Structure of the Shigella dysenteriae haem transport locus and its phylogenetic distribution in enteric bacteria

The ability to transport and use haemin as an iron source is frequently observed in clinical isolates of Shigella spp. and pathogenic Escherichia coli . We found that many of these haem-utilizing E. coli strains contain a gene that hybridizes at high stringency to the S. dysenteriae type 1 haem receptor gene, shuA . These shuA -positive strains belong to multiple phylogenetic groups and include clinical isolates from enteric, urinary tract and systemic infections. The distribution of shuA in these strains suggests horizontal transfer of the haem transport locus. Some haem-utilizing pathogenic E. coli strains did not hybridize with shuA , so at least one other haem transport system is present in this group. We also characterized the chromosomal region containing shuA in S. dysenteriae . The shuA gene is present in a discrete locus, designated the haem transport locus, containing eight open reading frames. Several of the proteins encoded in this locus participate with ShuA in haem transport, as a Salmonella typhimurium strain containing the entire haem transport locus used haem much more efficiently than the same strain containing only shuA . The haem transport locus is not present in E. coli K-12 strains, but the sequences flanking the haem transport locus in S. dysenteriae matched those at the 78.7 minute region of E. coli K-12. The junctions and flanking sequences in the shuA -positive pathogenic E. coli strains tested were nearly identical to those in S. dysenteriae , indicating that, in these strains, the haem transport locus has an organization similar to that in S. dysenteriae , and it is located in the same relative position on the chromosome.     

Stressful colours: corticosterone concentrations in a free-living songbird vary with the spectral composition of experimental illumination

Organisms have evolved under natural daily light/dark cycles for millions of years. These cycles have been disturbed as night-time darkness is increasingly replaced by artificial illumination. Investigating the physiological consequences of free-living organisms in artificially lit environments is crucial to determine whether nocturnal lighting disrupts circadian rhythms, changes behaviour, reduces fitness and ultimately affects population numbers. We make use of a unique, large-scale network of replicated field sites which were experimentally illuminated at night using lampposts emanating either red, green, white or no light to test effect on stress hormone concentrations (corticosterone) in a songbird, the great tit (Parus major). Adults nesting in white-light transects had higher corticosterone concentrations than in the other treatments. We also found a significant interaction between distance to the closest lamppost and treatment type: individuals in red light had higher corticosterone levels when they nested closer to the lamppost than individuals nesting farther away, a decline not observed in the green or dark treatment. Individuals with high corticosterone levels had fewer fledglings, irrespective of treatment. These results show that artificial light can induce changes in individual hormonal phenotype. As these effects vary considerably with light spectrum, it opens the possibility to mitigate these effects by selecting street lighting of specific spectra.     

Effects of temperature on circadian clock and chronotype: an experimental study on a passerine bird

Daily schedules of many organisms, including birds, are thought to affect fitness. Timing in birds is based on circadian clocks that have a heritable period length, but fitness consequences for individuals in natural environments depend on the scheduling of entrained clocks. This chronotype, i.e., timing of an individual relative to a zeitgeber, results from interactions between the endogenous circadian clock and environmental factors, including light conditions and ambient temperature. To understand contributions of these factors to timing, we studied daily activity patterns of a captive songbird, the great tit (Parus major), under different temperature and light conditions. Birds were kept in a light (L)-dark (D) cycle (12.5?L:11.5 D) at either 8°C or 18°C with ad libitum access to food and water. We assessed chronotype and subsequently tested birds at the same temperature under constant dim light (LL(dim)) to determine period length of their circadian clock. Thermal conditions were then reversed so that period length was measured under both temperatures. We found that under constant dim light conditions individuals lengthened their free-running period at higher temperatures by 5.7?±?2.1?min (p?=?.002). Under LD, birds kept at 18°C started activity later and terminated it much earlier in the day than those kept under 8°C. Overall, chronotype was slightly earlier under higher temperature, and duration of activity was shorter. Furthermore, individuals timed their activities consistently on different days under LD and over the two test series under LL(dim) (repeatability from .38 to .60). Surprisingly, period length and chronotype did not show the correlation that had been previously found in other avian species. Our study shows that body clocks of birds are precise and repeatable, but are, nonetheless, affected by ambient temperature. (Author correspondence: marina.lehmann@uni-konstanz.de ).     

