Molecular Evidence for the Ancient Origin of the Ribosomal Protection Protein That Mediates Tetracycline Resistance in Bacteria

The ribosomal protection proteins (RPPs) mediate the resistance to tetracycline (TC) in Gram-positive and Gram-negative bacteria. The RPPs display sequence similarity to translation elongation factors, EF-G/EF-2 and EF-Tu/EF-1α. To determine the evolutionary origin of the RPPs, we constructed a composite phylogenetic tree of the RPPs, EF-G/EF-2 and EF-Tu/EF-1α. This tree includes two universal trees for the EF-G/EF-2 and EF-Tu/EF-1α, which form clusters corresponding to the respective two groups of proteins from three superkingdoms. The cluster of RPPs was placed at a point between the EF-G/EF-2 and EF-Tu/EF-1α clusters. The branch length (substitutions/site) between the node for the RPP cluster and the primary divergence of the RPPs was statistically shorter than that between the node for this cluster and the primary divergence in the EF-G/EF-2 cluster. This indicates that the RPPs derived through duplication and divergence of the ancient GTPase before the divergence of the three superkingdoms. Furthermore, this suggests the RPPs’ extant function occurred before the streptomycetes that include the TC-producing strains. Therefore, the RPPs evolved independent of the presence of TCs and serve a function other than antibiotic resistance. The RPPs may provide ribosomal protection against other chemical substances in the environment. Reviewing Editor: Dr. Margaret Riley Takeshi Kobayashi, Lisa Nonaka have contributed significantly to the research and preparation of this article.     

Human Salivary Micro-RNA in Patients with Parotid Salivary Gland Neoplasms

Background

Currently, clinical examination, ultrasound scanning (with or without fine needle aspiration cytology), preoperative CT-scan and MRI are available for the differential diagnosis of parotid gland swelling. A preliminary non-invasive salivary diagnostic tool may be helpful in the clinical decision making process. Altered salivary micro-RNA (miRNA) expression levels have been observed in saliva from patients with various cancers. Therefore, we investigated miRNA expression levels in saliva samples from patients with a parotid gland neoplasm using Human miRNA cards in comparison to controls.

Results

In the discovery phase, eight miRNAs were identified having different expression levels in patients compared to controls. In the validation phase, the differences in miRNA expression levels between patients and controls were confirmed for seven out of eight discovered miRNAs (p < 0.001). A combination of two miRNAs yielded a receiver-operator-characteristics curve with an AUC of 0.94 (95% CI: 0.87–1.00; sensitivity 91%; specificity 86%). Validation of discovered miRNAs in segregated collected parotid saliva revealed that expression of these miRNAs differ between whole saliva and parotid saliva.

Conclusions

A two miRNA combination can predict the presence of a parotid gland neoplasm. Furthermore, this study suggested that the identified, patient-specific, salivary miRNAs were not derived from the parotid gland itself.     

Protein Profiling of the Medicinal Leech Salivary Gland Secretion by Proteomic Analytical Methods

Protein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One-dimensional (1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to 483 kD. 2D-electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values. SELDI-mass spectrometry analysis using the ProteinChip. System based on chromatography surfaces of strong anion or weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of SELDI-MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active components of leech saliva secretion.     

Interaction of 8-Hydroxyquinoline with Soil Environment Mediates Its Ecological Function

Background

Allelopathic functions of plant-released chemicals are often studied through growth bioassays assuming that these chemicals will directly impact plant growth. This overlooks the role of soil factors in mediating allelopathic activities of chemicals, particularly non-volatiles. Here we examined the allelopathic potential of 8-hydroxyquinoline (HQ), a chemical reported to be exuded from the roots of Centaurea diffusa.

Methodology/Principal Findings

Growth bioassays and HQ recovery experiments were performed in HQ-treated soils (non-sterile, sterile, organic matter-enriched and glucose-amended) and untreated control soil. Root growth of either Brassica campestris or Phalaris minor was not affected in HQ-treated non-sterile soil. Soil modifications (organic matter and glucose amendments) could not enhance the recovery of HQ in soil, which further supports the observation that HQ is not likely to be an allelopathic compound. Hydroxyquinoline-treated soil had lower values for the CO₂ release compared to untreated non-sterile soil. Soil sterilization significantly influenced the organic matter content, PO₄-P and total organic nitrogen levels.

Conclusion/Significance

Here, we concluded that evaluation of the effect of a chemical on plant growth is not enough in evaluating the ecological role of a chemical in plant-plant interactions. Interaction of the chemical with soil factors largely determines the impact of HQ on plant growth.     

