text:
A growing body of knowledge is revealing the critical role of circadian physiology in the development of specific pathological entities such as cancer. NK cell function participates in the immune response against infection and malignancy. We have reported previously the existence of a physiological circadian rhythm of NK cell cytolytic activity in rats, suggesting the existence of circadian mechanisms subjacent to NK cell function. At the cellular level, circadian rhythms are originated by the sustained transcriptional-translational oscillation of clock genes that form the cellular clock apparatus. Our aim in this study was to investigate the presence of molecular clock mechanisms in NK cells as well as the circadian expression of critical factors involved in NK cell function. For that purpose, we measured the circadian changes in the expression of clock genes (Per1, Per2, Bmal1, Clock), Dbp (a clock-controlled output gene), CREB (involved in clock signaling), cytolytic factors (granzyme B and perforin), and cytokines (IFN-γ and TNF-α) in NK cells enriched from the rat spleen. The results obtained from this study demonstrate for the first time the existence of functional molecular clock mechanisms in NK cells. Moreover, the circadian expression of cytolytic factors and cytokines in NK cells reported in this study emphasizes the circadian nature of NK cell function.
Circadian rhythms describe biological phenomena that oscillate within a 24-h cycle. These rhythms provide a temporal frame necessary for adequate homeostasis. By anticipating both environmental and internal changes, cells (and organisms) can efficiently program their physiological tasks. At the cellular level, circadian rhythms are originated by genetic elements (clock genes) organized in autoregulatory transcription-translation feedback loops. This oscillating machinery would ultimately generate the circadian rhythms in physiology and behavior. The core molecular clock components are: Per (Per1, Per2, and Per3) and Cry (Cry1 and Cry2) genes, which belong to the so-called negative limb of the clock; together with Clock and Bmal1 genes, which belong to the so-called positive limb of the clock. Downstream of the core clock components, the molecular clock machinery also comprises clock-controlled genes like the transcription factor Dbp, considered to be a direct regulator of clock outputs (1). In addition, other factors such as the Ca2+/CREB protein are being shown to play a central role in the clock molecular mechanism (2, 3). The basic oscillatory mechanism consists in the transcriptional activation of Per and Cry genes by CLOCK/BMAL1 heterodimers. Later accumulation of PER and CRY products suppresses the transcriptional activity driven by CLOCK/BMAL1, closing the feedback loop (4). Circadian oscillators (i.e., circadian rhythms in clock gene expression) have been detected in multiple peripheral tissues such as the heart (5), liver (6), spleen (7), bone marrow (8), endocrine tissues (9), pancreas (10), and others. To our knowledge, this is the first study addressing the existence of circadian oscillators in NK cells.
NK cells are a subset of lymphocytes characterized by the lack of CD3-TCR complex (11). NK cells are a critical component of the innate immunity against fungal, bacterial, and viral infections. They also play a vital role in cellular resistance to malignancy and tumor metastasis (12). NK cells can destroy infected and malignant cells by calcium-dependent release of cytolytic granules, by activation of the Fas (CD95) pathway or by TNF-α release (13). Among these mechanisms, the release of cytolytic granules containing granzymes (particularly granzyme B) and of perforin seems to be the major mechanism used for the killing of the target cell (14). Another vital function of NK cells is the production of cytokines such as IFN-γ and TNF-α (15). In addition to its essential role in effector functions against tumor metastases (16), IFN-γ acts as a self-activating molecule for NK cells (17). Similarly, TNF-α promotes NK cell-mediated killing (18).
Light and daily rhythms have a profound influence on immune function (19, 20). Many studies have described circadian variations of different immune parameters such as lymphocyte proliferation, Ag presentation, and cytokine gene expression (21). More specifically, previous works have pointed to changes in NK cell activity at different times of the day (22, 23, 24). We have recently published a conclusive study that describes the circadian rhythm in NK cell cytolytic activity in rats (25). Still, the underlying mechanisms governing NK cell circadian function are largely unknown. This uncertainty is not a trivial issue because alterations in circadian rhythms can lead to serious pathologies such as sleep disorders, cardiovascular diseases, depression, and even cancer (26, 27). Furthermore, emerging data in the human and animal literature suggest that circadian regulation may be crucial for the host defenses against cancer (28).
Under this perspective, our aim was to investigate the presence of molecular clock mechanisms in NK cells as well as the circadian expression of critical factors involved in NK cell function. For that purpose, we measured the circadian changes in the expression of clock genes (Per1, Per2, Bmal1, and Clock), Dbp (a clock-controlled output gene), CREB (involved in clock signaling), cytolytic factors (granzyme B and perforin), and cytokines (IFN-γ and TNF-α) in NK cells enriched from the rat spleen.
Sprague-Dawley male rats at 2 mo of age were maintained under constant environmental conditions on a 12 h light/dark cycle (light period from 7:00 a.m. to 7:00 p.m.) and were fed rodent chow ad libitum. Rats were euthanized by decapitation at 6:00 a.m., 10:00 a.m., 2:00 p.m., 6:00 p.m., 10:00 p.m., and 2:00 a.m. These time points correspond with Zeitgeber times (ZT)3 3, 7, 11, 15, 19, and 23, respectively. Spleens were immediately collected for enrichment of NK cells. Throughout the study, animal care and treatment complied with National Institutes of Health policy, were in accordance with institutional guidelines, and were approved by the Rutgers Animal Care and Facilities Committee.
Spleens were individually processed as previously described (29) to obtain homogeneous splenocyte suspensions in RPMI 1640 (Invitrogen Life Technologies). RBCs and granulocytes were separated by centrifugation (30 min at 400 × g) with Histopaque 1083 (Sigma-Aldrich). After washing with RPMI 1640, splenocytes (∼12 × 107 from one spleen) were resuspended in running buffer (PBS, 0.5% BSA). NK cells were then enriched by magnetic separation (negative selection) with an AutoMACS Magnetic Separator (Miltenyi Biotec) after incubating the splenocytes with anti-pan T cell Microbeads (OX52), anti CD45RA Microbeads (for mature B cells), and anti-MHC class II Microbeads (OX6) for dendritic cells and macrophages, following manufacturer’s indications. All Microbeads were purchased from Miltenyi Biotec. The yield in the negative (enriched) fraction was consistently ∼5 × 106 cells per spleen. The purity of the enriched fraction was assessed by flow cytometry using the FITC-conjugated anti-rat CD161a (NKR-P1A) mAb (BD Biosciences) together with appropriate isotypic IgG controls. This enrichment method, which is completed within 60–80 min, consistently yields a purity of ∼70% of untouched NKR-P1A-positive cells. The percentage of CD3- and CD8-positive cells in the NK-enriched fraction was also assessed by flow cytometry with PE-conjugated anti-rat CD3 and anti-rat CD8 mAbs (BD Biosciences) and it was consistently below 3%. Immediately after the separation procedure, enriched NK cells were lysed in appropriate buffers for later RNA extraction and protein analysis.
RNA from 2 × 106 enriched NK cells (per spleen) was isolated with the RNeasy mini kit (Qiagen). A 100 ng of total RNA was reverse transcribed to the first strand cDNA using Superscript II First Strand Synthesis System for RT-PCR (Invitrogen Life Technologies). Relative quantitation of mRNA levels was performed by real-time PCR (SYBR Green Assay; Applied Biosystems) using an ABI prism 7700 Sequence Detector. The following primer sequences were used: Per1 sense 5′-CCAGGCCCGGAGAACCTTTTT-3′, antisense 5′-CGAAGTTTGAGCTCCCGAAGT-3′ (10); Per2 sense 5′-GCAGCCTTTCGATTATTCTTC-3′, antisense 5′-GCTCCACGGGTTGATGAAG-3′; Bmal1 sense 5′-TCCGATGACGAACTGAAACAC-3′, antisense 5′-CTCGGTCACATCCTACGACAA-3′; Clock sense 5′-GAACTTGGCGTTGAGGAGTCT-3′, antisense 5′-GTGATCGAACCTTTCCAGTGC-3′; Dbp sense 5′-AGGCAAGGAAAGTCCAGGTG-3′, antisense 5′-TCTTGCGTCTCTCGACCTCTT-3′ (5); CREB sense 5′-ACTCAGCCGGGTACTACCATTCT-3′, antisense 5′-GGTGCTGTGCGAATCTGGTAT-3′; granzyme B sense 5′-CGTGCATCAGAAGTGGGTGTTG-3′, antisense 5′-GAGGCTGTTGTTACACATCCGG-3′; perforin sense 5′-GCATCGGTGCCCAAGCCAGTG-3′, antisense 5′-GCCAGCGAGCCCCTGCTCATCA-3′; IFN-γ sense 5′-AGAGCCTCCTCTTGGATATCTGG-3′, antisense 5′-GCTTCCTTAGGCTAGATTCTGGTG-3′ (30); TNF-α sense 5′-CCAGGTTCTCTTCAAGGGACAA-3′, antisense 5′-CTCCTGCTATGAAATGGCAAATC-3′ (31). Analyses were performed using the standard curve method with GAPDH (rodent GAPDH primers; Applied Biosystems) as the normalizing endogenous control. Relative mRNA levels at each time point were calculated as the percentage of the maximum value over the 24-h period.
