Cuticular control of diffusional water permeability

The diffusional water permeability (P_d) in various gut structures of the fleshfly, Sarcophaga bullata, was measured using tritiated water. Water P_d in the larval hindgut was 3.91 × 10^?6 cm/sec, whereas in the adult hindgut it was 4.4 × 10^?4 cm/sec. The presence of cuticle in various parts of the gut apparently controls the water permeabilities of these structures. Furthermore, the water permeability of the cuticle may be correlated with the mechanism for the production of a hyperosmotic excretion in the hindgut.     

Stabilization of neurotoxic soluble beta-sheet-rich conformations of the Alzheimer's disease amyloid-beta peptide

An emerging paradigm for degenerative diseases associated with protein misfolding, such as Alzheimer's disease, is the formation of a toxic species due to structural transitions accompanied by oligomerization. Increasingly, the focus in Alzheimer's disease is on soluble oligomeric forms of the amyloid-β peptide (Aβ) as the potential toxic species. Using a variety of methods, we have analyzed how sodium dodecyl sulphate (SDS) modulates the folding of Aβ40 and 42 and found that submicellar concentrations of SDS solubilize Aβ and induce structural transitions. Under these conditions, Aβ40 and 42 are interconverting oligomeric ensembles with a predominantly β-sheet structure. The Aβ42 soluble oligomers form β-sheet structures more readily and have increased stability compared with Aβ40 under identical conditions. The presence of added Cu²⁺ significantly promotes and stabilizes the formation of the soluble oligomeric β-sheet structures but these structures are nonamyloidogenic. In contrast, in the absence of added Cu²⁺, these β-sheet oligomers possess the hallmarks of amyloidogenic structures. These SDS-induced β-sheet forms of Aβ, both in the presence and absence of Cu²⁺, are toxic to neuronal cells.     

Evidence for sequence preferences in the intercalative binding of ethidium bromide to dinucleoside monophosphates.

The rate of production of tandem duplications in phage λ has been measured in the presence and absence of known recombination systems. Two deletion phages have been used: tdel33, a deletion derivative of a φ80-λ hybrid phage, and λb221, which carries a large deletion of the central portion of the λ chromosome. Both phages are int⁻, and tdel33 is also red⁻, by virtue of their deletions. Stocks of these phages can be prepared free of long tandem duplication derivatives by CsCl density gradient purification. After a single cycle of lytic growth, lysates from these purified phage stocks contain tandem duplications at a frequency of 10⁻³ in the case of tdel33 and 10⁻⁵ in the case of λb221. These frequencies are unaffected by the presence of mutations in the host Rec system or the phage Red system. To investigate the difference in duplication frequency between tdel33 and λb221, the phages were grown in mixed infection. The result indicates that a trans-active product of tdel33 is responsible for its high frequency of duplication production.Tandem duplications have been detected by banding the phage lysates in CsCl density gradients. Long DNA addition mutants can be detected in this way if they arise with a frequency of at least 10⁻⁵ and if the duplication length is at least 0.14 λ lengths. To accomplish this it is necessary to distinguish them from contaminating parental phage and from dense phages with aberrant structures which arise at roughly comparable frequencies. The former can be done by rebanding and the latter by growth and rebanding. To distinguish these types we have also made use of a new mutant of Escherichia coli which does not plate λ deletion phages. All of the DNA addition mutants we have detected in this way are tandem duplications; evidently mutants with long insertions arise more rarely.     

Deletion Analysis of the Tumorous-Head (tuh–3) Gene in DROSOPHILA MELANOGASTER

In the presence of the naturally occurring maternal-effect alleles tuh-1^h or tuh-1^g, the tuh-3 mutant gene can cause the tumorous-head trait or the sac-testis trait. The tuh-3 gene functions as a semidominant in the presence of the tuh-1^h maternal effect. Eye-antennal structures are replaced by posterior abdominal tergites and genital structures. If tuh-1^h is replaced by its naturally occurring allele tuh-1^g, tuh-3 functions as a recessive hypomorph and the defect switches from anterior to posterior structures, with a male genital-disc defect appearing with variable penetrance. Function and regulation of tuh-3⁺ may better be understood in light of the cytological localization of tuh-3 either adjacent to or as part of the bithorax complex. The tuh-3⁺ gene product appears to be essential for normal development, at least in the posterior end of the embryo.     

