Glucose exchange parameters in a subset of physiological conditions.

The chemical exchange of labile protons of the hydroxyl groups can be exploited in a variety of magnetic resonance experiments to gain information about the groups and their physicochemical environment. The exchangeable -OH protons provide important contributions to the T2 of water signals thus contributing to the T2-weighted contrast of MRI images. This exchange can be exploited more specifically and sensitively in chemical exchange saturation transfer (CEST) or longitudinal rotating frame relaxation (T1,ρ) experiments. Since glucose is omnipresent in living organisms, it may be seen as a rather universal probe. Even though the potential was first recognized many years ago, practical use has remained scarce due to numerous challenges. The major limitation is the rather low glucose concentration in most tissues. The other obstacles are related to multiple dependencies of the exchange parameters, such as temperature, pH, and concentration of various ions that are not known in sufficient detail for glucose. Thus, we embarked on evaluating the exchange parameters of a model that included every relevant chemical site for all -OH protons in both dominant enantiomers of glucose. We have (1) obtained conventional one-dimensional proton NMR spectra of glucose solutions in suitable temperature ranges, (2) we have iterated through several exchange models with various degrees of freedom determined by the number of distinguishable -OH proton sites and compared their performance, (3) we extrapolated the parameters of the best model of physiological temperature and (4) we demonstrated the use of the parameters in virtual experiments. As the main results, (1) we have obtained the temperature dependence of exchange parameters with reliable confidence intervals in three different pH values, with two of them reaching physiological temperature, and (2) we show how the parameters can be used in virtual experiments, helping to develop new applications for glucose as an NMR/MRI probe.

Effect of collagen cross-linking on quantitative MRI parameters of articular cartilage.

OBJECTIVE: To investigate the sensitivity of quantitative magnetic resonance imaging (MRI) parameters to increase of collagen cross-linking in articular cartilage, a factor possibly contributing to the aging-related development of osteoarthritis (OA). The issue has not been widely studied although collagen cross-links may significantly affect the evaluation of cartilage imaging outcome. DESIGN: Osteochondral samples (n = 14) were prepared from seven bovine patellae. To induce cross-linking, seven samples were incubated in threose while the other seven served as non-treated controls. The specimens were scanned at 9.4 T for T1, T1Gd (dGEMRIC), T2, adiabatic and continuous wave (CW) T1ρ, adiabatic T2ρ and T1sat relaxation times. Specimens from adjacent tissue were identically treated and used for reference to determine biomechanical properties, collagen, proteoglycan and cross-link contents, fixed charge density (FCD), collagen fibril anisotropy and water concentration of cartilage. RESULTS: In the threose-treated sample group, cross-links (pentosidine, lysyl pyridinoline (LP)), FCD and equilibrium modulus were significantly (P < 0.05) higher as compared to the non-treated group. Threose treatment resulted in significantly greater T1Gd relaxation time constant (+26%, P < 0.05), although proteoglycan content was not altered. Adiabatic and CW-T1ρ were also significantly increased (+16%, +28%, P < 0.05) while pre-contrast T1 was significantly decreased (-10%, P < 0.05) in the threose group. T2, T2ρ and T1sat did not change significantly. CONCLUSION: Threose treatment induced collagen cross-linking and changes in the properties of articular cartilage, which were detected by T1, T1Gd and T1ρ relaxation time constants. Cross-linking should be considered especially when interpreting the outcome of contrast-enhanced MRI in aging populations.

Capturing fast relaxing spins with SWIFT adiabatic rotating frame spin-lattice relaxation (T1ρ) mapping.