For protein analysis, 2 × 106 enriched NK cells (per spleen) were subjected to standard SDS-PAGE electrophoresis, and proteins were transferred to polyvinylidene difluoride membranes following usual procedures. Blots were probed with granzyme B mAb (BD Biosciences), perforin polyclonal Ab (Torrey Pines Biolabs), IFN-γ polyclonal Ab (Chemicon International), TNF-α polyclonal Ab (Chemicon International), phospho-CREB polyclonal Ab (Upstate Biotechnology) and mAb (Oncogene Research Products) for normalizing purposes. After chemiluminiscence detection (ECL Plus; Amersham), densitometric analyses were performed using Scion Image software. Relative protein levels were calculated as a percentage of the maximum value observed in each blot.
One-way ANOVA with the Newman-Keuls posttest was used to assess differences between means at different time points. A value of p < 0.05 was considered statistically significant.
We first investigated the presence of canonical components of the cellular clock in NK cells. RT-PCR analysis revealed that both negative (Per1 and Per2) and positive (Bmal1 and Clock) components of the molecular clock are expressed in enriched NK cells and in the rat NK cell line RNK16 (Fig. 1⇓). As a control, clock gene expression was codetected in the suprachiasmatic nuclei (SCN) and the spleen.
Canonical clock genes are expressed in enriched NK cells and in the RNK16 cell line. RT-PCR bands (arrows) corresponding to rat clock genes Per1, Per2, Bmal1, and Clock. Detection of clock gene expression in the SCN and in the spleen served as positive control. DNA mass ladder (left lane) is shown, together with the negative control (no reverse transcription).
Our second aim was to know whether splenic NK cells present clock gene oscillations in vivo. For that purpose, we performed relative quantitation of clock genes mRNA by real-time PCR in enriched splenic NK cells obtained at different time points. Per1 mRNA levels peaked at ZT11 and were minimum from ZT23 to ZT7 (p < 0.05; Fig. 2⇓A). Per2 mRNA levels were found to be lowest at ZT23, and were consistently high from ZT11 to ZT15 (p < 0.05; Fig. 2⇓B). The mRNA oscillations of the positive clock genes Bmal1 and Clock were very similar, showing two maxima at ZT3 and ZT7, and steady low levels from ZT11 to ZT23 (p < 0.05; Fig. 2⇓, C and D). These time points coincided with rising mRNA levels of Per1 and Per2, their negative counterparts in the molecular clock. Dbp, a clock-controlled transcription factor, also presented circadian oscillations in mRNA levels, which peaked at ZT11 and were minimum from ZT19 to ZT23 (p < 0.05; Fig. 2⇓E).
Canonical clock genes oscillate in enriched NK cells. Circadian rhythms in mRNA levels of Per1 (A), Per2 (B), Bmal1 (C), Clock (D), and Dbp (C) detected in NK cells enriched from rat spleen at 6:00 a.m., 10:00 a.m., 2:00 p.m., 6:00 p.m., 10:00 p.m., and 2:00 a.m. These time points correspond with ZT 3, 7, 11, 15, 19, and 23, respectively. Total RNA from the enriched fraction was reverse transcribed to cDNA and then subjected to real-time PCR. Relative mRNA levels at each time point were determined as a percentage of the maximum value. Data are mean ± SEM of four animals per time point. a, Significantly different (p at least <0.05) from the lowest value; b, significantly different (p at least <0.05) from the second lowest value; c, significantly different (p at least <0.05) from the third lowest value. The dark period (▪) in the cycle is shown.
If the cellular clock is somehow involved in NK cell function it would be expected to see circadian oscillations in the expression of cytolytic factors and cytokines produced by these lymphocytes. To investigate this possibility, we performed relative mRNA quantitation of granzyme B, perforin, IFN-γ, and TNF-α by real-time PCR in enriched splenic NK cells obtained at different time points. Circadian oscillations of transcripts levels were found in all the mentioned factors. Granzyme B mRNA levels peaked at ZT7 (p < 0.05; Fig. 3⇓A), perforin mRNA presented maximum levels from ZT3 to ZT7 (p < 0.05; Fig. 3⇓B), IFN-γ mRNA levels showed steady high levels from ZT3 to ZT11 (p < 0.05; Fig. 3⇓C), and TNF-α mRNA levels peaked at ZT7 (p < 0.05; Fig. 3⇓D). Interestingly, the circadian rhythms in mRNA levels of these four factors were coordinated, showing high levels at the beginning of the light phase (ZT3-ZT7) and low levels during the dark phase.
mRNA levels of cytolytic factors and cytokines show circadian oscillations in enriched NK cells. Circadian rhythms in mRNA levels of granzyme B (A), perforin (B), IFN-γ (C), and TNF-α (D) detected in NK cells enriched from rat spleen at 6:00 a.m., 10:00 a.m., 2:00 p.m., 6:00 p.m., 10:00 p.m., and 2:00 a.m. These time points correspond with ZT 3, 7, 11, 15, 19, and 23, respectively. Total RNA from the enriched fraction was reverse transcribed to cDNA and then subjected to real-time PCR. Relative mRNA levels at each time point were determined as a percentage of the maximum value. Data are mean ± SEM of four animals per time point. a, Significantly different (p at least <0.05) from the lowest value. The dark period (▪) in cycle is shown.
We also examined whether the circadian variations in the mRNA of cytolytic factors and cytokines were translated to oscillations in their protein levels. Enriched NK cells presented circadian oscillations in the protein levels of granzyme B, perforin, IFN-γ, and TNF-α. Granzyme B protein levels peaked at ZT19 (p < 0.05; Fig. 4⇓A), perforin protein presented maximum levels from ZT19 to Z23 (p < 0.05; Fig. 4⇓B), IFN-γ protein levels also showed two maxima at ZT19 and ZT23 (p < 0.05; Fig. 4⇓C), and TNF-α protein levels peaked at ZT15 (p < 0.05; Fig. 4⇓D). Similarly to mRNA oscillations, the circadian rhythms in protein levels of these four factors were also coordinated, showing high levels during the dark phase and low levels during the light phase. It is worth noting that the rhythms in protein levels of cytolytic factors and cytokines run in antiphase to those of mRNA levels. Furthermore, granzyme B, perforin, and IFN-γ all showed a significant peak in protein levels at ZT19, exactly when the peak in NK cell cytolytic activity has been reported (25).
Protein levels of cytolytic factors and cytokines show circadian oscillations in enriched NK cells. Circadian rhythms in protein levels of granzyme B (A), perforin (B), IFN-γ (C), and TNF-α (D) detected in NK cells enriched from rat spleen at 6:00 a.m., 10:00 a.m., 2:00 p.m., 6:00 p.m., 10:00 p.m., and 2:00 a.m. These time points correspond with ZT 3, 7, 11, 15, 19, and 23, respectively. Enriched fraction lysates were subjected to standard SDS-PAGE electrophoresis and blotting procedures. After densitometric quantitation, relative protein content at each time point was determined as a percentage of the maximum value. Data are mean ± SEM of four animals per time point. a, Significantly different (p at least <0.05) from the lowest value. The dark period (▪) in the cycle is shown. Representative immunoblots are shown.
As suggested, the cAMP response element (CRE)/CREB pathway appears to be involved in the clock molecular mechanism. Although there were no significant circadian variations in CREB mRNA levels in enriched NK cells (p > 0.05; Fig. 5⇓A), we found a significant peak in phosphorylated CREB levels at ZT7 (p < 0.05; Fig. 5⇓B). Interestingly enough, phosphorylated CREB peak coincides with maximum mRNA levels of granzyme B, perforin, IFN-γ, and TNF-α.
Circadian rhythms in CREB mRNA levels (A) and phosphorylated CREB (p-CREB) protein levels (B) detected in NK cells enriched from rat spleen at 6:00 a.m., 10:00 a.m., 2:00 p.m., 6:00 p.m., 10:00 p.m., and 2:00 a.m. These time points correspond with ZT 3, 7, 11, 15, 19, and 23, respectively. Total RNA from the enriched fraction was reverse transcribed to cDNA and then subjected to real-time PCR. Relative mRNA levels at each time point were determined as a percentage of the maximum value. Enriched fraction lysates were subjected to standard SDS-PAGE electrophoresis and blotting procedures. After densitometric quantitation, relative protein content at each time point was determined as a percentage of the maximum value. Data are mean ± SEM of four animals per time point. a, Significantly different (p at least <0.05) from the lowest value. The dark period (▪) in cycle is shown. Representative immunoblots are also shown.
In this study we report for the first time the existence of clock gene oscillations in splenic-enriched NK cells. In addition, we demonstrate circadian expression of granzyme B, perforin, IFN-γ, and TNF-α, which emphasizes the circadian nature of NK cell function.
We have used previously whole rat spleens to identify the mRNA and protein rhythms of granzyme B, perforin, and IFN-γ (25). In contrast, in this study we used enriched NK cells from rat spleens to analyze the rhythms of granzyme B, perforin, IFN-γ, and TNF-α. Other splenic lymphocytes can also express cytolytic factors and/or IFN-γ (CD8 lymphocytes, NKT cells, APCs, and others). Hence, a thorough study in an enriched NK cell population was mandatory because granzyme B, perforin, and IFN-γ rhythms coming from non-NK cell sources may mask, at least in part, the actual rhythms in the expression of cytolytic factors and IFN-γ in splenic NK cells. Although some differences exist when comparing the circadian patterns, both studies coincide in showing the highest mRNA levels of granzyme B, perforin, and IFN-γ during the light period and the highest protein levels of granzyme B, perforin, and IFN-γ during the dark period. The fact that the circadian rhythms in both mRNA and protein levels of granzyme B, perforin, and IFN-γ were found to be similar (±4 h shifts) in both enriched NK cells and in the spleen (25) leads to several conclusions: 1) these circadian rhythms are conserved and reproducible in rats, 2) NK cells and other splenocytes may share similar rhythms in the expression of granzyme B, perforin, and IFN-γ, or 3) the circadian nature of the cytolytic function may be exclusive of NK cells. Regarding the latest, it would be expected for innate immune mechanisms such as NK cell function to have developed a circadian regulation to optimize their activity when most needed.
