of both tungstate and molybdate from the media prevented growth and ethanol utilization. Tungstate-deprived cultures that were grown on lactate had much lower aldehyde dehydrogenase (benzylviologen as acceptor; BV-AIDH) levels than tungstate-supplemented cultures. These data suggest that tungstate is required for the synthesis of active BV-AIDH. The characteristics of the enzyme activities in cell-free extracts show that the BV-AIDH activity present in tungstate-supplemented cultures is not due to the recently characterized molybdenum-containing aldehyde dehydrogenase of D. gigas. Out of 13 other strains of ethanol-oxidizing, gram-negative, sulfate-reducing bacteria tested, most strains grew well with either tungstate or molybdate supplementation. In contrast to a recent report, good growth on ethanol of two D. baculatus (Desulfomicrobium) strains (DSM 1741 and DSM 1743) was observed.Abbreviations BV-AIDH Benzylviologen-linked aldehyde dehydrogenase - DCPIP-AIDH 2,6-dichlorophenolindophenol-linked aldehyde dehydrogenase - DTT dithiothreitol     

The influence of growth conditions on the synthesis of molybdenum cofactor in Proteus mirabilis

Cell-free extracts of Proteus mirabilis were able to reconstitute NADPH-dependent assimilatory nitrate reductase in crude extracts of the Neurospora crassa mutant strain nit-1, lacking molybdenum cofactor. Molybdenum cofactor was formed in the cytoplasm of the bacterium even in the presence of oxygen during growth though under these conditions no molybdo enzymes are formed. As a consequence no cofactor could be released by acid treatment from membranes of cells grown aerobically. The amount of cofactor released from membranes of cells grown anaerobically under various conditions was proportional to the amount of molybdo enzymes formed. During growth in the presence of tungstate a cofactor, which lacks molybdenum, was found in the cytoplasm. For detection of this so-called demolybdo cofactor the presence of molybdate during reconstitution was essential. Moreover, the cytoplasmic cofactor pool in cells grown in the presence of tungstate appeared to be two to three times higher than in cells grown under similar conditions without tungstate. After anaerobic growth in the presence of tungstate, the inactive demolybdo reductases were shown to contain partly no cofactor and partly a demolybdo cofactor. The P. mirabilis chlorate resistant mutant S 556 did not contain molybdenum cofactor. In two other chl-mutants the cofactor activity was the same as in the wild type.     

Some properties of formate dehydrogenase,accumulation and incorporation of 185W-tungsten into proteins of Clostridium formicoaceticum

Formate dehydrogenase of Clostridium formicoaceticum used only methyl and benzyl viologen, but not NAD as electron acceptor. The S_0.5 values were 0.9×10^-4 M for formate and 5.8×10^-3 M for methyl viologen. Using potassium phosphate buffer a pH-optimum of 7.9 was observed. The initial velocity of the formate dehydrogenase activity reached a maximum at 70°C, whereas the activity was stable only up to 50°C. The level of formate dehydrogenase in C. formicoaceticum was increased to its maximum when 10^-6 M selenite and 10^-7 M tungstate were added to a synthetic medium. Addition of molybdate instead of tungstate did not increase the level of formate dehydrogenase. ¹⁸⁵W-tungsten was concentrated about 100-fold by C. formicoaceticum; molybdate had no major effect on the uptake of tungsten. ¹⁸⁵W-tungsten was found almost exclusively in the soluble fluid and was predominantly recovered after chromatography in a protein of about 88000 molecular weight. Occasionally a labelled protein of low molecular weight was observed. Again molybdate added even in high molar excess did not influence the labelling pattern. No radioactivity peak could be obtained at the elution peak of formate dehydrogenase activity. The extreme instability of formate dehydrogenase prevented further purification.Abbreviations FDH formate dehydrogenase - DTE dithioerythritol - HEPES hydroxyethylpiperazine N-2-ethane sulconic acid - TEA triethylamine - DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulfate - TTC triphenyltetrazolium     