Rotating frame spin-lattice relaxation, with the characteristic time constant T1ρ, provides a means to access motion-restricted (slow) spin dynamics in MRI. As a result of their restricted motion, these spins are sometimes characterized by a short transverse relaxation time constant T2 and thus can be difficult to detect directly with conventional image acquisition techniques. Here, we introduce an approach for three-dimensional adiabatic T1ρ mapping based on a magnetization-prepared sweep imaging with Fourier transformation (MP-SWIFT) sequence, which captures signal from almost all water spin populations, including the extremely fast relaxing pool. A semi-analytical procedure for T1ρ mapping is described. Experiments on phantoms and musculoskeletal tissue specimens (tendon, articular and epiphyseal cartilages) were performed at 9.4 T for both the MP-SWIFT and fast spin echo (FSE) read outs. In the phantom with liquids having fast molecular tumbling and a single-valued T1ρ time constant, the measured T1ρ values obtained with MP-SWIFT and FSE were similar. Conversely, in normal musculoskeletal tissues, T1ρ values measured with MP-SWIFT were much shorter than the values obtained with FSE. Studies of biological tissue specimens demonstrated that T1ρ-weighted SWIFT provides higher contrast between normal and diseased tissues relative to conventional acquisitions. Adiabatic T1ρ mapping with SWIFT readout captures contributions from the otherwise undetected fast relaxing spins, allowing more informative T1ρ measurements of normal and diseased states.

MagRET Nanoparticles: An Iron Oxide Nanocomposite Platform for Gene Silencing from MicroRNAs to Long Noncoding RNAs.

Silencing of RNA to knock down genes is currently one of the top priorities in gene therapies for cancer. However, to become practical the obstacle of RNA delivery needs to be solved. In this study, we used innovative maghemite (γ-Fe2O3) nanoparticles, termed magnetic reagent for efficient transfection (MagRET), which are composed of a maghemite core that is surface-doped by lanthanide Ce(3/4+) cations using sonochemistry. Thereafter, a polycationic polyethylenimine (PEI) polymer phase is bound to the maghemite core via coordinative chemistry enabled by the [CeL(n)](3/4+)cations/complex. PEI oxidation was used to mitigate the in vivo toxicity. Using this approach, silencing of 80-100% was observed for mRNAs, microRNAs, and lncRNA in a variety of cancer cells. MagRET NPs are advantageous in hard to transfect leukemias. This versatile nanoscale carrier can silence all known types of RNAs and these MagRET NPs with oxidized PEI are not lethal upon injection, thus holding promise for therapeutic applications, as a theranostic tool.

Downregulation of Fer induces PP1 activation and cell-cycle arrest in malignant cells.

Fer is a nuclear and cytoplasmic intracellular tyrosine kinase. Herein we show that Fer is required for cell-cycle progression in malignant cells. Decreasing the level of Fer using the RNA interference (RNAi) approach impeded the proliferation of prostate and breast carcinoma cells and led to their arrest at the G0/G1 phase. At the molecular level, knockdown of Fer resulted in the activation of the retinoblastoma protein (pRB), and this was reflected by profound hypo-phosphorylation of pRB on both cyclin-dependent kinase CDK4 and CDK2 phosphorylation sites. Dephosphorylation of pRB was not seen upon the direct targeting of either CDK4 or CDK2 expression, and was only partially achieved by the simultaneous depletion of these two kinases. Amino-acid sequence analysis revealed two protein phosphatase 1 (PP1) binding motifs in the kinase domain of Fer and the association of Fer with the pRB phosphatase PP1alpha was verified using co-immunoprecipitation analysis. Downregulation of Fer potentiated the activation of PP1alpha and overexpression of Fer decreased the enzymatic activity of that phosphatase. Our findings portray Fer as a regulator of cell-cycle progression in malignant cells and as a potential target for cancer intervention.

The U6 snRNA-encoding gene of the monogenetic trypanosomatid Leptomonas collosoma.

The U6 snRNA (U6) is the most conserved small nuclear RNA (snRNA) and apparently plays a central role in catalysis of the cis-splicing reaction. In trans-splicing, U6 may have an additional function. In the nematode trans-splicing system, a direct interaction between the U6 and spliced leader (SL) RNAs has been demonstrated, suggesting that U6 may serve as a bridge between the SL RNA and the acceptor pre-mRNA. To examine possible phylogenetic conservation of trypanosomatid U6 sequences that may interact with spliceosomal RNAs, we have cloned and sequenced the U6 gene from the monogenetic trypanosomatid Leptomonas collosoma (Lc). The Lc U6 deviates from the Trypanosoma brucei (Tb) RNA only in four positions located in the 5' stem-loop and the central domains. As in Tb, U6 is a single-copy gene and two tRNA genes, tRNAGln and tRNAIle, are found upstream to the gene. The tRNAs are differentially expressed; tRNAGln is transcribed in the opposite direction to U6, whereas tRNAIle is not transcribed. Possible base-pairing between U6 and the U2 and SL RNAs, similar to the interactions that take place in the nematode trans-splicing system, are proposed.