Perforin and granzymes have been largely recognized as fundamental components of the granule-mediated cytolytic activity (32). IFN-γ produced by NK cells acts as a self-activating molecule that promotes NK-cell-mediated killing (17). In this work we have included the circadian analysis of TNF-α, also relevant for NK cell function. TNF-α promotes NK-cell mediated killing (33) and there is direct evidence for its role as a cytotoxic molecule during the effector phase of the NK-mediated and lymphokine-activated killer-mediated antitumor cytolytic pathway (34). Interestingly enough, TNF-α rhythms in mRNA and protein matched with those found for cytolytic factors and IFN-γ. This finding further suggests a circadian coordination of the molecular logistic necessary to perform NK cell function. Indeed, granzyme B, perforin, and IFN-γ, all showed a significant peak in protein levels at ZT19, exactly when the peak in NK cell cytolytic activity in rat spleen has been recently reported (25). The circadian oscillations of cytolytic factors and cytokines may compose an anticipation mechanism that would allow NK cells to follow their physiological rhythm in cytolytic activity. Indeed, a disruption in the rhythm of NK cell cytolytic activity in ethanol-treated rats was correlated with alterations in the circadian rhythms of granzyme B, perforin, and IFN-γ (25).
Recurrent evidences for circadian NK functionality point inescapably to the existence of molecular clock mechanisms within NK cells. In this study we demonstrate for the first time the expression of canonical components of the cellular clock in NK cells as well as their circadian oscillations in NK cells enriched from the rat spleen. Moreover, clock gene transcripts oscillate in enriched NK cells with many properties similar to those seen in other peripheral tissues: Per1 transcript peaks at the time of the light-dark transition (5, 35, 36). So does Per2, with a longer acrophase than Per1 (5, 10). Bmal1 runs near antiphase with Per genes (37) and Clock has been shown to oscillate in phase with Bmal1 in the heart (5) and in the liver (38). Finally, Dbp, a clock-controlled transcription factor, also showed a strong circadian oscillation. Dbp is thought to generate upstream signals for physiological processes under circadian regulation (39), as it has been proven for locomotor activity (40) and the expression of some liver enzymes (41). Moreover, the circadian rhythm of Dbp transcript levels matches with Per1 rhythm, as seen in other peripheral tissues (42, 43). Circadian oscillations of Dbp indicate ultimately that the NK cell clock is able to generate functional outputs.
The aforementioned properties suggest all together the existence of functional molecular clock machinery in NK cells. In addition, antiphasic mRNA and protein rhythms in cytolytic factors and cytokines point to a negative feedback regulation, which is a hallmark of any oscillatory mechanism. Recent studies prove that circadian clock mechanisms are involved in key physiological processes such as glucose homeostasis (44), insulin secretion (39), prolactin secretion (45), and reproductive activity (46). Similarly, the results obtained in this study yield a possible connection between NK cell function and the molecular clock machinery.
Per gene expression has been reported in human lymphocytes (47, 48). Furthermore, human Per1 gene daily variations have been shown to have a similar acrophase and peak in both PBMCs and peripheral polymorphonuclear cells (49). Oishi et al. (50) reported a circadian Per2 mRNA expression in rat peripheral mononuclear lymphocytes that peaks at the early dark phase (ZT14), in accordance to our results in enriched NK cells. Thus, the existence of molecular clock machinery may be conserved across different lymphocyte subsets and peripheral blood cells. Moreover, they may share common entrainment signals. In this sense, it is yet to be deciphered whether the SCN, the central circadian pacemaker, is involved in the entrainment of NK cell function, as well as the coordinating signals by which circadian information may be conveyed to NK cells and other lymphocytes. Rhythms in the number of circulating T cells persisted in rats with disrupted circadian outputs (51). Similarly, SCN ablation did not affect the 24-h rhythms in cell cycle phase distribution in bone marrow cells (52), suggesting that some rhythms in the immune system may be SCN-independent. It is known that circadian gene expression can be maintained in vitro (6, 53). Thus, some peripheral clocks may be able to independently generate circadian oscillations and this could be also the case for NK cells. Rather than a mere rhythm generator for the periphery, the SCN should be envisioned as a transducer for light entrainment. However, there are entrainment signals other than light that may be coordinating the rhythm in NK cell function and other immunological parameters. For example, feeding is an important ZT for peripheral clock gene expression (54), and interestingly enough, internal desynchronization produced by restricted feeding during the light period slowed down tumor progression in mice (55). Daily activity rhythms are also considered to act as entrainment cues for peripheral tissues (56) and may as well influence the molecular clock in NK cells. In addition, intrinsic immunological outputs such a cytokine secretion could function as entrainment factors for immune cells. Indeed, IL-6 has been shown to induce Per1 expression in vitro (57).
Recent studies are presenting CREB itself as a key component of the molecular clock mechanism. CREB-dependent signaling regulates Per promoters (58), being regulation necessary for light-induced signaling and phase shifts in the SCN (2, 59). In addition, CREB activation was shown to be involved in the generation of circadian clock gene expression in rat fibroblasts (43), suggesting a role for the CRE/CREB pathway also in peripheral tissues. In contrast, it has been reported that CRE sequences are necessary and sufficient for transcriptional activation of the granzyme B promoter in immortalized T cell lines and primary T cells (60). Indeed, a decreased CREB phosphorylation was correlated with a decrease in granzyme B transcription in NK cells (61). CRE sequences are also found in the perforin promoter (62). In addition, CREB has been proven to modulate IFN-γ and TNF-α gene transcription in several systems (63, 64, 65, 66). We show in this study a temporal correlation between the peak in phosphorylated CREB levels and the peaks in transcript levels of cytolytic factors and cytokines. Therefore, the CRE/CREB pathway appears as a suggestive link between the NK cell clock and NK cell circadian function.
Finally, the attention given by the scientific community to circadian-related pathology has been augmenting in the last few years (67). Circadian rhythms seem to be particularly relevant in cardiovascular diseases (68), immune responses (69), and cancer (70, 71). It has been shown in mice that chronic jet lag disrupts circadian gene expression and accelerates tumor growth (72). Similarly, a disruption of the molecular clock machinery by mutation of Per2 increases the susceptibility of mice to the development of tumors (73). Alterations in circadian NK cell function may underlie some of these circadian-related pathologies. For example, NK cell cytolytic activity is significantly reduced after partial sleep deprivation (74) and correlates with a faster progression of breast cancer metastases (28). Indeed, an increased incidence of breast cancer and colorectal cancer has been documented in women working during night shift hours (75, 76). However, further research will be necessary to unveil the plausible connection between the NK cell clock machinery and NK cell function. The information on the mechanisms orchestrating this interaction will be extremely helpful for the development of chronotherapeutic approaches, which are undergoing lately an extensive development, especially in cancer therapy (77).
We thank Dr. J. P. Advis and Dr. C. P. Chen for providing SCN samples. We also thank Petra Pham and Theresa H. Choi from the Analytical Cytometry/Image Analysis Core Facility of the Environmental and Occupational Health Sciences Institute, Rutgers, The State University of New Jersey and Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey.
↵1 This work was supported by National Institutes of Health Grants AA00220 and AA12642.
↵3 Abbreviations used in this paper: ZT, Zeitgeber time; SCN, suprachiasmatic nucleus; CRE, cAMP response element.
Ripperger, J. A., L. P. Shearman, S. M. Reppert, U. Schibler. 2000. CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev. 14: 679-689.
Gau, D., T. Lemberger, C. von Gall, O. Kretz, N. Le Minh, P. Gass, W. Schmid, U. Schibler, H. W. Korf, G. Schutz. 2002. Phosphorylation of CREB Ser142 regulates light-induced phase shifts of the circadian clock. Neuron 34: 245-253.
Alvarez-Lopez, C., R. Cernuda-Cernuda, E. Alcorta, M. Alvarez-Viejo, J. Manuel Garcia-Fernandez. 2004. Altered endogenous activation of CREB in the suprachiasmatic nucleus of mice with retinal degeneration. Brain Res. 1024: 137-145.
Reppert, S. M., D. R. Weaver. 2002. Coordination of circadian timing in mammals. Nature 418: 935-941.
Young, M. E., P. Razeghi, H. Taegtmeyer. 2001. Clock genes in the heart: characterization and attenuation with hypertrophy. Circ. Res. 88: 1142-1150.
Yoo, S. H., S. Yamazaki, P. L. Lowrey, K. Shimomura, C. H. Ko, E. D. Buhr, S. M. Siepka, H. K. Hong, W. J. Oh, O. J. Yoo, et al 2004. PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc. Natl. Acad. Sci. USA 101: 5339-5346.
Sakamoto, K., T. Nagase, H. Fukui, K. Horikawa, T. Okada, H. Tanaka, K. Sato, Y. Miyake, O. Ohara, K. Kako, N. Ishida. 1998. Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J. Biol. Chem. 273: 27039-27042.
Chen, Y. G., A. Mantalaris, P. Bourne, P. Keng, J. H. Wu. 2000. Expression of mPer1 and mPer2, two mammalian clock genes, in murine bone marrow. Biochem. Biophys. Res. Commun. 276: 724-728.
