T2 distribution profiles are a good way to show cartilage regional variabilities and cartilage insufficiency.

OBJECTIVE: To use T2 relaxation time distribution profiles to assess inter-group regional differences along articular surfaces and to evaluate the feasibility of this analysis for comparison of cartilage insufficiency. MATERIALS AND METHODS: Twelve pairs matched according to age and gender (12 healthy volunteers and 12 patients after anterior cruciate ligament reconstruction (ACLR)) underwent 3-T MRI. T2 maps were calculated from six time echo images of the mid-sagittal slice in the lateral and medial compartment. The femoral and tibial cartilage was analyzed by measuring T2 distribution profiles along the articular surfaces. RESULTS: T2 distribution profiles were generated along the length of the articular surface in the femorotibial compartments. Differences in the T2 distribution profiles between the tibial and femoral cartilage as well as between the cartilage of the femoral condyles were identified in healthy individuals. T2 distribution profiles clearly demonstrated cartilage insufficiency in the weight-bearing areas for subjects in the ACLR group. CONCLUSIONS: T2 distribution profiles can identify regional differences in femoral and tibial cartilage. The T2 distribution profile pattern is preserved with cartilage insufficiency, however, with important differences in T2 values for the ACLR group in weight-bearing areas.

Hybrid FePt/SiO2/Au nanoparticles as a theranostic tool: in vitro photo-thermal treatment and MRI imaging.

We have produced an innovative, theranostic material based on FePt/SiO2/Au hybrid nanoparticles (NPs) for both, photo-thermal therapy and magnetic resonance imaging (MRI). Furthermore, a new synthesis approach, i.e., Au double seeding, for the preparation of Au nanoshells around the FePt/SiO2 cores, is proposed. The photo-thermal and the MRI response were first demonstrated on an aqueous suspension of hybrid FePt/SiO2/Au NPs. The cytotoxicity together with the internalization mechanism and the intracellular fate of the hybrid NPs were evaluated in vitro on a normal (NPU) and a half-differentiated cancerous cell line (RT4). The control samples as well as the normal cell line incubated with the NPs showed no significant temperature increase during the in vitro photo-thermal treatment (ΔT < 0.8 °C) and thus the cell viability remained high (∼90%). In contrast, due to the high NP uptake by the cancerous RT4 cell line, significant heating of the sample was observed (ΔT = 4 °C) and, consequently, after laser irradiation the cell viability dropped significantly to ∼60%. These results further confirm that the hybrid FePt/SiO2/Au NPs developed in the scope of this work were not only efficient but also highly selective photo-thermal agents. Furthermore, the improvement in the contrast and the easier distinction between the healthy and the cancerous tissues were clearly demonstrated with in vitro MRI experiments, proving that hybrid NPs have an excellent potential to be used as contrast agents.

Magnetic interactions and in vitro study of biocompatible hydrocaffeic acid-stabilized Fe-Pt clusters as MRI contrast agents.

A detailed magnetic study of separated Fe-Pt NPs and Fe-Pt clusters was performed to predict their optimal size and morphology for the maximum saturation magnetization, a factor that is known to influence the performance of a magnetic-resonance-imaging (MRI) contrast agent. Excellent stability and biocompatibility of the nanoparticle suspension was achieved using a novel coating based on hydrocaffeic acid (HCA), which was confirmed with a detailed Fourier-transform infrared spectroscopy (FTIR) study. An in vitro study on a human-bladder papillary urothelial neoplasm RT4 cell line confirmed that HCA-Fe-Pt nanoparticles showed no cytotoxicity, even at a very high concentration (550 µg Fe-Pt per mL), with no delayed cytotoxic effect being detected. This indicates that the HCA coating provides excellent biocompatibility of the nanoparticles, which is a prerequisite for the material to be used as a safe contrast agent for MRI. The cellular uptake and internalization mechanism were studied using ICP-MS and TEM analyses. Furthermore, it was shown that even a very low concentration of Fe-Pt nanoparticles (<10 µg mL-1) in the cells is enough to decrease the T 2 relaxation times by 70%. In terms of the MRI imaging, this means a large improvement in the contrast, even at a low nanoparticle concentration and an easier visualization of the tissues containing nanoparticles, proving that HCA-coated Fe-Pt nanoparticles have the potential to be used as an efficient and safe MRI contrast agent.

