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To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.

Fin clipping is gaining momentum for stable isotope analysis in fish as a nonlethal alternative to lethally collecting dorsal white muscle tissue. The main advantage of fin clipping is the elimination of lethal collection, which thus increases the potential for stable isotope research involving threatened or endangered species. To make comparisons across studies using different methods, the degree to which fin clips and dorsal muscle are correlated for both δ15N and δ13C across time and space must be quantified. We evaluated the efficacy of using caudal fin clips by comparing tissues collected across time (2003-2005), space (21 streams), and body size (fork length, 40-140 mm) for juvenile Chinook salmon Oncorhynchus tshawytscha and O. mykiss (rainbow trout and steelhead [anadromous rainbow trout]). We also addressed several analytical and sampling issues to assess the potential benefits and limitations of using fin clips for δ15N and δ13C determination. We found that the relationship between fin ...

Stenotrophomonas maltophilia and Achromobacter ( Alcaligenes ) xylosoxidans have been increasingly recognized as a cause of respiratory tract colonization in cystic fibrosis (CF). Although both organisms have been associated with progressive deterioration of pulmonary function, demonstration of causality is lacking. To examine the molecular epidemiology of S. maltophilia and A. xylosoxidans in CF, isolates from patients monitored for up to 2 years were fingerprinted using a PCR-based randomly amplified polymorphic DNA (RAPD-PCR) method. Sixty-one of 69 CF centers screened had 183 S. maltophilia culture-positive patients, and 46 centers had 92 A. xylosoxidans -positive patients. At least one isolate from each patient was genotyped, and patients with ≥10 positive cultures (12 S . maltophilia cultures, 15 A. xylosoxidans cultures) had serial isolates genotyped. In addition, centers with multiple culture-positive patients were examined for evidence of shared clones. There were no instances of shared genotypes among different CF centers. Some patients demonstrated isolates with a single genotype throughout the observation period, and others had intervening or sequential genotypes. At the six centers with multiple S. maltophilia culture-positive patients and the seven centers with multiple A. xylosoxidans -positive patients, there were three and five instances of shared genotypes, respectively. The majority of shared isolates were from pairs who were siblings or otherwise epidemiologically linked. These findings suggest RAPD-PCR typing can distinguish unique CF isolates of S. maltophilia and A. xylosoxidans , person-to-person transmission may occur, there are not a small number of clones infecting CF airways, and patients with long-term colonization may either have a persistent organism or may acquire additional organisms over time.

Enhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid:plasmid DNA (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanolamine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphatidylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid:pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.

Recently, we identified the major in vivo phosphorylation site on v-Mos as Ser-56, which is phosphorylated by cyclic AMP dependent protein kinase (PKA). Others have shown that c-Mos phosphorylation at Ser-3 (equivalent to Ser-34 in v-Mos) is important for the interaction of c-Mos with its substrate MEK and for its stability and cytostatic factor activity in eggs. To investigate the role of Ser-56 phosphorylation, we generated site-directed mutants of v-Mos that would mimic phosphorylation in terms of charge at positions 56 and 34. After mutating serine (S) residues with alanine (A) or glutamic acid (E) in different combinations, various v-Mos mutants were expressed in a rabbit reticulocyte lysate in vitro translation system and in COS-1 or NIH/3T3 cells. The effect of mutations on Mos function was evaluated by in vitro protein kinase assays and by the ability of Mos to cause neoplastic transformation of NIH/3T3 cells. The S56E but not the S56A mutation inhibited v-Mos kinase activity suggesting that Ser-56 phosphorylation has an inhibitory role. As predicted from Xenopus c-Mos studies, S34A but not S34E mutation inhibited v-Mos activity. Studies with the double mutants showed that the S56E mutation but not S56A mutation inhibited v-Mos kinase activity of both S34A and S34E mutants. Interestingly, the S56A mutation blocked the inhibitory effect of the S34A mutation on v-Mos kinase suggesting that in c-Mos the corresponding serine (Ser-25) can influence the regulation of c-Mos by Ser-3. Results showing inhibition of v-Mos kinase activity of the S34E mutant by the S56E mutation is significant as it suggests that doubly phosphorylated Mos at these residues would be inactive. Because residues corresponding to both v-Mos Ser-34 and Ser-56 are evolutionarily conserved in c-Mos, the kinase activity of c-Mos during meiosis may also be regulated in the same manner as v-Mos kinase activity.

We investigated the normal physiological role of the c-mos gene by generating antisense mos transgenic mice. To allow ubiquitous expression of the transgene, it was placed under the control of phosphoglycerate kinase I promoter and polyadenylation signal. Antisense mos RNA expression had not effect on the normal development. However, consistent with the results obtained with the c-mos-/- mice in two different laboratories, antisense mos RNA expression caused a dramatic reduction in female fertility. Antisense mos RNA expression also result-ed in ovarian tumor due to parthenogenetic activation of eggs. Significantly, the tumor was observed at the age of 27 months in transgenic female in contrast to 4–8 months in c-mos-/- mice. Overall, our results support the conclusions drawn from the gene knock out experiments regarding the essential role of c-Mos in eggs.