Bittman, E. L., L. Doherty, L. Huang, A. Paroskie. 2003. Period gene expression in mouse endocrine tissues. Am. J. Physiol. Regul. Integr. Comp. Physiol. 285: R561-R569.
Muhlbauer, E., S. Wolgast, U. Finckh, D. Peschke, E. Peschke. 2004. Indication of circadian oscillations in the rat pancreas. FEBS Lett. 564: 91-96.
Lotzova, E.. 1989. Cytotoxicity and clinical application of activated NK cells. Med. Oncol. Tumor Pharmacother. 6: 93-98.
Colucci, F., M. A. Caligiuri, J. P. Di Santo. 2003. What does it take to make a natural killer?. Nat. Rev. Immunol. 3: 413-425.
Austin Taylor, M., M. Bennett, V. Kumar, J. D. Schatzle. 2000. Functional defects of NK cells treated with chloroquine mimic the lytic defects observed in perforin-deficient mice. J. Immunol. 165: 5048-5053.
Street, S. E., E. Cretney, M. J. Smyth. 2001. Perforin and interferon-γ activities independently control tumor initiation, growth, and metastasis. Blood 97: 192-197.
Cifone, M. G., S. D’Alo, R. Parroni, D. Millimaggi, L. Biordi, S. Martinotti, A. Santoni. 1999. Interleukin-2-activated rat natural killer cells express inducible nitric oxide synthase that contributes to cytotoxic function and interferon-γ production. Blood 93: 3876-3884.
Papamichail, M., S. A. Perez, A. D. Gritzapis, C. N. Baxevanis. 2004. Natural killer lymphocytes: biology, development, and function. Cancer Immunol. Immunother. 53: 176-186.
Roberts, J. E.. 2000. Light and immunomodulation. Ann. NY Acad. Sci. 917: 435-445.
Chacón, F., P. Cano, S. Lopez-Varela, V. Jiménez, A. Marcos, A. I. Esquifino. 2002. Chronobiological features of the immune system: effect of calorie restriction. Eur. J. Clin. Nutr. 56:(Suppl. 3): S69-S72.
Levi, F., C. Canon, M. Dipalma, I. Florentin, J. L. Misset. 1991. When should the immune clock be reset: from circadian pharmacodynamics to temporally optimized drug delivery. Ann. NY Acad. Sci. 618: 312-329.
Fernandes, G., F. Carandente, E. Halberg, F. Halberg, R. A. Good. 1979. Circadian rhythm in activity of lympholytic natural killer cells from spleens of Fischer rats. J. Immunol. 123: 622-625.
Esquifino, A. I., L. Selgas, A. Arce, V. D. Maggiore, D. P. Cardinali. 1996. Twenty-four-hour rhythms in immune responses in rat submaxillary lymph nodes and spleen: effect of cyclosporine. Brain Behav. Immun. 10: 92-102.
Shakhar, G., I. Bar-Ziv, S. Ben-Eliyahu. 2001. Diurnal changes in lung tumor clearance and their relation to NK cell cytotoxicity in the blood and spleen. Int. J. Cancer 94: 401-406.
Arjona, A., N. Boyadjieva, D. K. Sarkar. 2004. Circadian rhythms of granzyme B, perforin, IFN-γ, and NK cell cytolytic activity in the spleen: effects of chronic ethanol. J. Immunol. 172: 2811-2817.
Bovbjerg, D. H.. 2003. Circadian disruption and cancer: sleep and immune regulation. Brain Behav. Immun. 17:(Suppl. 1): S48-S50.
Fu, L., C. C. Lee. 2003. The circadian clock: pacemaker and tumour suppressor. Nat. Rev. Cancer 3: 350-361.
Sephton, S. E., R. M. Sapolsky, H. C. Kraemer, D. Spiegel. 2000. Diurnal cortisol rhythm as a predictor of breast cancer survival. J. Natl. Cancer Inst. 92: 994-1000.
Boyadjieva, N., M. Dokur, J. P. Advis, G. G. Meadows, D. K. Sarkar. 2001. Chronic ethanol inhibits NK cell cytolytic activity: role of opioid peptide β-endorphin. J. Immunol. 167: 5645-5652.
Jiang, H., X. Yang, R. N. Soriano, T. Fujimura, K. Krishnan, M. Kobayashi. 2000. Distinct patterns of cytokine gene suppression by the equivalent effective doses of cyclosporine and tacrolimus in rat heart allografts. Immunobiology 202: 280-292.
Raja, S. M., B. Wang, M. Dantuluri, U. R. Desai, B. Demeler, K. Spiegel, S. S. Metkar, C. J. Froelich. 2002. Cytotoxic cell granule-mediated apoptosis: characterization of the macromolecular complex of granzyme B with serglycin. J. Biol. Chem. 277: 49523-49530.
Lee, R. K., J. Spielman, D. Y. Zhao, K. J. Olsen, E. R. Podack. 1996. Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells. J. Immunol. 157: 1919-1925.
Baxevanis, C. N., I. F. Voutsas, O. E. Tsitsilonis, M. L. Tsiatas, A. D. Gritzapis, M. Papamichail. 2000. Compromised anti-tumor responses in tumor necrosis factor-α knockout mice. Eur. J. Immunol. 30: 1957-1966.
Oishi, K., H. Fukui, K. Sakamoto, K. Miyazaki, H. Kobayashi, N. Ishida. 2002. Differential expressions of mPer1 and mPer2 mRNAs under a skeleton photoperiod and a complete light-dark cycle. Brain Res. Mol. Brain Res. 109: 11-17.
Iwasaki, T., K. Nakahama, M. Nagano, A. Fujioka, H. Ohyanagi, Y. Shigeyoshi. 2004. A partial hepatectomy results in altered expression of clock-related and cyclic glyceraldehyde 3-phosphate dehydrogenase (GAPDH) genes. Life Sci. 74: 3093-3102.
Oishi, K., K. Sakamoto, T. Okada, T. Nagase, N. Ishida. 1998. Antiphase circadian expression between BMAL1 and period homologue mRNA in the suprachiasmatic nucleus and peripheral tissues of rats. Biochem. Biophys. Res. Commun. 253: 199-203.
Preitner, N., F. Damiola, L. Lopez-Molina, J. Zakany, D. Duboule, U. Albrecht, U. Schibler. 2002. The orphan nuclear receptor REV-ERBα controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110: 251-260.
Allaman-Pillet, N., R. Roduit, A. Oberson, S. Abdelli, J. Ruiz, J. S. Beckmann, D. F. Schorderet, C. Bonny. 2004. Circadian regulation of islet genes involved in insulin production and secretion. Mol. Cell. Endocrinol. 226: 59-66.
Lopez-Molina, L., F. Conquet, M. Dubois-Dauphin, U. Schibler. 1997. The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior. EMBO J. 16: 6762-6771.
Lavery, D. J., L. Lopez-Molina, R. Margueron, F. Fleury-Olela, F. Conquet, U. Schibler, C. Bonfils. 1999. Circadian expression of the steroid 15 α-hydroxylase (Cyp2a4) and coumarin 7-hydroxylase (Cyp2a5) genes in mouse liver is regulated by the PAR leucine zipper transcription factor DBP. Mol. Cell. Biol. 19: 6488-6499.
Damiola, F., N. Le Minh, N. Preitner, B. Kornmann, F. Fleury-Olela, U. Schibler. 2000. Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus. Genes Dev. 14: 2950-2961.
Yagita, K., H. Okamura. 2000. Forskolin induces circadian gene expression of rPer1, rPer2 and dbp in mammalian rat-1 fibroblasts. FEBS Lett. 465: 79-82.
Rudic, R. D., P. McNamara, A. M. Curtis, R. C. Boston, S. Panda, J. B. Hogenesch, G. A. Fitzgerald. 2004. BMAL1 and CLOCK, two essential components of the circadian clock, are involved in glucose homeostasis. PLoS Biol. 2: e377.
Lincoln, G. A., H. Andersson, D. Hazlerigg. 2003. Clock genes and the long-term regulation of prolactin secretion: evidence for a photoperiod/circannual timer in the pars tuberalis. J. Neuroendocrinol. 15: 390-397.
Chappell, P. E., R. S. White, P. L. Mellon. 2003. Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1–7 cell line. J. Neurosci. 23: 11202-11213.
Takata, M., N. Burioka, S. Ohdo, H. Takane, H. Terazono, M. Miyata, T. Sako, H. Suyama, Y. Fukuoka, K. Tomita, E. Shimizu. 2002. Daily expression of mRNAs for the mammalian Clock genes Per2 and Clock in mouse suprachiasmatic nuclei and liver and human peripheral blood mononuclear cells. Jpn. J. Pharmacol. 90: 263-269.
Boivin, D. B., F. O. James, A. Wu, P. F. Cho-Park, H. Xiong, Z. S. Sun. 2003. Circadian clock genes oscillate in human peripheral blood mononuclear cells. Blood 102: 4143-4145.
Kusanagi, H., K. Mishima, K. Satoh, M. Echizenya, T. Katoh, T. Shimizu. 2004. Similar profiles in human period1 gene expression in peripheral mononuclear and polymorphonuclear cells. Neurosci. Lett. 365: 124-127.
Oishi, K., K. Sakamoto, T. Okada, T. Nagase, N. Ishida. 1998. Humoral signals mediate the circadian expression of rat period homologue (rPer2) mRNA in peripheral tissues. Neurosci. Lett. 256: 117-119.
Depres-Brummer, P., P. Bourin, N. Pages, G. Metzger, F. Levi. 1997. Persistent T lymphocyte rhythms despite suppressed circadian clock outputs in rats. Am. J. Physiol. 273: R1891-R1899.
