Enamel thickness and microstructure in pitheciin primates, with comments on dietary adaptations of the middle Miocene hominoid Kenyapithecus

Many living primates that feed on hard food have been observed to have thick-enameled molars. Among platyrrhine primates, members of the tribe Pitheciini (Cacajao, Chiropotes, and Pithecia) are the most specialized seed and nut predators, and Cebus apella also includes exceptionally hard foods in its diet. To examine the hypothesized relationship between thick enamel and hard-object feeding, we sectioned small samples of molars from the platyrrhine primates Aotus trivergatus, Ateles paniscus, Callicebus moloch, Cebus apella, Cacajao calvus, Chiropotes satanas, Pithecia monachus, and Pithecia pithecia. We measured relative enamel thickness and examined enamel microstructure, paying special attention to the development of prism decussation and its optical manifestation, Hunter-Schreger Bands (HSB). Cebus apella has thick enamel with well-defined but sinuous HSB overlain by a substantial layer of radial prisms. Aotus and Callicebus have thin enamel consisting primarily of radial enamel with no HSB, Ateles has thin enamel with moderately developed HSB and an outer layer of radial prisms, and the thin enamel of the pitheciins (Cacajao, Chiropotes, and Pithecia) has extremely well-defined HSB. Among platyrrhines, two groups that feed on hard objects process these hard foods in different ways. Cebus apella masticates hard and brittle seeds with its thick-enameled cheek teeth. Pitheciin sclerocarpic foragers open hard husks with their canines but chew relatively soft and pliable seeds with their molars. These results reveal that thick enamel per se is not a prerequisite for hard object feeding. The Miocene hominoid Kenyapithecus may have included hard objects in its diet, but its thick-enameled molars indicate that its feeding adaptations differed from those of the pitheciins. The morphology of both the anterior and posterior dentition, including enamel thickness and microstructure, should be taken into consideration when inferring the dietary regime of fossil species.     

Nontyphoidal salmonellae in United Kingdom badgers: prevalence and spatial distribution

Eighteen (72%) of 25 badger social groups were found to excrete Salmonella enterica serovar Ried, S. enterica serovar Binza, S. enterica serovar Agama, or S. enterica serovar Lomita. Each serovar was susceptible to a panel of antimicrobials. Based on results of pulsed-field gel electrophoresis, the S. enterica serovar Agama and S. enterica serovar Binza isolates were very similar, but two clones each of S. enterica serovar Lomita and S. enterica serovar Ried were found. Badgers excreting S. enterica serovar Agama were spatially clustered.     

Yeast genome-wide expression analysis identifies a strong ergosterol and oxidative stress response during the initial stages of an industrial lager fermentation

Genome-wide expression analysis of an industrial strain of Saccharomyces cerevisiae during the initial stages of an industrial lager fermentation identified a strong response from genes involved in the biosynthesis of ergosterol and oxidative stress protection. The induction of the ERG genes was confirmed by Northern analysis and was found to be complemented by a rapid accumulation of ergosterol over the initial 6-h fermentation period. From a test of the metabolic activity of deletion mutants in the ergosterol biosynthesis pathway, it was found that ergosterol is an important factor in restoring the fermentative capacity of the cell after storage. Additionally, similar ERG10 and TRR1 gene expression patterns over the initial 24-h fermentation period highlighted a possible interaction between ergosterol biosynthesis and the oxidative stress response. Further analysis showed that erg mutants producing altered sterols were highly sensitive to oxidative stress-generating compounds. Here we show that genome-wide expression analysis can be used in the commercial environment and was successful in identifying environmental conditions that are important in industrial yeast fermentation.     

Shotgun proteomic analysis of a chromatophore-enriched preparation from the purple phototrophic bacterium Rhodopseudomonas palustris

A proteomics approach was evaluated for analysis of photosyntheis-related proteins that are characteristic of chromatophores, particles derived from purple phototrophic bacterial intracytoplasmic membranes. Proteins of purified chromatophores from Rhodopseudomonas palustris were solubilized and digested with trypsin, to create a collection of peptides that were fractionated by liquid chromatography. Peptide sequences were determined and assigned to specific proteins by analysis of tandem mass spectra of peptides, and comparison to a library derived from the recently determined R. palustris genome sequence. A total of 300 proteins were detected with a probability value >/=0.9, and the number of proteins detected increased to 345 when the minimum probability value was reduced to 0.5. Membrane-integral proteins of the reaction center, cytochrome b/c (1), light-harvesting and ATPase complexes were used as controls to assess how well this approach performs with hydrophobic proteins. New genes were identified, and tentatively designated as encoding photosynthesis-related proteins. We conclude that this approach is a powerful method to evaluate the possible existence of new photosynthesis-related proteins (and genes), although alternative methods are needed to evaluate the exact functions of newly discovered genes.     