An Exported Inducer Peptide Regulates Bacteriocin Production in Enterococcus faecium CTC492

Production of the bacteriocins enterocin A and enterocin B in Enterococcus faecium CTC492 was dependent on the presence of an extracellular peptide produced by the strain itself. This induction factor (EntF) was purified, and amino acid sequencing combined with DNA sequencing of the corresponding gene identified it as a peptide of 25 amino acids. The gene encodes a prepeptide of 41 amino acids, including a 16-amino-acid leader peptide of the double-glycine type. Environmental factors influenced the level of bacteriocin production in E. faecium CTC492. The optimal pH for bacteriocin production was 6.2. At pH 5.5, growth was slow, and very little bacteriocin was formed. The presence of NaCl or ethanol (EtOH) was also inhibitory to bacteriocin production, and at high concentrations of these solutes, no bacteriocin production was observed. The induction factor induced its own synthesis, and by dilution of the culture 10⁶ times or more, nonproducing cultures were obtained. Bacteriocin production was induced in these cultures by addition of EntF. The response was linear, and low bacteriocin production could be induced by about 10⁻¹⁷ M EntF. This response was attenuated by low pH or the presence of high concentrations of NaCl or EtOH, and 300 times more EntF was needed to induce detectable bacteriocin production in the presence of 6.5% NaCl. High levels of bacteriocin production in cultures grown at low pH or in the presence of high concentrations of NaCl or EtOH were obtained by addition of sufficient amounts of EntF.     

Auxiliary Ca2+ binding sites can influence the structure of CIB1

Recent X-ray crystal structures and solution NMR spectroscopy data for calcium- and integrin-binding protein 1 (CIB1) have all revealed a common EF-hand domain structure for the protein. However, the orientation of the two protein domains, the oligomerization state, and the conformations of the N- and C-terminal extensions differ among the structures. In this study, we examine whether the binding of glutathione or auxiliary Ca²⁺ ions as observed in the crystal structures, occur in solution, and whether these interactions can influence the structure or dimerization of CIB1. In addition, we test the potential phosphatase activity of CIB1, which was hypothesized based on the glutathione binding site geometry observed in one of the crystal structures of the protein. Biophysical and biochemical experiments failed to detect glutathione binding, protein dimerization, or phosphatase activity for CIB1 under several solution conditions. However, our data identify low affinity (K_d, 10⁻²M) Ca²⁺ binding events that influence the structures of the N- and C-terminal extensions of CIB1 under high (300 mM) Ca²⁺ crystallization conditions. In addition to providing a rationale for differences amongst the various solution and crystal structures of CIB1, our results show that the impact of low affinity Ca²⁺ binding events should be considered when analyzing and interpreting protein crystallographic structures determined in the presence of very high Ca²⁺ concentrations.     

Membrane activity of a C-reactive protein

C-reactive protein (CRP) from the American horseshoe crab, Limulus polyphemus, exhibits complex membrane activities. Here, we describe the behavior of protein and lipid as CRP interacts with model liposomes and bacterial membranes. Limulus C-reactive protein (L-CRP) forms extended fibrilar structures that encapsulate liposomes in the presence of Ca²⁺. We have observed structures consistent in size and shape with these fibers bound to the surface of Gram-negative bacteria. The membranes of Limulus CRP-treated bacteria exhibit significantly different mechano-elastic properties than those of untreated bacteria. In vitro, bilayer lipids undergo a rigidification and reorganization of small domains. We suggest that these interactions reflect the protein’s role as a primary defense molecule, functioning in the entrapment and killing of potential pathogens.     

