Nerve-dependent histone phosphorylation in the regenerating forelimb of the newt

Regenerating tissue of the newt forelimb extensively incorporates radioactive phosphate into the H2A histone subfraction, with little or no apparent labeling of the H1 protein. Denervation results in a decrease in the level of labeling. The amount of total inorganic phosphate in the stump is unchanged by denervation. However, the rate of uptake of labeled phosphate by the denervated tissue is decreased by about 50%. This leads to a twofold difference between the specific activities of the phosphate pools in the innervated and denervated tissues. Measurements of the specific activities of the ATP pools show a drop of about 40%, which is similar to the decrease in the rates of macromolecular syntheses. Since the major effect of denervation appears to be on phosphate uptake, a nerve-dependent change in the properties of the cell membranes is suggested.     

Assay of cyclic 3'-5'-monophosphate in the regenerating forelimb of the newt, Triturus.

The cyclic adenosine 3′-5′-monophosphate content of four regenerate stages in the forelimb of the newt, Triturus viridescens, was assayed using the Gilman method and compared to the content in the normal, unamputated, forelimb. The concentration was found to be highest in the earliest stages of regeneration, followed by a sharp drop and then a rise to a plateau approximately that of the unamputated limb. The possibility that cyclic AMP acts as a second messenger for nervous and hormonal influences on regeneration is discussed.     

Identification of potentially involved proteins in levofloxacin resistance mechanisms in Coxiella burnetii

The etiological agent of Q fever, Coxiella burnetii, is an obligate intracellular bacterium that multiplies within a phagosome-like parasitophorous vacuole. Fluoroquinolones have been used as an alternative therapy for Q fever. Resistance to fluoroquinolones can arise via several mechanisms utilized by pathogens to avoid killing. Until today, genome-based studies have shown that the main mechanism of C. burnetii to resist inhibition by fluoroquinolones is based on mutations in quinolone-resistance-determining region (QRDR). In this study, in a broader search at the protein level for C. burnetii mechanisms that confer resistance to fluoroquinolones, the proteomes of in vitro developed fluoroquinolone resistant bacteria and susceptible bacteria were compared using the MS-driven combined fractional diagonal chromatography (COFRADIC) proteomics technique. Quantitative comparison of the 381 proteins identified in both strains indicated the different expression of 15 bacterial proteins. These proteins are involved in different cellular processes indicating that the antibiotic resistance mechanism of the bacterium is a multifaceted process.     

Identification of up-regulated proteins potentially involved in the antagonism mechanism of Bacillus amyloliquefaciens G1

The use of Bacillus probiotics has been demonstrated as a promising method in the biocontrol of bacterial diseases in aquaculture. However, the molecular antibacterial mechanism of Bacillus still remains unclear. In order to explore the antibacterial mechanism of the potential antagonistic Bacillus amyloliquefaciens strain G1, comparative proteomics between B. amyloliquefaciens strain G1 and its non-antagonistic mutant strain was investigated. The 2-dimensional electrophoresis gel maps of their total extracted proteins were described and 42 different proteins were found to be highly expressed in strain G1 in comparison with those in the mutant strain. 35 of these up-regulated proteins were successfully identified using MALDI-TOF-TOF MS and databank analysis, and their biological functions were analyzed through the KEGG database. The increased expression of these proteins suggested that high levels of energy metabolism, biosynthesis and stress resistance could play important roles in strain G1’s antagonism. To our knowledge, this is the first report on the proteins involved in the antagonism mechanism of B. amyloliquefaciens using a proteomic approach and the proteomic data also contribute to a better understanding of the molecular basis for the antagonism of B. amyloliquefaciens.     

