A CD determination of the alpha-helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3

The CD spectra of four filamentous bacteriophages--fd, IKe, Pf1, and Pf3--were analyzed to determine the alpha-helix contents of their major coat proteins. Measured spectra included the 192-nm band so that analyses could be carried out over the full wavelength range of the reference spectra for protein secondary structures available (a) from globular proteins [J.T. Yang, C.S.C. Wu, and H.M. Martinez (1986) Methods in Enzymology 130, 208-269] and (b) from poly(L-lysine) [N. Greenfield and G.D. Fasman (1960) Biochemistry 8, 4108-4116]. Extended analyses were also performed with the addition of the spectrum of a model beta-turn to the Greenfield and Fasman reference set, with the spectrum of a short alpha-helix in the Yang et al. reference set, and with an estimate of the spectrum of Trp added to both reference sets. The reference set based on the simple poly(L-lysine) polypeptide, plus a spectrum of a model beta-turn or of Trp, gave reasonably good fits to the measured spectra for all four phages and yielded the largest percentages of alpha-helix. The class I phages--fd and IKe--had large percentages of alpha-helix of 98 +/- 2 and 97 +/- 5%, respectively, while the two class II phages--Pf1 and Pf3--had similar but smaller alpha-helix contents of 83 +/- 6 and 84 +/- 2, respectively. While these alpha-helix contents were within the ranges previously reported from CD spectra of these phages in solution, they were more precise, and they indicated that the coat proteins of the intact phages have CD spectra that are probably modeled better by the reference spectra of polypeptides than by those of globular proteins.     

An Ustilago maydis Mutant Partially Blocked in P45014DM Activity Is Hypersensitive to Azole Fungicides

An Ustilago maydis ergosterol biosynthesis mutant (A14) which is partially blocked in sterol 14alpha-demethylase (P45014DM) activity is described. This mutant accumulated the abnormal 14alpha-methyl sterols, eburicol, 14alpha-methylfecosterol, and obtusifoliol, along with significant amounts of ergosterol. Although the A14 mutant grew nearly as well as the wild type, it was impaired in cell extension growth, which indicated a dysfunction in apical cell wall synthesis. The mutant was also found to be hypersensitive to the azole fungicides penconazole and tebuconazole.     

Calcium release from rod outer segments: evidence for a cGMP-sensitive calcium binding protein.

Numerous studies investigating the cGMP-gated cation conductance in rod disk membranes have purported to measure efflux of Ca2+ entrapped in rod disk membrane vesicles. We have utilized sonication and osmotic shock as additional tests for sensitivity of cGMP- and A23187-induced Ca2+ release to elimination of the transvesicular Ca2+ gradient. We find that 1) Treatment with sonication or osmotic shock in low Ca2+ medium does not release Ca2+ from either native cGMP/Ca2(+)-loaded vesicles or solubilized, reconstituted "Ca2(+)-loaded" vesicles, 2) 70-100% of the cGMP-induced "flux" and 90-100% of the A23187-induced Ca2+ "flux" is insensitive to elimination of the Ca2+ gradient by sonication or osmotic shock in low Ca2+ medium, and 3) total amount of releasable Ca2+ is related to membrane surface area rather than vesicle entrapment volume. We conclude that 1) A23187 disrupts binding of Ca2+ to proteins and phospholipids as well as releasing entrapped Ca2+ and 2) a large fraction of the cGMP-induced release observed in rod disk vesicles is due to release of bound Ca2+.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

Economic evaluation of water loss saving due to the biological control of water hyacinth at New Year’s Dam,Eastern Cape province,South Africa

Water hyacinth Eichhornia crassipes is considered the most damaging aquatic weed in the world. However, few studies have quantified the impact of this weed economically and ecologically, and even fewer studies have quantified the benefits of its control. This paper focuses on water loss saving as the benefit derived from biological control of this plant between 1990 and 2013 at New Year’s Dam, Alicedale, Eastern Cape, South Africa. Estimates of water loss due to evapotranspiration from water hyacinth vary significantly; therefore, the study used three different rates, high, medium and low. A conservative raw agriculture value of R 0.26 per m³ was used to calculate the benefits derived by the water saved. The present benefit and cost values were determined using 10% and 5% discount rates. The benefit/cost ratio at the low evapotranspiration rate was less than one, implying that biological control was not economically viable but, at the higher evapotranspiration rates, the return justified the costs of biological control. However, at the marginal value product of water, the inclusion of the costs of damage to infrastructure, or the adverse effects of water hyacinth on biodiversity, would justify the use of biological control, even at the low transpiration rate.     

The Tau/A152T mutation,a risk factor for frontotemporal‐spectrum disorders,leads to NR2B receptor‐mediated excitotoxicity

We report on a novel transgenic mouse model expressing human full‐length Tau with the Tau mutation A152T (hTau^AT), a risk factor for FTD‐spectrum disorders including PSP and CBD. Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis‐sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short‐ or long‐term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage‐gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau^AT causes excitotoxicity mediated by NR2B‐containing NMDA receptors due to enhanced extracellular glutamate.