Interleukin-2 dose,blood monocyte and CD25+ lymphocyte counts as predictors of clinical response to interleukin-2 therapy in patients with renal cell carcinoma

Summary The purpose of this study was to determine immunological parameters in the peripheral blood that correlate with the clinical effect of interleukin-2 (IL-2) in patients with metastatic renal cell cancer. A group of 26 patients with metastatic renal cell cancer underwent IL-2 treatment using a 36-day schedule with continuous intravenous IL-2 infusion (3 × 10⁶ units m^–2 day^–1) administered from days 1 to 5 and days 12 to 16. The white blood cell count and the absolute and relative number of neutrophils, lymphocytes, eosinophils and monocytes were recorded six times in peripheral blood during the treatment. Also the blood counts of T cell and NK cell subsets and cells expressing the T cell activation markers IL-2R and VLA-1 were measured. The lymphokine-activated killer (LAK) cell cytotoxicity was measured either with or without additional in vitro stimulation by IL-2. Multivariate statistical analysis showed that the clinical responses were related to the administered dose of IL-2, to a low number of blood cells expressing IL-2 receptors and to a reduction in the blood monocyte count (P <0.05).     

Longitudinal immune monitoring of patients receiving intratumoral injection of a MART-1 T-cell receptor-transduced cell line (C-Cure 709)

Background aimsAdoptive transfer of tumor-specific lymphocytes is a promising strategy in the treatment of cancer. We conducted intratumoral administration of an allogeneic irradiated continuous T-cell line (C-Cure 709) expressing an HLA-A2-restricted MART-1-specific T-cell receptor (TCR) into HLA-A2⁺ melanoma patients. The C-Cure 709 cell line is cytotoxic against MART-1⁺ HLA-A2⁺ melanoma cell lines and secretes several immune stimulatory cytokines upon stimulation.MethodsAnti-tumor immune responses against the commonly expressed tumor antigen (Ag) MART-1 were longitudinally analyzed in peripheral blood by fluorescence-activated cell sorting (FACS) before and after intratumoral injection of C-Cure 709.ResultsNo treatment-induced increase in Ag-specific T-cell frequencies was observed in peripheral blood, and the phenotype of MART-1-specific T cells was very stable during the treatment. Interestingly, despite a very stable frequency of MART-1-specific T cells over the course of treatment, clonotype mapping revealed that the response was in fact highly diverse and dynamic, with new clonotypes emerging during treatment. Only a few clonotypes were recurrently detected in consecutive samples. One MART-1-specific T-cell clone disappearing from peripheral blood was later detected in a metastatic lesion.ConclusionsSequence analyzes of the CDR3 region revealed conserved structural characteristics in the MART-1-specific TCR used by T-cell clones.     

Effects of constant light on circadian rhythmicity in mice lacking functional <Emphasis Type="Italic">cry</Emphasis> genes: dissimilar from <Emphasis Type="Italic">per</Emphasis> mutants

Mutations in each of the genes mPer1, mPer2, mCry1 and mCry2 separately cause deviations from the wild type circadian system. Differences between these mutant strains have inspired the hypothesis that the duality of circadian genes (two mPer and two mCry genes involved) is related to the existence of two components in the circadian oscillator (Daan et al., J Biol Rhythms 16:105–116, 2001). We tested the predictions from this theory that the circadian period (τ) lengthens under constant illumination (LL) in mCry1 and mPer1 mutant mice, while it shortens in mCry2 and mPer2 mutants. mCry1 ^−/− and mCry2 ^−/− knockout mice both consistently increased τ with increasing light intensity, as did wild type mice. With increasing illumination, rhythmicity is reduced in mCry1, mCry2 and mPer1, but not in mPer2 deficient mice. Results for mPer mutant mice are in agreement with data reported on these strains earlier by Steinlechner et al. (J Biol Rhythms 17:202–209, 2002), and also with the predictions from the model. The increase in cycle length of the circadian system by light in the mCry2 deficient mice violates the predictions. The model is thereby rejected: the mCry genes do not play a differential role, although the opposite responses of mPer mutants to light remain consistent with a functional Evening–Morning differentiation.     

