Chronic warming stimulates growth of marsh grasses more than mangroves in a coastal wetland ecotone.

Increasing temperatures and a reduction in the frequency and severity of freezing events have been linked to species distribution shifts. Across the globe, mangrove ranges are expanding toward higher latitudes, likely due to diminishing frequency of freezing events associated with climate change. Continued warming will alter coastal wetland plant dynamics both above- and belowground, potentially altering plant capacity to keep up with sea level rise. We conducted an in situ warming experiment, in northeast Florida, to determine how increased temperature (+2°C) influences co-occurring mangrove and salt marsh plants. Warming was achieved using passive warming with three treatment levels (ambient, shade control, warmed). Avicennia germinans, the black mangrove, exhibited no differences in growth or height due to experimental warming, but displayed a warming-induced increase in leaf production (48%). Surprisingly, Distichlis spicata, the dominant salt marsh grass, increased in biomass (53% in 2013 and 70% in 2014), density (41%) and height (18%) with warming during summer months. Warming decreased plant root mass at depth and changed abundances of anaerobic bacterial taxa. Even while the poleward shift of mangroves is clearly controlled by the occurrences of severe freezes, chronic warming between these freeze events may slow the progression of mangrove dominance within ecotones.

Mechanisms for regulating electron transfer in multi-centre redox proteins.

Protein-mediated electron transfer is a key process in nature. Many of the proteins involved in such electron transfers are complex and contain a number of redox-active cofactors. The very complexity of these multi-centre redox proteins has made it difficult to fully understand the various electron transfer events they catalyse. This is sometimes because the electron transfer steps themselves are gated or coupled to other processes such as proton transfer. However, with the molecular structures of many of these proteins now available it is possible to probe these electron transfer reactions at the molecular level. It is becoming apparent that many of these multi-centre redox proteins have rather subtle and elegant ways for regulating electron transfer. The purpose of this article is to illustrate how nature has used different approaches to control electron transfer in a number of different systems. Illustrative examples include: thermodynamic control of electron transfer in flavocytochromes b(2) and P450 BM3; a novel control mechanism involving calmodulin-binding-dependent electron transfer in neuronal nitric oxide synthase; the probable gating of electron transfer by ATP hydrolysis in nitrogenase; conformational gating of electron transfer in cytochrome cd(1); the regulation of electron transfer by protein dynamics in the cytochrome bc(1) complex; and finally the coupling of electron transfer to proton transfer in cytochrome c oxidase.

A search for the function of human carbonic anhydrase B.

Because of the very high activity and abundance of human red cell carbonic anhydrase C (carbamate hydrolase, EC 4.2.1.1), it seemed likely that the second isozyme, B, might not be essential for CO2 metabolism. It was then found that physiological concentrations of Cl- inhibited catalysis of CO2 hydration by the B enzyme (but not by type C), suggesting further that type B does not function in vivo as a carbonic anhydrase. The versatility of the catalytic activity of carbonic anhydrase for a number of 'artificial' substrates suggested that enzyme B may be utilized in reactions of intermediary metabolism. A number of hydration, dehydration, decarboxylation, kinase, and phosphatase systems were tested to determine a possible physiological function for the enzyme. Results with eighteen possible substrates were negative and the possibility is discussed that mammalian carbonic anhydrase B is an evolutionary accident.

Substitution of a haem-iron axial ligand in flavocytochrome b2.

The importance of haem-iron axial coordination in flavocytochrome b2 (L-lactate: cytochrome-c oxidoreductase) has been examined by replacing one of the ligating histidines, His-43, with methionine. The His-43-->Met mutation (H43M) results in a distinct colour change from red in the wild-type enzyme to green in the mutant enzyme. The electronic absorption spectrum indicates that only approx. 5% of the haem binding sites are occupied. There is no evidence of any absorption band at 695 nm (characteristic of methionine ligation) suggesting that methionine does not act as an axial ligand in the mutant enzyme. The H43M-mutant enzyme shows a band around 640-650 nm which is usually associated with high-spin ferric-haem proteins, either five coordinate or with a weak-field ligand in the sixth position. The EPR spectrum of the H43M-enzyme at 7 K shows a g-value near 6.0, indicating that the haem-iron is high-spin in contrast to its low-spin state in the wild-type enzyme. The His-43-->Met mutation has only a small effect on the lactate dehydrogenase activity of the enzyme as measured with ferricyanide as external electron acceptor, but greatly reduces its cytochrome-c reductase activity.

Catalysis in fumarate reductase.

In the absence of oxygen many bacteria are able to utilise fumarate as a terminal oxidant for respiration. In most known organisms the fumarate reductases are membrane-bound iron-sulfur flavoproteins but Shewanella species produce a soluble, periplasmic flavocytochrome c(3) that catalyses this reaction. The active sites of all fumarate reductases are clearly conserved at the structural level, indicating a common mechanism. The structures of fumarate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarate to succinate requires hydride transfer from FAD and protonation by an active site acid. The identity of the proton donor has been open to question but we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally by analysing the effects of site-directed mutations on enzyme activity. Substitutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis.