A Trypanosoma brucei small RNP particle containing the 5S rRNA.

In mammalian cells, approximately 50% of the 5S rRNA is found in ribosomes, and the remainder in a small particle, the 5S rRNA/ribosomal protein L5 complex, which is thought to be a precursor in ribosome assembly. Trypanosoma brucei, an African trypanosome, is one of the most primitive eukaryotic organisms which have been studied, and it likewise possesses a 5S rRNA species, a small proportion of which is found in an apparent ribonucleoprotein-(RNP) complex. Like the mammalian RNP particle, the T. brucei particle has a sedimentation coefficient of about 7S in sucrose gradients; unlike its mammalian counterpart, the complex is not disrupted by high salt and can be fractionated in cesium sulfate density gradients at a density characteristic of RNP complexes (1.45 g ml-1). Our studies demonstrate the the T. brucei 7S RNP contains 5S rRNA in association with a 36-kDa rRNA binding protein which not only shares molecular size, but also immunological determinants, with the yeast ribosomal protein YL3, and its mammalian homologue, L5. These results indicate that the RNP complex formed between the 5S rRNA and the 36-kDa ribosomal protein is conserved throughout great evolutionary distances between eukaryotic species.

Karyotype analysis of the monogenetic trypanosomatid Leptomonas collosoma.

In order to develop a genetic system for the monogenetic trypanosomatids, we have analyzed the molecular karyotype of Leptomonas collosoma based on chromosome separation by clamped homogeneous electric field (CHEF) gel electrophoresis. The chromosome location of 5 RNA coding genes (SL, U6, 5S, 7SL and rRNA) and 2 protein coding genes (for HSP83 and alpha-tubulin) was determined. All of the L. collosoma genes examined were found on at least 2 chromosomes, which differ in size in the range of 100-500 kb, suggesting that the organism is diploid. The weighted sum of L. collosoma chromosomes separated by CHEF analysis was approximately 62 +/- 3 Mb, whereas the genome size determined by FACS was estimated at approx. 80 Mb. This suggests that some of the homologous chromosomes differ in their size. The analysis presented here may facilitate studies on the function of individual genes, and on the genetic stability of this organism.

Identification of a tRNA-like molecule that copurifies with the 7SL RNA of Trypanosoma brucei.

During the purification of Trypanosoma brucei 7SL RNA, we detected a small RNA, 76 nucleotide-long (sRNA-76), that copurified with the 7SL RNP through several different separation steps. In this study, a partial RNA sequence of sRNA-76 was obtained and a complementary oligonucleotide to the RNA sequence was used to clone the corresponding gene. sRNA-76 is very similar to a tRNA molecule and is encoded by a single copy gene. The gene is located next to a tRNA(Val) which has 75.3% homology to T. brucei tRNA(Val) that exists in a different chromosomal locus. The highest homology of sRNA-76 is to mouse and rat tRNA(Asp) (69%), to mouse tRNA(Gly) (68.1%) and to yeast suppressor tRNA(Gly) (69.5%). However, sRNA-76 is neither a tRNA(Asp) nor a tRNA(Gly), since it has a Leu anticodon. In addition, sRNA-76 deviates from the canonical tRNA structure in 3 positions. A potential for base pairing between sRNA-76 and 7SL RNA was found in the 100 nt region of 7SL RNA, which is a highly conserved region in all 7SL RNAs.

The 7SL RNA homologue of Trypanosoma brucei is closely related to mammalian 7SL RNA.

