Time-dependent upregulation of electron transport with concomitant induction of regulated excitation dissipation in Haslea diatoms.

Photoacclimation by strains of Haslea "blue" diatom species H. ostrearia and H. silbo sp. nov. ined. was investigated with rapid light curves and induction-recovery curves using fast repetition rate fluorescence. Cultures were grown to exponential phase under 50 µmol m-2 s-1 photosynthetic available radiation (PAR) and then exposed to non-sequential rapid light curves where, once electron transport rate (ETR) had reached saturation, light intensity was decreased and then further increased prior to returning to near growth light intensity. The non-sequential rapid light curve revealed that ETR was not proportional to the instantaneously applied light intensity, due to rapid photoacclimation. Changes in the effective absorption cross sections for open PSII reaction centres (σPSII') or reaction centre connectivity (ρ) did not account for the observed increases in ETR under extended high light. σPSII' in fact decreased as a function of a time-dependent induction of regulated excitation dissipation Y(NPQ), once cells were at or above a PAR coinciding with saturation of ETR. Instead, the observed increases in ETR under extended high light were explained by an increase in the rate of PSII reopening, i.e. QA- oxidation. This acceleration of electron transport was strictly light dependent and relaxed within seconds after a return to low light or darkness. The time-dependent nature of ETR upregulation and regulated NPQ induction was verified using induction-recovery curves. Our findings show a time-dependent induction of excitation dissipation, in parallel with very rapid photoacclimation of electron transport, which combine to make ETR independent of short-term changes in PAR. This supports a selective advantage for these diatoms when exposed to fluctuating light in their environment.

Osteoarthritis of the thumb carpometacarpal joint: correlation of ultrasound appearances to disability and treatment response.

AIM: To evaluate grading of thumb carpometacarpal joint (CMCJ) osteoarthritis (OA) using ultrasound, correlating findings with disability and treatment response. MATERIALS AND METHODS: Patients with symptomatic thumb OA attending for ultrasound-guided CMCJ steroid injection and a group of asymptomatic controls were recruited prospectively. Thumb CMCJ ultrasound was graded (osteophytes, joint-space narrowing, capsule size, and measured capsule size), and a Disabilities of the Arm Shoulder and Hand (DASH) questionnaire was completed for each patient. Symptomatic patients then underwent injection with DASH repeated 6 weeks post-treatment. Ultrasound features were correlated with the initial DASH disability score and response as defined by change in DASH 6 weeks after treatment. RESULTS: Thirty-one patients with symptomatic OA and 37 asymptomatic controls were recruited. With the exception of osteophytes (p = 0.017), no statistically significant correlation was demonstrated between severity of ultrasound features and patient disability. However, all features demonstrated statistically significant higher grades in the symptomatic group compared to controls. Ultrasound grading did not have statistical correlation with treatment response. CONCLUSION: No correlation was found between the majority of ultrasound features and the clinical severity of OA or likely response to treatment. However, these features are significantly more common in the symptomatic population.

Immunosuppressive activity of a subline of the mouse EL-4 lymphoma. Evidence for minute virus of mice causing the inhibition.

Filtered culture fluids from the early in vitro passages of a subline of the C57BL/6 mouse EL-4 lymphoma, EL-4(G-), were strongly inhibitory for BABL/c vs. C57BL/6 mixed lymphocyte cultures (MLC). The inhibitory activity could be preserved by storage at -75 degrees C or 4 degrees C, thus allowing its further characterization. The inhibitory factor was particulate (nondialyzable, sedimentable at 100,000 g for 1 h), very small (recovered after 0.10 mum filtration), sensitive to UV irradiation, but heat stable (56 degrees C, 1 h) and resistant to chloroform. It was infectious, since later, noninhibitory passages of EL-4(G-) tissue culture cells became strongly inhibitory upon inoculation with the culture fluid. This data was consistent with the inhibitory factor being an infectious virus. Virus analysis by mouse antibody production tests revealed that viruses were indeed present in EL-4(G-) ascites cells and in the culture fluid, and not in a late passage of EL-4(G-) tissue culture cells which were not inhibitory. Neutralization of the inhibitory factor was achieved by pretreatment with ascitic fluid or with the sera raised against those (EL-4(G-)-derived materials which contained viruses. Mouse reference immune sera against minute virus of mice (MVM) completely neutralized the inhibitory factor in the culture fluid or in EL-4(G-) ascites cells. Two prototype MVM strains, and one Kilham rat virus preparation, did not inhibit the mouse MLC. Thus, the possibility exists that a variant of MVM, or an unidentified virus, has been grown and selected for in EL-4(G-) cells and recognized, due to its immunosuppressive characteristics. In any event, immunosuppression by EL-4(G-) cells was not mediated by the tumor cells, their metabolic products, or associated endogenous type C viruses, but by an exogenous virus, most likely a variant MVM with immunosuppressive characteristics. This adds weight to a parallel observation from our laboratory on the immunosuppressive effects of Kilham rat virus in rat lymphocyte cultures.