In order to determine the physiological significance of c-mos RNA expression in somatic cells, we introduced antisense c-mos under the control of an inducible promoter. NIH/3T3 cells were stably transfected with antisense mos under the control of the mouse mammary tumor virus long terminal repeat (MMTV-LTR). Positive transfectants were selected under G418 conditions. Following selection, NIH/3T3 cells that received the antisense mos failed to form foci, whereas sense mos transfected cells grew normally. Moreover, v-mos-transformed cells were unaffected by antisense mos transfection. Of: interest, NIH/3T3 antisense mos transfectants that survived selection were growth-arrested. Nuclear abnormalities and the extrusion of microvesicles containing cellular material were observed in these cells. In order to rescue these cells from growth inhibition, the v-mos gene was introduced into cells by acute infection with Moloney murine sarcoma virus. Following infection, these cells resumed growth and became rapidly transformed. In other experiments, mouse C2 cells stably transfected with antisense mos showed a slower growth rate and gross morphological changes. C2 cells containing antisense mos under the control of mouse metallothionein-1 promoter had a large and flattened morphology and a relatively high percentage (30%) of binucleated cells. Our results indicate that basal level expression of antisense mos (under uninduced conditions) results in either arrested or retarded cell growth. The phenotypes exhibited in both cell lines leads us to suggest that the c-mos expression may play a role in mitotic progression in some somatic cells, in particular affecting cytokinesis.

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, 39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis.

The c-Mos serine/threonine protein kinase is an essential component of cytostatic factor (CSF), which is required for metaphase II arrest of eggs in vertebrates. Previously, we showed that c-Mos residue Ser-16 is phosphorylated in the ts110 Mo-MuSV-encoded Gag-Mos fusion protein. Here we provide evidence that Mos is phosphorylated at Ser-16 in transfected COS-1 cells. To investigate the role of this phosphorylation, Ser-16 was substituted with alanine or glutamic acid in full-length v-Mos (an Env-Mos fusion protein that contains 31 additional amino acids at the amino terminus of c-Mos), its mouse c-Mos equivalent version (v-Mos residues 32-374, hereafter referred to as Mos), and mouse c-Mos. Constructs expressing mutant versions of Mos were transfected into COS-1 and NIH3T3 cells in a transient and stable manner, respectively. Synthesis and proteolysis of Mos were evaluated by pulse-chase analysis of 35S-methionine-labeled proteins. Our findings indicate that the S16A mutant of Mos was highly unstable. It accumulated to approximately 10% of the level of wild-type Mos or its S16E mutant. In addition, the S16A mutation but not the S16E mutation inhibited Mos interaction with a cellular protein, p35, suggesting that phosphorylation at Ser-16 may promote Mos interaction with p35. As expected from its destabilizing effect, the S16A mutation caused a dramatic decrease in the cellular transforming activity of Mos (determined by soft-agar colony-formation assay with the stably transfected NIH3T3 cells), which is known to correlate with its CSF function. Efficient ubiquitin-mediated proteolysis of c-Mos requires proline as the second residue from the amino-terminus. In contrast to Mos, neither the stability nor protein kinase activity of v-Mos (in which c-Mos residue Pro-2 becomes Pro-33) was affected by the S16A mutation. To provide further proof that, similar to c-Mos, the S16A mutant is recognized by the proteolysis system through Pro-2, we show that the effect of the S16A mutation is reversed by the Pro-2-Ala mutation. Thus, our results indicate that Ser-16 has an important role in the regulation of c-Mos and that phosphorylation at Ser-16 may inhibit proteolysis of c-Mos.

c-Mos is a germ cell-specific MAP kinase kinase kinase (MAPKKK) that plays an essential role during meiotic divisions of oocytes. c-Mos is a key component of an activity, cytostatic factor, required for metaphase II arrest of unfertilized eggs in vertebrates. To understand the regulation of c-Mos, we are investigating c-Mos-interacting proteins. We provide evidence that mouse c-Mos binds to Hsp70, a molecular chaperone. Hsp70 was found to associate with Mos ectopically expressed in COS-1 cells. Mos-Hsp70 complexes could be immunoprecipitated with both Mos and Hsp70 antibodies. Despite a low-abundance of Mos, the Hsp70 antibody immunoprecipitated Mos as the major protein. Of importance, the Mos protein present in anti-Hsp70 immunoprecipitates functioned as an active MAPKKK indicating that it is not grossly misfolded. It is known that c-Mos protein kinase activity in cell extracts of transfected COS-1 or NIH3T3 cells is labile. We found that the inclusion of adenosine triphosphate (ATP) in cell extracts protected against the loss of Mos kinase activity. In the absence of ATP from cell extracts, protein kinase activity of Mos was lost within 6 h on ice even though the Mos protein was not degraded and remained bound to Hsp70. Based on our identification of c-Mos-Hsp70 interaction, one of the roles of ATP may be to assist the regulation of c-Mos via ATP involvement in the protein-folding function of Hsp70 and possibly other molecular chaperones. We also detected by coimmunoprecipitation a physical association between endogenous c-Mos and Hsp70 in Xenopus eggs. To provide further evidence for the functional significance of Hsp70 interaction to Mos function, we show that the residue serine 3 in Mos, which is important for the regulation of protein kinase activity of Mos is also important for Hsp70 association.

Mos is a germ cell-specific serine/threonine protein kinase that plays an important role during meiotic divisions of oocytes. Upon expression in somatic cells, Mos causes cell cycle perturbations leading to neoplastic transformation. Mos activates the MAP kinase pathway in both oocytes and transformed somatic cells. To determine the mechanism of cell cycle perturbation in mos-transformed cells, we examined the status of some key regulators of G1 phase. We provide evidence that Mos causes an elevation in the level of cyclin D1 in NIH/3T3 cells. As expected from the increased cyclin D1 level, mos transformation of NIH/3T3 cells caused an increase in the protein kinase activities of cyclin D1-Cdk4 and cyclin E-Cdk2 and induced hyperphosphorylation of the retinoblastoma protein. Of importance, the level of cyclin D1 was also elevated in eye lens of the c-mos-transgenic mice compared to normal mice. Our results indicate that the mechanism of cellular transformation by Mos involves an elevation in the level of cyclin D1 in somatic cells.