Screening for carcinoma in situ of the contralateral testis in patients with germinal testicular cancer.

Two hundred and fifty biopsy specimens from the contralateral testis in patients with unilateral germinal testicular cancer were analysed by light microscopy for carcinoma-in-situ changes. Changes were found in 13 (5.2%) patients. One-third of patients with an atrophic contralateral testis (volume less than or equal to 12 ml) and one-fifth of patients with a history of cryptorchidism had changes in the remaining testis. In the present series 85% of cases with carcinoma-in-situ changes would have been diagnosed if the one-fifth of the patients having an atrophic testis or a history of cryptorchidism or both had been screened. Since the natural course of carcinoma in situ in the contralateral testis of patients with germinal testicular cancer has not been established, the patients are being re-evaluated frequently. To date two patients with carcinoma in situ have developed a second cancer.     

When the sun never sets: diverse activity rhythms under continuous daylight in free-living arctic-breeding birds

Circadian clocks are centrally involved in the regulation of daily behavioural and physiological processes. These clocks are synchronized to the 24 h day by external cues (Zeitgeber), the most important of which is the light–dark cycle. In polar environments, however, the strength of the Zeitgeber is greatly reduced around the summer and winter solstices (continuous daylight or continuous darkness). How animals time their behaviour under such conditions has rarely been studied in the wild. Using a radio-telemetry-based system, we investigated daily activity rhythms under continuous daylight in Barrow, Alaska, throughout the breeding season in four bird species that differ in mating system and parental behaviour. We found substantial diversity in daily activity rhythms depending on species, sex and breeding stage. Individuals exhibited either robust, entrained 24 h activity cycles, were continuously active (arrhythmic) or showed ‘free-running’ activity cycles. In semipalmated sandpipers, a shorebird with biparental incubation, we show that the free-running rhythm is synchronized between pair mates. The diversity of diel time-keeping under continuous daylight emphasizes the plasticity of the circadian system, and the importance of the social and life-history context. Our results support the idea that circadian behaviour can be adaptively modified to enable species-specific time-keeping under polar conditions.     

Interactions of drugs and radiation in haemopoietic tissue assessed by lethality of mice after whole-body irradiation

Drug-radiation interactions in haemopoietic tissue were assessed as the lethality of mice within 7-28 days after whole-body irradiation. The investigated drugs were adriamycin (ADM), bleomycin (BLM), cyclophosphamide (CTX), 5-fluorouracil (5-FU), methotrexate (MTX), mitomycin C (MM-C) and cis-diamminedichloroplatinum II (cis-DDP). The drugs were administered as single doses 15 min before graded doses of whole-body irradiation or at different intervals from 7 days before to 7 days after fixed radiation doses. ADM, CTX, 5-FU, MM-C and cis-DDP enhanced the radiation response when administered 15 min before irradiation. The dose effect factor (DEF) was 9.11 for 5-FU and in the range 1.25-1.59 for the other drugs. MTX administration 15 min before irradiation had no effect (DEF 1.00). However, MTX increased lethality if given 1-3 days after irradiation (DEF 1.21-1.76) and protected against lethality if given 1-3 days before irradiation (DEF 0.83). A similar time dependence was observed for ADM, CTX, 5-FU, MM-C and cis-DDP. Protection against lethality was not observed but in all these cases the lethality was significantly lower at administration 1-3 days before than 1-3 days after irradiation. A proper investigation of the effect of BLM was not possible as the combination of this drug and whole-body irradiation caused a high rate of gastrointestinal deaths.     

The circadian cycle: daily rhythms from behaviour to genes

The daily recurrence of activity and rest are so common as to seem trivial. However, they reflect a ubiquitous temporal programme called the circadian clock. In the absence of either anatomical clock structures or clock genes, the timing of sleep and wakefulness is disrupted. The complex nature of circadian behaviour is evident in the fact that phasing of the cycle during the day varies widely for individuals, resulting in extremes colloquially called 'larks' and 'owls'. These behavioural oscillations are mirrored in the levels of physiology and gene expression. Deciphering the underlying mechanisms will provide important insights into how the circadian clock affects health and disease.