A new consensus for Trypanosoma cruzi intraspecific nomenclature: second revision meeting recommends TcI to TcVI.

In an effort to unify the nomenclature of Trypanosoma cruzi, the causative agent of Chagas disease, an updated system was agreed upon at the Second Satellite Meeting. A consensus was reached that T. cruzi strains should be referred to by six discrete typing units (T. cruzi I-VI). The goal of a unified nomenclature is to improve communication within the scientific community involved in T. cruzi research. The justification and implications will be presented in a subsequent detailed report.

Correction: Yes and Lyn play a role in nuclear translocation of the epidermal growth factor receptor.

In Figure 4C, it was identified that the Histone H3 and α-Tubulin purification control blots for YES and LYN overexpressing cells were duplicated. The original Histone H3 control blot was found and confirmed the published results, however, the α-Tubulin control blot was not found. This error was determined to not impact the scientific findings of this figure. The authors regret this error.

Comparison of histidine-tryptophan-ketoglutarate and University of Wisconsin preservation in renal transplantation.

Histidine-tryptophan-ketoglutarate (HTK) is replacing University of Wisconsin (UW) solution as the preservation fluid for renal allografts in many centers, but recent large-scale data to support this transition are lacking. We conducted a retrospective analysis of patient and graft outcomes after renal transplantation at our center, comparing 475 consecutive living donor and 317 deceased donor transplants since the adoption of HTK with equal numbers of grafts preserved using UW solution. Data collected included donor and recipient age, race, sex, comorbidities and graft ischemia time. Graft and patient survival, as well as the incidence of delayed graft function (DGF), were studied by Kaplan-Meier and Cox regression analysis. No significant difference was seen in either patient or graft survival. Deceased donor kidneys in the HTK group had a higher incidence of DGF than the UW cohort, whereas this trend was reversed in the case of living donor organs. In multivariate analysis, HTK was associated with a significant risk reduction on the incidence of DGF. Prolonged preservation with HTK compared to UW was not associated with excess risk to the graft or patient. In summary, HTK demonstrated efficacy similar to UW in terms of patient and graft survival.

Role of tumor necrosis factor-alpha in the pathophysiologic alterations after hepatic ischemia/reperfusion injury in the rat.

Cytokines are recognized as critical early mediators of organ injury. We attempted to determine whether or not severe hepatic ischemia/reperfusion injury results in tumor necrosis factor-alpha (TNF-alpha) release with subsequent local and systemic tissue injury. After 90 min of lobar hepatic ischemia, TNF was measurable during the reperfusion period in the plasma of all 14 experimental animals, with levels peaking between 9 and 352 pg/ml. Endotoxin was undetectable in the plasma of these animals. Pulmonary injury, as evidenced by a neutrophilic infiltrate, edema and intra-alveolar hemorrhage developed after hepatic reperfusion. The neutrophilic infiltrate was quantitated using a myeloperoxidase (MPO) assay; this demonstrated a significant increase in MPO after only 1 h of reperfusion. Anti-TNF antiserum pretreatment significantly reduced the pulmonary MPO after hepatic reperfusion. After a 12-h reperfusion period, there was histologic evidence of intra-alveolar hemorrhage and pulmonary edema. Morphometric assessment showed that pretreatment with anti-TNF antiserum was able to completely inhibit the development of pulmonary edema. Liver injury was quantitated by measuring serum glutamic pyruvic transaminase which showed peaks at 3 and 24 h. Anti-TNF antiserum pretreatment was able to significantly reduce both of these peak elevations. These data show that hepatic ischemia/reperfusion results in TNF production, and that this TNF is intimately associated with pulmonary and hepatic injury.