Study of different nitrogen sources on glucose uptake and production of melanin precursors and fungal mass of Fonsecaea pedrosoi cultured in tricyclazole

The influence of tricyclazole on the production of phenolic precursors, glucose uptake and production of fungal mass of Fonsecaea pedrosoi was assessed in the presence of phenylalanine, sodium nitrate and tryptophan. Photocolorimetry was used for the quantitation of glucose and total phenol, and the dry weight method for biomass. The glucose uptake with phenylalanine was 99.85×10³ mg/ml; with tryptophan, 99.88×10³ mg/ml and with sodium nitrate, 99.90×10³ mg/ml. Production of biomass: with tryptophan, 2.7×10⁻¹ mg; with sodium nitrate, 3.0×10⁻¹ mg and with phenylalanine, 3.4×10⁻¹ mg. Tricyclazole induced higher phenol accumulation, lower glucose uptake, inhibition of melanin deposition on the cell and higher production and higher biomass production in relation to glucose uptake for all nitrogen sources tested under these conditions. A negative correlation between biomass production and glucose uptake was observed in the presence of secondary metabolism.     

α1-Adrenergic inositol trisphosphate production in brown adipocytes is Na+ dependent

In order to investigate the ionic requirements for inositol trisphosphate production, brown adipocytes were prelabelled with myo-[³H]inositol and the formation of inositol trisphosphates and inositol bisphosphates as a consequence of α₁-adrenergic stimulation was monitored. Omission of Ca²⁺ from the incubation medium diminished the norepinephrine-induced increase in inositol trisphosphate levels, but it would seem that this reduction can be fully accounted for by a decreased level of the ‘inactive’ isomer inositol 1,3,4-trisphosphate. Omission of Na⁺ fully abolished the norepinephrine-induced inositol trisphosphate response. However, it was observed that the presence of Li⁺ in the incubation medium could fully reconstitute the ability of the cells to yield the early response of inositol trisphosphate production; Li⁺ could, however, not substitute for Na⁺ in the entire α₁-adrenergic cellular pathway. It was concluded that the Na⁺-dependent step is found in the coupling mechanism between the α₁-receptor and the activation of the phosphodiesterase responsible for inositol trisphosphate production. Thus, all events in the α₁-adrenergic pathway which are consequences of IP₃ production should appear to be Na⁺-dependent in these cells.     

cis-benzeneglycol production using a mutant Pseudomonas strain

The biotransformation of commodity aromatic chemicals into dihydroxy derivatives was studied. A strain isolated from the invironment, Pseudomonas JI104, used benzene, toluene, and other hydrocarbons as sole carbon and energy sources. We selected mutants unable to grow with benzene, and among these, screened for strains with deficient cis-benzenglycol dehydrogenase able to stably produce cis-benzeneglycol when another carbon source was co-metabolized.We exained the possibility of cis-benzeneglycol production by growing the mutant strain in the presence of benzene vapor. Ethanol was the carbon and energy source most adapted to the cis-benzeneglycol production phase, and lactate or propanol could also be used. Glucose inhibited the production of the metabolite.The growth rates were barely affected by the presence of benzene at a reduced partial pressure (less than 20% of saturation), showing that continuous culture is possible. In a batch process, 0.54g·1⁻¹ of a cell suspension produced 5.1 mmol·1⁻¹cis-benzeneglycol in 27 h, using ethanol as the energy source.     

Factors and Selenocysteine Insertion Sequence Requirements for the Synthesis of Selenoproteins from a Gram-Positive Anaerobe in Escherichia coli

Selenoprotein synthesis in Escherichia coli strictly depends on the presence of a specific selenocysteine insertion sequence (SECIS) following the selenocysteine-encoding UGA codon of the respective mRNA. It is recognized by the selenocysteine-specific elongation factor SelB, leading to cotranslational insertion of selenocysteine into the nascent polypeptide chain. The synthesis of three different selenoproteins from the gram-positive anaerobe Eubacterium acidaminophilum in E. coli was studied. Incorporation of ⁷⁵Se into glycine reductase protein B (GrdB1), the peroxiredoxin PrxU, and selenophosphate synthetase (SelD1) was negligible in an E. coli wild-type strain and was fully absent in an E. coli SelB mutant. Selenoprotein synthesis, however, was strongly increased if selB and selC (tRNA^Sec) from E. acidaminophilum were coexpressed. Putative secondary structures downstream of the UGA codons did not show any sequence similarity to each other or to the E. coli SECIS element. However, mutations in these structures strongly reduced the amount of ⁷⁵Se-labeled protein, indicating that they indeed act as SECIS elements. UGA readthrough mediated by the three different SECIS elements was further analyzed using gst-lacZ translational fusions. In the presence of selB and selC from E. acidaminophilum, UGA readthrough was 36 to 64% compared to the respective cysteine-encoding UGC variant. UGA readthrough of SECIS elements present in Desulfomicrobium baculatum (hydV), Treponema denticola (selD), and Campylobacter jejuni (selW-like gene) was also considerably enhanced in the presence of E. acidaminophilum selB and selC. This indicates recognition of these SECIS elements and might open new perspectives for heterologous selenoprotein synthesis in E. coli.     

