Elastin V: Syntheses, Characterization, and Staining Properties of Amidol Black I

One of the monoazo dyes reported by us in an earlier study (Lillie et at. 1972), amidol brown NAP, yielded an oxidation product, amidol black I, which was an excellent elastin stain. The present study revises the synthesis of amidol black I to a more convenient form giving much larger yields. Briefly, diazotized 4-nitro-2-aminophenol is azo-coupled into 2, 4-diaminophenol (amidol) and the product is oxidized to a quinoneimine with nitric. The product stains elastin black from an acid alcohol bath according to a Taenzer-Unna (1890) orcein type technic. The stain may be combined with a Van Gibson collagen stain or with an oil red O fat stain. The synthesis is presented in sufficient detail to permit its repetition in a hospital pathology laboratory.     

Major capsid reinforcement by a minor protein in herpesviruses and phage

Herpes simplex type 1 virus (HSV-1) and bacteriophage λ capsids undergo considerable structural changes during self-assembly and DNA packaging. The initial steps of viral capsid self-assembly require weak, non-covalent interactions between the capsid subunits to ensure free energy minimization and error-free assembly. In the final stages of DNA packaging, however, the internal genome pressure dramatically increases, requiring significant capsid strength to withstand high internal genome pressures of tens of atmospheres. Our data reveal that the loosely formed capsid structure is reinforced post-assembly by the minor capsid protein UL25 in HSV-1 and gpD in bacteriophage λ. Using atomic force microscopy nano-indentation analysis, we show that the capsid becomes stiffer upon binding of UL25 and gpD due to increased structural stability. At the same time the force required to break the capsid increases by ∼70% for both herpes and phage. This demonstrates a universal and evolutionarily conserved function of the minor capsid protein: facilitating the retention of the pressurized viral genome in the capsid. Since all eight human herpesviruses have UL25 orthologs, this discovery offers new opportunities to interfere with herpes replication by disrupting the precise force balance between the encapsidated DNA and the capsid proteins crucial for viral replication.     

Cloning, expression, and biochemical characterization of hexahistidine-tagged terminase proteins.

The terminase enzyme from bacteriophage lambda is composed of two viral proteins (gpA, 73.2 kDa; gpNu1, 20.4 kDa) and is responsible for packaging viral DNA into the confines of an empty procapsid. We are interested in the genetic, biochemical, and biophysical properties of DNA packaging in phage lambda and, in particular, the nucleoprotein complexes involved in these processes. These studies require the routine purification of large quantities of wild-type and mutant proteins in order to probe the molecular mechanism of DNA packaging. Toward this end, we have constructed a hexahistidine (hexa-His)-tagged terminase holoenzyme as well as hexa-His-tagged gpNu1 and gpA subunits. We present a simple, one-step purification scheme for the purification of large quantities of the holoenzyme and the individual subunits directly from the crude cell lysate. Importantly, we have developed a method to purify the highly insoluble gpNu1 subunit from inclusion bodies in a single step. Hexa-His terminase holoenzyme is functional in vivo and possesses steady-state and single-turnover ATPase activity that is indistinguishable from wild-type enzyme. The nuclease activity of the modified holoenzyme is near wild type, but the reaction exhibits a greater dependence on Escherichia coli integration host factor, a result that is mirrored in vivo. These results suggest that the hexa-His-tagged holoenzyme possesses a mild DNA-binding defect that is masked, at least in part, by integration host factor. The mild defect in hexa-His terminase holoenzyme is more significant in the isolated gpA-hexa-His subunit that does not appear to bind DNA. Moreover, whereas the hexa-His-tagged gpNu1 subunit may be reconstituted into a holoenzyme complex with wild-type catalytic activities, gpA-hexa-His is impaired in its interactions with the gpNu1 subunit of the enzyme. The results reported here underscore that a complete biochemical characterization of the effects of purification tags on enzyme function must be performed prior to their use in mechanistic studies.     

Phylogenetic inference based on landmark data in 41 empirical data sets

The inference of phylogenetic hypotheses from landmark data has been questioned during the last two decades. Besides theoretical concerns, one of the limitations pointed out for the use of landmark data in phylogenetics is its (supposed) lack of information relevant to the inference of phylogenetic relationships. However, empirical analyses are scarce; there exists no previous study that systematically evaluates the phylogenetic performance of landmark data in a series of data sets. In the present study, we analysed 41 published data sets in order to assess the correspondence between the phylogenetic trees derived from landmark data and those obtained with alternative and independent sources of evidence, and determined the main factors that might affect this inference. The data sets presented a variable number of terminals (5–200) and configurations (1–14), belonging to different taxonomic groups. The results showed that for most of the data sets analysed, the trees derived from landmark data presented a low correspondence with the reference phylogenies. The results were similar irrespective of the phylogenetic method considered. Complementary analyses strongly suggested that the limited amount of evidence included in each data set (one or a few landmark configurations) is the main cause for that low correspondence: the phylogenetic analysis of eight data sets that presented three or more configurations clearly showed that the inclusion of several landmark configurations improves the results. In addition, the analyses indicated that the inclusion of landmark data from different configurations is more important than the inclusion of more landmarks from the same configuration. Based on the results presented here, we consider that the poor results previously obtained in phylogenetic analyses based on landmark data were not caused by methodological limitations, but rather due to the limited amount of evidence included in the data sets.     