Transcription termination and 3'-End processing of the spliced leader RNA in kinetoplastids.

Addition of a 39-nucleotide (nt) spliced leader (SL) by trans splicing is a basic requirement for all trypanosome nuclear mRNAs. The SL RNA in Leishmania tarentolae is a 96-nt precursor transcript synthesized by a polymerase that resembles polymerase II most closely. To analyze SL RNA genesis, we mutated SL RNA intron structures and sequence elements: stem-loops II and III, the Sm-binding site, and the downstream T tract. Using an exon-tagged SL RNA gene, we examined the phenotypes produced by a second-site 10-bp linker scan mutagenic series and directed mutagenesis. Here we report that transcription is terminated by the T tract, which is common to the 3' end of all kinetoplastid SL RNA genes, and that more than six T's are required for efficient termination in vivo. We describe mutants whose SL RNAs end in the T tract or appear to lack efficient termination but can generate wild-type 3' ends. Transcriptionally active nuclear extracts show staggered products in the T tract, directed by eight or more T's. The in vivo and in vitro data suggest that SL RNA transcription termination is staggered in the T tract and is followed by nucleolytic processing to generate the mature 3' end. We show that the Sm-binding site and stem-loop III structures are necessary for correct 3'-end formation. Thus, we have defined the transcription termination element for the SL RNA gene. The termination mechanism differs from that of vertebrate small nuclear RNA genes and the SL RNA homologue in Ascaris.

Genomic and transcriptional linkage of the genes for calmodulin, EF-hand 5 protein, and ubiquitin extension protein 52 in Trypanosoma brucei.

We report genomic linkage of a pair of tandem, identical ubiquitin-extension protein 52 (EP52) genes, a novel EF-hand superfamily member gene (EFH5), and the calmodulin gene cluster in Trypanosoma brucei. The intergenic regions of these four genes are short: about 108 bp between the calmodulin gene C and the EFH5 gene, about 111 bp between the EFH5 gene and the ubiquitin-EP52/1 gene, and about 116 bp between the ubiquitin-EP52/1 and -EP52/2 genes. RNA molecules that span these three intergenic regions have been detected by polymerase chain reaction, which suggests that the genes are transcribed in a polycistronic manner. Transcription of the calmodulin, EFH5, and ubiquitin-EP52 genes in isolated nuclei is rapidly inactivated by UV irradiation, which further strengthens the hypothesis that this cluster of three different genes is transcribed in a polycistronic manner and suggests that they are under the control of a single distant upstream promoter. These results suggest that polycistronic transcription is common in trypanosomes and will probably be found for most, if not all, protein-encoding genes. The presence of at least three housekeeping genes with different known or potential regulatory functions within a polycistronic unit suggests that regulation of transcription initiation plays an important role in the coordinated expression of housekeeping genes in trypanosomes.

Three small nucleolar RNAs identified from the spliced leader-associated RNA locus in kinetoplastid protozoans.

First characterized in Trypanosoma brucei, the spliced leader-associated (SLA) RNA gene locus has now been isolated from the kinetoplastids Leishmania tarentolae and Trypanosoma cruzi. In addition to the T. brucei SLA RNA, both L. tarentolae and T. cruzi SLA RNA repeat units also yield RNAs of 75 or 76 nucleotides (nt), 92 or 94 nt, and approximately 450 or approximately 350 nt, respectively, each with significant sequence identity to transcripts previously described from the T. brucei SLA RNA locus. Cell fractionation studies localize the three additional RNAs to the nucleolus; the presence of box C/D-like elements in two of the transcripts suggests that they are members of a class of small nucleolar RNAs (snoRNAs) that guide modification and cleavage of rRNAs. Candidate rRNA-snoRNA interactions can be found for one domain in each of the C/D element-containing RNAs. The putative target site for the 75/76-nt RNA is a highly conserved portion of the small subunit rRNA that contains 2'-O-ribose methylation at a conserved position (Gm1830) in L. tarentolae and in vertebrates. The 92/94-nt RNA has the potential to form base pairs near a conserved methylation site in the large subunit rRNA, which corresponds to position Gm4141 of small rRNA 2 in T. brucei. These data suggest that trypanosomatids do not obey the general 5-bp rule for snoRNA-mediated methylation.