Histofluorescence of monoamines in newt forelimb regenerates

Summary Newt forelimb regenerates were studied at various stages of development using the histofluorescent method of Falck and Hillarp. A green formaldehyde-induced fluorescence was found in nerve fibres, large dendritic cells, skin gland cells and skin gland cell secretions. To ascertain the nature of the fluorescent material, animals were submitted to treatments with L-dopa, nialamide, benserazide and reserpine, used separately or in combination and administered before cutting off the regenerates. The modifications of the fluorescence after the various treatments confirmed the monoaminic nature of the fluorophores. Catecholaminic fibres were numerous in tissues of fast-growing stages while in dedifferentiated cell areas as well as in prochondral cell condensations and in cartilage they were completely absent. Fluorescent dendritic cells that have never been described before in regenerating limbs were observed and, from their localisation and cytological appearance, classed as promelanophores (or melanoblasts).     

Identification of proteins involved in inhibition of spheroid formation under microgravity

Many types of cells transit in vitro from a two‐ to a three‐dimensional growth, when they are exposed to microgravity. The underlying mechanisms are not yet understood. Hence, we investigated the impact of microgravity on protein content and growth behavior. For this purpose, the human thyroid cancer cells FTC‐133 were seeded either in recently developed cell containers that can endure enhanced physical forces and perform media changes and cell harvesting automatically or in T‐25 culture flasks. All cells were cultured for five days at 1g. Afterwards, a part of the cell containers were flown to the International Space Station, while another part was kept on the ground. T‐25 flasks were mounted on and next to a Random Positioning Machine. The cells were cultured for 12 days under the various conditions, before they were fixed with RNAlater. All fixed cultures showed monolayers, but three‐dimensional aggregates were not detected. In a subsequent protein analysis, 180 proteins were identified by mass spectrometry. These proteins did not indicate significant differences between cells exposed to microgravity and their 1g controls. However, they suggest that an enhanced production of proteins related to the extracellular matrix could detain the cells from spheroid formation, while profilin‐1 is phosphorylated.     

Potential morphogens involved in pattern formation during Dictyostelium differentiation.

Upon starvation, Dictyostelium amoebae aggregate together and then differentiate into either the stalk or spore cells that, respectively, form the stalk and sorus of the fruiting body. During differentiation, the prestalk and prespore cells become spatially segregated in a clearly defined developmental pattern. Several low molecular weight molecules that influence cell type determination during in vitro differentiation have been identified. The possible role of these molecules as morphogens, responsible for the formation of the developmental pattern, is discussed.     

Identification two novel nacrein-like proteins involved in the shell formation of the Pacific oyster Crassostrea gigas

Nacrein-like proteins have carbonic anhydrase (CA)-like domains, but their coding regions are flanked by inserted repeat sequence, such as Gly-X-Asn. Reportedly, nacrein-like proteins show the highest similarity to human carbonic anhydrase 1(α-CA1), possess CA catalytic functions, and play a key role in shell biomineralization. In the present study, two novel nacrein-like proteins were firstly identified from the shell-forming mantle of the Pacific oyster Crassostrea gigas. With numerous analyses, it was identified and characterized that both the nacrein-like proteins F1 and F2 were secreted and most closely related to the nacrein-like protein of California mussel Mytilus californianus via phylogenetic analysis. RT-PCR analysis showed that the nacrein-like proteins F1 and F2 were expressed in multiple tissues and the expression levels remarkably rose after entering the spat stage, which were basically consistent with the increase of calcite fractions in the total shell volume. Surprisingly, the Gly-X-Asn repeat domain, which is distinctive in most nacrein-like proteins, was absent in the two newly identified nacrein-like proteins in C. gigas and replaced with a series of acidic amino acids (D/E). Regardless, nacrein-like proteins in mollusks seem to be vital to the deposition of calcium carbonate and likely perform diverse functions.     

Identification of the vitellogenin proteins in the newt Pleurodeles waltl (urodele amphibian)

Vitellogenin derived from the blood of estrogen-treated Pleurodeles waltl was identified by immunochemical and electrophoretic analyses, using an antiserum against plasma vitellogenin isolated by dimethylformamide precipitation. Pleurodeles vitellogenin migrates as four bands on native PAGE, designated alpha-, beta-, gamma- and delta- VTG, with apparent mol. wts of 250,000, 270,000, 280,000 and 520,000 respectively. In the plasma, from estrogen-treated males like from ovariectomized estrogen-treated females, an additional band (mu-VTG) was found by native PAGE, never observed in estrogen-treated female plasma. It has a mol. wt of about 380,000 and shows complete immunological cross-reactivity with the vitellogenin antiserum. At least two polypeptides, termed VTG-I and VTG-II (mol. wt = 180,000 and 210,000) were identified by SDS-PAGE. Rocket immunoelectrophoresis displays three distinct precipitate lines indicating major immunological differences between the plasma vitellogenins.     

