Immunogold identification of the somatotrophs of domestic fowl of different ages

Summary The somatotrophs of the pituitary gland of the male domestic fowl were identified by means of an immuno-electron-microscopic method based on gold as the electron-opaque label and an antibody to growth hormone. Gold particles indicating sites of growth hormone were restricted to cells in which virtually all of the granules were labelled. Little, if any, gold label was found outside the granules in these cells designated as somatotrophs, or at sites outside these cells. The size of these gold-labelled secretory granules presumed to contain growth hormone decreased with age, from a mean sectional diameter of 256±6.2 nm (SEM) at 4–6 weeks to 221±5.7 nm at 11–18 weeks and 205±8.6 nm at 24–30 weeks of age. On the basis of these values for mean sectional diameters the change between the first two periods represents a decrease in granule volume of about 36%. However, during the same period the growth hormone concentration of the granules increased. Accordingly, growth hormone content per granule changed little if at all. In contrast, from 11–18 weeks to 24–30 weeks of age there was a decrease of 31% in growth hormone content per granule. These data indicate that growth hormone packaging in the chicken somatotroph changes with age. The first change results in the production of smaller granules of higher growth hormone concentration. During this period growth hormone content per granule remains relatively constant. The later change results in the production of granules of lower growth hormone content than that of younger animals.This is a paper of the Journal Sciences, New Jersey Agriculture Experimental Station supported in part by the State and Hatch Act Funds and a grant from the National Science Foundation (PMC-8022727)     

Methods for peptide identification by spectral comparison

Background  

Tandem mass spectrometry followed by database search is currently the predominant technology for peptide sequencing in shotgun proteomics experiments. Most methods compare experimentally observed spectra to the theoretical spectra predicted from the sequences in protein databases. There is a growing interest, however, in comparing unknown experimental spectra to a library of previously identified spectra. This approach has the advantage of taking into account instrument-dependent factors and peptide-specific differences in fragmentation probabilities. It is also computationally more efficient for high-throughput proteomics studies.     

Disruption of reducing pathways is not essential for efficient disulfide bond formation in the cytoplasm of <Emphasis Type="Italic">E. coli</Emphasis>

Background  

The formation of native disulfide bonds is a complex and essential post-translational modification for many proteins. The large scale production of these proteins can be difficult and depends on targeting the protein to a compartment in which disulfide bond formation naturally occurs, usually the endoplasmic reticulum of eukaryotes or the periplasm of prokaryotes. It is currently thought to be impossible to produce large amounts of disulfide bond containing protein in the cytoplasm of wild-type bacteria such as E. coli due to the presence of multiple pathways for their reduction.     

Modeling brewers' yeast flocculation

Flocculation of yeast cells occurs during the fermentation of beer. Partway through the fermentation the cells become flocculent and start to form flocs. If the environmental conditions, such as medium composition and fluid velocities in the tank, are optimal, the flocs will grow in size large enough to settle. After settling of the main part of the yeast the green beer is left, containing only a small amount of yeast necessary for rest conversions during the next process step, the lagering. The physical process of flocculation is a dynamic equilibrium of floc formation and floc breakup resulting in a bimodal size distribution containing single cells and flocs. The floc size distribution and the single cell amount were measured under the different conditions that occur during full scale fermentation. Influences on flocculation such as floc strength, specific power input, and total number of yeast cells in suspension were studied. A flocculation model was developed, and the measured data used for validation. Yeast floc formation can be described with the collision theory assuming a constant collision efficiency. The breakup of flocs appears to occur mainly via two mechanisms, the splitting of flocs and the erosion of yeast cells from the floc surface. The splitting rate determines the average floc size and the erosion rate determines the number of single cells. Regarding the size of the flocs with respect to the scale of turbulence, only the viscous subrange needs to be considered. With the model, the floc size distribution and the number of single cells can be predicted at a certain point during the fermentation. For this, the bond strength between the cells, the fractal dimension of the yeast, the specific power input in the tank and the number of yeast cells that are in suspension in the tank have to be known. Copyright 1998 John Wiley & Sons, Inc.     

