A model for light adaptation: Producing Weber's law with bleaching-type kinetics

An adaptation model having two stages is introduced and its mathematical properties are examined. The two stages are the adaptive process (parameter K _b), which has bleaching-type kinetics, and the response function (parameters K _r and n), which incorporates response saturation. In order to study the increment threshold functions generated by the adaptation model the concept of a detector is required. It is demonstrated that without an adaptive process the compression hypothesis, in the form of the difference equation, produces increment threshold functions which saturate and do not obey Weber's law. It is then shown that an adaptive process with bleaching-type kinetics can prevent saturation and produce Weber's law behavior provided that the adaptive strength of the system exceeds the detector sensitivity.     

Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes

Summary The dinucleotide CpG is a hotspot for mutation in the human genome as a result of (1) the modification of the 5 cytosine by cellular DNA methyltransferases and (2) the consequent high frequency of spontaneous deamination of 5-methyl cytosine (5mC) to thymidine. DNA methylation thus contributes significantly, albeit indirectly, to the incidence of human genetic disease. We have attempted to estimate for the first time the in vivo rate of deamination of 5mC from the measured rate of 5mC deamination in vitro and the known error frequency of the cellular G/T mismatch-repair system. The accuracy and utility of this estimate (m _d ) was then assessed by comparison with clinical data, and an improved estimate of m _d (1.66x10^-16 s^-1) was derived. Comparison of the CpG mutation rates exibited by globin gene and pseudogene sequences from human, chimpanzee and macaque provided further estimates of m _d , all of which were consistent with the first. Use of this value in a mathematical model then permitted the estimation of the length of time required to produce the level of CpG suppression currently found in the bulk DNA of vertebrate genomes. This time span, approximately 450 million years, corresponds closely to the estimated time since the emergence and adaptive radiation of the vertebrates and thus coincides with the probable advent of heavily methylated genomes. An accurate estimate of the 5mC deamination rate is important not only for clinical medicine but also for studies of gene evolution. Our data suggest both that patterns of vertebrate gene methylation may be comparatively stable over relatively long periods of evolutionary time, and that the rate of CpG deamination can, under certain limited conditions, serve as a molecular clock.     

Enumeration of iron-oxidizing bacteria by the membrane filter technique

This work presents a simple methodology to enumerate ferrous-iron-oxidizing bacteria in solution, easily applicable in bioleaching industrial plants, because it does not require expertise or specific equipment. The enumeration is based on bacterial concentration by microfiltration through a membrane filter. The filter containing the bacteria is placed on an agarose plate containing ferrous sulphate for bacterial growth. No difference was observed for the enumeration of Acidithiobacillus ferrooxidans ATCC 19859 when either 0.1 or 0.22 m pore size membrane filters were used. However, when the technique was applied to bacteria present in pregnant leaching solution, the smaller bacteria present in these solutions passed through the 0.22 m pore size membrane. Therefore the number of bacteria could be underestimated if they are monitored and filtered using a filter with pore size greater than 0.1 m. The limit of detection of this technique was one ferrous-iron-oxidizing bacterium in the filtered solutions.     

A mathematical model for the growth of bacterial microcolonies on marine sediment

Counts of bacterial microcolonies attached to deep-sea sediment particles showed 4-, 8-, 16-, and 32-celled microcolonies to be very rare. This was investigated with a mathematical model in which microcolonies grew from single cells at a constant growth rate (), detached from particles at constant rate (), and reattached as single cells. Terms for attachment of foreign bacteria (a) and death of single cells (d) were also included. The best method of fitting the model to the microcolony counts was a weighted least-squares approach by which(0.83 hour^–1) was estimated to be about 20 times greater than(0.038 hour^–1). This showed that the bacteria were very mobile between sediment particles and this mobility was explained in terms of attachment by reversible sorption. The implications of the results for the frequency of dividing cell method for estimating growth rates of sediment bacteria are discussed. The ratio of and was found to be very robust both in terms of the errors associated with the microcolony counts and the range of microcolony sizes used to obtain the solution.     

