Laser-ultraviolet-A-induced ultraweak photon emission in mammalian cells

Photobiological research in the last 30 yr has shown the existence of ultraweak photon emission in biological tissue, which can be detected with sophisticated photomultiplier systems. Although the emission of this ultraweak radiation, often termed biophotons, is extremely low in mammalian cells, it can be efficiently increased by ultraviolet light. Most recently it was shown that UV-A (330 to 380 nm) releases such very weak cell radiation in differentiated human skin fibroblasts. Based on these findings, a new and powerful tool in the form of UV-A-laser-induced biophotonic emission of cultured cells was developed with the intention to detect biophysical changes between carcinogenic and normal cells. With suspension densities ranging from 1 to 8 x 10(6) cells/mL, it was evident that an increase of the UV-A-laser-light induced photon emission intensity could be observed in normal as well as melanoma cells. Using this new detection procedure of ultraweak light emission, photons in cell suspensions as low as 100 microL could be determined, which is a factor of 100 lower compared to previous procedures. Moreover, the detection procedure has been further refined by turning off the photomultiplier system electronically during irradiation leading to the first measurements of induced light emission in the cells after less than 10 micros instead of 150 ms, as reported in previous procedures. This improvement leads to measurements of light bursts up 10(7) photons/s instead of several hundred as found with classical designs. Overall, we find decreasing induction ratings between normal and melanoma cells as well as cancer-prone and melanoma cells. Therefore, it turns out that this highly sensitive and noninvasive device enables us to detect high levels of ultraweak photon emission following UV-A-laser-induced light stimulation within the cells, which enables future development of new biophysical strategies in cell research.     

Inhibition of protein kinase C-dependent protein phosphorylation correlates with increased polarity and locomotion in Walker 256 carcinosarcoma cells

Signal transduction pathways controlling tumor cell locomotion are not yet well understood. We have studied the role of protein kinase C (PKC)-dependent protein phosphorylation associated with changes in cell shape and locomotor activity of Walker carcinosarcoma cells in culture. We show that the inhibitory effect of phorbol-12-myristate-13-acetate (PMA), an activator of PKC, on cell polarity and locomotion can be suppressed by the PKC-selective inhibitor Ro 31-8220. PMA induces increased phosphorylation of at least 2 proteins, of 65 and 80 kDa, in intact Walker carcinosarcoma cells. These bands are enriched in cytosolic fractions isolated from cells incubated with ³²PO₄. Pre-incubation with Ro 31-8220 inhibits the PMA-induced phosphorylation of both bands in a concentration-dependent manner. This effect is very likely not due to inhibition of translocation of PKC to the membrane as Ro 31-8220 enhances, rather than inhibits, PMA-induced transfer of PKC β₁₁ to the particulate fraction. We have carried out a quantitative analysis of phosphorylation of the 80-kDa band. Ro 31-8220 reverses both PMA-induced phosphorylation of this band and PMA-induced suppression of cell polarity in parallel. Increased phosphorylation of proteins via PKC may thus be a stop signal for locomoting Walker carcinosarcoma cells. © 1996 Wiley-Liss, Inc.     

Paradoxical SR Ca2+ release in guinea-pig cardiac myocytes after β-adrenergic stimulation revealed by two-photon photolysis of caged Ca2+

In heart muscle the amplification and shaping of Ca²⁺ signals governing contraction are orchestrated by recruiting a variable number of Ca²⁺ sparks. Sparks reflect Ca²⁺ release from the sarcoplasmic reticulum (SR) via Ca²⁺ release channels (ryanodine receptors, RyRs). RyRs are activated by Ca²⁺ influx via L-type Ca²⁺ channels with a specific probability that may depend on regulatory mechanisms (e.g. β-adrenergic stimulation) or diseased states (e.g. heart failure). Changes of RyR phosphorylation may be critical for both regulation and impaired function in disease. Using UV flash photolysis of caged Ca²⁺ and short applications of caffeine in guinea-pig ventricular myocytes, we found that Ca²⁺ release signals on the cellular level were largely governed by global SR content. During β-adrenergic stimulation resting myocytes exhibited smaller SR Ca²⁺ release signals when activated by photolysis (62.3% of control), resulting from reduced SR Ca²⁺ content under these conditions (58.6% of control). In contrast, local signals triggered with diffraction limited two-photon photolysis displayed the opposite behaviour, exhibiting a larger Ca²⁺ release (164% of control) despite reduced global and local SR Ca²⁺ content. This apparent paradox implies changes of RyR open probabilities after β-adrenergic stimulation, enhancing local regenerativity and reliability of Ca²⁺ signalling. Thus, our results underscore the importance of phosphorylation of RyRs (or of a related protein), as a regulatory physiological mechanism that may also provide new therapeutic avenues to recover impaired Ca²⁺ signalling during cardiac disease.     

The CTLA-4 gene region of chromosome 2q33 is linked to, and associated with, type 1 diabetes. Belgian Diabetes Registry

Susceptibility to autoimmune insulin-dependent (type 1) diabetes mellitus is determined by a combination of environmental and genetic factors, which include variation in MHC genes on chromosome 6p21 (IDDM1) and the insulin gene on chromosome 11p15 (IDDM2). However, linkage to IDDM1 and IDDM2 cannot explain the clustering of type 1 diabetes in families, and a role for other genes is inferred. In the present report we describe linkage and association of type 1 diabetes to the CTLA-4 gene (cytotoxic T lymphocyte associated-4) on chromosome 2q33 (designated IDDM12). CTLA-4 is a strong candidate gene for T cell- mediated autoimmune disease because it encodes a T cell receptor that mediates T cell apoptosis and is a vital negative regulator of T cell activation. In addition, we provide supporting evidence that CTLA-4 is associated with susceptibility to Graves' disease, another organ- specific autoimmune disease.      

Trisomy 1q generating translocations in Wilms tumor

Unbalanced translocations generating trisomy of 1q are common in Wilms tumor (WT). We present eight unbalanced 1q translocations from seven tumors and a review of the literature. An unbalanced translocation that results in a der(16)t(1q;16q) chromosome represents more than half of the published +1q generating translocations in WT. This translocation is also common to many other tumor types. Four of the tumors presented here contained this chromosome and,in two cases, it was the primary acquired cytogenetic abnormality within the tumor. The other four translocations involved 9q31, 9q34, 17p1?, and 21p11 as the partner to 1q. The chromosome 17 and 21 translocations occurred within the same tumor as apparently independent events. In contrast with the 16q translocations, these other translocations were secondary cytogenetic events, thereby indicating a role in tumor progression rather than initiation. Probes mapping to 1q12 and 1q21 were employed for fluorescence in situ hybridization and it was demonstrated that different 1q breakpoints are possible. In this series, the majority of breakpoints either mapped to 1q12 or were centromeric to this region.