Trichoderma harzianum Produces a New Thermally Stable Acid Phosphatase,with Potential for Biotechnological Application

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Differentiation between Clostridium acidiurici and Clostridium cylindrosporum on the basis of specific metal requirements for formate dehydrogenase formation

The formate dehydrogenases of Clostridium acidiurici and of C. cylindrosporum coupled the oxidation of formate with the reduction of viologen dyes. The basal activity level was about 0.85 moles/min s mg of protein for both species. The level of formate dehydrogenase of C. acidiurici increased 12-fold when 10^-7 M tungstate and selenite were present during growth. Molybdate exerted no effect. On the other hand, molybdate and selenite were required to increase the formate dehydrogenase of C. cylindrosporum, and tungstate exhibited an antagonistic effect in this organism.Growth on hypoxanthine generally depended on the addition of bicarbonate. Supplementation with tungstate and selenite accelerated growth of C. acidiurici and increased again the level of formate dehydrogenase. The addition of both, molybdate and selenite was necessary to initiate growth of C. cylindrosporum and to form an active formate dehydrogenase.The differences in the requirement for metal ion supplementation to form high levels of formate dehydrogenase and their involvement in hypoxanthine degradation can be used to differentiate between C. acidiurici and C. cylindrosporum.Abbreviation FDH formate dehydrogenase     

Biosorption of tungstate by a Bacillus sp. isolated from Anzali lagoon

An attempt was made to isolate bacterial strains capable of biologically removing tungstate (WO₄²⁻). Thirty-eight water samples were collected from various areas of Anzali lagoon, Iran. Initial screening of a total of 100 bacterial isolates at pH 5, resulted in the selection of one isolate with maximum adsorption capacity of 65.4 mg tungstate/g dry weight. It was tentatively identified as Bacillus sp. according to morphological and biochemical properties and named strain MGG-83. Tungsten concentration was measured spectrophotometrically using the dithiol method. Higher adsorption capacity was observed in the acidic pH ranging from 1 to 3. At pH 2, the strain removed 274.4 mg tungstate/g dry weight within 5 min from the solution with 300 mg WO₄²⁻/l initial concentration and thereafter adsorption rate decreased remarkably. The applicability of the Freundlich isotherm for representation of the experimental data was investigated. Using 1 mM sodium azide and 10 mM 2,4−dinitrophenol, it was shown that only 20% reduction occurred in adsorption and steam sterilization of the bacterial cells resulted in 11% decrease in tungstate uptake. Temperature variations (20–40°C) had no significant effect on tungstate uptake. Pretreatment with the cations had no effect in uptake but pretreatment with anions decreased the tungstate uptake as indicated: sulfate > chromate > nitrate > molybdate > selenate > rhenate. Tungstate was removed from metal-laden biomass after desorption treatments by addition of different desorbing solutions with the results sodium acetate > EDTA > NaCl > KOH > H₂SO₄.     

The effect of phosphate and flavin adenine dinucleotide on nitrate reductase activity of some unicellular marine algae

Nitrate reductase (NR) activity was measured in cell-free extracts from seven species of unicellular, marine algae. For most species Tris buffer was not a satisfactory assay buffer unless supplemented with phosphate. With some species (e.g. Brachiomonas submarina Bohlin, Nannochloropsis oculata Droop, Phaeodactylum tricomutum Bohlin) maximum NR activity required the addition of phosphate and flavin adenine dinucleotide (FAD) to assays; with extracts of P. tricornutum, the requirement for FAD increased after partial purification of extracts. Like phosphate, arsenate stimulated NR activity in extracts, but selenate, sulphate, and tungstate were less effective. Addition of manganese to partially purified extracts of P. tricomutum resulted in an inhibition of NR activity which was alleviated by phosphate or EDTA.     