In eukaryotes, protein translocation across the endoplasmic reticulum is mediated by a signal recognition particle, a small ribonucleoprotein (RNP) containing 7SL RNA. We have cloned and sequenced the gene coding for the Trypanosoma brucei 7SL RNA homologue and found that its sequence shows the highest degree of similarity to the human 7SL RNA sequence. In keeping with the prototype secondary structure of eukaryotic 7SL RNA, the trypanosome 7SL RNA secondary structure can be folded into four domains. The 7SL RNP, which sediments at approximately 11S on sucrose density gradients, was partially purified using column chromatography. A particle containing a 76-nucleotide-long RNA co-purified with the 7SL RNP; however, these particles did not co-fractionate by non-denaturing polyacrylamide gel electrophoresis.

Molecular conservation of MSP4 and MSP5 in Anaplasma marginale and A. centrale vaccine strain.

Anaplasma centrale msp4 and msp5 genes were cloned and sequenced, and the recombinant proteins were expressed. The identity between Anaplasma marginale and A. centrale MSP4 was 83% in the nucleotide sequences and 91.7% in the encoded protein sequences. A. centrale msp5 nucleotide sequences shared 86.8% identity with A. marginale msp5, and there was 92.9% homology between A. centrale and A. marginale encoded amino acids of the MSP5 protein. Southern blots hybridized with probes derived from the msp4 and msp5 central regions indicate that msp4 and msp5 of A. centrale are encoded by single copy genes. Recombinant MSP4 and MSP5 fusion proteins reacted with anti-A. marginale monoclonal antibodies ANAR76A1 and ANAF16C, respectively, demonstrating the conservation of conformation-sensitive B-cell epitopes between A. centrale and A. marginale. These data demonstrate the structural and antigenic conservation of MSP4 and MSP5 in A. centrale and A. marginale. This conservation is consistent with the cross-protective immunity between A. marginale and A. centrale and supports the development of improved vaccines based upon common outer membrane proteins.

The value of right atrial emptying rate in predicting reduction in pulmonary artery pressure in patients with chronic obstructive pulmonary disease.

We used an improved noninvasive radionuclide method, recently developed by us, to evaluate changes in pulmonary artery pressure induced by sublingual nifedipine in patients with chronic obstructive pulmonary disease and pulmonary hypertension. The new method enhances the predictive power of right ventricular ejection fraction by using the right atrial emptying rate as an index of reduced right ventricular compliance. The results were compared to those of invasively measured pulmonary arterial pressure. In the 21 patients studied 20 mg of nifedipine sublingually reduced pulmonary arterial pressure by 13.35% from 36.95 +/- 13.95/12.71 +/- 6.24 (mean 20.79 +/- 8.19) mmHg to 32.67 +/- 12.17/10.9 +/- 6.2 (mean 18.16 +/- 7.3) mmHg (p less than 0.05 for all pressures). Cardiac index increased and the pulmonary and systemic resistances decreased. The percent changes in right atrial emptying rate showed an excellent correlation with the percent change in pulmonary pressure. An increase of 12% or more in right atrial emptying rate predicted in all patients a reduction in pulmonary arterial pressure of at least 8%, the specificity and positive predictive accuracy being 100%. The sensitivity and the predictive accuracy of a negative test were 93% and 80%, respectively. The new method is useful for long-term evaluation of drug therapy in patients with pulmonary hypertension.

Novel trypanosomatid small nucleolar RNAs that guide methylation: their genome organization, expression and potential use to direct specific methylation on target RNA molecules.

Trypanosomatids are the causative agent of several major parasitic diseases including African trypanosomiasis, American trypanosomiasis, and leishmaniasis. These parasites possess unique RNA-processing mechanisms including trans-splicing of pre-mRNA and RNA editing of mitochondrial transcripts. In this study, we identified a trypanosomatid novel group of small nucleolar RNAs that belong to the box C/D snoRNA, which were shown to guide ribose methylation on rRNA. Three snoRNA genes were identified; snoRNA-2 carrying a single snoRNA and g2 and b2 coding for a single or multiple snoRNAs, respectively. Mapping of the methylation sites guided by snoRNA-2 using two different approaches suggest that snoRNA-2 has the potential to guide methylation on both 5.8S and 18S rRNAs. The trypanosomes follow the same guide-methylation rule established for yeast and for mammals. As a first attempt to change the methylation pattern of target RNAs, we generated transgenic parasites carrying the B2 and snoRNA-2, which were engineered to shift the methylation site on rRNA. Despite efficient expression of these tagged snoRNAs, the novel methylation site was not generated. However, efficient expression of tagged snoRNAs in transgenic parasites opens the possibility of engineering novel methylation sites on different target RNAs in vivo.