N-Sulfation of Heparan Sulfate Regulates Early Branching Events in the Developing Mammary Gland

Branching morphogenesis, a fundamental process in the development of epithelial organs (e.g. breast, kidney, lung, salivary gland, prostate, pancreas), is in part dependent on sulfation of heparan sulfate proteoglycans. Proper sulfation is mediated by biosynthetic enzymes, including exostosin-2 (Ext2), N-deacetylase/N-sulfotransferases and heparan sulfate O-sulfotransferases. Recent conditional knockouts indicate that whereas primary branching is dependent on heparan sulfate, other stages are dependent upon selective addition of N-sulfate and/or 2-O sulfation (Crawford, B .E., Garner, O. B., Bishop, J. R., Zhang, D. Y., Bush, K. T., Nigam, S. K., and Esko, J. D. (2010) PLoS One 5, e10691; Garner, O .B., Bush, K. T., Nigam, S .K., Yamaguchi, Y., Xu, D., Esko, J. D., and Nigam, S. K. (2011) Dev. Biol. 355, 394–403). Here, we analyzed the effect of deleting both Ndst2 and Ndst1. Whereas deletion of Ndst1 has no major effect on primary or secondary branching, deletion of Ndst2 appears to result in a mild increase in branching. When both genes were deleted, ductal growth was variably diminished (likely due to variable Cre-recombinase activity), but an overabundance of branched structures was evident irrespective of the extent of gland growth or postnatal age. “Hyperbranching” is an unusual phenotype. The effects on N-sulfation and growth factor binding were confirmed biochemically. The results indicate that N-sulfation or a factor requiring N-sulfation regulates primary and secondary branching events in the developing mammary gland. Together with previous work, the data indicate that different stages of ductal branching and lobuloalveolar formation are regulated by distinct sets of heparan sulfate biosynthetic enzymes in an appropriate growth factor context.     

Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Early Events in the Interaction of Melanotropin with Cloudman S91 Melanoma Cells

Melanotropin stimulates melanin synthesis in Cloudman S91 melanomacells, the accumulation of melanin being preceded by a numberof events which are understood to various degrees. In this reviewI will discuss the control of melanogenesis by melanotropin;emphasis will be on the early events of the process, althoughother aspects will be discussed. The probes (fluorescent andradioactive labels) used to examine the binding of the hormoneto the cells will be described, as will be the results of initialbinding studies. One of the events immediately following thebinding of the hormone is the activation of adenylate cyclase:the evidence supporting this, plus the role of cyclic AMP instimulating melanin synthesis, will be discussed. Also examinedis the role which internalization of the melanotropin-receptor-adenylatecyclase complex may play in mediating hormone action.     

AQP and the Control of Fluid Transport in a Salivary Gland

Experiments were performed with the perfused rat submandibular gland in vitro to investigate the nature of the coupling between transported salt and water by varying the osmolarity of the source bath and observing the changes in secretory volume flow. Glands were submitted to hypertonic step changes by changing the saline perfusate to one containing different levels of sucrose. The flow rate responded by falling to a lower value, establishing a new steady-state flow. The rate changes did not correspond to those expected from a system in which fluid production is due to simple osmotic equilibration, but were much larger. The changes were fitted to a model in which fluid production is largely paracellular, the rate of which is controlled by an osmosensor system in the basal membrane. The same experiments were done with glands from rats that had been bred to have very low levels of AQP5 (the principal aquaporin of the salivary acinar cell) in which little AQP5 is expressed at the basal membrane. In these rats, salivary secretion rates after hypertonic challenges were small and best modelled by simple osmotic equilibration. In rats which had intermediate AQP5 levels the changes in flow rate were similar to those of normal rats although their AQP5 levels were reduced. Finally, perfused normal glands were subject to retrograde ductal injection of salines containing different levels of Hg²⁺ ions (0, 10 and 100 μM) which would act as inhibitors of AQP5 at the apical acinar membrane. The overall flow rates were progressively diminished with rising Hg²⁺ concentration, but after hypertonic challenge the changes in flow rates were unchanged and similar to those of normal rats. All these results are difficult to explain by a cellular osmotic model but can be explained by a model in which paracellular flow is controlled by an osmosensor (presumably AQP5) present on the basal membrane.     