Modelling the effect of laminar axially directed blood flow on the dissolution of non-occlusive blood clots.

Axially directed blood plasma flow can significantly accelerate thrombolysis of non-occlusive blood clots. Viscous forces caused by shearing of blood play an essential role in this process, in addition to biochemical fibrinolytic reactions. An analytical mathematical model based on the hypothesis that clot dissolution dynamics is proportional to the power of the flowing blood plasma dissipated along the clot is presented. The model assumes cylindrical non-occlusive blood clots with the flow channel in the centre, in which the flow is assumed to be laminar and flow rate constant at all times during dissolution. Effects of sudden constriction on the flow and its impact on the dissolution rate are also considered. The model was verified experimentally by dynamic magnetic resonance (MR) microscopy of artificial blood clots dissolving in an in vitro circulation system, containing plasma with a magnetic resonance imaging contrast agent and recombinant tissue-type plasminogen activator (rt-PA). Sequences of dynamically acquired 3D low resolution MR images of entire clots and 2D high resolution MR images of clots in the axial cross-section were used to evaluate the dissolution model by fitting it to the experimental data. The experimental data fitted well to the model and confirmed our hypothesis.

[Aortic valve insufficiency due to rupture of the cusp in a patient with multiple trauma]. / Insuffizienz der Aortenklappe aufgrund einer Ruptur des Klappensegels bei einem polytraumatisierten Patienten.

Lesions of the heart valve caused by blunt chest trauma is rare, but when it does occur it can significantly injure the patient. On the basis of autopsy studies, research shows that heart valves are injured in less than 5% of patients who have died due to impact thoracic trauma. Among the heart valves, the aortic valve is the most often lacerated, which has been proved by relevant autopsy and clinical studies. Aortic valve lesions can be the only injury, but it is possible that additional heart or large vessel injuries are also present (myocardial contusion, rupture of the atrial septum, aortic rupture, rupture of the left common carotid artery). The force that causes such an injury is often great and often causes injuries to other organs and organ systems. In a multiple trauma patient, it is very important to specifically look for heart-related injuries because it is possible that they may be overlooked or missed by the surgeon, because of other obvious injuries. We describe the case of a 41-year-old man with multiple trauma who was diagnosed with aortic valve insufficiency due to rupture of the left coronary cusp 6 weeks after a road accident. Valvuloplasty was performed. Seven years later the patient is free of symptoms and is in good physical condition. Echocardiography showed normal dimensions of the heart chambers, a normal thickness of the heart walls, and normal systolic and diastolic function of the left ventricle. Heart valves are morphologically normal, and only an unimportant aortic insufficiency was noticed by echocardiography.

Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: responses to nitrate, amino acids and developmental stage.

The NR72.1 gene codes for a high-affinity nitrate transporter in Arabidopsis thaliana. To examine the regulation of NRT2.1 gene expression, we used a promoter-beta-glucuronidase (GUS) fusion and found that the NRT2.1 promoter directs expression to the epidermal, cortical and endodermal cell layers of mature root parts. The gene appeared to be expressed essentially in roots, but was also present in the leaf hydathodes. Investigation of NRT2.1 expression pattern during the plant developmental cycle showed that it increased rapidly during early vegetative growth, peaked prior to floral stem emergence, and decreased to very low levels in flowering and silique-bearing plants. Experiments with various nitrogen supply regimes demonstrated the induction of NRT2.1 expression by nitrate and repression by amino acids. Amino acid analysis showed that this repression was specifically related to increased internal glutamine, suggesting a role for this particular amino acid in nitrogen signalling responsible for nitrate uptake regulation. Taken together, our results support the hypothesis that the NRT2.1 gene codes for a major component of the inducible high-affinity transport system for nitrate, which is spatially and developmentally controlled at the transcriptional level. Surprisingly, NRT2.1 was not expressed in younger root parts, although a similar rate of nitrate influx was observed in both young and old root samples. This lack of correlation between nitrate influx and NRT2.1 expression suggests that another high-affinity nitrate transporter operates in root tips.