Protein engineering of cytochromes P-450.

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.

Randomized, double-blind comparison of two nelfinavir doses plus nucleosides in HIV-infected patients (Agouron study 511).

OBJECTIVE: To evaluate the safety and antiretroviral activity of nelfinavir mesylate at two doses as part of a combination regimen in HIV-infected, antiretroviral-naive patients. DESIGN: Phase III, multicenter, double-blind, placebo-controlled trial. PATIENTS AND METHODS: Two-hundred and ninety-seven patients were randomized to one of three treatment groups: nelfinavir 750 mg three times daily (tid), nelfinavir 500 mg tid, or matching placebo, each in combination with open-label zidovudine (ZDV) 200 mg tid and lamivudine (3TC) 150 mg twice daily (bid). Data were analyzed on an intent-to-treat basis. RESULTS: Sixty-seven percent of patients receiving nelfinavir 750 mg tid, and 50% receiving nelfinavir 500 mg tid in combination with ZDV/3TC achieved HIV RNA < 400 copies/ml compared to 7% receiving ZDV/3TC plus placebo (P < 0.001); 55% and 30% of patients in the nelfinavir-containing arms achieved HIV RNA < 50 copies/ml at week 24. This compared with 4% in the placebo-containing arm. For patients continuing nelfinavir treatment (750 mg or 500 mg tid as treated) for a further 6 months, the proportions achieving < 400 copies/ml at week 48 were 75% and 54% (P = 0.001) and < 50 copies/ml 61% and 37%, respectively (P = 0.004). The mean increases from baseline in CD4 cell counts were also durable in patients receiving the triple combination nelfinavir therapy. The range and incidence of adverse events was similar for the two nelfinavir-containing arms, with diarrhea being the most common adverse event. CONCLUSIONS: Nelfinavir plus ZDV/3TC was superior to ZDV/3TC/placebo. In addition, the 750 mg tid nelfinavir dose was better than the 500 mg tid dose. Virologic responses were sustained over 12 months.

On the lack of coordination between protein folding and flavin insertion in Escherichia coli for flavocytochrome b2 mutant forms Y254L and D282N.

Wild-type flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae, as well as a number of its point mutants, can be expressed to a reasonable level as recombinant proteins in Escherichia coli (20-25 mg per liter culture) with a full complement of prosthetic groups. At the same expression level, active-site mutants Y254L and D282N, on the other hand, were obtained with an FMN/heme ratio significantly less than unity, which could not be raised by addition of free FMN. Evidence is provided that the flavin deficit is due to incomplete prosthetic group incorporation during biosynthesis. Flavin-free and holo-forms for both mutants could be separated on a Blue-Trisacryl M column. The far-UV CD spectra of the two forms of each mutant protein were very similar to one another and to that of the wild-type enzyme, suggesting the existence of only local conformational differences between the active holo-enzymes and the nonreconstitutable flavin-free forms. Selective proteolysis with chymotrypsin attacked the same bond for the two mutant holo-enzymes as in the wild-type one, in the protease-sensitive loop. In contrast, for the flavin-free forms of both mutants, cleavage occurred at more than a single bond. Identification of the cleaved bonds suggested that the structural differences between the mutant flavin-free and holo-forms are located mostly at the C-terminal end of the barrel, which carries the prosthetic group and the active site. Altogether, these findings suggest that the two mutations induce an alteration of the protein-folding process during biosynthesis in E. coli; as a result, the synchrony between folding and flavin insertion is lost. Finally, a preliminary kinetic characterization of the mutant holo-forms showed the Km value for lactate to be little affected; kcat values fell by a factor of about 70 for the D282N mutant and of more than 500 for the Y254L mutant, compared to the wild-type enzyme.

Rational re-design of the substrate binding site of flavocytochrome P450 BM3.

Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C(12)-C(20)). Binding or activity with substrates of chain length 13-fold with butyrate, while the L75T/L181K double mutant has k(cat)/K(M) increased >15-fold with hexanoate and binding (K(d)) improved >28-fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.

NMR structure of the haem core of a novel tetrahaem cytochrome isolated from Shewanella frigidimarina: identification of the haem-specific axial ligands and order of oxidation.

The tetrahaem cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 is a small protein (86 residues) involved in electron transfer to Fe(III), which can be used as a terminal respiratory oxidant by this bacterium. A 3D solution structure model of the reduced form of the cytochrome has been determined using NMR data in order to determine the relative orientation of the haems. The haem core architecture of S. frigidimarina tetrahaem cytochrome differs from that found in all small tetrahaem cytochromes c(3) so far isolated from strict anaerobes, but has some similarity to the N-terminal cytochrome domain of flavocytochrome c(3) isolated from the same bacterium. NMR signals obtained for the four haems of S. frigidimarina tetrahaem cytochrome at all stages of oxidation were cross-assigned to the solution structure using the complete network of chemical exchange connectivities. Thus, the order in which each haem in the structure becomes oxidised was determined.