Early Events of Virus-Cell Interaction in an Adenovirus System

The interaction of (32)P-labeled adenovirus type 2 and HeLa or KB cells has been examined during early infection. The kinetics of virus uncoating to deoxyribonuclease-sensitive products, the partial characterization of three such products by gradient centrifugation, and the distribution of these products in the extranuclear and nuclear portions of infected cells are reported. The results are compatible with the following model. Extracellular virus attaches to a receptor on the plasma membrane. The membrane-bound virus has a half-life of less than 15 min and is transformed to a partly uncoated product which is free inside the cell and about half of which rapidly enters the cell nucleus. This is rapidly transformed, in both cytoplasm and nucleus, to a membrane-bound virion "core." The proteins of the bound "core" are then removed from the intact virus deoxyribonucleic acid (DNA). In the nucleus, viral DNA is the main product and there the overall sequence is completed in about 2 hr.     

实验室果蝇(Drosophil melanogaster)唾腺染色体制备前期技术优化

经过多年的实践积累,从果蝇(Drosophil melanogaster)品系的选择、培养基的配制和果蝇的饲养等方面摸索出了一套改进的制备果蝇唾腺染色体的经验方法。     

四川短尾唾液腺的组织学观察

利用生物显微技术观察和研究了四川短尾鼩(Anourosorex squamipes)唾液腺的组织结构。结果表明,腮腺属纯浆液腺,有闰管和分泌管,无颗粒曲管;颌下腺属混合腺,以混合性腺泡为主,有少量浆液性腺泡和黏液性腺泡,有闰管、颗粒曲管和分泌管;舌下腺属纯黏液腺,有闰管和分泌管,无颗粒曲管,但在分泌管上存在有颗粒曲管细胞。     

Evidence for a Lectin Specific for Sulfated Glycans in the Salivary Gland of the Malaria Vector,Anopheles gambiae

Salivary gland homogenate (SGH) from the female mosquitoes Anopheles gambiae, An. stephensi, An. freeborni, An. dirus and An. albimanus were found to exhibit hemagglutinating (lectin) activity. Lectin activity was not found for male An. gambiae, or female Ae aegypti, Culex quinquefasciatus, Phlebotomus duboscqi, and Lutzomyia longipalpis. With respect to species-specificity, An. gambiae SGH agglutinates red blood cells (RBC) from humans, horse, sheep, goat, pig, and cow; it is less active for rats RBC, and not detectable for guinea-pigs or chicken RBC. Notably, lectin activity was inhibited by low concentrations of dextran sulfate 50–500 K, fucoidan, heparin, laminin, heparin sulfate proteoglycan, sialyl-containing glycans (e.g. 3′-sialyl Lewis X, and 6′-sialyl lactose), and gangliosides (e.g. GM3, GD1, GD1b, GTB1, GM1, GQ1B), but not by simple sugars. These results imply that molecule(s) in the salivary gland target sulfated glycans. SGH from An. gambiae was also found to promote agglutination of HL-60 cells which are rich in sialyl Lewis X, a glycan that decorates PSGL-1, the neutrophils receptor that interacts with endothelial cell P-selectin. Accordingly, SGH interferes with HL-60 cells adhesion to immobilized P-selectin. Because An. gambiae SGH expresses galectins, one member of this family (herein named Agalectin) was expressed in E. coli. Recombinant Agalectin behaves as a non-covalent homodimer. It does not display lectin activity, and does not interact with 500 candidates tested in a Glycan microarray. Gel-filtration chromatography of the SGH of An. gambiae identified a fraction with hemagglutinating activity, which was analyzed by 1D PAGE followed by in-gel tryptic digestion, and nano-LC MS/MS. This approach identified several genes which emerge as candidates for a lectin targeting sulfated glycans, the first with this selectivity to be reported in the SGH of a blood-sucking arthropod. The role of salivary molecules (sialogenins) with lectin activity is discussed in the context of inflammation, and parasite-vector-host interactions.     

Evidence That Selenium Binding Protein 1 Is a Tumor Suppressor in Prostate Cancer

Selenium-Binding Protein 1 (SBP1, SELENBP1, hSP56) is a selenium-associated protein shown to be at lower levels in tumors, and its lower levels are frequently predictive of a poor clinical outcome. Distinguishing indolent from aggressive prostate cancer is a major challenge in disease management. Associations between SBP1 levels, tumor grade, and disease recurrence following prostatectomy were investigated by duplex immunofluorescence imaging using a tissue microarray containing tissue from 202 prostate cancer patients who experienced biochemical (PSA) recurrence after prostatectomy and 202 matched control patients whose cancer did not recur. Samples were matched by age, ethnicity, pathological stage and Gleason grade, and images were quantified using the Vectra multispectral imaging system. Fluorescent labels were targeted for SBP1 and cytokeratins 8/18 to restrict scoring to tumor cells, and cell-by-cell quantification of SBP1 in the nucleus and cytoplasm was performed. Nuclear SBP1 levels and the nuclear to cytoplasm ratio were inversely associated with tumor grade using linear regression analysis. Following classification of samples into quartiles based on the SBP1 levels among controls, tumors in the lowest quartile were more than twice as likely to recur compared to those in any other quartile. Inducible ectopic SBP1 expression reduced the ability of HCT-116 human tumor cells to grow in soft agar, a measure of transformation, without affecting proliferation. Cells expressing SBP1 also demonstrated a robust induction in the phosphorylation of the p53 tumor suppressor at serine 15. These data indicate that loss of SBP1 may play an independent contributing role in prostate cancer progression and its levels might be useful in distinguishing indolent from aggressive disease.     