An iatrogenic enchondroma? An unusual X-ray finding.

In this unusual case, accumulation of silver nitrate used to treat over-granulation in a finger injury led to a near-misdiagnosis of a bony tumour on X-ray. This underlines the need to support X-ray results with a full clinical assessment.

Regulated tRNA import in Leishmania mitochondria.

The genes for three new tRNA and a 5S RNA were identified from a genomic DNA clone of 917 nucleotide pairs from the protozoon Leishmania tarentolae. They were encoded in the following order. The transcriptional directions and anticodons are in parentheses: tRNA(Val) (CAC-->)-5SRNA (-->)-tRNA(His) (<--GUG)-tRNA(Phe) (GAA-->). The tRNA(His) and tRNA(Phe) sequences have not been reported previously in trypanosomatid organisms. By northern analysis, tRNA(Val) and tRNA(Phe) were equally distributed between the cytosol and mitochondria, while tRNA(His) was less abundant in mitochondria than in the cytosol. Accordingly, the latter tRNA is classified as Import restricted (Impr). As shown before, 5S RNA was not imported. Recently, Mahapatra and Adhya [S. Mahapatra, T. Ghosh, S. Adhya, Nucl. Acids Res. 22 (1994) 3381-3386; S. Mahapatra, S. Adhya, J. Biol. Chem. 271 (1996) 20432-20437] have developed an in vitro import system in Leishmania and suggested that the D-loop sequence could serve as the import determinant. We examined all available tRNA gene sequences in trypanosomatids but found no apparent consensus within the D-loop that might account for tRNA-import regulation.

Characterization of a GTP-binding protein in the ADP-ribosylation factor subfamily from Leishmania tarentolae.

We report the cloning and characterization of a gene, LtARL, which encodes a small GTP-binding protein, from the protozoan Leishmania tarentolae. Hybridization analysis of genomic DNA under high stringency conditions indicates the single-copy nature of LtARL. LtARL is transcribed and yields a approximately 0.9 kb mRNA that is processed at the 5' end by trans-splicing. When expressed in Escherichia coli, LtArl binds GTP with a low stoichiometry and in a phospholipid-independent manner. Based on the greatest sequence identity with Homo sapiens Arl3 and lipid-independent binding of guanine nucleotides we designate this gene LtARL and the encoded protein LtArl.

The human glutathione S-transferases: comparison of isoenzyme expression in normal and astrocytoma brain.

We describe expression of alpha, mu and pi class glutathione S-transferases (GST) in brain tissue from 21 controls and uninfiltrated and tumour tissue from 17 glioma patients. GST were sequentially resolved by chromatofocusing into the GST2, GST1, GST5, GST2 (5.5), GST3, GST6 sets and the contribution of each to total activity determined. The immunological identity of these isoforms was studied using immunoblotting. The pi class GST3 isoform was the major contributor to activity in control tissue (70.9%) and, uninfiltrated (75.1%) and tumour samples (82.4%). Expression was significantly greater in the tumours (P less than 0.05). Expression of alpha isoforms GST2 and GST2 (5.5) was variable with most subjects demonstrating no detectable GST2 (B1 and B2 chromatofocused monomers). An isoform termed GST2 (5.5) chromatofocussed at pH 5.5 and cross-reacted with antisera to B1. It was detected in most control and glioma patients and comprised about 5% of total activity. The contribution of GST2 and GST2 (5.5) to activity was similar in control, uninfiltrated and tumour tissue. Two mu class enzymes, GST1 and GST5, were identified. GST1 isoforms were detected in 9 of 21 control samples, the phenotype of these and matched liver samples were identical. GST1 isoforms were detected in 4 of 16 tumour samples, a significantly lower incidence than in a previously established control group. GST5 was expressed in most samples, the contribution of this locus to activity was significantly reduced in the tumours (5.2%) compared with control samples (14.5%).

Precise identification of cleavage sites involved in the unusual processing of trypanosome ribosomal RNA.