T1rho and T2rho MRI in the evaluation of Parkinson's disease.

Prior work has shown that adiabatic T(1rho) and T(2rho) relaxation time constants may have sensitivity to cellular changes and the presence of iron, respectively, in Parkinson's disease (PD). Further understanding of these magnetic resonance imaging (MRI) methods and how they relate to measures of disease severity and progression in PD is needed. Using T(1rho) and T(2rho) on a 4T MRI scanner, we assessed the substantia nigra (SN) of nine non-demented moderately affected PD and ten gender- and age-matched control participants. When compared to controls, the SN of PD subjects had increased T(1rho) and reduced T(2rho). We also found a significant correlation between asymmetric motor features and asymmetry based on T(1rho). This study provides additional validation of T(1rho) and T(2rho) as a means to separate PD from control subjects, and T(1rho) may be a useful marker of asymmetry in PD.

Water spin dynamics during apoptotic cell death in glioma gene therapy probed by T1rho and T2rho.

Longitudinal and transverse relaxations in the rotating frame, with characteristic time constants T1rho and T2rho, respectively, have potential to provide unique MRI contrast in vivo. On-resonance spin-lock T1rho with different spin-lock field strengths and adiabatic T2rho with different radiofrequency-modulation functions were measured in BT4C gliomas treated with Herpes Simplex Virus thymidine kinase (HVS-tk) gene therapy causing apoptotic cell death. These NMR tools were able to discriminate different treatment responses in tumor tissue from day 4 onward. An equilibrium two-site exchange model was used to calculate intrinsic parameters describing changes in water dynamics. Observed changes included increased correlation time of water associated with macromolecules and a decreased fractional population of this pool. These results are consistent with destructive intracellular processes associated with cell death and the increase of extracellular space during the treatment. Furthermore, association between longer exchange correlation time and decreased pH during apoptosis is discussed. In this study, we demonstrated that T1rho and T2rho MR imaging are useful tools to quantify early changes in water dynamics reflecting treatment response during gene therapy.

Functional analyses of positions across the 5' splice site of the trypanosomatid spliced leader RNA. Implications for base-pair interaction with U5 and U6 snRNAs.

In this study, we have used a genetic compensatory approach to examine the functional significance of the previously proposed interaction of spliced leader (SL) RNA with U5 small nuclear RNA (snRNA) (Dungan, J. D., Watkins, K. P., and Agabian, N. (1996) EMBO J. 15, 4016-4029; Xu, Y.-X., Ben Shlomo, H., and Michaeli, S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8473-8478) and the interaction of the SL RNA intron with U6 snRNA analogous to cis-splicing. Mutations were introduced at positions -4, -1, +1, +4, +5, and +7/+8 relative to the SL RNA 5' splice site that were proposed to interact with U5 and U6 snRNAs. All mutants exhibited altered splicing phenotypes compared with the parental strain, showing the importance of these intron and exon positions for trans-splicing. Surprisingly, mutation at invariant +1 position did not abolish splicing completely, unlike cis-splicing, but position +2 had the most severe effect on trans-splicing. Compensatory mutations were introduced in U5 and U6 snRNAs to examine whether the defects resulted from failure to interact with these snRNAs by base pairing. Suppression was observed only for positions +5 and +7/+8 with U5 compensatory mutations and for position +5 with a U6 compensatory mutation, supporting the existence of a base pair interaction of U5 and U6 with the SL RNA intron region. The failure to suppress the other SL RNA mutants by the U5 compensatory mutations suggests that another factor(s) interacts with these key SL RNA positions.

Leishmania mexicana amazonensis: effect of heat shock on the spliced leader RNA and its ribonucleoprotein particle SL RNP.