The antigenicity of regenerating tail tissue in the newt Diemictylus viridescens

Young adult male rabbits were inoculated with antigens prepared from regenerating (blastema stage) and nonregenerating tail tissues of the newtDiemictylus viridescens. Blood was collected from these rabbits after six weeks of semiweekly injection, two weeks of respite, and two more weeks of injections. A Freund adjuvant was added to the antigen preparations at the time of injection in order to elicit the anamnestic effect.Ouchterlony agar diffusions of the newt antigen preparations vs. the rabbit antisera were carried out. The resulting patterns of precipitation bands were compared and photographed.The strongest precipitation reactions of a given series were those between the antigen preparations made from nonregenerating tissue and their homologous antisera. The weakest reactions occurred between regenerating tissue antigens and regenerating tissue antisera. The strength of the antigen-antibody reactions was judged by the number of bands appearing in the diffusion plate and by the distinctness of these bands. Reactions of intermediate strength occurred between regenerating antigens and nonregenerating antisera, between nonregenerating antigens and regenerating antisera, and between antigens and antisera of different series.The loss of antigenicity during the blastemal period was considered to be related to the destruction of tissue in the wound areas at this time, and to indicate a quantitative rather than a qualitative loss of protein in regenerating tissue.This work is taken from data submitted to the Department of Biology of Fordham University in partial fulfillment of the requirements for the degree of Doctor of Philosophy, and was supported in part by a grant from the New York City Cancer Committee of the American Cancer Society.The author acknowledges his indebtedness to Dr.Alexander Wolsky of Fordham University, under whose direction this investigation was carried out, and Dr.John J. Corbett of Manhattan College and Lederle Laboratories.     

Selection of a chemically defined medium for culturing adult newt forelimb regenerates.

Three commercially available tissue-culture media were evaluated for their ability to support continued growth and differentiation of 14-day regenerates of adult newt forelimbs. Serums, embryo extracts, egg ultrafiltrates, and antimicrobial agents were avoided in this analysis. The hormones insulin and L-thyroxine were added to these chemically defined media to enhance continued cellular metabolism and growth. The optimum conditions appeared to be cultivated at 25 degrees C (pH 7.2 to 7.4) in media osmotically adjusted to conditions approximating amphibian blood values (i.e. 225 m0SM for 199, 244 m0SM for CMRL-1066, and 262 m0SM FOR L-15).     

Identification of key proteins involved in the anammox reaction

Bacteria performing anaerobic ammonium oxidation (anammox) are key players in the global nitrogen cycle due to their inherent ability to convert biologically available nitrogen to N₂. Anammox is increasingly being exploited during wastewater treatment worldwide, and about 50% of the total N₂ production in marine environments is estimated to proceed by the anammox pathway. To fully understand the microbial functionality and mechanisms that control environmental feedbacks of the anammox reaction, key proteins involved in the reaction must be identified. In this study we have utilized an analytical protocol that facilitates detection of proteins associated with the anammoxosome, an intracellular membrane compartment within the anammox bacterium. The protocol enabled us to identify several key proteins of the anammox reaction including a hydrazine hydrolase producing hydrazine, a hydrazine-oxidizing enzyme converting hydrazine to N₂ and a membrane-bound ATP synthase generating ATP from the gradients of protons formed in the anammox reaction. We also performed immunogold labelling electron microscopy to determine the subcellular location of the hydrazine hydrolase. The results from our study support the hypothesis that proteins associated with the anammoxosome host the complete suite of reactions during anammox.