Regulation of NRT1 and NRT2 gene families of Arabidopsis thaliana: responses to nitrate provision.

Four low-affinity (NRT1), and seven high-affinity (NRT2) nitrate transporter gene homologues have been identified in Arabidopsis thaliana. We investigated the transcript abundances of all eleven genes in shoot and root tissues in response to the provision of 1 mM NO(3)(-), using relative quantitative RT-PCR. Based upon this criterion, genes were classified as nitrate-inducible, nitrate-repressible, or nitrate-constitutive. AtNRT1.1, 2.1, and 2.2 were strongly induced by NO(3)(-), peaking at 3-12 h and subsequently declining. By contrast AtNRT2.4 showed only modest induction both in shoots and roots. Expression of AtNRT2.5, one of the nitrate-repressible genes, was strongly suppressed by nitrate provision in both roots and shoots. The last group, characterized by a constitutive expression pattern, included AtNRT1.2, 1.4, 2.3, 2.6, and 2.7. Correlation coefficients between (13)NO(3)(-) influx from 100 micro M and 5 mM [NO(3)(-)], suggest that high- and low-affinity transport systems are mediated primarily by AtNRT2.1 and AtNRT1.1, respectively. Functional roles for the other members of these families remain uncertain.

[Traumatic jejunal entrapment in a patient with Bechterew disease]. / Traumatische Jejunumeinklemmung bei einem Patienten mit M. Bechterew.

We report an unusual case of incarceration of small bowel between the 4th and 5th lumbar vertebrae in a patient with rheumatoid spondylitis. Incarceration of the loop of jejunum within the fracture or between dislocated lumbar vertebrae is an uncommon entity. The cases of traumatic jejunal entrapment have been previously reported in the literature [1, 2, 3, 4, 5, 6, 7, 8, 9, 11]: 2 cases occurred in adults and 8 in children. There has been no report on this injury in patients with rheumatoid spondylitis (Bechterew arthritis).

Cloning and characterization of a root specific high-affinity sulfate transporter from Arabidopsis thaliana.

Hst1At (accession number AB018695) was identified from the Arabidopsis thaliana sequencing project on BAC T3F12, and the corresponding cDNA was isolated by reverse transcription-PCR. Southern blot analysis reveals a single copy of this gene. The cDNA encodes a root specific sulfate transporter of 649 amino acids. Heterologous expression of hst1At in a sulfate transport deficient yeast mutant shows that this gene encodes a high-affinity transport system ( approximately 2 microM). The transcript relative abundance increases in roots in response to sulfate deprivation, which correlated with increased root SO(4)(2-) influx capacity. These patterns were reversed upon sulfate addition to the medium and were accompanied by an increased glutathione level in roots. Feeding plants with cysteine or glutathione led to similar responses. Using buthionine sulfoximine, an inhibitor of glutathione synthesis, we show that glutathione rather than cysteine controls hst1At expression.

Regulation of high-affinity nitrate transporter genes and high-affinity nitrate influx by nitrogen pools in roots of barley.