The large subunit ribosomal RNA (LSRNA) of Trypanosoma brucei is unusual in being cleaved at multiple sites to yield six stable fragments of RNA. We report here the complete nucleotide sequence of two regions of the ribosomal DNA repeat unit. The first sequence includes all of the processing sites involved in the generation of one of the small LSRNA fragments. The second region encodes the trypanosome 5.8 S RNA. By RNA sequencing and S1 nuclease mapping, we have identified the processing sites involved in the generation of both of these small RNAs. On the basis of predicted secondary structure models, we infer that all the cleavages apparently occur near the junction of single- and double-stranded regions. The sites involved in the novel LSRNA processing show a clear symmetry with respect to a conserved region of ten base-pairs. No such signals are evident for the processing sites that generate the 5.8 S RNA.

The Induction of Mitotic Gene Conversion by X-Irradiation of Haploid SACCHAROMYCES CEREVISIAE.

Mitotic recombination in Saccharomyces cerevisiae was examined by means of experiments in which one of the haploid parents was X-irradiated prior to zygote formation. By this method radiation-induced lesions are restricted to only one of the two non-sister chromatids that may be expected to undergo mitotic exchange in the diploid. The principal results of this work are: (1) X-irradiated haploid cells that are incapable of further vegetative growth (colony formation) are efficiently rescued into viable diploids by mating with unirradiated haploid cells. (2) X-rays delivered to only one of the two haploid parents are recombinogenic in the resultant diploid. The frequency of detected recombinational events increases as a probable linear function of the X-ray dose. (3) A majority of the induced recombinational events are nonreciprocal in nature (mitotic gene conversion). These results complement those obtained from X-irradiation of the vegetative diploid itself, where the induced genetic exchanges are principally reciprocal.

Association of chromosome loss with centromere-adjacent mitotic recombination in a yeast disomic haploid.

Experiments designed to characterize the association between disomic chromosome loss and centromere-adjacent mitotic recombination were performed. Mitotic gene convertants were selected at two heteroallelic sites on the left arm of disomic chromosome III and tested for coincident chromosome loss. The principal results are: (1) Disomic chromosome loss is markedly enhanced (nearly 40-fold) over basal levels among mitotic gene convertants selected to arise close to the centromere; no such enhancement is observed among convertants selected to arise relatively far from the centromere. (2) Chromosome loss is primarily associated with proximal allele conversion at the centromere-adjacent site, and many of these convertants are reciprocally recombined in the adjacent proximal interval. (3) Partial aneuploid exceptions provisionally identified as carrying left arm telocentrics have been found. A testable model is proposed suggesting that centromere involvement in genetic recombination may precipitate segregational disfunction leading to mitotic chromosome loss.

Mitotic chromosome loss in a disomic haploid of Saccharomyces cerevisiae.

Experiments designed to characterize the incidence of mitotic chromosome loss in a yeast disomic haploid were performed; The selective methods employed utulize the non-mating property of strains disomic for linkage group III and heterozygous at the mating type locus. The principal findings are: (1) The grequency of spontaneous chromosome loss in the disome is of the order 10- minus 4 per cell; this value approximates the frequency in the same population of spontaneous mitotic exchange resulting in homozygosity at the mating type locus. (2) The recovered diploids are pure clones, and thus represent unique events in the disomic haploid. (3) Of the euploid chromosomes recovered after events leading to chromosome loss, approximately 90% retain the parental marker configuration expected from segregation alone; however, the remainder are recombinant for marker genes, and are the result of mitotic exchanges in the disome, especially in regions near the centromere. The recombinant proportion significantly exceeds that expected if chromosome loss and mitotic exchange in the disome were independent events. The data are consistent with a model proposing mitotic nondisjunction as the event responsible for chromosome loss in the disomic haploid.

Mutational removal of the major site of serine phosphorylation of the epidermal growth factor receptor causes potentiation of signal transduction: role of receptor down-regulation.

The major site of epidermal growth factor receptor (EGF-R) serine phosphorylation is located within the COOH-terminal domain of the receptor at Ser1046/7. We have previously demonstrated that this phosphorylation site accounts for the acute desensitization of the EGF-R observed in EGF-treated cells. Here we show that the mutational removal of this negative regulatory phosphorylation site causes potentiation of signal transduction by the EGF-R. This potentiation can be accounted for in part by a block in the EGF-stimulated down-regulation of the EGF-R. These data indicate that the SER1046/7 phosphorylation site may have a regulatory role during long term incubation of cells with mitogenic concentrations of EGF.