Proteins derived from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, which performs plant-type oxygenic photosynthesis, are suitable for biochemical, biophysical, and X-ray crystallographic studies. We developed an automated bioluminescence real-time monitoring system for the circadian clock in the thermophilic cyanobacterium T. elongatus BP-1 that uses a bacterial luciferase gene set (Xl luxAB) derived from Xenorhabdus luminescens as a bioluminescence reporter gene. A promoter region of the psbA1 gene of T. elongatus was fused to the Xl luxAB gene set and inserted into a specific targeting site in the genome of T. elongatus. The bioluminescence from the cells of the psbA1-reporting strain was measured by an automated monitoring apparatus with photomultiplier tubes. The strain exhibited the circadian rhythms of bioluminescence with a 25-h period length for at least 10 days in constant light and temperature. The rhythms were reset by light-dark cycle, and their period length was almost constant over a wide range of temperatures (30 to 60°C). Theses results indicate that T. elongatus has the circadian clock that is widely temperature compensated.Circadian rhythms are endogenous daily fluctuations in physiological activities that have the following three characteristics: they are sustained under constant conditions with a period length of about 24 h; the phase of the rhythm is reset by environmental cues such as light-dark (LD) cycles and lowhigh temperature cycles; and the period of the rhythm is almost constant at physiological temperatures (5, 26). Thus far, circadian rhythms have been observed only in mesophilic organisms; we have found no reports of circadian rhythms in thermophilic organisms.The clock gene cluster kaiABC generates circadian rhythms in the mesophilic cyanobacterium Synechococcus sp. strain PCC 7942 (hereafter called Synechococcus) (8). KaiA and KaiC proteins regulate the transcription of the kaiBC operon positively and negatively, respectively, and this feedback regulation is important for generating circadian oscillations (8). Kai proteins interact with each other in all possible combinations (9), and KaiA enhances the phosphorylation of KaiC (10,28,29). These mesophilic clock proteins are unstable, however, and their structure and function, their mutual interactions, and their reactions with their substrates in the circadian feedback processes remain unknown.The thermophilic cyanobacteria Thermosynechococcus elongatus (30) and T. vulcanus (13), which were isolated from a Japanese hot spring, grow optimally at ca. 57°C, and their proteins are highly stable. The proteins can be easily purified, characterized for biochemical and biophysical properties, and crystallized. The photosynthetic reaction center protein complexes of photosystems I and II have been X-ray analyzed in T. elongatus (11, 31) and T. vulcanus (12). Recently, we cloned the gene cluster kaiABC from both species (7; T. Uzumaki and M. Ishiura, unpublished data), and both showed high homology with the correspo...
Background Urinary excretion of megalin, a proximal tubular endocytic receptor, may be associated with the development and progression of diabetic kidney disease (DKD). However, no studies have assessed whether the levels of the urinary ectodomain (A-megalin) and full-length (C-megalin) forms of megalin can predict DKD progression. Methods We evaluated the correlation of urinary A-megalin levels of 34 patients with type 2 diabetes as measured by novel reducing and previous methods. Then, we retrospectively analyzed 188 type 2 diabetes patients not taking sodium glucose cotransporter 2 (SGLT2) inhibitors in order to investigate whether urinary A- and C-megalin might be used as predictors of kidney outcomes. The median observation period was 3.96 years. The associations between the baseline urinary A-megalin measured by the novel method and/or C-megalin levels and the subsequent estimated glomerular filtration rate (eGFR) slope were analyzed using a generalized estimating equation. Patients were categorized into higher or lower groups based on the optimal cutoff values, obtained from a receiver operating characteristic (ROC) curve, of the two forms of urinary megalin. Results Urinary A-megalin levels measured by the two methods were strongly correlated. The eGFR slopes of the higher A-megalin and C-megalin groups were −0.904 (95% confidence interval [CI] −1.584, −0.224) and −0.749 (95% CI −1.312, −0.186) ml/min/1.73 m2 per year steeper than those of the lower groups, respectively. Moreover, the eGFR slope was −1.888 (95% CI −2.764, −1.011) ml/min/1.73 m2 per year steeper in the group with both higher A- and higher C-megalin than in the other groups. These results remained significant when adjusted for albuminuria or known tubular injury markers. Conclusions Our novel method allows urinary A-megalin measurements to be performed more easily. Baseline urinary megalin levels were associated with the subsequent eGFR slope independently of known biomarkers in type 2 diabetes patients not receiving SGLT2 inhibitors. These two forms of megalin may be distinct urinary biomarkers of the progression of DKD.
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