Primary steps in the energy conversion reaction of the cytochrome bc1 complex Qo site

The primary energy conversion (Qo) site of the cytochrome bc1 complex is flanked by both high- and low-potential redox cofactors, the [2Fe-2S] cluster and cytochrome bL, respectively. From the sensitivity of the reduced [2Fe-2S] cluster electron paramagnetic resonance (EPR) spectral g(x)-band and line shape to the degree and type of Qo site occupants, we have proposed a double-occupancy model for the Qo site by ubiquinone in Rhodobacter capsulatus membrane vesicles containing the cytochrome bc1 complex. Biophysical and biochemical experiments have confirmed the double occupancy model and from a combination of these results and the available cytochrome bc1 crystal structures we suggest that the two ubiquinone molecules in the Qo site serve distinct catalytic roles. We propose that the strongly bound ubiquinone, termed Qos, is close to the [2Fe-2S] cluster, where it remains tightly associated with the Qo site during turnover, serving as a catalytic cofactor; and the weaker bound ubiquinone, Qow, is distal to the [2Fe-2S] cluster and can exchange with the membrane Qpool on a time scale much faster than the turnover, acting as the substrate. The crystallographic data demonstrates that the FeS subunit can adopt different positions. Our own observations show that the equilibrium position of the reduced FeS subunit is proximal to the Qo site. On the basis of this, we also report preliminary results modeling the electron transfer reactions that can occur in the cytochrome bc1 complex and show that because of the strong distance dependence of electron transfer, significant movement of the FeS subunit must occur in order for the complex to be able to turn over at the experimental observed rates.     

X-ray structure of a maquette scaffold

Maquettes are de novo designed mimicries of nature used to test the construction and engineering criteria of oxidoreductases. One type of scaffold used in maquette construction is a four-alpha-helical bundle. The sequence of the four-alpha-helix bundle maquettes follows a heptad repeat pattern typical of left-handed coiled-coils. Initial designs were molten globular due partly to the minimalist approach taken by the designers. Subsequent iterative redesign generated several structured scaffolds with similar heme binding properties. Variant [I(6)F(13)](2), a structured scaffold, was partially resolved with NMR spectroscopy and found to have a set of mobile inter-helical packing interfaces. Here, the X-ray structure of a similar peptide ([I(6)F(13)M(31)](2) i.e. ([CGGG EIWKL HEEFLKK FEELLKL HEERLKKM](2))(2) which we call L31M), has been solved using MAD phasing and refined to 2.8A resolution. The structure shows that the maquette scaffold is an anti-parallel four-helix bundle with "up-up-down-down" topology. No pre-formed heme-binding pocket exists in the protein scaffold. We report unexpected inter-helical crossing angles, residue positions and translations between the helices. The crossing angles between the parallel helices are -5 degrees rather than the expected +20 degrees for typical left-handed coiled-coils. Deviation of the scaffold from the design is likely due to the distribution and size of hydrophobic residues. The structure of L31M points out that four identical helices may interact differently in a bundle and heptad repeats with an alternating [HPPHHPP]/[HPPHHPH] (H: hydrophobic, P: polar) pattern are not a sufficient design criterion to generate left-hand coiled-coils.     

Nitric oxide regulation of human peripheral blood mononuclear cells: critical time dependence and selectivity for cytokine versus chemokine expression

NO is antiproliferative for T cells and other immune cells, but there is debate over whether it influences cytokine expression and if so whether it shows cytokine selectivity. Furthermore, the NO effect may depend on exposure time. To address these issues, we precultured human PBMC with the NO donors S-nitrosoglutathione (a natural storage form of NO) or S-nitroso-N-acetyl-D-penicillamine for up to 48 h before cell activation and then monitored proliferation and cytokine and chemokine expression. S-nitrosoglutathione or S-nitroso-N-acetyl-D-penicillamine, but not their non-NO-releasing analogues, inhibited proliferation induced by PHA or IL-2, the effect declining progressively from 48 to 0 h pre-exposure to the mitogen. This was accompanied by reduced PHA-induced IL-2 release and reduced IL-2, IFN-gamma, and IL-13 mRNA expression. In contrast, NO did not influence PHA-induced expression of mRNA for the chemokines lymphotactin, RANTES, IFN-gamma-inducible protein, macrophage-inhibitory protein-1alpha, macrophage-inhibitory protein-1beta, macrophage chemoattractant protein-1, and IL-8 or release of RANTES or IL-8. The NO effects were not toxic and were not accompanied by changes in PHA-induced CD25 expression. We conclude that exposure time to NO is critical to altered PBMC responsiveness and that NO inhibits expression of both Th1 and Th2 cytokines but not chemokines.     