Assimilatory nitrate uptake in Pseudomonas fluorescens studied using nitrogen-13

The mechanism of nitrate uptake for assimilation in procaryotes is not known. We used the radioactive isotope, ¹³N as NO₃ ^-, to study this process in a prevalent soil bacterium, Pseudomonas fluorescens. Cultures grown on ammonium sulfate or ammonium nitrate failed to take up labeled nitrate, indicating ammonium repressed synthesis of the assimilatory enzymes. Cultures grown on nitrite or under ammonium limitation had measurable nitrate reductase activity, indicating that the assimilatory enzymes need not be induced by nitrate. In cultures with an active nitrate reductase, the form of ¹³N internally was ammonium and amino acids; the amino acid labeling pattern indicated that ¹³NO₃ ^- was assimilated via glutamine synthetase and glutamate synthase. Cultures grown on tungstate to inactivate the reductase concentrated NO₃ ^- at least sixfold. Chlorate had no effect on nitrate transport or assimilation, nor on reduction in cell-free extracts. Ammonium inhibited nitrate uptake in cells with and without active nitrate reductases, but had no effect on cell-free nitrate reduction, indicating the site of inhibition was nitrate transport into the cytoplasm. Nitrate assimilation in cells grown on nitrate and nitrate uptake into cells grown with tungstate on nitrite both followed Michaelis-Menten kinetics with similar K _mvalues, 7 M. Both azide and cyanide inhibited nitrate assimilation. Our findings suggest that Pseudomonas fluorescens can take up nitrate via active transport and that nitrate assimilation is both inhibited and repressed by ammonium.     

Identification of two tungstate-sensitive molybdenum cofactor mutants, chl2 and chl7, of Arabidopsis thaliana.

Summary The characterization of mutants that are resistant to the herbicide chlorate has greatly increased our understanding of the structure and function of the genes required for the assimilation of nitrate. Hundreds of chlorate-resistant mutants have been identified in plants, and almost all have been found to be defective in nitrate reduction due to mutations in either nitrate reductase (NR) structural genes or genes required for the synthesis of the NR cofactor molybdenum-pterin (MoCo). The chlorate-resistant mutant ofArabidopsis thaliana, ch12, is also impaired in nitrate reduction, but the defect responsible for this phenotype has yet to be explained.chl2 plants have low levels of NR activity, yet the map position of thechl2 mutation is clearly distinct from that of the two NR structural genes that have been identified inArabidopsis. In addition,chl2 plants are not thought to be defective in MoCo, as they have near wild-type levels of xanthine dehydrogenase activity, which has been used as a measure of MoCo in other organisms. These results suggest thatchl2 may be a NR regulatory mutant. We have examinedchl2 plants and have found that they have as much NR (NIA2) mRNA as wild type a variable but often reduced level of NR protein, and one-eighth the NR activity of wild-type plants. It is difficult to explain these results by a simple regulatory model; therefore, we reexamined the MoCo levels inchl2 plants using a sensitive, specific assay for MoCo: complementation ofNeurospora MoCo mutant extracts. We found thatchl2 has low levels of MoCo — about one-eighth the wild-type level and less than the level in anotherArabidopsis MoCo mutantchl6 (B73). To confirm this result we developed a new diagnostic assay for MoCo mutants, growth inhibition by tungstate. Bothchl2 andchl6 are sensitive to tungstate at concentrations that have no effect on wildtype plants. The tungstate sensitivity as well as the chlorate resistance, low NR activity and low MoCo levels all cosegregate, indicating that all are due to a single mutation that maps to thechl2 locus, 10 centimorgans fromerecta on chromosome 2. We also report on the isolation of a new chlorate-resistant mutant ofArabidopsis, ch17, which is a MoCo mutant with the same phenotypes aschl2 andchl6.     

