Review physical and chemical aspects of stabilization of compounds in silk

The challenge of stabilization of small molecules and proteins has received considerable interest. The biological activity of small molecules can be lost as a consequence of chemical modifications, while protein activity may be lost due to chemical or structural degradation, such as a change in macromolecular conformation or aggregation. In these cases, stabilization is required to preserve therapeutic and bioactivity efficacy and safety. In addition to use in therapeutic applications, strategies to stabilize small molecules and proteins also have applications in industrial processes, diagnostics, and consumer products like food and cosmetics. Traditionally, therapeutic drug formulation efforts have focused on maintaining stability during product preparation and storage. However, with growing interest in the fields of encapsulation, tissue engineering, and controlled release drug delivery systems, new stabilization challenges are being addressed; the compounds or protein of interest must be stabilized during: (1) fabrication of the protein or small molecule-loaded carrier, (2) device storage, and (3) for the duration of intended release needs in vitro or in vivo. We review common mechanisms of compound degradation for small molecules and proteins during biomaterial preparation (including tissue engineering scaffolds and drug delivery systems), storage, and in vivo implantation. We also review the physical and chemical aspects of polymer-based stabilization approaches, with a particular focus on the stabilizing properties of silk fibroin biomaterials.     

Restricted use of nitrate and a strong preference for ammonium reflects the nitrogen ecophysiology of a light‐limited red alga

Ammonium and nitrate are important sources of inorganic nitrogen for coastal primary producers. Nitrate has higher energy requirement for uptake and assimilation, compared with ammonium, suggesting that it might be a more efficient nitrogen source for slow‐growing, light‐limited macroalgae. To address this hypothesis, we examined the nitrogen ecophysiology of Anotrichium crinitum, a rhodophyte macroalgae common in low‐light habitats in New Zealand. We measured seasonal changes in seawater nitrate and ammonium concentrations and the concentration of nitrate and ammonium stored internally by A. crinitum. We determined the maximal uptake rates of nitrate and ammonium seasonally and grew A. crinitum in the laboratory with these nitrogen sources under two ecologically relevant saturating light levels. Our results show that field‐harvested A. crinitum has a high affinity for ammonium and although it will grow when supplied exclusively with nitrate, internal nitrate pools are low and it is unable to take up nitrate without several days of acclimation to saturating light. Our data predict that A. crinitum would be able to sustain growth with ammonium as the sole source of nitrogen, a strategy that would help it survive under low‐light conditions that prevail in the field.     

Exploration of peptides bound to MHC class I molecules in melanoma

Advancements in high‐resolution HPLC and mass spectrometry have reinvigorated the application of this technology to identify peptides eluted from immunopurified MHC class I molecules. Three melanoma cell lines were assessed using w6/32 isolation, peptide elution and HPLC purification; peptides were identified by mass spectrometry. A total of 13 829 peptides were identified; 83–87% of these were 8–11 mers. Only approximately 15% have been described before. Subcellular locations of the source proteins showed even sampling; mRNA expression and total protein length were predictive of the number of peptides detected from a single protein. HLA‐type binding prediction for 10 078 9/10 mer peptides assigned 88–95% to a patient‐specific HLA subtype, revealing a disparity in strength of predicted binding. HLA‐B*27‐specific isolation successfully identified some peptides not found using w6/32. Sixty peptides were selected for immune screening, based on source protein and predicted HLA binding; no new peptides recognized by antimelanoma T cells were discovered. Additionally, mass spectrometry was unable to identify several epitopes targeted ex vivo by one patient's T cells.     

CD5 is dissociated from the B-cell receptor in B cells from bovine leukemia virus-infected, persistently lymphocytotic cattle: consequences to B-cell receptor-mediated apoptosis

Bovine leukemia virus (BLV), a retrovirus related to human T-cell leukemia virus types 1 and 2, can induce persistent nonneoplastic expansion of the CD5(+) B-cell population, termed persistent lymphocytosis (PL). As in human CD5(+) B cells, we report here that CD5 was physically associated with the B-cell receptor (BCR) in normal bovine CD5(+) B cells. In contrast, in CD5(+) B cells from BLV-infected PL cattle, CD5 was dissociated from the BCR. In B cells from PL cattle, apoptosis decreased when cells were stimulated with antibody to surface immunoglobulin M (sIgM), while in B cells from uninfected cattle, apoptosis increased after sIgM stimulation. The functional significance of the CD5-BCR association was suggested by experimental dissociation of the CD5-BCR interaction by cross-linking of CD5. This caused CD5(+) B cells from uninfected animals to decrease apoptosis when stimulated with anti-sIgM. In contrast, in CD5(+) B cells from PL animals, in which CD5 was already dissociated from the BCR, there was no statistically significant change in apoptosis when CD5 was cross-linked and the cells were stimulated with anti-sIgM. Disruption of CD5-BCR interactions and subsequent decreased apoptosis and increased survival in antigenically stimulated B cells may be a mechanism of BLV-induced PL.     