Filipski, E., V. M. King, M. C. Etienne, X. Li, B. Claustrat, T. G. Granda, G. Milano, M. H. Hastings, F. Levi. 2004. Persistent twenty-four hour changes in liver and bone marrow despite suprachiasmatic nuclei ablation in mice. Am. J. Physiol. Regul. Integr. Comp. Physiol. 287: R844-R851.
Balsalobre, A., L. Marcacci, U. Schibler. 2000. Multiple signaling pathways elicit circadian gene expression in cultured Rat-1 fibroblasts. Curr. Biol. 10: 1291-1294.
Kobayashi, H., K. Oishi, S. Hanai, N. Ishida. 2004. Effect of feeding on peripheral circadian rhythms and behaviour in mammals. Genes Cells 9: 857-864.
Wu, M. W., X. M. Li, L. J. Xian, F. Levi. 2004. Effects of meal timing on tumor progression in mice. Life Sci. 75: 1181-1193.
Schibler, U., J. Ripperger, S. A. Brown. 2003. Peripheral circadian oscillators in mammals: time and food. J. Biol. Rhythms 18: 250-260.
Motzkus, D., U. Albrecht, E. Maronde. 2002. The human PER1 gene is inducible by interleukin-6. J. Mol. Neurosci. 18: 105-109.
Travnickova-Bendova, Z., N. Cermakian, S. M. Reppert, P. Sassone-Corsi. 2002. Bimodal regulation of mPeriod promoters by CREB-dependent signaling and CLOCK/BMAL1 activity. Proc. Natl. Acad. Sci. USA 99: 7728-7733.
Tischkau, S. A., J. W. Mitchell, S. H. Tyan, G. F. Buchanan, M. U. Gillette. 2003. Ca2+/cAMP response element-binding protein (CREB)-dependent activation of Per1 is required for light-induced signaling in the suprachiasmatic nucleus circadian clock. J. Biol. Chem. 278: 718-723.
Babichuk, C. K., B. L. Duggan, R. C. Bleackley. 1996. In vivo regulation of murine granzyme B gene transcription in activated primary T cells. J. Biol. Chem. 271: 16485-16493.
Thomas, L. D., H. Shah, S. A. Green, A. D. Bankhurst, M. M. Whalen. 2004. Tributyltin exposure causes decreased granzyme B and perforin levels in human natural killer cells. Toxicology 200: 221-233.
Youn, B. S., C. C. Liu, K. K. Kim, J. D. Young, M. H. Kwon, B. S. Kwon. 1991. Structure of the mouse pore-forming protein (perforin) gene: analysis of transcription initiation site, 5′ flanking sequence, and alternative splicing of 5′ untranslated regions. J. Exp. Med. 173: 813-822.
Cippitelli, M., A. Sica, V. Viggiano, J. Ye, P. Ghosh, M. J. Birrer, H. A. Young. 1995. Negative transcriptional regulation of the interferon-γ promoter by glucocorticoids and dominant negative mutants of c-Jun. J. Biol. Chem. 270: 12548-12556.
Penix, L. A., M. T. Sweetser, W. M. Weaver, J. P. Hoeffler, T. K. Kerppola, C. B. Wilson. 1996. The proximal regulatory element of the interferon-γ promoter mediates selective expression in T cells. J. Biol. Chem. 271: 31964-31972.
Becker, C., K. Barbulescu, S. Wirtz, K. H. Meyer zum Buschenfelde, S. Pettersson, M. F. Neurath. 1999. Constitutive and inducible in vivo protein-DNA interactions at the tumor necrosis factor-α promoter in primary human T lymphocytes. Gene Expr. 8: 115-127.
Chong, Y. H., Y. J. Shin, Y. H. Suh. 2003. Cyclic AMP inhibition of tumor necrosis factor α production induced by amyloidogenic C-terminal peptide of Alzheimer’s amyloid precursor protein in macrophages: involvement of multiple intracellular pathways and cyclic AMP response element binding protein. Mol. Pharmacol. 63: 690-698.
Hastings, M. H., A. B. Reddy, E. S. Maywood. 2003. A clockwork web: circadian timing in brain and periphery, in health and disease. Nat. Rev. Neurosci. 4: 649-661.
Casetta, I., E. Granieri, F. Portaluppi, R. Manfredini. 2002. Circadian variability in hemorrhagic stroke. J. Am. Med. Assoc. 287: 1266-1267.
Uthgenannt, D., D. Schoolmann, R. Pietrowsky, H. L. Fehm, J. Born. 1995. Effects of sleep on the production of cytokines in humans. Psychosom. Med. 57: 97-104.
Schernhammer, E. S., K. Schulmeister. 2004. Melatonin and cancer risk: does light at night compromise physiologic cancer protection by lowering serum melatonin levels?. Br. J. Cancer 90: 941-943.
Stevens, R. G.. 2005. Circadian disruption and breast cancer: from melatonin to clock genes. Epidemiology 16: 254-258.
Filipski, E., F. Delaunay, V. M. King, M. W. Wu, B. Claustrat, A. Grechez-Cassiau, C. Guettier, M. H. Hastings, L. Francis. 2004. Effects of chronic jet lag on tumor progression in mice. Cancer Res. 64: 7879-7885.
Fu, L., H. Pelicano, J. Liu, P. Huang, C. Lee. 2002. The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111: 41-50.
Irwin, M., A. Mascovich, J. C. Gillin, R. Willoughby, J. Pike, T. L. Smith. 1994. Partial sleep deprivation reduces natural killer cell activity in humans. Psychosom. Med. 56: 493-498.
Schernhammer, E. S., F. Laden, F. E. Speizer, W. C. Willett, D. J. Hunter, I. Kawachi, C. S. Fuchs, G. A. Colditz. 2003. Night-shift work and risk of colorectal cancer in the nurses’ health study. J. Natl. Cancer Inst. 95: 825-828.
Hansen, J.. 2001. Increased breast cancer risk among women who work predominantly at night. Epidemiology 12: 74-77.
Mormont, M. C., F. Levi. 2003. Cancer chronotherapy: principles, applications, and perspectives. Cancer 97: 155-169.

matches: V. V. V. V. V. V.


Al. Piłsudskiego 44, 35-001 Rzeszów.
The Client has the right to negotiate the provisions of the contract with the Seller prior to placing an order.
In the event the Client abandons contract conclusion possibility by negotiation, these Terms & Conditions and general provisions of law shall be applicable.
The online Store available at: selectshop.eu is run by Robert Zapora, operating under Przedsiębiorstwo Wielobranżowe „RAPU” Robert Zapora name - detailed information related to the business activity is located above.
The Terms & Conditions are drawn up in Polish and constitute stipulated contract model being a distant contract, according to the general provisions of the Polish law.
In order to conclude a Contract with the Seller, the Buyer can use the negotiating rights regarding Contract conditions or conclude a Contract with the Seller based on these Terms & Conditions.
The content of the Terms & Conditions, in case of separate decision of the Buyer, shall constitute the content of the Contract concluded between the Parties. The content of Contract shall be established and made available to the Buyer on a durable medium according to binding provisions in order to invoke the Contract by the Buyer, if necessary.
The selling is performed within the Republic of Poland and the European Union.
All items offered at the selectshop.eu Store are brand new, free from physical and legal defects, and were legally placed on the Polish market. This information shall be tantamount to the Buyer’s obligation to provide the Client with deliveries without defects.
Prices given at selectshop.eu Store website are expressed in Polish Zloty and are VAT inclusive.
The Buyer may place orders at the Store 24 hours per day and 7 days per week through www.selectshop.eu website.
Any communication with the Seller performed by the Buyer will cause costs incurred by the Buyer resulting from the contracts concluded between the Buyer and the third parties. In the event of using given forms of distant communication the Seller shall not charge any additional fees or performance for the communication possibility with him.
The business activity of the selectshop.eu online Store shall be retail and wholesale selling of clothes and other items through the Internet.
For matters not covered by these Terms & Conditions the relevant law regulations binding in the Republic of Poland shall apply, and in particular provisions of the Act of April 23, 1964 - the Civil Code (Dz. U. [Polish Journey of Laws] No. 16, item 93, as amended), the Act on consumer rights of May 30, 2014 (Dz. U. 2014, item 827) and the Act on personal data protection of September 29, 1997 (consolidated text: Dz. U. of 2002 No. 101 item 926, as amended).
The provisions of these Terms & Conditions shall not exclude or limit any rights of the Buyer who is at one time the Consumer within the meaning of Act of April 23, 1964 - the Civil Code (Dz. U. No. 16, item 93, as amended), to which the Buyer is entitled under the mandatory provisions of law. The aforementioned regulations shall take precedence over these Terms & Conditions in any event of non-compliance.
SELLER – Robert Zapora, operating under Przedsiębiorstwo Wielobranżowe „RAPU” Robert Zapora name with a registered office in Rzeszów, at the address: ul. Tadeusza Boya-Żeleńskiego 11, 35 – 305 Rzeszów, NIP [Tax Identification Number]: 9222181461 REGON number: 950325689 entered into the CEIDG (Business Activity Register). The business is operated also at additional addresses, i.e. respectively: Al. Kopisto 1, 35-315 Rzeszów, Al. Piłsudskiego 44, 35-001 Rzeszów and Al. Jerozolimskie 179, 02-222 Warszawa.