Photodynamic Inactivation of Candida albicans with Imidazoacridinones: Influence of Irradiance,Photosensitizer Uptake and Reactive Oxygen Species Generation

The increasing applicability of antifungal treatments, the limited range of available drug classes and the emergence of drug resistance in Candida spp. suggest the need for new treatment options. To explore the applicability of C. albicans photoinactivation, we examined nine structurally different imidazoacridinone derivatives as photosensitizing agents. The most effective derivatives showed a >10⁴-fold reduction of viable cell numbers. The fungicidal action of the three most active compounds was compared at different radiant powers(3.5 to 63 mW/cm²), and this analysis indicated that 7 mW/cm² was the most efficient. The intracellular accumulation of these compounds in fungal cells correlated with the fungicidal activity of all 9 derivatives. The lack of effect of verapamil, an inhibitor targeting Candida ABC efflux pumps, suggests that these imidazoacridinones are not substrates for ABC transporters. Thus, unlike azoles, a major class of antifungals used against Candida, ABC transporter-mediated resistance is unlikely. Electron paramagnetic resonance (EPR)-spin trapping data suggested that the fungicidal light-induced action of these derivatives might depend on the production of superoxide anion. The highest generation rate of superoxide anion was observed for 1330H, 1610H, and 1611. Singlet oxygen production was also detected upon the irradiation of imidazoacridinone derivatives with UV laser light, with a low to moderate yield, depending on the type of compound. Thus, imidazoacridinone derivatives examined in the present study might act via mixed type I/type II photodynamic mechanism. The presented data indicate lack of direct correlation between the structures of studied imidazoacridinones, cell killing ability, and ROS production. However, we showed for the first time that for imidazoacridinones not only intracellular accumulation is necessary prerequisite of lethal photosensitization of C. albicans, but also localization within particular cellular structures. Our findings present IA derivatives as efficient antifungal photosensitizers with a potential to be used in local treatment of Candida infection.     

The effect of Mn2+ on ethanol production from lactose using Kluyveromyces marxianus IMB3 immobilized in magnetically responsive matrices

The thermotolerant, ethanol producing yeast strain, K. marxianus IMB3 was immobilized in calcium alginate containing magnetically responsive Fe₃O₄ particles. In these studies the β-galactosidase derived from K. marxianus IMB3 was immobilized onto the Fe₃O₄ particles prior to inclusion into the alginate matrix. Ethanol production by the immobilized microorganism in the presence of Fe₃O₄ reached a maximum of 16?g/L on 40?g/L lactose whereas prior immobilization of the enzyme to the particles and inclusion into the alginate matrix increased ethanol production to a maximum concentration of 18 g/L. When Mn²⁺ was incorporated into fermentations containing the immobilized enzyme in the alginate matrix, ethanol production increased further to a maximum concentration of 20?g/L. In addition, the behaviour of the magnetically responsive biocatalyst containing the co-immobilized enzyme was examined in a batch-fed system in the presence and absence of Mn²⁺.     