Purification and characterization of cyclic 3′5′-nucleotide phosphodiesterase D3 from Sinorhizobium fredii MAR-1

The cyclic 35-nucleotide phosphodiesterase D3 was purified from Sinorhizobium fredii MAR-1. The native enzyme had a molecular weight of approximately 44.5kDa and a subunit molecular weight of approximately 21kDa as judged by SDS-gel electrophoresis. The pH optimum of the enzyme for the hydrolysis of cyclic AMP was approximately 6.0 with both acetate and Tris-maleate buffers. The optimum temperature for hydrolysing cyclic AMP was approximately 50C. No metal ion was required for activity and EDTA up to 2.5mM did not markedly affect the enzyme. However, methylxanthines, adenine and adenosine as well as 5-AMP, ATP, ADP and metal ions like Zn²⁺, Fe²⁺, Pb²⁺, Al³⁺ and Fe³⁺, were strongly inhibitory at 2.5mM.The D3 enzyme could hydrolyse both cyclic AMP and cyclic GMP with the apparent K _m for cyclic AMP of approximately 0.23M.     

Orthopaedic implant related metal toxicity in terms of human lymphocyte reactivity to metal‐protein complexes produced from cobalt‐base and titanium‐base implant alloy degradation

Metal toxicity from sources such as orthopaedic implants was investigated in terms of immune system hyper-reactivity to metal implant alloy degradation products. Lymphocyte response to serum protein complexed with metal from implant alloy degradation was investigated in this in vitro study using primary human lymphocytes from healthy volunteers (n = 10). Cobalt chromium molybdenum alloy (CoCrMo, ASTM F75) and titanium alloy (Ti6Al4V, ASTM F136) beads (70 m) were incubated in agitated human serum at 37 degrees Celsius to simulate naturally occurring metal implant alloy degradation processes. Particulate free serum samples, which were incubated with metal, were then separated into molecular weight based fractions. The amounts of soluble Cr and Ti within each serum fraction were measured and correlated with lymphocyte proliferation response to the individual serum fractions. Lymphocytes from each subject were cultured with 11 autologous molecular weight based serum fractions either with or without added metal. Two molecular weight ranges of human serum proteins were associated with the binding of Cr and Ti from CoCrMo and Ti implant alloy degradation (at < 30 and 180–330 kDa). High molecular weight serum proteins ( 180 kDa) demonstrated greater lymphocyte reactivity when complexed with metal released from CoCrMo alloy and Ti alloy than with low (5–30 kDa) and midrange (30–77 kDa) serum proteins. When the amount of lymphocyte stimulation was normalized to both the moles of metal and the moles of protein within each fraction (MetalProtein Complex Reactivity Index, MPCRI), Cr from CoCrMo alloy degradation demonstrated approximately 10 fold greater reactivity than Ti in the higher molecular weight serum proteins ( 180–250 kDa). This in vitro study demonstrated a lymphocyte proliferative response to both CoCrMo and Ti alloy metalloprotein degradation products. This response was greatest when the metals were complexed with high molecular weight proteins, and with metalprotein complexes formed from CoCrMo alloy degradation.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Oxidative detoxification of sulfide by mitochondria of the California killifish Fundulus parvipinnis and the speckled sanddab Citharichthys sitgmaeus