SERVICE PROVIDER – Robert Zapora, operating under name Przedsiębiorstwo Wielobranżowe „RAPU” Robert Zapora with a registered office in Rzeszów, ul. Tadeusza Boya-Żeleńskiego 11, 35 – 305 Rzeszów, NIP 9222181461, REGON 950325689 entered into the CEIDG (Business Activity Register). The business is operated also at additional addresses, i.e. respectively: ul. Boya-Żeleńskiego 11, 35-105 Rzeszów, Al. Kopisto 1, 35-315 Rzeszów, Al. Piłsudskiego 44, 35-001 Rzeszów and Al. Jerozolimskie 179 02-222 Warszawa.
1. The Registration at the selectshop.eu online Store is voluntary and cost-free.
2. The User should fill in the registration form located on the Store website, giving his/her factual data in it in order to register at the Store.
4. Consent to receive Newsletter about the promotions and new releases at the store to the email address provided during registration can be optionally expressed at time of registration.
5. The User Account will be created the time the registration form fields are filled with the required data.
6. In the event of a change of the User’s data required for registration after the registration, the User is asked to update the data so it reflects the reality.
7. In the event of a change of the User’s data required for registration after the implementation of a contract, the User is asked to update it and inform the Store about such event.
9. Optionally, there is a possibility to provide payer data, according to the above-mentioned indications in subparagraph 3.
10. The shipment method shall be determined after the User fills the order form fields.
11. The Client may optionally enter his notes to the order he/she places.
2. The information about items presented on the Store websites shall not constitute an offer under provisions of the Civil Code; they shall mean an invitation to enter into the Contract.
3. Placing of an Order shall constitute an offer placed by the Client to the Seller under the Civil Code.
4. The Registration and setting-up of an Account shall not constitute a requirement for placing an order in the selectshop.eu Store. The Buyer shall have the possibility to place an order through the online Store without prior registration using the order form.
5. The Buyer should add the item he/she wants to purchase to the “Basket” to place an order. Adding the item to the “Basket” shall not be equivalent of placing an order. The items can be freely added and removed from the “Basket”.
6. The “Basket” shall enable the Buyer to manage the ordered items and calculate the value of the order.
7. Chosen items should be added to the “Basket” to finalize the order. The next step shall be the selection of the options determining respectively: payment and delivery method. The buyer can place an order after logging into his/her Account or should they be unregistered, by providing the necessary data for the Seller to complete the contract into the form.
8. They Buyer while placing an order can optionally enter the notes to the order by allocating them in the given space. Other ship-to address than the billing data can be selected at this space.
9. Determination of the bill of sale form shall be the final stage (receipt or VAT invoice). After the necessary fields of the form are filled, the “acceptance” button shall be selected.
10. The Buyer shall be shown the purchaser and address data, as well as the order value and its realization method after placing the proper order. In the event of the acceptance of all the chosen elements, “Order and Pay” button should be selected in order to place an Order successfully. It shall be deemed that the time of contract conclusion is the time of confirmation by the online Store sent by e-mail.
11. The Buyer can make changes in the order or withdraw the order entirely by means of e-mail or telephone.
12. Ordered goods shall be shipped to the address indicated by the Buyer.
It shall be deemed that the time of contract full performance is the day of reception of the shipment by the Buyer.
1.	All the prices in the Store are gross prices, inclusive Goods and Services Tax (VAT).
3. The Buyer shall do the shopping and order a service according to the prices and shipment costs valid at the time of the order placement.
4. The Seller shall reserve the right to change prices and shipment costs, in particular in the event of the amendments to the price lists of the entities making the deliveries. This provision shall not apply to the orders already under way.
5. Actual prices and shipment costs shall be present on the subpage of the online Store and visible at the Basket level.
1. The delivery shall be made to the address set out by the Buyer in the order.
2. Deliveries are made through DPD courier company. Dispatch of goods shall be made immediately after reception and confirmation of an order by the selectshop.eu online Store and realized within 48 hours, within working days in Poland.
4. The orders shall be realized within the working days from 9.00 – 17:00.
5. The Buyer shall have the possibility to select other delivery method (carrier) than offered on the online Store Website. Whereas in such a case, the risk of losing, damage and the responsibility for the shipment at the time of its consignment by the Seller shall lie with the Buyer.
6. In the event of the above described situation in subparagraph 5, the information on the responsibility for the shipment shall be transferred to the Buyer by the Seller via email to the provided contact address after the order placement.
7. In case the Seller is not capable of making a performance due to the goods unavailability, immediately, and in 30 days from the Contract conclusion at the latest, he/she shall inform the Buyer who then will decide on the further proceedings about the placed order.
8. In case the Seller is not capable of making a performance with the Buyer’s individually ordered features due to the temporary incapability, the Seller can make a supplementary performance that corresponds the same quality and destination and at the same price or remuneration or in other manner agreed upon by the Parties prior to the consent of the Buyer.
1. In case of the contracts concluded with the Consumers under the art. 22¹ of the Civil Code, the Seller shall be liable towards the Consumer on principles set out in art. 556 and further of the Civil Code for the physical or legal defects (warranty).
2. The Seller shall remain liable under warranty in case the physical defect is discovered before two years have elapsed from the moment of the item release to the Consumer, and in case the item of a sale performance was a used item - before one year has elapsed from the moment of the item release to the Consumer.
3. Consumer’s claim for the defect removal or the replacement of an item into the free of defects one, shall terminate once one year has elapsed, starting from the day of the defect discovery, however not before two years have elapsed from the moment of the item release to the Consumer, and in case the item of a sale was a used item - before one year has elapsed from the moment of the item release to the Consumer.
4. In case the date of shelf-life set out by the Seller or producer elapses after two years have elapsed from the moment of the item release to the Consumer, the Seller shall remain liable under warranty for physical defects of the said item discovered before this term termination.
6. The public assurance of a producer or its representative or that of a person who places the item on the market within his/her business activity, and of a person who by putting his/her business name, trademark or other distinguishing feature on the item presents himself as its producer shall be equally treated with the Seller’s assurance.
8. The sold item shall have defect also in case of its inappropriate installation and commissioning if those activities were performed by the Seller or a third party for which the Seller is liable or by the Buyer who complied with the manual given by the Seller.
9. In the event of a Contract with the Consumer, when the physical defect was discovered before one year from the moment of the item release, it shall be deemed that it existed by the time of the risk transfer to the Consumer. The Consumer shall be liable for indication that the defect existed at the time of the purchase in case the defect was discovered after the year from the moment of the item release.
11. The Consumer shall not be liable to back out of a Contract in case the defect is insignificant.
12. The Consumer that exercises his/her powers under statutory warranty shall be obliged to provide the defective item to the Complaint Address at the Seller’s expense, and in case that due to the item kind or the method of its installation the delivery by the Consumer would be excessively difficult, Consumer shall be obliged to make the item available for the Seller at the place where the said item is located. In case of non-performance of the obligation by the Seller, the Consumer shall be entitled to return the item at the expense and risk of the Seller.
13. The Seller kindly asks to attach the description of the discrepancy between the item and the Contract aiming at the complaint process facilitation.
14. The Seller in 14 (fourteen) days shall take a position to the Client’s notification.
Otherwise, it shall be considered that the Seller acknowledged the Client’s statement or request reasoned.
15. The Seller in the event of the Consumer’s request defined in subparagraph 10 a or b can replace the defective item into the free of defects one or remove a defect, provided it is done immediately and without significant inconvenience for the Consumer.
16. However, when the item has been already replaced or fixed by the Seller or the Seller has not complied with his obligation to replace an item into the free of defects or remove the defect, he shall not have the right to replace or fix the item.
17. The Consumer can, instead of the Seller’s proposed defect removal, request the replacement of the item into the free of defects one or instead of replacement, request the defect removal, unless bringing the item into line with the Contract in the way selected by the Consumer is impossible or this would require excessive costs compared to the way proposed by the Seller, whereas by the evaluation of the excessive costs the value of the free of defects item, its kind and meaning of the discovered defect, as well as inconvenience on which the Client could be exposed to by other way of adjustment of claims shall be included.
18. The Seller can refuse the Consumer’s request reimbursement when bringing the defective item into line with the Contract in the way chosen by the Consumer is impossible or compared to the other possible way requires excessive costs.
19. In the event of the price reduction, the reduced price should remain in such proportion to the price, resulting from the Contract, in which the defective item value remains to the defect free item.
20. The Seller shall be obliged to replace the defective item into the free of defects one or remove the defect at any reasonable time without excessive inconvenience to the Consumer.
21. In the situation when the defective item was installed, the Consumer may request dismantlement from the Seller and re-installation after the replacement into the free of defects item or defect removal. In case of non-performance of the obligation by the Seller, the Consumer shall be entitled to do these actions at the expense and risk of the Seller.
22. The Seller can refuse the dismantlement or re-installation provided that the cost of such actions overpasses the value of the item sold.
23. The Consumer, in the case referred to in above, may request dismantlement and re-installation from the Seller, however he/she shall be obliged to bear part of the costs related to it at the value overpassing the sold item price or may request payment of the part of the costs of the dismantlement or re-installation from the Seller up to the sold item price.
24. In case of the contracts concluded between the Client, not being the consumer under the art. 22¹ of the Civil Code, pursuant to art. 558 section 1 of the Civil Code, the Seller’s liability under warranty shall be excluded.
25. In compliance with the terms set out in subparagraph 2,3 and 4 of this section of the Terms & Conditions the Consumer may make a statement concerning his/her backing out of the contract or price reduction due to the physical defect of the sold item, and when the Consumer requested the replacement of the item into the free of defects one or the defect removal, the time limits to the submission of the statement on backing out of the contract or price reduction run with the moment of the ineffective expiry of a term to the item replacement or defect removal.