The 3120 +1G-->A splicing mutation in CFTR is common in Brazilian cystic fibrosis patients.

Cystic fibrosis patients from Rio de Janeiro, Brazil, were screened for mutations in exons 11 and 16 of the cystic fibrosis transmembrane conductance regulator gene (CFTR) by a nonradioactive single-stranded conformational polymorphism (SSCP) analysis technique. This procedure was used to evaluate the undefined mutations in one or both alleles of 64 cystic fibrosis patients. Unusual SSCP profiles were investigated further by sequence analysis. Two patients were shown to carry the G542X mutation (exon 11) and five had the splicing mutation 3120+1G-->A(intron 16), one of them being homozygous for the mutation. This is the first report of the 3120+ IG-->A mutation in Brazil. where it appears to be a frequent disease-associated molecular alteration in the CFTR gene.     

Histidine placement in de novo-designed heme proteins.

The effects of histidine residue placement in a de novo-designed four-alpha-helix bundle are investigated by placement of histidine residues at coiled coil heptad a positions in two distinct heptads and at each position within a single heptad repeat of our prototype heme protein maquette, [H10H24]2 [[Ac-CGGGELWKL x HEELLKK x FEELLKL x HEERLKK x L-CONH2]2]2 composed of a generic (alpha-SS-alpha)2 peptide architecture. The heme to peptide stoichiometry of variants of [H10H24]2 with either or both histidines on each helix replaced with noncoordinating alanine residues ([H10A24]2, [A10H24]2, and [A10A24]2) demonstrates the obligate requirement of histidine for biologically significant heme affinity. Variants of [A10A24]2, [[Ac-CGGGELWKL x AEELLKK x FEELLKL x AEERLKK x L-CONH2]2]2, containing a single histidine per helix in positions 9 to 15 were evaluated to verify the design based on molecular modeling. The bis-histidine site formed between heptad positions a at 10 and 10' bound ferric hemes with the highest affinity, Kd1 and Kd2 values of 1.5 and 800 nM, respectively. Placement of histidine at position 11 (heptad position b) resulted in a protein that bound a single heme with moderate affinity, Kd1 of 9.5 microM, whereas the other peptides had no measurable apparent affinity for ferric heme with Kd1 values >200 microM. The bis-histidine ligation of heme to [H10A24]2 and [H11A24]2 was confirmed by electron paramagnetic resonance spectroscopy. The protein design rules derived from this study, together with the narrow tolerances revealed, are applicable for improving future heme protein designs, for analyzing the results of randomized heme protein combinatorial libraries, as well as for implementation in automated protein design.     

A novel immunocytochemistry technique to measure hsp70 induction in L929 cells exposed to cadmium chloride

The heat shock response has been suggested as a potential biomarker in toxicology. A vast amount of stimuli have been shown to induce heat shock proteins and new techniques to measure the response are constantly being assessed. In this study we use a novel immunocytochemistry technique to measure heat shock protein 70 (hsp70) induction in L929 cells exposed to cadmium chloride. Hsp70 induction was quantifiably measured using a soluble coloured substrate and qualitatively measured using a coloured substrate that precipitated at the location of hsp70. Using the insoluble coloured substrate hsp70 was identified predominantly within the cytoplasm of control cells. At intermediate cadmium concentrations hsp70 was observed to translocate to the nucleus. At these intermediate concentrations a heterogeneous heat shock response was observed. At lethal concentrations a strong heat shock response was observed with a widespread cellular response. This study demonstrates the potential of this immunocytochemistry technique to measure toxicological effects in cells by identifying the response quantitatively and qualitatively.