Summary Earlier whole-animal experiments have shown that the California killifish (Fundulus parvipinnis) from tidal marshes is highly tolerant to sulfide while the speckled sanddab (Citharichtys stigmaeus) from the open coast is intolerant to sulfide. In the present paper, we demonstrate that the liver mitochondria of the California killifish detoxify sulfide by oxidizing it to thiosulfate and produce ATP in the process. Sulfide oxidation is obligately and stoichiometrically linked to mitochondrial electron transport to oxygen. Concentrations up to 20 M sulfide stimulate mitochondrial respiration while 50 M sulfide causes half-inhibition. Sulfide oxidation by mitochondria is adversely affected at pH<7.4. ATP production is maximal at 10 M sulfide. The finding of sulfide oxidation coupled to ATP production by killifish mitochondria is unprecedented among vertebrates. In comparison, mitochondria of the specked sanddab oxidize sulfide at a much lower rate. This is the first demonstration of biochemical adaptation to sulfide among coastal marine fishes.Abbreviations ADP adenosine diphosphate - APHA American Public Health Association - ATP adenosine triphosphate - BSA bovine serum albumin - EGTA ethyleneglycol-bis-(-aminoethyl-ether) N,N,N,N-tetraacetic acid - G-6-PDH glucose-6-phosphate dehydrogenase - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - HPLC high-performance liquid chromatography; mBBr monobromobimane - NADP nicotinamide adenine dinucleotide phosphate, oxidized form - NADPH reduced form - RCR respiratory control ratio     

AT-1 Receptor and Phospholipase C Are Involved in Angiotensin III Modulation of Hypothalamic Noradrenergic Transmission

SUMMARY 1. We previously reported that angiotensin III modulates noradrenergic neurotransmission in the hypothalamus of the rat. In the present work we studied the effects of angiotensin III on norepinephrine release and tyrosine hydroxylase activity. We also investigated the receptors and intracellular pathways involved in angiotensin III modulation of noradrenergic transmission.2. In rat hypothalamic tissue labeled with [³H]norepinephrine 1, 10, and 100 nM and 1 M losartan (AT1 receptor antagonist) had no effect on basal neuronal norepinephrine release, whereas 10 and 100 nM and 1 M losartan partially diminished norepinephrine secretion evoked by 25 mM KCl. The AT2 receptor antagonist PD 123319 showed no effect either on basal or evoked norepinephrine release. The increase in both basal and evoked norepinephrine output induced by 1 M angiotensin III was blocked by 1 M losartan, but not by 1 M PD 123319.3. The phospholipase C inhibitor 5 M neomicin inhibited the increase in basal and evoked norepinephrine release produced by 1 M angiotensin III.4. Tyrosine hydroxylase activity was increased by 1 M angiotensin III and this effect was blocked by 1 M LST and 5 M neomicin, but not by PD 123319. On the other hand, 1 M angiotensin III enhanced phosphatidyl inositol hydrolysis that was blocked by 1 M losartan and 5 M neomicin. PD 123319 (1 M) did not affect ANG III-induced phosphatidyl inositol hydrolysis enhancement.5. Our results confirm that angiotensin III acts as a modulator of noradrenergic transmission at the hypothalamic level through the AT1-phospholipase C pathway. This enhancement of hypothalamic noradrenergic activity suggests that angiotensin III may act as a central modulator of several biological processes regulated at this level by catecholamines, such as cardiovascular, endocrine, and autonomic functions as well as water and saline homeostasis.     

Organ-specific crystalline structures of ferritin cores inβ-thalassemia/hemoglobin E

Summary The cores of ferritins isolated from different organs of human subjects with-thalassemia/hemoglobin E (-thal/HbE) disease have different size distributions and crystallinities depending on the source organ. These patients have not been treated by hypertransfusion regimen or iron chelation therapy.-Thal/HbE spleens and livers yield ferritin cores which are less crystalline than those isolated from normal spleens and livers, reflecting the more rapid deposition of iron in the diseased state. Ferritins isolated from the hearts and pancreases of-thal/HbE subjects were found to have larger, more crystalline cores than those from the-thal/HbE livers and spleens, possibly as a consequence of the role of the heart and pancreas as long-term iron deposition sites in this iron overload pathology.     

Expression of gloshedobin,a thrombin-like enzyme from the venom of Gloydius shedaoensis,in Escherichia coli

Gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, was expressed in Escherichia coli using expression vector pET-32a(+). The gene was expressed under T7 promotor with a fusion partner of Thx.Tag and a 6xHis.Tag at its 5 terminal. After induction by IPTG for 6 h, the recombinant enzyme was expressed in the cytoplasm. Expression at 25°C gave twice the amount of recombinant gloshedobin in cytoplasm than at 37°C.     