Inhibition of heat shock protein 90 (hsp90) in proliferating endothelial cells uncouples endothelial nitric oxide synthase activity

Dual increases in nitric oxide ((*)NO) and superoxide anion (O(2)(*-)) production are one of the hallmarks of endothelial cell proliferation. Increased expression of endothelial nitric oxide synthase (eNOS) has been shown to play an important role in maintaining high levels of (*)NO generation to offset the increase in O(2)(*-) that occurs during proliferation. Although recent reports indicate that heat shock protein 90 (hsp90) associates with eNOS to increase (*)NO generation, the role of hsp90 association with eNOS during endothelial cell proliferation remains unknown. In this report, we examine the effects of endothelial cell proliferation on eNOS expression, hsp90 association with eNOS, and the mechanisms governing eNOS generation of (*)NO and O(2)(*-). Western analysis revealed that endothelial cells not only increased eNOS expression during proliferation but also hsp90 interactions with the enzyme. Pretreatment of cultures with radicicol (RAD, 20 microM), a specific inhibitor that does not redox cycle, decreased A23187-stimulated (*)NO production and increased L(omega)-nitroargininemethylester (L-NAME)-inhibitable O(2)(*-) generation. In contrast, A23187 stimulation of controls in the presence of L-NAME increased O(2)(*-) generation, confirming that during proliferation eNOS generates (*)NO. Our findings demonstrate that hsp90 plays an important role in maintaining (*)NO generation during proliferation. Inhibition of hsp90 in vascular endothelium provides a convenient mechanism for uncoupling eNOS activity to inhibit (*)NO production. This study provides new understanding of the mechanisms by which ansamycin antibiotics inhibit endothelial cell proliferation. Such information may be useful in the development and design of new antineoplastic agents in the future.     

Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine α s1-casein

Summary The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with _s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the aminp acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of _s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the _s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No cleat consensus sequence of amino acid residues surrounding the cleavage sites could be identified.Offprint requests to: G. G. Pritchard     

Interchangeable effects of gibberellic acid and temperature on embryo growth, seed germination and epicotyl emergence in Ribes multiflorum ssp. sandalioticum (Grossulariaceae)

Morphophysiological dormancy was investigated in seeds of Ribes multiflorum Kit ex Roem et Schult. ssp. sandalioticum Arrigoni, a rare mountain species endemic to Sardinia (Italy). There were no differences in imbibition rates between intact and scarified seeds, suggesting a lack of physical dormancy, while methylene blue solution (0.5%) highlighted a preferential pathway for solution entrance through the raphe. Embryos were small at seed dispersal, with an initial embryo:seed ratio (E:S) of ca. 0.2 (embryo length, ca. 0.5 mm), whereas the critical E:S ratio for germination was three times longer (ca. 0.6). Gibberellic acid (GA(3), 250 mg · l(-1)) and warm stratification (25 °C for 3 months) followed by low temperature (<15 °C) enhanced embryo growth rate (maximum of ca. 0.04 mm · day(-1) at 10 °C) and subsequent seed germination (radicle emergence; ca. 80% at 10 °C). Low germination occurred at warmer temperatures, and cold stratification (5 °C for 3 months) induced secondary dormancy. After radicle emergence, epicotyl emergence was delayed for ca. 2 months for seeds from three different populations. Mean time of epicotyl emergence was affected by GA(3) . Seeds of this species showed non-deep simple (root) - non-deep simple (epicotyl) morphophysiological dormancy, highlighting a high synchronisation with Mediterranean seasonality in all the investigated populations.     

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.     

Statistical tests for admixture mapping with case-control and cases-only data

Admixture mapping is a promising new tool for discovering genes that contribute to complex traits. This mapping approach uses samples from recently admixed populations to detect susceptibility loci at which the risk alleles have different frequencies in the original contributing populations. Although the idea for admixture mapping has been around for more than a decade, the genomic tools are only now becoming available to make this a feasible and attractive option for complex-trait mapping. In this article, we describe new statistical methods for analyzing multipoint data from admixture-mapping studies to detect "ancestry association." The new test statistics do not assume a particular disease model; instead, they are based simply on the extent to which the sample's ancestry proportions at a locus deviate from the genome average. Our power calculations show that, for loci at which the underlying risk-allele frequencies are substantially different in the ancestral populations, the power of admixture mapping can be comparable to that of association mapping but with a far smaller number of markers. We also show that, although "ancestry informative markers" (AIMs) are superior to random single-nucleotide polymorphisms (SNPs), random SNPs can perform quite well when AIMs are not available. Hence, researchers who study admixed populations in which AIMs are not available can perform admixture mapping with the use of modestly higher densities of random markers. Software to perform the gene-mapping calculations, "MALDsoft," is freely available on the Pritchard Lab Web site.     

Full Genome Characterization of the Culicoides-Borne Marsupial Orbiviruses: Wallal Virus,Mudjinbarry Virus and Warrego Viruses

Viruses belonging to the species Wallal virus and Warrego virus of the genus Orbivirus were identified as causative agents of blindness in marsupials in Australia during 1994/5. Recent comparisons of nucleotide (nt) and amino acid (aa) sequences have provided a basis for the grouping and classification of orbivirus isolates. However, full-genome sequence data are not available for representatives of all Orbivirus species. We report full-genome sequence data for three additional orbiviruses: Wallal virus (WALV); Mudjinabarry virus (MUDV) and Warrego virus (WARV). Comparisons of conserved polymerase (Pol), sub-core-shell ‘T2’ and core-surface ‘T13’ proteins show that these viruses group with other Culicoides borne orbiviruses, clustering with Eubenangee virus (EUBV), another orbivirus infecting marsupials. WARV shares <70% aa identity in all three conserved proteins (Pol, T2 and T13) with other orbiviruses, consistent with its classification within a distinct Orbivirus species. Although WALV and MUDV share <72.86%/67.93% aa/nt identity with other orbiviruses in Pol, T2 and T13, they share >99%/90% aa/nt identities with each other (consistent with membership of the same virus species - Wallal virus). However, WALV and MUDV share <68% aa identity in their larger outer capsid protein VP2(OC1), consistent with membership of different serotypes within the species - WALV-1 and WALV-2 respectively.