Enhanced root growth in phosphate‐starved Arabidopsis by stimulating de novo phospholipid biosynthesis through the overexpression of LYSOPHOSPHATIDIC ACID ACYLTRANSFERASE 2 (LPAT2)

Upon phosphate starvation, plants retard shoot growth but promote root development presumably to enhance phosphate assimilation from the ground. Membrane lipid remodelling is a metabolic adaptation that replaces membrane phospholipids by non‐phosphorous galactolipids, thereby allowing plants to obtain scarce phosphate yet maintain the membrane structure. However, stoichiometry of this phospholipid‐to‐galactolipid conversion may not account for the massive demand of membrane lipids that enables active growth of roots under phosphate starvation, thereby suggesting the involvement of de novo phospholipid biosynthesis, which is not represented in the current model. We overexpressed an endoplasmic reticulum‐localized lysophosphatidic acid acyltransferase, LPAT2, a key enzyme that catalyses the last step of de novo phospholipid biosynthesis. Two independent LPAT2 overexpression lines showed no visible phenotype under normal conditions but showed increased root length under phosphate starvation, with no effect on phosphate starvation response including marker gene expression, root hair development and anthocyanin accumulation. Accompanying membrane glycerolipid profiling of LPAT2‐overexpressing plants revealed an increased content of major phospholipid classes and distinct responses to phosphate starvation between shoot and root. The findings propose a revised model of membrane lipid remodelling, in which de novo phospholipid biosynthesis mediated by LPAT2 contributes significantly to root development under phosphate starvation.     

Impact of MtrA on phosphate metabolism genes and the response to altered phosphate conditions in Streptomyces

Phosphate metabolism is known to be regulated by the PhoPR regulatory system in Streptomyces and some other bacteria. In this study, we report that MtrA also regulates phosphate metabolism in Streptomyces. Our data showed that, in Streptomyces coelicolor, MtrA regulates not only phosphate metabolism genes such as phoA but also phoP under different phosphate conditions, including growth on rich complex media without added inorganic phosphate and on defined media with low or high concentrations of inorganic phosphate. Cross-regulation was also observed among mtrA, phoP and glnR under these conditions. We demonstrated both in vitro and in vivo binding of MtrA to the promoter regions of genes associated with phosphate metabolism and to the intergenic region between phoR and phoU, indicating that these phosphate metabolism genes are targets of MtrA. We further showed that MtrA in S. lividans and S. venezuelae has detectable regulatory effects on expression of phosphate metabolism genes. Additionally, the MtrA homologue from Corynebacterium glutamicum bound predicted MtrA sites of multiple phosphate metabolism genes, implying its potential for regulating phosphate metabolism in this species. Overall, our findings support MtrA as a major regulator for phosphate metabolism in Streptomyces and also potentially in other actinobacteria.     

Nitrogen and Phosphorus in a Stretch of the Guadalupe River,Texas, with Five Main-Stream Impoundments by

Nitrogen and phosphorus were studied in a 168-km stretch of the Guadalupe River that had five main-stream impoundments. Flow through the study area was controlled by releases from these five reservoirs and from Canyon Reservoir, a deep-storage reservoir, located 30 km upstream. Parameters measured monthly on a diel basis at 16 stations were nitrate nitrogen, nitrite nitrogen, ammonia nitrogen, Kjeldahl nitrogen, inorganic phosphate phosphorus, organic phosphate phosphorus, and total phosphate phosphorus.Inorganic nitrogen concentrations observed in this study were as high or higher than that previously reported for other bodies of water. Nitrate nitrogen entered the study area in relatively high concentrations from Comal Springs which was a major source of water for the Guadalupe River. Water from Canyon Reservoir, the other major source of water, was relatively low in nitrate nitrogen. The concentration of nitrate nitrogen was, therefore, dependent in part upon the portion of the total river flow originating from the two sources. Increased discharge from Canyon Reservoir and utilization by plants in areas of high chlorophyll a resulted in low nitrate-nitrogen levels. Retention of water in reservoirs reduced the concentration of nitrate nitrogen due to increased utilization by plants in areas of low flow. Nitrate nitrogen, in general, reached seasonal minima in summer and maxima in winter. Nitrite nitrogen showed considerable variation with no meaningful pattern except that higher concentrations occurred in association with high chlorophyll a and high Kjeldahl nitrogen, regions and periods of low river flow, and large phytoplankton populations. There was no increase in concentration of any form of nitrogen in the vicinity of sewage outfalls and no downstream accrual.Phosphorus levels in the study area were as high or higher than those reported in studies of other bodies of water. Sewage treatment plants at New Braunfels and Seguin, Texas, were major sources of phosphorus to the Guadelupe River. Total phosphate phosphorus was determined to be the most critical phosphate parameter in assessing eutrophication. Seasonally, it ranged from a winter high to a summer low. Concentrations were highest immediately below sewage outfalls and decreased as water progressed downstream. Inorganic-phosphate-phosphorus concentrations showed no clear seasonal trend but were clearly associated with sewage outfalls. Since large standing crops of phytoplankton were observed in areas of low inorganic phosphate phosphorus, it was not considered to limit photosynthesis. Total organic phosphate phosphorus varied seasonally, with high concentrations occurring during the spring and low concentrations in the fall. Total organic phosphate phosphorus showed no correlation with sewage outfalls, but was correlated to a degree with total Kjeldahl nitrogen and chlorophyll a. No consistent pattern of diel fluctuations was evident for any phosphorus or nitrogen compounds analyzed.     