Evolutionary origin of the class A and class C β-lactamases

Summary The protein sequences of 18 class A -lactamases and 2 class C -lactamases were analyzed to produce a rooted phylogenetic tree using the DD peptidase of Streptomyces R61 as an outgroup. This tree supports the penicillin-binding proteins as the most likely candidate for the ancestoral origin of the class A and class C -lactamases, these proteins diverging from a common evolutionary origin close to the DD peptidase. The actinomycetes are clearly shown as the origin of the class A -lactamases found in other non-actinomycete species. The tree also divides the -lactamases from the Streptomyces into two subgroups. One subgroup is closer to the DD peptidase root. The other Streptomyces subgroup shares a common branch point with the rest of the class A -lactamases, showing this subgroup as the origin of the non-actinomycete class A -lactamases. The non-actinomycete class A -lactamase phylogenetic tree suggests a spread of these -lactamases by horizontal transfer from the Streptomyces into the non-actinomycete gram-positive bacteria and thence into the gram-negative bacteria. The phylogenetic tree of the Streptomyces class A -lactamases supports the possibility that horizontal transfer of class A -lactamases occurred within the Streptomyces.     

Killing ofLegionella pneumophila by human serum and iron-binding agents

The inhibitory effect of serum on the growth and survival ofLegionella pneumophila Bloomington 2 was investigated. When incubated in the presence of 20%–50% normal human serum for 10 h, viability was decreased by >99%. Heat-inactivated or <40% normal serum supplemented with 50 M iron was not inhibitory. The addition of guinea pig complement to heat-inactivated serum resulted in killing of approximately 98% of the cells. Growth in buffered yeast extract broth was inhibited by the addition of ferric iron-binding compounds. Minimum bactericidal concentrations at 37°C were 10 M apotransferrin, 35 M 1,10-phenanthroline, and 50 M deferoxamine. Addition of iron chelators to normal serum did not accelerate killing. Egg yolk-passaged virulent strains and agar-grown avirulent strains exhibited similar serum sensitivity. Results of this study indicate that complement and serum transferrin are antagonistic to the growth ofLegionella in serum.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Adhesion of bacteria from mixed cell suspension to solid surfaces

The attachment of four species of bacteria to solid surfaces was investigated to determine whether the attachment of one species of bacterium could be influenced by the presence of other attaching or attached species. Three types of experiment were done: (i) attachment of bacteria from suspensions containing two species (termed simultaneous attachment) was compared to attachment of each species in pure culture, (ii) the attachment of one species of bacterium to surfaces already colonized by a second species (termed sequential attachment) was compared to attachment of the bacteria to clean, uncolonized surfaces, and (iii) bacteria were allowed to attach to a surface already colonized by a second strain, and their effect on the stabilization of adhesion of the initial colonizing strain was determined. The bacteria were Acinetobacter calcoaceticus, a Staphylococcus sp., a coryneform (isolates from a canning factory), and Staphylococcus aureus. The surfaces were tin plate, glass, and nylon. The attachment of each species was either increased, decreased or not affected by the simultaneous or sequential attachment of another species. The results depended upon the species combination, the surface composition, and the sequence of attachment. The detachment of a primary colonizing species was either increased, decreased or not affected by the subsequent attachment of a second species, depending on the species combination and surface. The results demonstrate that bacterial attachment to a surface can be influenced by the composition of the attaching population and can differ considerably from the attachment of the component species in pure culture. This has implications for the control and removal of biofilms in food processing plants, as well as a wider significance for the composition and dynamics of biofilms in industrial and natural environments.Abbreviation PYE Peptone/yeast extract medium     

Electrophoretic variation between class II molecules expressed on HLA-DRw8 homozygous typing cells reveals multiple distinct haplotypes