Akt and PI 3-kinase signaling in cardiomyocyte hypertrophy and survival

In many systems, activation of the "protein and lipid kinase" phosphoinositide 3-kinase (PI 3-kinase) and its downstream serine-threonine kinase effector, Akt (or Protein Kinase B), provide a potent stimulus for cell proliferation, growth, and survival. In the heart, constrained by the limited proliferative capacity of cardiomyocytes, this pathway plays a key role in regulating cardiomyocyte growth and survival, with little effect on proliferation. Simultaneously, PI 3-kinase and Akt are important modulators of metabolic substrate utilization and cardiomyocyte function. Thus, the convergent signaling pathways controlling so many clinically important phenotypes of the cardiomyocyte suggest it holds promise as a therapeutic target in a variety of cardiac diseases. However, the similar role of PI 3-kinase/Akt signaling in neoplasia suggests the difficulty of activating this pathway in the heart without invoking adverse consequences elsewhere. Here we review evidence regarding the role of PI 3-kinase/Akt in controlling cardiomyocyte growth and survival, and discuss the implications for therapeutic strategies.     

Mechanism for multiple ligand recognition by the human transferrin receptor

Transferrin receptor 1 (TfR) plays a critical role in cellular iron import for most higher organisms. Cell surface TfR binds to circulating iron-loaded transferrin (Fe-Tf) and transports it to acidic endosomes, where low pH promotes iron to dissociate from transferrin (Tf) in a TfR-assisted process. The iron-free form of Tf (apo-Tf) remains bound to TfR and is recycled to the cell surface, where the complex dissociates upon exposure to the slightly basic pH of the blood. Fe-Tf competes for binding to TfR with HFE, the protein mutated in the iron-overload disease hereditary hemochromatosis. We used a quantitative surface plasmon resonance assay to determine the binding affinities of an extensive set of site-directed TfR mutants to HFE and Fe-Tf at pH 7.4 and to apo-Tf at pH 6.3. These results confirm the previous finding that Fe-Tf and HFE compete for the receptor by binding to an overlapping site on the TfR helical domain. Spatially distant mutations in the TfR protease-like domain affect binding of Fe-Tf, but not iron-loaded Tf C-lobe, apo-Tf, or HFE, and mutations at the edge of the TfR helical domain affect binding of apo-Tf, but not Fe-Tf or HFE. The binding data presented here reveal the binding footprints on TfR for Fe-Tf and apo-Tf. These data support a model in which the Tf C-lobe contacts the TfR helical domain and the Tf N-lobe contacts the base of the TfR protease-like domain. The differential effects of some TfR mutations on binding to Fe-Tf and apo-Tf suggest differences in the contact points between TfR and the two forms of Tf that could be caused by pH-dependent conformational changes in Tf, TfR, or both. From these data, we propose a structure-based model for the mechanism of TfR-assisted iron release from Fe-Tf.     

The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins

Plasma, the soluble component of the human blood, is believed to harbor thousands of distinct proteins, which originate from a variety of cells and tissues through either active secretion or leakage from blood cells or tissues. The dynamic range of plasma protein concentrations comprises at least nine orders of magnitude. Proteins involved in coagulation, immune defense, small molecule transport, and protease inhibition, many of them present in high abundance in this body fluid, have been functionally characterized and associated with disease processes. For example, protein sequence mutations in coagulation factors cause various serious disease states. Diagnosing and monitoring such diseases in blood plasma of affected individuals has typically been conducted by use of enzyme-linked immunosorbent assays, which using a specific antibody quantitatively measure only the affected protein in the tested plasma samples. The discovery of protein biomarkers in plasma for diseases with no known correlations to genetic mutations is challenging. It requires a highly parallel display and quantitation strategy for proteins. We fractionated blood serum proteins prior to display on two-dimensional electrophoresis (2-DE) gels using immunoaffinity chromatography to remove the most abundant serum proteins, followed by sequential anion-exchange and size-exclusion chromatography. Serum proteins from 74 fractions were displayed on 2-DE gels. This approach succeeded in resolving approximately 3700 distinct protein spots, many of them post-translationally modified variants of plasma proteins. About 1800 distinct serum protein spots were identified by mass spectrometry. They collapsed into 325 distinct proteins, after sequence homology and similarity searches were carried out to eliminate redundant protein annotations. Although a relatively insensitive dye, Coomassie Brilliant Blue G-250, was used to visualize protein spots, several proteins known to be present in serum in < 10 ng/mL concentrations were identified such as interleukin-6, cathepsins, and peptide hormones. Considering that our strategy allows highly parallel protein quantitation on 2-DE gels, it holds promise to accelerate the discovery of novel serum protein biomarkers.     

