Failure of highly purified lysyl hydroxylase to hydroxylate lysyl residues in the non-helical regions of collagen.

The activity of highly purified lysyl hydroxylase towards lysyl residues within both the helical and the N-terminal non-helical telopeptide regions of chick type I collagen has been examined. The peptides alpha 1(I)-CB1 and alpha 2(I)-CB1, isolated from protocollagen following CNBr digestion and containing the N-terminal telopeptidyl lysyl residues, failed themselves to act as substrates. With protocollagen as substrate, analysis of products obtained following bacterial collagenase digestion of the reaction mixture showed that overall 37% hydroxylation of lysyl residues within the helical region of collagen had been obtained, which may be maximal. No hydroxylation, however, of the single lysyl residue in either alpha 1(I)-CB1 or alpha 2(I)-CB1, isolated following CNBr digestion of the reaction mixture, was observed, despite the known susceptibility of these residues to hydroxylation. These findings provide strong circumstantial evidence for the suggestion that a lysyl hydroxylase specific for the telopeptidyl residues and distinct from that active towards lysyl residues in the helical portion of the molecule may exist [Barnes, Constable, Morton & Royce (1974) Biochem. J. 139, 461-468].     

Structure-function relations in flavodoxins

Summary Flavodoxins are low molecular weight, FMN containing, proteins which function as electron transfer agents in a variety of microbial metabolic processes, including nitrogen fixation. Utilizing structural information obtained from x-ray crystal analysis, it has been possible to derive some new and important insights into the relationships which exist between flavin properties and protein environment by comparing the spectroscopic, thermodynamic and kinetic behavior of the flavodoxins with that of free flavin. Thus, for example, a qualitative understanding of the contribution of the protein to flavin redox potentials, semiquinone reactivity and mechanism of electron transfer is beginning to emerge. The highly negative redox potential required for the biochemical activity of the flavodoxins is accomplished by stabilizing the semiquinone via a hydrogen bond to the N-5 position of the flavin and destabilizing the fully-reduced form by constraining it to assume an unfavorable planar conformation. The reactivity of the semiquinone form is lowered by the aforementioned hydrogen bond, as well as by an interaction with a tryptophan residue in the binding site. Electron transfer is accomplished through the exposed dimethylbenzene ring of the bound coenzyme. Although it is not possible at present to determine the extent to which this understanding can be generalized to other flavoproteins, it is clear that a study of the flavodoxins will provide us with at least some of the principles which biological systems have used to modify flavin properties to fulfill a biochemical need.     

Coupling of M3 Acetylcholine Receptor to Gq16 Activates a Natriuretic Peptide Receptor Guanylyl Cyclase

Muscarinic activation of tracheal smooth muscle (TSM) involves a M₃AChR/heterotrimeric-G protein/NPR-GC coupling mechanism. G protein activators Mastoparan (MAS) and Mastoparan-7 stimulated 4- and 10-fold the NPR-GC respectively, being insensitive to PTX and antibodies against Gα_i/o subfamily. Muscarinic and MAS stimulation of NPR-GC was blocked by antibodies against C-terminal of Gα_q16, whose expression was confirmed by RT-PCR. However, synthetic peptides from C-terminal of Gα_q15/16 stimulated the NPR-GC. Coupling of α_q16 to M₃AChR is supported by MAS decreased [³H]QNB binding, being abolished after M₃AChR-4-DAMP-alkylation. Anti-i₃M₃AChR antibodies blocked the muscarinic activation of NPR-GC, and synthetic peptide from i₃M₃AChR (M₃P) was more potent than MAS increasing GTPγ [³⁵S] and decreasing the [³H]QNB activities. Coupling between NPR-GC and Gα_q16 was evaluated by using trypsin-solubilized-fraction from TSM membranes, which displayed a MAS-sensitive-NPR-GC activity, being immunoprecipitated with anti-Gα_q16, also showing an immunoreactive heterotrimeric-G-β -subunit. These data support the existence of a novel transducing cascade, involving Gα_q16β γ coupling M₃AChR to NPR-GC.     

A holistic high-throughput screening framework for biofuel feedstock assessment that characterises variations in soluble sugars and cell wall composition in <Emphasis Type="Italic">Sorghum bicolor</Emphasis>

Background

A major hindrance to the development of high yielding biofuel feedstocks is the ability to rapidly assess large populations for fermentable sugar yields. Whilst recent advances have outlined methods for the rapid assessment of biomass saccharification efficiency, none take into account the total biomass, or the soluble sugar fraction of the plant. Here we present a holistic high-throughput methodology for assessing sweet Sorghum bicolor feedstocks at 10 days post-anthesis for total fermentable sugar yields including stalk biomass, soluble sugar concentrations, and cell wall saccharification efficiency.