Two-dimensional (2D) gel electrophoresis of immunoprecipitated HLA-DR antigens from eight homozygous typing cells (HTC) expressing the HLA-DRw8 specificity revealed a clustering of polymorphic chain patterns into distinct electrophoretic variants. The variant patterns correlate with three discrete HLA-D clusters that are defined in the mixed leukocyte culture reaction (MLR) using DRw8-positive HTC. These HLA-D clusters have been provisionally designated Dw8.1, detected primarily in Caucasoids, Dw8.2, detected primarily in American Indians, and Dw8.3, detected predominantly in Orientals. All three HLA-Dw8.1 cell lines express a single DR-locus product as defined by immunoprecipitation with a DR-specific monoclonal antibody, P4.1. This DR chain is identical among the Dw8.1 cell lines and different from the DR chains of the Dw8.2 and Dw8.3 cell lines. Two separate Dw8.2 HTC express a shared DR chain that is slightly more basic than the 8.1 DR molecule; interestingly, one of these lines also expresses an additional DR-like chain not found in the other cells. Thus, the two lines defining the Dw8.2 cluster share one distinct class 11 molecule, but differ in another and therefore are not biochemically HLA-identical. Cells from the Dw8.3 cluster are likewise distinct from all other Dw8 clusters. One additional DRw8-positive HTC has been analyzed and found to be distinct from the Dw8.1, 8.2 and 8.3 clusters by both MLR and 2D gels. lmmunoprecipitates using monoclonal antibody 1B5 [anti-DR and anti-DQ(DS)] identify additional polymorphic class II variants among the cell lines tested. These data indicate that HLA-DRw8 is a public serologic specificity present on class II molecules expressed on multiple distinct haplotypes. These haplotypes differ from each other in expression of polymorphic class II molecules encoded by at least two HLA loci. They also differ in HLA-D, even though they all type as HLA-DRw8 homozygous. In Dw8.2, variation in expressed chains is not reflected in variation in HLA-D, indicating that MLR, as well as serologic typing, does not detect the full degree of allelic polymorphism within HLA.     

Ferredoxin degradation in growing Clostridium pasteurianum during periods of iron deprivation

Clostridium pasteurianum was grown in batch cultures on media with an initial iron concentration of 10 M. The uptake of iron and the synthesis of ferredoxin was followed. All the iron present in the medium was taken up by the cells before 50% of the final cell density was attained. The bacteria then continued to grow in the complete absence of exogenous iron. Ferredoxin was synthesized during growth until the exogenous iron concentration dropped below 1 M. During growth in the absence of iron ferredoxin was degraded with the result that at the end of growth the cells did not contain ferredoxin. The specific activity of the iron sulfur protein, pyruvate synthase (E.C. 1.2.7.1), remained constant during growth of C. pasteurianum in the absence of exogenous iron. This finding suggests that ferredoxin was used as an endogenous source of iron for the synthesis of essential iron proteins during periods of iron deprivation.The term ferredoxin degradation is used here to indicate that the ferredoxin content in the growing cells decreased more than could be accounted for by repeated cell division. Ferredoxin = holoferredoxin = protein containing iron and sulfide; apoferredoxin = protein free of iron and sulfide     

Meiotic studies ofElymus nutans andE. jacquemontii (Poaceae,Triticeae) and their hybrids withPseudoroegneria spicata and seventeenElymus species

Hybridizations ofElymus nutans andE. jacquemontii were carried out with one species ofPseudoroegneria (S genome), and 20Elymus species, each containing either of the SH, SY, SYH, or SYW genomes. Chromosome configurations were analysed at metaphase I of the two target taxa and their interspecific hybrids. It is concluded that (i)E. nutans is an allohexaploid containing the SYH genomes, andE. jacquemontii is an allotetraploid having the SY genomes; (ii) the genomic affinity is associated with the geographic distance between the species studied; (iii) minor genomic structural rearrangements have occurred within the hexaploid taxon ofE. nutans.