Comparative aspects of the calcium-sensitive photoproteins aequorin and obelin

1. The calcium-dependency of the process of light emission has been investigated for the photoproteins aequorin and obelin.2. The experimental curves of light production, expressed as a percentage of the maximal rate of utilisation, versus pCa are accurately predicted by the cooperative action of at least 2Ca²⁺ for aequorin and at least 3Ca²⁺ for obelin.3. At low total monovalent cation concentrations, a pH change from 6.8 to 7.1 shifts the light production vs pCa curve by approx. 0.2 pCa units to the right for aequorin, while that for obelin is shifted by some 0.37 pCa units.4. Other monovalent cations, such as Na⁺ are able to compete with Ca²⁺ for the active sites of aequorin and also shift the light production vs pCa curve to the right. There is no apparent change in the calcium stoichiometry for light production under these conditions.5. The same calcium stoichiometry for light emission was also obtained for aequorin or obelin in the presence of either unbuffered Ca²⁺ solutions or of calcium/EGTA buffers.     

Iron uptake from ferric citrate by Vibrio anguillarum

We report here that Vibrio anguillarum possesses a non-inducible active transport system which can efficiently supply iron to the cell from ferric citrate, independently of the siderophore-based mechanisms. The strains tested were able to grow in CM9 medium in iron-restricted conditions when ferric citrate was present in the medium. Moreover, the presence of ferric citrate inhibited the production of siderophores in the strains tested. V. anguillarum cells and isolated membranes could incorporate ⁵⁵Fe³⁺ complexed by citrate, without a difference between cells grown in the presence or absence of ferric citrate. The presence of 2,4-dinitrophenol, ferrozine, ferricyanide, trypsin, as well as low temperature produced a marked decrease or total inhibition of ⁵⁵Fe³⁺ uptake by the cells. All these results suggest that iron uptake from ferric citrate in V. anguillarum must be an energy-dependent process not induced by the presence of iron or citrate in the medium, mediated by a membrane protein(s), which may require an iron reduction step to function.     

Flavonoids of Penthorum sedoides

In an attempt to elucidate the familial affinities of Penthorum, the flavonoids and other phenolic compounds of P. sedoides were examined. The species is characterized by a relatively simple array of kaempferol and quercetin 3-O-mono-, di- and triglycosides. Flavonoid variation was detected between populations. Also present was a group of phenolic compounds that gave positive color reactions for the existence of gallic acid-like components. Although structures were not determined for these compounds, the presence of gallic acid-like moieties in two of the compounds was confirmed by ¹H NMR. The array of these compounds and flavonoids detected in P. sedoides does not readily support inclusion of Penthorum in the Saxifragaceae.     

Influence of substrate concentration and bicarbonate addition on the anaerobic digestion of an inulin-rich substrate as the ending stage in a dandelion biorefinery scheme

This study was focused on the suitability of dandelion plant as part of a biorefinery scheme in which anaerobic degradation (biomethanization) for biogas production is the ending process. Inulin as the main compound in the tuberous root was selected as substrate and tested at different concentrations (5, 10 and 15 g TS L⁻¹). The presence of bicarbonate as a buffer on anaerobic digestion was assessed as well. Methane production curves were fitted with the modified Gompertz equation. Methane production yield (Y_CH4), specific maximum methane production rate (SR_MAX) and the first-order hydrolysis constant (k_H) were affected by the substrate concentration. Bicarbonate was found not to be determinant for the overall biomethanization process because no significant effect over the Y_CH4was achieved in the presence or absence of bicarbonate; between 0.19 to 0.23 m³ kg^-1 VS added. However, k_Hand SR_MAX were affected by the presence of this inorganic compound. Without the addition of bicarbonate, k_Hand SR_MAX decreased as the TS increased. These results will be key for a dandelion biorefinery proof-of-concept under the proposed conditions.     

Studies on the metabolic interactions between 4-methylpyrazole and methanol using the monkey as an animal model

Bovine adrenal chromaffin granules have been shown to contain [Met]enkephalin and [Leu]enkephalin and at least seven other small peptides that exhibit specific binding to opiate receptors. Six of these peptides have been characterized and their structures established as (O)-[Met]enkephalin, [Met]enkephalin-Arg⁶-Arg⁷, [Met]enkephalin-Lys⁶, [Met]enkephalin-Arg⁶, [Leu]enkephalin-Arg⁶, and [Met]enkephalin-Arg⁶-Phe⁷. Many of these hexa- and heptapeptides are also present in bovine and human brain. It is suggested that the presence of these peptides in a tissue is evidence of a common biosynthetic pathway of the enkephalins from a large precursor protein.