Efficacy of desciclovir in the treatment of Epstein-Barr virus infection in oral hairy leukoplakia.

The efficacy of desciclovir, an analog of acyclovir, in eliminating lesions of oral hairy leukoplakia (HL) and suppressing Epstein-Barr virus (EBV) infection was evaluated in a double-blind, placebo-controlled study of 14 patients. Patients were randomized to receive either the active drug, 250 mg three times a day for 14 days, or placebo. In all eight patients receiving desciclovir, lesions of HL were either completely resolved or significantly reduced during the treatment period, whereas lesions in patients receiving placebo showed no change. The histological features of HL were significantly diminished in patients on desciclovir, and cytochemical, in situ hybridization, and ultrastructural studies showed that EBV infection was eliminated or dramatically reduced in the desciclovir group only. Four patients on desciclovir reported side effects, but none required withdrawal from the study. The reappearance of HL in all eight subjects on desciclovir within 1-4 months after therapy was discontinued suggests the need for additional study.

Mutation to glutamine of histidine 373, the catalytic base of flavocytochrome b2 (L-lactate dehydrogenase).

Flavocytochrome b2 catalyzes the two-electron oxidation of L-lactate. Reducing equivalents are transferred first to FMN then to heme b2 in the same subunit, finally to cytochrome c or a non-physiological acceptor. The enzyme's three-dimensional structure, when analyzed in the light of existing mechanistic knowledge, suggested that His 373 is the active site base which initiates the substrate chemical transformation by abstracting the lactate alpha-proton. We report here the properties of a mutant enzyme with glutamine substituted histidine at position 373. The mutated enzyme preparations show a 10(4)-fold decrease in catalytic activity. We find that most of this residual activity can be eliminated by treatments with: 1) fluoropyruvate, an affinity label for His 373; and 2) 2- hydroxy-3-butynoate, a suicide reagent which normally forms an adduct with FMN but in this case leaves the bulk of the prosthetic group intact. Furthermore, although spectral titrations do not detect any binding of oxalate, this reagent inhibits the mutant enzyme with the same kinetic behaviour as for the wild-type enzyme. We conclude that the enzyme preparations contain about 1 in 10(4) molecules of wild-type flavocytochrome b2; this is probably due to codon misreading during biosynthesis. Thus the H373Q enzyme displays at most 10(5)-fold less activity than the wild-type enzyme. We report values for the spectrally determined binding constants of sulfite, pyruvate and D-lactate for the mutant enzyme. Finally, we show that 2,6-dichlorophenol indophenol, which is a 10-fold more sensitive routine electron acceptor than ferricyanide, accepts electrons only from heme b2 and not from the flavin.

Combined light and electron microscope in routine histopathology.

We report our experience with a prototype combined light and electron microscope (the LEM 2000) with particular reference to its application to routine surgical histopathology. We found its major advantages over conventional transmission electron microscopies were due to the large grid size (7 mm diameter), low magnification capacity (x 250), and the built-in microprocessor for recording areas of interest. These features combine to reduce sampling errors and greatly facilitate orientation and relocation of fields of diagnostic importance.

Antiproliferative effects of inhibitors of polyamine synthesis in tumors of neural origin.

The antiproliferative properties of inhibitors of polyamine synthesis were evaluated in cultured neuroblastoma and glioma cells. The diamines (1,3-propanediamine, 1,5-pentanediamine, and 1,6-hexanediamine) dramatically decreased neuroblastoma replication and inhibited the rate-limiting enzyme, ornithine decarboxylase. Glioma cells were less sensitive to the diamines in spite of significant drug-induced decreases in enzyme activity. The fact that ornithine decarboxylase was inhibited in both cell lines with different effects on proliferation suggests that the activity of other enzymes in polyamine biosynthesis may be altered selectively by these inhibitors.

Developmental screening of Afro-American infants using the Wolanski Gross Motor Evaluation.

The purpose of this study was to assess the appropriateness of using the Wolanski Gross Motor Evaluation (WGME) to screen for development delay in Afro-American infants of low socioeconomic status. Screening of 122 Afro-American infants at the Jefferson County (Alabama, USA) Department of Health well-child clinics was performed using the WGME. No differences were noted in the performance scores of males and females. The Afro-American infants of low socioeconomic status received higher performance scores at each given age than did white, middle-class U.S. infants tested in another study. The Afro-American infants also scored higher at each given age than the Polish infants on whom the WGME norms were established. The WGME does seem to be an appropriate screening tool in terms of practicality and the items are appropriate prior to onset of walking for the population studied. Normative data on different populations is needed in developing grids that can be used appropriately. Further studies are needed to establish reliability and validity.