High-throughput proteomic analysis of human infiltrating ductal carcinoma of the breast

Large-scale proteomics will play a critical role in the rapid display, identification and validation of new protein targets, and elucidation of the underlying molecular events that are associated with disease development, progression and severity. However, because the proteome of most organisms are significantly more complex than the genome, the comprehensive analysis of protein expression changes will require an analytical effort beyond the capacity of standard laboratory equipment. We describe the first high-throughput proteomic analysis of human breast infiltrating ductal carcinoma (IDCA) using OCT (optimal cutting temperature) embedded biopsies, two-dimensional difference gel electrophoresis (2-D DIGE) technology and a fully automated spot handling workstation. Total proteins from four breast IDCAs (Stage I, IIA, IIB and IIIA) were individually compared to protein from non-neoplastic tissue obtained from a female donor with no personal or family history of breast cancer. We detected differences in protein abundance that ranged from 14.8% in stage I IDCA versus normal, to 30.6% in stage IIB IDCA versus normal. A total of 524 proteins that showed > or = three-fold difference in abundance between IDCA and normal tissue were picked, processed and identified by mass spectrometry. Out of the proteins picked, approximately 80% were unambiguously assigned identities by matrix-assisted laser desorbtion/ionization-time of flight mass spectrometry or liquid chromatography-tandem mass spectrometry in the first pass. Bioinformatics tools were also used to mine databases to determine if the identified proteins are involved in important pathways and/or interact with other proteins. Gelsolin, vinculin, lumican, alpha-1-antitrypsin, heat shock protein-60, cytokeratin-18, transferrin, enolase-1 and beta-actin, showed differential abundance between IDCA and normal tissue, but the trend was not consistent in all samples. Out of the proteins with database hits, only heat shock protein-70 (more abundant) and peroxiredoxin-2 (less abundant) displayed the same trend in all the IDCAs examined. This preliminary study demonstrates quantitative and qualitative differences in protein abundance between breast IDCAs and reveals 2-D DIGE portraits that may be a reflection of the histological and pathological status of breast IDCA.     

Novel approaches for regulating gas supply to plant systems <Emphasis Type="Italic">in vitro</Emphasis>: Application and benefits of artificial gas carriers

Summary The composition of the gaseous environment within tissue culture vessels is a critical factor in determining in vitro plant growth and morphogenic responsiveness. Consequently, the provision of an adequate and sustainable oxygen supply for cultured plant cells (including isolated protoplasts), tissues and organs is a crucial prerequisite for optimization and regulation of such cultural responses. During the past decade, research has focused on improving growth and development using artificial gas carriers based on inert perfluorocarbon (PFC) liquids and hemoglobin (Hb) solution. Supplementation of culture media with such artificial oxygen carriers has demonstrated beneficial effects of increased and sustainable cellular mitotic division and subsequent biomass production in a diverse range of plant species, during both short- and longer-term culture. Studies have targeted systems where oxygen availability is actually or potentially a major growth-limiting factor. Undoubtedly, gas carrier-facilitated improvements in regulating the supply of respiratory gases to cultured cells, tissues and organs will have increasingly important biotechnological and practical implications in the context of plant micropropagation, somatic hybridization, transgenic plant production, and secondary product biosynthesis.     

A new identity for MLK3 as an NIMA-related,cell cycle-regulated kinase that is localized near centrosomes and influences microtubule organization

Although conserved counterparts for most proteins involved in the G(2)/M transition of the cell cycle have been found in all eukaryotes, a notable exception is the essential but functionally enigmatic fungal kinase NIMA. While a number of vertebrate kinases have been identified with catalytic domain homology to NIMA, none of these resemble NIMA within its extensive noncatalytic region, a region critical for NIMA function in Aspergillus nidulans. We used a bioinformatics approach to search for proteins with homology to the noncatalytic region of NIMA and identified mixed lineage kinase 3 (MLK3). MLK3 has been proposed to serve as a component in MAP kinase cascades, particularly those resulting in the activation of the c-Jun N-terminal kinase (JNK). Here we describe the first in-depth study of endogenous MLK3 and report that, like NIMA, MLK3 phosphorylation and activity are enhanced during G(2)/M, whereas JNK remains inactive. Coincident with the G(2)/M transition, a period marked by dramatic reorganization of the cytoplasmic microtubule network, endogenous MLK3 transiently disperses away from the centrosome and centrosomal-proximal sites where it is localized during interphase. Furthermore, when overexpressed, MLK3, like NIMA, localizes to the centrosomal region, induces profound disruption of cytoplasmic microtubules and a nuclear distortion phenotype that differs from mitotic chromosome condensation. Cellular depletion of MLK3 protein using siRNA technology results in an increased sensitivity to the microtubule-stabilizing agent taxol. Our studies suggest a new role for MLK3, separable from its function in the JNK pathway, that may contribute to promoting microtubule instability, a hallmark of M phase entry.