Dissemination of a Highly Virulent Pathogen: Tracking The Early Events That Define Infection

The series of events that occurs immediately after pathogen entrance into the body is largely speculative. Key aspects of these events are pathogen dissemination and pathogen interactions with the immune response as the invader moves into deeper tissues. We sought to define major events that occur early during infection of a highly virulent pathogen. To this end, we tracked early dissemination of Yersinia pestis, a highly pathogenic bacterium that causes bubonic plague in mammals. Specifically, we addressed two fundamental questions: (1) do the bacteria encounter barriers in disseminating to draining lymph nodes (LN), and (2) what mechanism does this nonmotile bacterium use to reach the LN compartment, as the prevailing model predicts trafficking in association with host cells. Infection was followed through microscopy imaging in addition to assessing bacterial population dynamics during dissemination from the skin. We found and characterized an unexpected bottleneck that severely restricts bacterial dissemination to LNs. The bacteria that do not pass through this bottleneck are confined to the skin, where large numbers of neutrophils arrive and efficiently control bacterial proliferation. Notably, bottleneck formation is route dependent, as it is abrogated after subcutaneous inoculation. Using a combination of approaches, including microscopy imaging, we tested the prevailing model of bacterial dissemination from the skin into LNs and found no evidence of involvement of migrating phagocytes in dissemination. Thus, early stages of infection are defined by a bottleneck that restricts bacterial dissemination and by neutrophil-dependent control of bacterial proliferation in the skin. Furthermore, and as opposed to current models, our data indicate an intracellular stage is not required by Y. pestis to disseminate from the skin to draining LNs. Because our findings address events that occur during early encounters of pathogen with the immune response, this work can inform efforts to prevent or control infection.     

The Journey of Malaria Sporozoites in the Mosquito Salivary Gland

The life cycle of malaria parasites in the mosquito vector is completed when the sporozoites infect the salivary gland and are ready to be injected into the vertebrate host. This paper describes the fine structure of the invasive process of mosquito salivary glands by malaria parasites. Plasmodium gallinaceum sporozoites start the invasion process by attaching to and crossing the basal lamina and then penetrating the host plasma membrane of the salivary cells. The penetration process appears to involve the formation of membrane junctions. Once inside the host cells, the sporozoites are seen within vacuoles attached by their anterior end to the vacuolar membrane. Mitochondria surround, and are closely associated with, the invading sporozoites. After the disruption of the membrane vacuole, the parasites traverse the cytoplasm, attach to, and invade the secretory cavity through the apical plasma membrane of the cells. Inside the secretory cavity, sporozoites are seen again inside vacuoles. Upon escaping from these vacuoles, sporozoites are positioned in parallel arrays forming large bundles attached by multilammelar membrane junctions. Several sporozoites are seen around and inside the secretory duct. Except for the penetration of the chitinous salivary duct, our observations have morphologically characterized the entire process of sporozoite passage through the salivary gland.     

Malaria Parasite Invasion of the Mosquito Salivary Gland Requires Interaction between the Plasmodium TRAP and the Anopheles Saglin Proteins

SM1 is a twelve-amino-acid peptide that binds tightly to the Anopheles salivary gland and inhibits its invasion by Plasmodium sporozoites. By use of UV-crosslinking experiments between the peptide and its salivary gland target protein, we have identified the Anopheles salivary protein, saglin, as the receptor for SM1. Furthermore, by use of an anti-SM1 antibody, we have determined that the peptide is a mimotope of the Plasmodium sporozoite Thrombospondin Related Anonymous Protein (TRAP). TRAP binds to saglin with high specificity. Point mutations in TRAP''s binding domain A abrogate binding, and binding is competed for by the SM1 peptide. Importantly, in vivo down-regulation of saglin expression results in strong inhibition of salivary gland invasion. Together, the results suggest that saglin/TRAP interaction is crucial for salivary gland invasion by Plasmodium sporozoites.