Utilization of yeast grown on n-paraffins in the feeding of heavy pigs,from weaning to slaughter

The effects of the substitution of n-paraffin-grown yeast for soya bean meal or herring meal in pig diets have been studied.Fifty-four pigs, subdivided into three groups of 18 each (9 male and 9 female) were used. Between 9 and 155 kg live-weight, one group was given a control diet without yeast, and the other two received diets containing 5–10% or 6.5–15% yeast, according to the different phases.The results for the groups fed with yeast were the same as those for the control group, for growth, food conversion, quality of carcass, and for health conditions and hygienic characteristics of the carcass.These results show that n-paraffin-grown yeast can be used successfully in pig feeding.     

Optimal selection on two quantitative trait loci with linkage

A mathematical approach to optimize selection on multiple quantitative trait loci (QTL) and an estimate of residual polygenic effects was applied to selection on two linked or unlinked additive QTL. Strategies to maximize total or cumulative discounted response over ten generations were compared to standard QTL selection on the sum of breeding values for the QTL and an estimated breeding value for polygenes, and to phenotypic selection. Optimal selection resulted in greater response to selection than standard QTL or phenotypic selection. Tight linkage between the QTL (recombination rate 0.05) resulted in a slightly lower response for standard QTL and phenotypic selection but in a greater response for optimal selection. Optimal selection capitalized on linkage by emphasizing selection on favorable haplotypes. When the objective was to maximize total response after ten generations and QTL were unlinked, optimal selection increased QTL frequencies to fixation in a near linear manner. When starting frequencies were equal for the two QTL, equal emphasis was given to each QTL, regardless of the difference in effects of the QTL and regardless of the linkage, but the emphasis given to each of the two QTL was not additive. These results demonstrate the ability of optimal selection to capitalize on information on the complex genetic basis of quantitative traits that is forthcoming.     

Spermatocyte chromosome analysis of Helicella virgata (Pulmonata: Helicidae): silver-stained and C-banded chromosomes.

Chromosome numbers of the snail Helicella virgata from the fields of Castellammare del Golfo (Sicily) are n = 26 and 2n = 52. Silver-staining analyses of testicular cells suggest that both mitotic and meiotic chromosomes are involved in nucleolus organization. A within-individual variability in NOR-banding pattern is present in each of the 20 specimens analyzed.     

Lycopene prevention of oxysterol-induced proinflammatory cytokine cascade in human macrophages: inhibition of NF-κB nuclear binding and increase in PPARγ expression

It is now well accepted that oxysterols play important roles in the formation of atherosclerotic plaque, involving cytotoxic, pro-oxidant and proinflammatory processes. It has been recently suggested that tomato lycopene may act as a preventive agent in atherosclerosis, although the exact mechanism of such a protection is not clarified. The main aim of this study was to investigate whether lycopene is able to counteract oxysterol-induced proinflammatory cytokines cascade in human macrophages, limiting the formation of atherosclerotic plaque. Therefore, THP-1 macrophages were exposed to two different oxysterols, such as 7-keto-cholesterol (4-16 μM) and 25-hydroxycholesterol (2-4 μM), alone and in combination with lycopene (0.5-2 μM). Both oxysterols enhanced pro-inflammatory cytokine [interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor α) secretion and mRNA levels in a dose-dependent manner, although at different extent. These effects were associated with an increased reactive oxygen species (ROS) production through an enhanced expression of NAD(P)H oxidase. Moreover, a net increment of phosphorylation of extracellular regulated kinase 1/2, p-38 and Jun N-terminal kinase and of nuclear factor kB (NF-κB) nuclear binding was observed. Lycopene prevented oxysterol-induced increase in pro-inflammatory cytokine secretion and expression. Such an effect was accompanied by an inhibition of oxysterol-induced ROS production, mitogen-activated protein kinase phosphorylation and NF-κB activation. The inhibition of oxysterol-induced cytokine stimulation was also mimicked by the specific NF-κB inhibitor pyrrolidine dithiocarbamate. Moreover, the carotenoid increased peroxisome proliferator-activated receptor γ levels in THP-1 macrophages. Taken all together, these data bring new information on the anti-atherogenic properties of lycopene, and on its mechanisms of action in atherosclerosis prevention.     

Antibody-mediated phagocytosis contributes to the anti-tumor activity of the therapeutic antibody daratumumab in lymphoma and multiple myeloma

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA''s mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt''s lymphoma cell lines. Phagocytosis contributed to DARA''s anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.