To investigate the regulation of HvNRT2, genes that encode high-affinity NO(3)(-) transporters in barley (Hordeum vulgare) roots, seedlings were treated with 10 mM NO(3)(-) in the presence or absence of amino acids (aspartate, asparagine, glutamate [Glu], and glutamine [Gln]), NH(4)(+), and/or inhibitors of N assimilation. Although all amino acids decreased high-affinity (13)NO(3)(-) influx and HvNRT2 transcript abundance, there was substantial interconversion of administered amino acids, making it impossible to determine which amino acid(s) were responsible for the observed effects. To clarify the role of individual amino acids, plants were separately treated with tungstate, methionine sulfoximine, or azaserine (inhibitors of nitrate reductase, Gln synthetase, and Glu synthase, respectively). Tungstate increased the HvNRT2 transcript by 20% to 30% and decreased NO(3)(-) influx by 50%, indicating that NO(3)(-) itself does not regulate transcript abundance, but may exert post-transcriptional effects. Experiments with methionine sulfoximine suggested that NH(4)(+) may down-regulate HvNRT2 gene expression and high-affinity NO(3)(-) influx by effects operating at the transcriptional and post-transcriptional levels. Azaserine decreased HvNRT2 transcript levels and NO(3)(-) influx by 97% and 95%, respectively, while decreasing Glu and increasing Gln levels. This suggests that Gln (and not Glu) is responsible for down-regulating HvNRT2 expression, although it does not preclude a contributory effect of other amino acids.

Isolation and characterization of HvNRT2.3 and HvNRT2.4, cDNAs encoding high-affinity nitrate transporters from roots of barley.

Two full-length cDNAs, HvNRT2.3 and HvNRT2.4, were isolated from roots of barley (Hordeum vulgare), using reverse transcriptase-PCR and RACE-PCR. The corresponding polypeptides, consisting of 507 amino acids (molecular masses of 54.6 kD), belong to the major facilitator superfamily (MFS), and are closely related (>87% identity) to those encoded by HvNRT2.1 and HvNRT2.2 (formerly BCH1 and BCH2, respectively) from roots of barley. The latter are considered to encode inducible high-affinity NO(3)(-) transporters (Trueman et al., 1996). HvNRT2 transcripts were undetectable in NO(3)(-)-deprived plants. Following exposure to either NO(3)(-) or NO(2)(-), transcript abundance and (13)NO(3)(-) influx increased to a maximum by 6 to 12 h, then declined in HvNRT2.1, HvNRT2.2, and HvNRT2.3. The pattern of HvNRT2.4 transcript abundance was different, remaining high after achieving peak abundance. When external NO(3)(-) concentrations were varied from 0 to 500 microM under steady-state conditions of NO(3)(-) supply, HvNRT2 transcript accumulation and (13)NO(3)(-) influx were highest in 50 microM NO(3)(-) -grown plants. When NH(4)(+) was provided together with NO(3)(-), transcript accumulation during the first 2 h was similar to that due to NO(3)(-) alone, but by 4 h the transcript level was significantly reduced. HvNRT2 transcript was undetectable in leaf tissues.

Regulation of the hvst1 gene encoding a high-affinity sulfate transporter from Hordeum vulgare.

A cDNA, hvst1, was isolated from Hordeum vulgare by heterologous complementation in Escherichia coli. This cDNA encodes a high-affinity sulfate transporter that is 2442 bp in length and consists of 660 amino acids. Under steady-state conditions of sulfate supply during culture, sulfate influx (measured at 100 microM external sulfate concentration) and hvst1 transcript level were inversely correlated with sulfate concentrations in the culture solution. Glutathione (GSH) concentrations increased as external sulfate was increased from 2.5 to 250 microM. A time-course study, designed to investigate effects of sulfate withdrawal on the abundance of hvst1 transcript, showed a 5-fold increase of the latter within the first two hours. This was followed by a further slight increase during the next 46 h. These changes were accompanied by a parallel increase in sulfate influx and a decrease of root GSH concentrations. When plants that had been deprived of sulfate for 24 h were exposed to L-cysteine (Cys) or GSH for 3 h, GSH was the more effective down-regulator, reducing hvst1 transcript level to below that of unstarved controls. The decrease in transcript abundance induced by sulfate or Cys was partially relieved by the addition of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis. Both hvst1 transcripts and sulfate influx increased as a function of N supply to N-starved plants. Amino oxyacetate acid (AOA), an aminotransferase inhibitor, when supplied with NO3-, increased transcript abundance of hvst1, while tungstate, methionine sulfoximine (MSO) and azaserine (AZA), inhibitors of nitrate reductase, glutamine synthetase and glutamate synthase (GOGAT), respectively, were without effect. AOA decreased root concentrations of aspartate (Asp), Cys and GSH; in contrast, glutamate (Glu) concentrations remained unchanged.

