Structural plasticity in RNA and its role in the regulation of protein translation in coliphage Q beta

We have analyzed both conformational and functional changes caused by two large cis-acting deletions (delta 159 and delta 549) located within the read-through domain, a 850 nucleotide hairpin, in coliphage Q beta genomic RNA. Studies in vivo show that co-translational regulation of the viral coat and replicase genes has been uncoupled in viral genomes carrying deletion delta 159. Translational regulation is restored in deletion delta 549, a naturally evolved pseudorevertant. Structural analysis by computer modeling shows that structural features within the read-through domain of delta 159 RNA are less well determined than they are in the read-through domain of wild-type RNA, whereas predicted structure in the read-through domain of evolved pseudorevertant delta 549 is unusually well determined. Structural analysis by electron microscopy of the genomic RNAs shows that several long range helices at the base of the read-through domain, that suppress translational initiation of the viral replicase gene in the wild-type genome, have been destabilized in delta 159 RNA. In addition, the structure of local hairpins within the read-through region is more variable in delta 159 RNA than in wild-type RNA. Stable RNA secondary structure is restored in the read-through domain of delta 549 RNA. Our analyses suggest that structure throughout the read-through domain affects the regulation of viral replicase expression by altering the likelihood that long-range interactions at the base of the domain will form. We discuss possible kinetic and equilibrium models that can explain this effect, and argue that observed changes in structural plasticity within the read-through domain of the mutant genomes are key in understanding the process. During the course of these studies, we became aware of the importance of the information contained in the energy dot plot produced by the RNA secondary structure prediction program mfold. As a result, we have improved the graphical representation of this information through the use of color annotation in the predicted optimal folding. The method is presented here for the first time.     

Cell signaling by the type IV pili of pathogenic Neisseria

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+]i) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+]i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.     

Surface enhanced Raman spectroscopy for characterization of structural characteristics of carbon chains in alpha1-acid glycoprotein and pseudoglycoproteins]

Surface-enhanced Raman scattering (SERS) spectroscopy was used to study the structure of carbohydrate chains in glycosylated forms of alpha 1-acid glycoprotein (AGP) and in pseudoglycoproteins obtained by transferring the carbohydrate chains of AGP to a polyacrylamide carrier. It was found that AGP-D glycoform and pseudoglycoproteins containing three or more glycans per molecule, which possess high immunomodulating activity, have a specific spatial organization of carbohydrate chains. This organization is maintained by the interaction of neighboring glycans with each other and does not depend on the nature of the carrier (whether it is polypeptide or polyacrylamide).     

Cytogenetics, in situ hybridization and molecular approaches in the diagnosis of cancer

BACKGROUND: Dysmotility in the gastrointestinal tract increases with age. Colonic endocrine cells play an important role in regulating intestinal secretion and motility. The objective was to study possible age-related changes in the colonic endocrine cells of an animal model. METHODS: The colonic endocrine cells in four different age groups of mice were investigated by immunocytochemistry and quantified by computerized image analysis. The ages of these groups were 1, 3, 12 and 24 months old. RESULTS: The numbers of peptide YY (PYY), enteroglucagon and serotonin immunoreactive (IR) cells in 1-month-old mice were significantly increased compared with those of 3-month-old mice. Similarly, the numbers of these cells were significantly greater in 12- and 24-month-old mice than in 3-month-old mice. The cell secretory index (CSI) of enteroglucagon and serotonin IR cells was higher in 1-, 12- and 24-month-old mice than in 3-month-old mice. There was no significant difference between the different age groups regarding the CSI of PYY IR cells, nor was there any statistical difference between females and males in all endocrine cell types regarding numbers and CSI. CONCLUSION: It is suggested that the increase in colonic endocrine cells prior to puberty might reflect the role of gut hormones in the development of the gastrointestinal tract. It is speculated further that the increase in colonic endocrine cells with ageing may compensate for increased receptor resistance and/or weakened response of effector organs. It is suggested that the increase in numbers and unchanged CSI of PYY cells with advancing age may be responsible for the slow colonic transit and constipation, both of which increase with age.     

Identification of rCop-1, a new member of the CCN protein family, as a negative regulator for cell transformation

By using a model system for cell transformation mediated by the cooperation of the activated H-ras oncogene and the inactivated p53 tumor suppressor gene, rCop-1 was identified by mRNA differential display as a gene whose expression became lost after cell transformation. Homology analysis indicates that rCop-1 belongs to an emerging cysteine-rich growth regulator family called CCN, which includes connective-tissue growth factor, CYR61, CEF10 (v-src inducible), and the product of the nov proto-oncogene. Unlike the other members of the CCN gene family, rCop-1 is not an immediate-early gene, it lacks the conserved C-terminal domain which was shown to confer both growth-stimulating and heparin-binding activities, and its expression is lost in cells transformed by a variety of mechanisms. Ectopic expression of rCop-1 by retroviral gene transfers led to cell death in a transformation-specific manner. These results suggest that rCop-1 represents a new class of CCN family proteins that have functions opposing those of the previously identified members.     

Interaction between a murine myeloma cell line and bone marrow stromal cells

Intravenous transplantation of an in vitro maintained murine myeloma cell line, 5T33, results in progressive growth in the bone marrow of C57Bl/KaLwRiJ mice. Concurrent with the growth of the tumor in vivo, the bone marrow stromal cells are inhibited, as assayed by their ability to form stromal cell foci and long-term monolayers in vitro. Inhibition of normal mouse marrow stromal cell growth also occurs when 5T33 cells are added to the marrow cells in vitro, and contact between the marrow and 5T33 cells is not necessary to achieve inhibition, indicating secretion of one or more diffusible inhibitory factors.     

Cloning and primary structure of the chiA gene from Aeromonas caviae

The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chitinase A of Serratia marcescens. Expression in Escherichia coli yielded an enzymatically active protein from which a leader sequence was removed, presumably during transport of the enzyme across the cell membrane.