26. In the event of judicial proceedings before the arbitration court of one of the warranty rights, due date of other rights, provided for by law, to which the Consumer is entitled pursuant to this shall be suspended until final termination of proceedings thereupon. These regulations are respectively applied for the mediation proceedings, whereas due date of other warranty rights, to which the Consumer is entitled, begins from the day of settlement agreed before a mediator acceptance refusal by court or ineffective mediation termination.
27. Time limits to the execution of warranty rights for the legal defects of item sold run from the day on which the Consumer discovered the defect and in case the Consumer discovered the defect only as a result of a third party complaint - from the day on which the predicate or other decision of the authority issued within a dispute with the third party became legally binding.
28. When due to of the sold item defect the Consumer submitted a statement on backing out of the contract or price reduction, he/she can request redressing of a damage incurred as a result of the Contract conclusion without being aware of the defect, even if the damage results from the circumstances of which the Seller is not liable for, and in particular he/she can request reimbursement of the costs of conclusion of Contract, collection, proceedings, items insurance storage, return of expenditures made to the extent to which he/she benefited and not received the aforementioned from a third party.
29. The foregoing shall not prejudice the generally applicable laws on duty to redress the damage under relevant laws and regulations.
30. Should the Seller conceal a defect, end of the term shall not exclude warranty rights.
33. The Store shall not accept cash on delivery shipments. All the costs of justified complaint shall be returned to the client to the bank account specified by the client.
1. Goods sold through the online Store selecthop.pl shall be covered by the Seller’s and Producer’s guarantee. Both the Producer and the Seller shall provide a one year guarantee for the purchased item. The Seller shall be liable for the item inconformity with the contract under warranty stipulated in section VII Complaint - Warranty.
2. Detailed conditions of the guarantee shall be determined in a guarantee card issued by the guarantor.
1. The Buyer shall be entitled to replace the given item for a different size or model within 30 days upon item reception.
2. Forwarding costs both directions shall be borne by the Buyer.
3. Returned item cannot show traces of use, it should have factory packaging and labels, in other case the item will be sent back on Buyer’s expense.
4. Replacement shall be done immediately upon the shipment reception by the Store not later than within 14 days from the reception.
5. We kindly ask you to arrange the replacements with the online Store selecthop.pl service.
2. Statement can be submitted on the form constituting Attachment to these Terms & Conditions and which was forwarded to the Consumer together with the item, also available at www.selectshop.eu or in a different form, in accordance with the act on consumer rights.
3. Alongside the statement referred to in subparagraph 1. Client shall be also asked to provide information on the current bank account for reimbursement.
to the Consumer immediately upon reception. Such confirmation shall be forwarded to the e-mail address, given alongside the Order or indicated as contact on the submitted statement on backing out of the contract.
5. The Contract shall be considered not concluded in case of backing out of the contract.
7. The Consumer shall return the item or items of the Contract of which he/she has backed out on their own risk and expense.
8. The Consumer shall be liable for the contractual item or items value reduction, resulting from the use of the item or items in the way extending beyond the ordinary item management in order to determine features, functionalities and the nature of the item.
10. In the event of backing out of the distant contract for the services commenced under the Consumer’s consent prior to the deadline of backing out of the Contract, the Consumer shall be obliged to pay the performance executed by the time of backing out of the Contract.
11. The payment amount shall be calculated proportionally to the extent of executed performance, including the price or remuneration included in the Contract. In case the price or remuneration is excessive, the basis for calculation of this amount shall be the market value of the performance executed.
12. The Consumer shall not bear expenses of the supply of digital content which is not saved on a tangible medium provided that he/she has not approved the performance execution prior to the deadline for backing out of the Contract expiry or has not been informed about the loss of the deadline of his/her right to back out of the Contract by the time of such approval or the entrepreneur has not provided confirmation under art. 15 para. 1 and art. 21 para 1 of the Act on consumer rights.
13. The Seller can withhold the reimbursement of the payment received from the Consumer until the item is returned or the evidence of shipping it back is provided, whichever is sooner.
14. The Consumer shall not be entitled to back out of the contract in cases indicated in art. 38 of the Act on consumer rights.
2. Provision of electronic Services by the Service Provider shall be cost-free.
3. Electronic Service contract for Account operation at the online Store and provision of the Newsletter Service shall be concluded for an indefinite duration.
4. Electronic service contract for allowing to place an order through the relevant Form shall be concluded for a definite period of time and shall be terminated as soon as the order is placed or order placement is ceased by the Service Recipient.
of an unlawful nature. The use of electronic Services in the way that unlawfully disturbs the functioning of the online Store through the use of specified software or devices or distribution or location of unsolicited commercial communication at the online Store shall not be allowed.
8. The Service Recipient can terminate the service not limited by time on provision of electronic Service of a continuous nature at any time and without indicating the reasons by sending appropriate statement by e-mail to the address: info@selectshop.eu or in writing to the address: Przedsiębiorstwo Wielobranżowe „RAPU” Robert Zapora, ul. Boya-Żeleńskiego 11, 35-105 Rzeszów.
9. The Service Provider can terminate the contract on provision of electronic Service not limited by time in case the Service Recipient infringes the Terms & Conditions objectively and persistently, and in particular supplies content of an unlawful nature, after ineffective, one-time call to stop with the relevant date determination. In this case, the contract shall come to an end after 7 days upon the submission of a statement of will of termination.
10. The Service Provider and Service Recipient may terminate the Contract on provision of electronic Service at any time by common agreement between the parties.
11. Termination of the Contract on provision of electronic service concluded for an indefinite period by the Service Provider and Service Recipient shall not prejudice rights or performances obtained by the Parties during the course of the contract.
12. The Service Provider shall respond in case of a Complaint to the e-mail address given by the Service Recipient, or other agreed-upon manner.
1. Amendments to this Terms & Conditions can be made upon prior notification of the Users about the scope of the envisaged amendments not later than within 14 days before the date of their entry into force.
2. Orders placed during the term of the previous Terms & Conditions version shall be realized according to its provisions. The User can remove his/her Account in case of his disagreement with the amendments to the Terms & Conditions to be implemented.
3. Any disputes between the Seller and the Client who is a Consumer under the art. 22¹ of the Civil Code shall be settled before the common court competent in compliance with provisions of the Code of Civil Procedure.
4. Any disputes between the Seller and the Client who is not a Consumer under the art. 22¹ of the Civil Code shall be settled before the common court competent over the seat of the Store.
6. Clients can gain access to this Terms & Conditions at any time through the link placed on the main page of the selectshop.eu online Store.
7. The Terms & Conditions can be consolidated, obtained and reproduced by printing or saving on appropriate data carrier.
8. Name of the selectshop.eu online Store, address where it may be found: www.selectshop.eu and all other materials included there are object of copyrights and are subject to legal protection. The use and distribution of them without the Store owner’s consent is forbidden.
9. This Terms & Conditions shall apply from September 30, 2016.
1. The Controller of the data gathered through the online store shall be the store owner.
2. The online store shall operate according to the regulations of the generally applicable law, in particular to the regulations of the act of August 29, 1997 on personal data protection (i.e. consolidated text of Dz.U. of 2002, no. 101, item 926, as amended) and of the Act of July 18, 2002 on provision of electronic services (Dz. U. no. 144, item 1204, as amended).
3. The Controller shall be entitled to make the personal data available only to the entities authorized by law.
4. The store owner shall be obliged to protect personal data transferred by the Users against loss, damage, disclosure, access of unauthorized persons or their improper use.
5. The data Controller shall process them with a view to the contracts being concluded through the online store and for the purposes relating to the DM of own products.
7. The User shall voluntarily provide his/her personal data required for registration.
8. According to the applicable law, in case of a request of Client’s personal data removal, the Client’s data located on the sales document stored for the fiscal purposes shall not be removed.
9. The Controller shall give Client’s gathered data to the selected forwarder in the scope necessary to perform product delivery to the Client.
2. Cookie files are text files saved on the User’s computer by his/her Internet browser allowing the User identification and consolidating his/her preferences on the basis of the history of visited sites.
3. Websites browsing software ( Internet browser ) usually allows storing cookies in the final User’s device by default. Users can change the settings in this regard. Internet browser allows cookie files deletion. Automatic cookie files blocking is possible. Detailed information concerning this matter are included in the help or documentation of the Internet browser.
4. Cookie type files allow maintenance of the User session without the need to re-entering the login and password on every subpage.
8. Above data shall not be associated with specified persons visiting websites.
9. All the employees of the Controller have been properly trained in the scope of personal data protection.
10. The access to personal data is given only to the persons who own relevant authorization for such access.


text:
The Supreme Court of Texas, deeming habeas corpus an appropriate method for challenging the validity of the statute as applied, 7 sustained the Act as a valid exercise of the State's police power, taken 'for the protection of the general welfare of the public, and particularly the laboring class,' (141 Tex. 591, 174 S.W.2d 961) with special reference to safeguarding laborers from imposture when approached by an alleged organizer. The provision, it was said, 'affects only the right of one to engage in the business as a paid organizer, and not the mere right of an individual to express his views on the merits of the union.' The court declared the act 'does not require a paid organizer to secure a license,' but makes mandatory the issuance of the card 'to all who come within the provisions of the Act upon their good-faith compliance therewith.' Accordingly it held that the regulation was not unreasonable.