Inter-organ signaling in plants: regulation of ATP sulfurylase and sulfate transporter genes expression in roots mediated by phloem-translocated compound.

Sulfate uptake and ATP sulfurylase activity in the roots of Arabidopsis thaliana and Brassica napus were enhanced by S deprivation and reduced following resupply of SO4(2-). Similar responses occurred in split-root experiments where only a portion of the root system was S-deprived, suggesting that the regulation involves inter-organ signaling. Phloem-translocated glutathione (GSH) was identified as the likely transducing molecule responsible for regulating SO4(2-) uptake rate and ATP sulfurylase activity in roots. The regulatory role of GSH was confirmed by the finding that ATP sulfurylase activity was inhibited by supplying Cys except in the presence of buthionine sulfoximine, an inhibitor of GSH synthesis. In direct and remote (split-root) exposures, levels of protein detected by antibodies against the Arabidopsis APS3 ATP sulfurylase increased in the roots of A. thaliana and B. napus during S starvation, decreased after SO4(2-) restoration, and declined after feeding GSH. RNA blot analysis revealed that the transcript level of APS1, which codes for ATP sulfurylase, was reduced by direct and remote GSH treatments. The abundance of AST68 (a gene encoding an SO4(2-) transporter) was similarly affected by altered sulfur status. This report presents the first evidence for the regulation of root genes involved in nutrient acquisition and assimilation by a signal that is translocated from shoot to root.

Regulation of a putative high-affinity nitrate transporter (Nrt2;1At) in roots of Arabidopsis thaliana.

Putative high-affinity nitrate (NO3-) transporter genes, designated Nrt2;1At and Nrt2;2At, were isolated from Arabidopsis thaliana by RT-PCR using degenerate primers. The genes shared 86% and 89% identity at the amino acid and nucleotide levels, respectively, while their proteins shared 30-73% identities with other eukaryotic high-affinity NO3- transporters. Both genes were induced by NO3-, but Nrt2;1At gene expression was not apparent in 2- and 5-day-old plants. By 10 days, and thereafter, Nrt2;1At gene expression in roots was substantially higher than for the Nrt2;2At gene. Root Nrt2;1At expression levels were strongly correlated with inducible high-affinity 13NO3- influx into intact roots under several treatment conditions. The use of inhibitors of N assimilation indicated that downregulation of Nrt2;1At expression was mediated by NH4+, gln and other amino acids.

A simple assay for monitoring the mutagenic effects of 5-methylcytosine deamination in Escherichia coli.

We have developed and tested a simple phenotypic assay which monitors C to T transition mutations at the second C of a CCAGG sequence in the lacZ gene of Escherichia coli. The assay is based on new data concerning amino acid requirements on either side of a crucial active site residue in beta-galactosidase, glutamic acid 461. We show that the frequency of occurrence of the mutation is influenced by two genes: dcm, the cytosine methylase gene, and vsr, one of the genes involved in very short patch repair. The assay has been used to evaluate the function of vsr cloned from a potential very short patch repair mutant.

MutY repair is mutagenic in mutT- strains of Escherichia coli.

We have determined the numbers and types of mutations that occur in strains of Escherichia coli defective in mutT and (or) mutY repair. High rates of C.G to A.T mutations in mutY- cells are unaffected by the status of mutT. However, mutT-/mutY+ strains have higher rates of A.T to C.G mutations than mutT-/mutY- strains. This result indicates that the high rates of A.T to C.G mutations seen in mutT- strains of E. coli are due in part to the activity of the mutY repair system. We conclude that mutY repair is mutagenic in a mutT- background.