Results

A mathematical method for assessing whole S. bicolor stalks using the fourth internode from the base of the plant proved to be an effective high-throughput strategy for assessing stalk biomass, soluble sugar concentrations, and cell wall composition and allowed calculation of total stalk fermentable sugars. A high-throughput method for measuring soluble sucrose, glucose, and fructose using partial least squares (PLS) modelling of juice Fourier transform infrared (FTIR) spectra was developed. The PLS prediction was shown to be highly accurate with each sugar attaining a coefficient of determination (R ² ) of 0.99 with a root mean squared error of prediction (RMSEP) of 11.93, 5.52, and 3.23 mM for sucrose, glucose, and fructose, respectively, which constitutes an error of <4% in each case. The sugar PLS model correlated well with gas chromatography–mass spectrometry (GC-MS) and brix measures. Similarly, a high-throughput method for predicting enzymatic cell wall digestibility using PLS modelling of FTIR spectra obtained from S. bicolor bagasse was developed. The PLS prediction was shown to be accurate with an R ² of 0.94 and RMSEP of 0.64 μg.mgDW^-1.h^-1.

Conclusions

This methodology has been demonstrated as an efficient and effective way to screen large biofuel feedstock populations for biomass, soluble sugar concentrations, and cell wall digestibility simultaneously allowing a total fermentable yield calculation. It unifies and simplifies previous screening methodologies to produce a holistic assessment of biofuel feedstock potential.

     

Epidemiology of honey bee parasites

The epidemiology of honey bee parasites has been somewhat neglected, but Lynn Royce and Philippe Rossignol describe their unique characteristics. Indeed, it appears that a parasite of social insects has in essence to adapt to two hosts: first, the individual worker within a colony, the numbers of which grow linearly and second, to the colony itself, the actual reproductive 'organism'. Transmission also has vertical and horizontal components. Analysis of tracheal mite populations in particular suggests that intracolony parasite levels are regulated by the swarming behavior of their hosts. Ironically, current and highly productive methods of honey bee management with movable hives curb swarming and may contribute to increasing the spread and the impact of some parasites. This insight may result in changing management practices to reduce the detrimental effects of bee parasites.     

Mediterranean climate effects. II. Conifer growth phenology across a Sierra Nevada ecotone

Growth and xylem water potential of the lower elevation conifers Pinus jeffreyi and Abies concolor and the higher elevation Pinus monticola and Abies magnifica were monitored in their montane Mediterranean habitat of the southernmost Sierra Nevada mountains of California. Measurements were made across the ecotone between the midmontane and upper montane forests and through light and heavy snowfall years.Radial stem growth, averaging ～1.5 mm/yr, started 2 wk after snow melt, providing that maximum air temperatures had reached 21°C, and ended when predawn water potentials fell rapidly at the onset of the summer drought. Leader growth started on or after a fixed date, providing that minimum air temperatures were above -4°C for Pinus species or +2.5°C for Abies species. The cue for leader growth was inferred to be photoperiodic. Leader growth ended when either a determinate internode length of ～1 mm was reached or predawn water potentials fell rapidly. Abies magnifica grew more rapidly than the low-elevation species, but had a shorter growth period; its annual leader growth, as a consequence, was only 35 mm/yr vs. 50 mm/yr for the low-elevation species. Needle growth was similarly determinate in the absence of early drought. This growth phenology contributes to determining species distribution across the ecotone.     

In vivo efficacy of anti-glycoprotein 41, but not anti-glycoprotein 120, immunotoxins in a mouse model of HIV infection

Immunotoxins (ITs) targeting the HIV envelope protein are among the most efficacious antiviral therapies when tested in vitro. Yet a first-generation IT targeted to gp120, CD4-PE40 (chimeric immunotoxin using CD4 and the translocation and enzymatic domains of Pseudomonas exotoxin A), showed limited promise in initial clinical testing, highlighting the need for improved ITs. We have used a new mouse model of HIV infection to test the comparative efficacy of anti-HIV ITs targeted to gp120 or to gp41. Irradiated SCID/nonobese diabetic mice are injected with a tumor of human CD4(+) cells susceptible to infection and at a separate site persistently HIV-infected cells. The spread of infection from infected to susceptible tumor is monitored by plasma p24 and the presence of HIV-infected cells in the spleen. Anti-gp41 ITs in combination with tetrameric CD4-human Ig fusion protein have pronounced anti-HIV effects. Little if any anti-HIV efficacy was found with either CD4-PE40 or an Ab-targeted anti-gp120 IT. These data support continued exploration of the utility of ITs for HIV infection, particularly the use of anti-gp41 ITs in combination with soluble CD4 derivatives.