Appellant first urges that the application of the statute amounted to the requirement of a license 'for the simple act of delivering an address to a group of workers.' He says the act penalized 'was simply and solely the act of addressing the workers on the * * * benefits of unionism, and concluding the address with a plea to the audience generally and to a named worker in the audience to join a union.' He points out that he did not parade on the streets, did not solicit or receive funds, did not 'sign up' workers, 9 engaged in no disturbance or breach of the peace, and that his sole purpose in going to Texas and his sole activity there were to make the address including the invitations which he extended at the end. There is no evidence that he solicited memberships or members for a union at any other time or occasion or intended to do so. His position necessarily maintains that the right to make the speech includes the right to ask members of the audience, both generally and by name, to join the union.
Finally, as the case is presented here, Texas apparently would rest the validity of the judgment exclusively upon the specific individual solicitation of O'Sullivan, and would throw out of account the general invitation, made at the same time, to all nonunion workers in the audience. 14 However, the case cannot be disposed of on such a basis. The Texas Supreme Court made no distinction between the general and the specific invitations. 15 Nor did the District Court. The record shows that the restraining order was issued in explicit anticipation of the speech and to restrain Thomas from uttering in its course any language which could be taken as solicitation. 16 The motion for the fiat in contempt was filed and the fiat itself was issued on account of both invitations. 17 The order adjudging Thomas in contempt was in general terms, finding that he had violated the restraining order, without distinction between the solicitations set forth in the petition and proved as violations. 18 The sentence was a single penalty. In this state of the record it must be taken that the order followed the prayer of the motion and the fiat's recital, and that the penalty was imposed on account of both invitations. The judgment therefore must be affirmed as to both or as to neither. Cf. Williams v. North Carolina, 317 U.S. 287, 292, 63 S.Ct. 207, 210, 87 L.Ed. 279, 143 A.L.R. 1273; Stromberg v. California, 283 U.S. 359, 368, 51 S.Ct. 532, 535, 75 L.Ed. 1117, 73 A.L.R. 1484. And it follows that the statute, as it was applied, restrained and punished Thomas for uttering, in the course of his address, the general as well as the specific invitation.
For these reasons any attempt to restrict those liberties must be justified by clear public interest, threatened not doubtfully or remotely, but by clear and present danger. 19 The rational connection between the remedy provided and the evil to be curbed, which in other contexts might support legislation against attack on due process grounds, will not suffice. These rights rest on firmer foundation. Accordingly, whatever occasion would restrain orderly discussion and persuasion, at appropriate time and place, must have clear support in public danger, actual or impending. Only the gravest abuses, endangering paramount interests, give occasion for permissible limitation. It is therefore in our tradition to allow the widest room for discussion, the narrowest range for its restriction, particularly when this right is exercised in conjunction with peaceable assembly. It was not by accident or coincidence that the rights to freedom in speech and press were coupled in a single guaranty with the rights of the people peaceably to assemble and to petition for redress of grievances. All these, though not identical, are inseparable. They are cognate rights, cf. De Jonge v. Oregon, 299 U.S. 353, 364, 57 S.Ct. 255, 259, 81 L.Ed. 278, and therefore are united in the First Article's assurance. Cf. 1 Annals of Congress 759760.
Thomas went to Texas for one purpose and one onlyto make the speech in question. Its whole object was publicly to proclaim the advantages of workers' organization and to persuade workmen to join Local No. 1002 as part of a campaign for members. These also were the sole objects of the meeting. The campaign, and the meeting, were incidents of an impending election for collective bargaining agent, previously ordered by national authority pursuant to the guaranties of national law. Those guaranties include the workers' right to organize freely for collective bargaining. And this comprehends whatever may be appropriate and lawful to accomplish and maintain such organization. It included, in this case, the right to designate Local No. 1002 or any other union or agency as the employees' representative. It included their right fully and freely to discuss and be informed concerning this choice, privately or in public assembly. Necessarily correlative was the right of the union, its members and officials, whether residents or nonresidents of Texas and, if the latter, whether there for a single occasion or sojourning longer, to discuss with and inform the employees concerning matters involved in their choice. These rights of assembly and discussion are protected by the First Amendment. Whatever would restrict them, without sufficient occasion, would infringe its safeguards. The occasion was clearly protected. The speech was an essential part of the occasion, unless all meaning and purpose were to be taken from it. And the invitations, both general and particular, were parts of the speech, inseparable incidents of the occasion and of all that was said or done.
Accordingly, decision here has recognized that employers' attempts to persuade to action with respect to joining or not joining unions are within the First Amendment's guaranty. National Labor Relations Board v. Virginia Electric & Power Co., 314 U.S. 469, 62 S.Ct. 344, 86 L.Ed. 348. Decisions of other courts have done likewise. 21 When to this persuasion other things are added which bring about coercion, or give it that character, the limit of the right has been passed. 22 Cf. National Labor Relations Board v. Virginia Electric & Power Co., supra. But short of that limit the employer's freedom cannot be impaired. The Constitution protects no less the employees' converse right. Of course espousal of the cause of labor is entitled to no higher constitutional protection than the espousal of any other lawful cause. It is entitled to the same protection.
How far the State can require previous identification by one who undertakes to exercise the rights secured by the First Amendment has been largely undetermined. It has arisen here chiefly, though only tangentally, in connection with license requirements involving the solicitation of funds, Cantwell v. Connecticut, supra; cf. Schneider v. State, 308 U.S. 147, 60 S.Ct. 146, 84 L.Ed. 155; Largent v. Texas, 318 U.S. 418, 63 S.Ct. 667, 87 L.Ed. 873, and other activities upon the public streets or in public places, cf. Lovell v. Griffin, 303 U.S. 444, 58 S.Ct. 666, 82 L.Ed. 949; Hague v. Committee for Industrial Organization, 307 U.S. 496, 59 S.Ct. 954, 83 L.Ed. 1423, or house-to-house canvassing, cf. Schneider v. State, supra. In these cases, however, the license requirements were for more than mere identification or previous registration and were held invalid because they vested discretion in the issuing authorities to censor the activity involved. Nevertheless, it was indicated by dictum in Cantwell v. Connecticut, 310 U.S. 296, 306, 60 S.Ct. 900, 904, 84 L.Ed. 1213, 128 A.L.R. 1352, 23 that a statute going no further than merely to require previous identification would be sustained in respect to the activities mentioned. Although those activities are not involved in this case, that dictum and the decision in People of State of New York ex rel. Bryant v. Zimmerman, 278 U.S. 63, 49 S.Ct. 61, 73 L.Ed. 184, 62 A.L.R. 785, furnish perhaps the instances of pronouncement or decision here nearest this phase of the question now presented.
As we think the requirement of registration, in the present circumstances, was in itself an invalid restriction, we have no occasion to consider whether the restraint as imposed goes beyond merely requiring previous identification or registration. 24 Nor do we undertake to determine the validity of Section 5 in any other application than that made upon the facts of this case. Neither do we ground our decision upon other contentions advanced in the briefs and argument. Upon the reargument attention was given particularly to the questions whether and to what extent the prohibitions of Section 5, or their application in this case, are consistent with the provisions of the National Labor Relations Act. Both the parties and the Government, which has appeared as amicus curiae, have advanced contentions on this issue independent of those put forward upon the question of constitutionality. Since a majority of the Court do not agree that Section 5 or its present application conflicts with the National Labor Relations Act, our decision rests exclusively upon the grounds we have stated for finding that the statute as applied contravenes the Constitution.
As frequently is the case, this controversy is determined as soon as it is decided which of two well-established, but at times overlapping, constitutional principles will be applied to it. The State of Texas stands on its well-settled right reasonably to regulate the pursuit of a vocation, includingwe may assumethe occupation of labor organizer. Thomas, on the other hand, stands on the equally clear proposition that Texas may not interfere with the right of any person peaceably and freely to address a lawful assemblage of workmen intent on considering labor grievances.
Fourthly, the Act and the injunction which he disobeyed say nothing of speech; they are aimed at a transactionthat of solicitation of members for a union. This, and this only, is the statutory object which is said to render it unconstitutional.
As respects the second, I see no reason to limit what was said in Cantwell v. Connecticut, 310 U.S. 296, 305, 60 S.Ct. 900, 904, 84 L.Ed. 1213, 128 A.L.R. 1352, to solicitation of money. The solicitation at which the Texas Act is aimed may or may not involve the payment of initiation fees or dues to the solicitor. But, in any case, it involves the assumption of business and financial liability by him who is persuaded to join a union. The transaction is in essence a business one. Labor unions are business associations; their object is generally business dealings and relationships as is manifest from the financial statements of some of the national unions. Men are persuaded to join them for business reasons, as employers are persuaded to join trade associations for like reasons. Other paid organizers, whether for business or for charity, could be required to identify themselves. There is no reason why labor organizers should not to likewise. I think that if anyone pursues solicitation as a business for profit, of members for any organization, religious, secular or business, his calling does not bar the state from requiring him to identify himself as what he is,a paid solicitor.
Without doubt a State may protect its citizens from fraudulent solicitation by requiring a stranger in the community, before permitting him publicly to solicit funds for any purpose, to establish his identity and his authority to act for the cause which he purports to represent,' (emphasis added) citing for comparison Lewis Publishing Co. v. Morgan, 229 U.S. 288, 306-310, 33 S.Ct. 867, 871873, 57 L.Ed. 1190; People of State of New York ex rel. Bryant v. Zimmerman, 278 U.S. 63, 72, 49 S.Ct. 61, 64, 73 L.Ed. 184, 62 A.L.R. 785. Cf. text infra at note 23.

matches: v. v. v. v. v. v. v. v. v. v. v. v. v. v. v. v.