Trypanosomatid parasites of the genus Leishmania experience a temperature shift from 22-38 degrees C to 33-37 degrees C while being transmitted from the invertebrate vector to the mammalian host. Expression of many protein-coding genes in protozoan parasites that cycle between two hosts have been shown to be thermosensitive, and temperature changes most probably serve as a major regulatory factor during stage differentiation. We present here our studies on effects of physiological temperature shift on the steady-state and nascent synthesis of the spliced leader SL RNA, as well as on its small RNP particle, SL RNP. Northern blot analysis showed no significant changes in the steady-state level of SL RNA at elevated temperatures. Neither were any alterations detected at the two different temperatures in nascent transcription of the SL RNA gene, examined in cells made permeable by treatment with lysolecithin. Fractionation of cell extracts on Cs2SO4 gradients indicated that temperature elevation led to changes in SL RNP particles. Alterations in these particles upon heat shock were also observed by separation on polyacrylamide gels, but only in the presence of urea, indicating that the differences caused by elevation of temperature were revealed exclusively under stringent fractionation conditions.

The trypanosomatid Leptomonas collosoma 7SL RNA gene. Analysis of elements controlling its expression.

We have previously reported the co-purification of a tRNA-like molecule with the Trypanosoma brucei SRP complex [Béjà et al . (1993) Mol. Biochem. Parasitol . 57, 223-230]. To examine whether the trypanosome SRP has a unique composition compared with that of other eukaryotes, we analyzed the 7SL RNA and the SRP complex of the monogenetic trypanosomatid Leptomonas collosoma. The 7SL RNA from L. collosoma was cloned, and its gene was sequenced. In contrast to T. brucei , two 7SL RNA transcripts were detected in L.collosoma that originate from a single-copy gene. Using stable cell lines expressing tagged 7SL RNA, we demonstrate that the tRNAArggene located 98 bp upstream to the 7SL RNA serves as part of the 7SL RNA extragenic promoter. The steady-state level of 7SL RNA was found to be tightly regulated, since the presence of the gene on the multi-copy plasmid repressed the synthesis of the chromosomal gene. Cell lines carrying truncated 7SL RNA genes were established and their expression indicated that domain I is essential for expressing the 7SL RNA. No constructs carrying portions of the 7SL RNA were expressed, except for a construct which lacked 23 nt from the 3'end of the RNA. This suggests that 90% of the 7SL RNA molecule is important for its maintenance as a stable small RNA. We propose that the repression phenomenon may originate from a regulatory mechanism that coordinates the level of the 7SL RNA by its binding proteins.

The U5 RNA of trypanosomes deviates from the canonical U5 RNA: the Leptomonas collosoma U5 RNA and its coding gene.

Fractionation of the abundant small ribonucleoproteins (RNPs) of the trypanosomatid Leptomonas collosoma revealed the existence of a group of unidentified small RNPs that were shown to fractionate differently than the well-characterized trans-spliceosomal RNPs. One of these RNAs, an 80-nt RNA, did not possess a trimethylguanosine (TMG) cap structure but did possess a 5' phosphate terminus and an invariant consensus U5 snRNA loop 1. The gene coding for the RNA was cloned, and the coding region showed 55% sequence identity to the recently described U5 homologue of Trypanosoma brucei [Dungan, J. D., Watkins, K. P. & Agabian, N. (1996) EMBO J. 15, 4016-4029]. The L. collosoma U5 homologue exists in multiple forms of RNP complexes, a 10S monoparticle, and two subgroups of 18S particles that either contain or lack the U4 and U6 small nuclear RNAs, suggesting the existence of a U4/U6.U5 tri-small nuclear RNP complex. In contrast to T. brucei U5 RNA (62 nt), the L. collosoma homologue is longer (80 nt) and possesses a second stem-loop. Like the trypanosome U3, U6, and 7SL RNA genes, a tRNA gene coding for tRNACys was found 98 nt upstream to the U5 gene. A potential for base pair interaction between U5 and SL RNA in the 5' splice site region (positions -1 and +1) and downstream from it is proposed. The presence of a U5-like RNA in trypanosomes suggests that the most essential small nuclear RNPs are ubiquitous for both cis- and trans-splicing, yet even among the trypanosomatids the U5 RNA is highly divergent.