Long-day flowering of Lemna perpusilla 6746 in Mo-deficient medium

Lemna perpusilla 6746, a short-day duckweed, flowered undercontinuous illumination if some of the SH inhibitors, such ascyanide or tungstate were added to the M-sucrose medium. Theeffect of tungstate was not overcome by simultaneous applicationof molybdate, but deletion of the Mo from the medium was enoughto induce the long-day flowering. In vivo assay of nitrate reductaseactivity suggested that nitrate reduction was not inhibitedby tungstate, CuSO₄ or AgNO₃ which induced longday flowering.The possibility was suggested that suppression of some Mo-requiringprocess other than nitrate reduction brings about the long-dayflowering in this plant. (Received November 12, 1975; )     

Ribosomes after infection with bacteriophage T4 and T7

Summary The synthesis of E. coli ribosomal proteins ceases after infection with bacteriophages T4 or T7 as does the synthesis of most other host proteins. The shut-off does not affect all ribosomal proteins to the same extent. After T7 infection no new proteins were detected in NH₄Cl-washed ribosomal particles. Bacteriophage T4, however, induces 3–4 new protein bands demonstrated by one-dimensional gel electrophoresis. The appearance of these bands is prevented by the addition of rifampicin at the time of infection but not when rifampicin is added one minute after infection. The NH₄Cl-washed ribosomal particles present at the time of T7 or T4 infection do not show any structural changes by sedimentation, subunit dissociation, or protein analysis on two-dimensional polyacrylamide gels. However, by labeling the T7 infected cells with ³²P-phosphate, it is seen that the ribosomes become phosphorylated. The ³²P-label comigrates with ribosomal proteins. This phosphorylating activity depends on a T7 gene. The T7 protein phosphokinase utilizes ribosomes as phosphate acceptor in vitro. The T7 ribosomes (NH₄Cl-washed) still function in vitro as do ribosomal particles from uninfected cells.Paper No. 83 on Ribosomal Proteins. Preceding paper is by Isono et al., Mol. gen. Genet. 127, 191–195 (1973).     

Triterpeneglycosides as Inhibitors of Fungal Growth and Metabolism

A venacin, the resistance factor in oat roots against Ophio-bolus graminis var. graminis, and a related triterpeneglycoside, aescin, induced a rapid release of K⁺ from mycelia of Opbio-bolus graminis and Neurospora crassa, suspended in phosphate buffer. N. crassa also released Mg²⁺ whereas no outflux of Mg²⁺ was found from O. graminis. The inhibitors induced a release of inorganic phosphate into acetate buffer from Neurospora crassa. The amount of inorganic phosphate in the mycelia decreased when O. graminis and N. crassa were treated with the inhibitors in phosphate buffer. In other media the inhibitors had weak or no effects on the ion contents of the mycelia. The effect of aescin was low in Aspergillus niger and nil in Pythium irregulare. However, high amounts of K⁺, Mg²⁺, and phosphate ions were lost to the medium when the mycelium of P. irregulare, washed with distilled water, was suspended in different buffers. The ions lost were reabsorbed during the experimental period. The leakage of ions indicates that the plasma membrane of the growth sensitive fungi is severely affected by the inhibitors, while a corresponding effect on the growth insensitive fungi does not take place.     

Resistance Transfer Factors in sensitive strains ofS. panama

Two natural strains ofS.panama with an incomplete Resistance Factor (R factor) are described:S.panama I carries a Resistance Transfer Factor (RTF) exerting restriction on phageS.panama 47, but no resistance determinants; andS.panama 219 has a tetracycline-resistance determinant but no RTF.A number of drug-sensitive strains ofS.panama belonging to various phage types, were found to carry a factor which is able to mobilise the tetracycline-resistance determinant ofS.panama 219 and also of one strain ofE.coli and to transfer them toS.panama andE.coli K 12. This factor is not identical with the F factor, it is not a bacteriocinogenic factor and can therefore be considered as an RTF. These RTFs exert no restriction on (spp) and phage 47, and some of them are fi whilst others are fi.     

A Comparison of Mitochondria Isolated from Male-Sterile and Nonserile Cytoplasm Etiolated Corn Seedlings

Toxins from Helminthosporium maydis race T and Phyllosticta maydis have been found to affect the functional processes of corn mitochondria isolated from Texas male-sterile (T) cytoplasm, but not of mitochondria isolated from nonsterile (N) cytoplasm. The effects of chemicals known to induce responses similar to those of the toxin were compared on mitochondria isolated from T and N cytoplasm inbreds (W64A, Zea mays L.). Valinomycin, gramicidin, and decenylsuccinic acid (DSA) each caused more swelling (measured by transmission changes in %) of N mitochondria than of T mitochondira. The stimulation of exogenous NADH oxidation was the same for N and T mitochondria in the valinomycin, DSA, and Ca²⁺ plus phosphate treatments, was greater for T mitochondria than for N mitochondria in the gramicidin and DNP treatments, and was greater for N mitochondrai than for T mitochondira in the Ca⁺² minus phosphate treatment. Sodium azide inhibited NADH oxidation equally for N and T mitochondria. In addition, N and T mitochondria had similar respiration rates for various substrates and equal efficiencies of oxidative phosphorylation. In contrast to the specificity of toxins for T mitochondria, none of the treatment effects were specific for N or T mitochondria. The results indicate that mitochondria isolated from N and T cytoplasm generally respond similarly to various conditions, but that there can be quantitative differences in the response. The extent to which these differences represent cytoplasmically controlled modification of mitochondrial physiology or structure is not known.     

Multicellular-vesicle-promoting polypeptide from Trichoplusia ni: Tissue distribution and N-terminal sequence

An N-terminal amino acid sequence of a 16.9 kDa hemolymph polypeptide, “Vesicle Promoting Factor” (VPF) from Trichoplusia ni, revealed a high sequence homology (70%) with Manduca sexta apolipophorin-III. A polyclonal antibody developed against VPF, however, was not immunoreactive with either purified M. sexta or T. ni apolipophorin-III. Immunoblots of tissue homogenates of T. ni indicated that VPF was present in imaginal wing discs, central nervous system (CNS), silk glands, midgut and hemocytes from fifth instar larvae, and also in the IAL-TND1 cell line which can grow as either fluid-filled multicellular vesicles or multicellular aggregates. VPF was also detected immunologically in the hemolymph of adults of T. ni, and in hemolymph of adults and larvae of Galleria mellonella and Heliothis virescens. Testes, midgut, hemocytes, and wing discs, but not Malpighian tubules, of T. ni released VPF into tissue culture medium during a 3 h incubation period. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.