Analysis of 1H NMR-detectable mobile lipid domains for assessment of apoptosis induced by inhibitors of DNA synthesis and replication

Cell membrane rearrangements coincident with apoptosis may contribute to the increase in the ratio of methylene (CH(2) at 1.3 ppm) to methyl (CH(3) at 0.9 ppm) resonance signal intensity as observed by proton nuclear magnetic resonance ((1)H NMR). We studied CH(2) and CH(3) resonances in cultured cell lines treated with etoposide and fludarabine or bioflavonoid quercetin. Etoposide treatment (10 microM, 18 h) resulted in 3.3 fold increase of the CH(2)/CH(3) signal intensity ratio and 6.4 fold decrease in choline signal of MT4 cells. Incubation of Namalwa cells with fludarabine (3 microM, 72 h) increased the CH(2)/CH(3) signal intensity ratio by 2.4 fold and choline resonance intensity was unchanged. Quercetin treatment (30 microM, 1.5 month) increased CH(2)/CH(3) ratio by 2.1 fold. Necrotic cell death upon ethanol (20%) or DMSO (30%) treatment did not change the CH(2)/CH(3) signal intensity ratio. (1)H NMR-based study of mobile lipid domains is sensitive for detection of early engagement into apoptosis.     

Temporal dynamics of 1H-NMR-visible metabolites during radiation-induced apoptosis in MG-63 human osteosarcoma spheroids

The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH(2) and CH(3) lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.     

Effects of TNF-alpha/colchicine combined treatment on Burkitt lymphoma cells: molecular and ultrastructural changes

Tumour necrosis factor alpha (TNF-alpha) kills Daudi cells (Human Burkitt Lymphoma), inducing either necrosis or apoptosis without DNA fragmentation. Therefore, we were interested in studying the molecular and ultrastructural events occurring when the nucleus is more accessible and cells are blocked in mitosis, following colchicine treatment. In fact, as early as after 1 h treatment a typical ladder pattern was shown by means of DNA gel electrophoresis. In parallel the quantitative analysis of the different morphological patterns observed gave evidence of an increased percentage of primary necrosis after 6 h treatment, and a higher incidence of cells in late apoptosis as well as in secondary necrosis after 24 h treatment. Our findings show that Daudi cells respond to the combined treatment with an increased formation of micronuclei and nuclear alterations which follow a number of early mitochondrial changes and result in enhanced cell death. These data imply that TNF-alpha-induced apoptosis of Daudi cells can be triggered by mitochondrial changes and is somehow related to microtubule organization.     

Apoptosis in canine corpus luteum during spontaneous and prostaglandin-induced luteal regression

Spontaneous luteal regression and prostaglandin-induced luteolysis in bitches were evaluated by measuring the apoptotic index for DNA fragmentation and the relative level of Bax gene expression in ovaries removed from nine untreated nonpregnant bitches at selected times during diestrus and in nine pregnant bitches after 1 day of administering abortive doses of a PGF-analog gel formulation given intravaginally at selected times during gestation. Nonpregnant diestrus was divided into three periods (early, mid and late) based on vaginal cytology and plasma progesterone concentration. Pregnant bitches were treated with a PGF-analog gel at corresponding stages of pregnancy (early, mid and late) and evaluated by ultrasound. Another eight pregnant bitches were similarly studied and serum progesterone concentrations were determined after 1, 2, 3 or 4 days of PGF-analog gel. Corpora lutea obtained by ovariohysterectomy were analyzed for apoptotic internucleosomal DNA fragmentation relative to that in a control cell line (U937), using an apoptotic DNA ladder kit and gel electrophoresis and for relative expression of the pro-apoptotic Bax gene by RT-PCR and electrophoresis. In nonpregnant bitches, the DNA fragmentation apoptotic index was greater in late than in early diestrus (P < 0.01). The index after 1 day of PGF-analog gel was higher in early pregnant bitches than in early diestrus bitches (P < 0.05); it was highest in midpregnancy (P < 0.05). The degree of apoptosis was related to the number of times PGF-analog gel was administered. Bax mRNA was detected in the corpus luteum (CL) and Bax expression increased from early to middiestrus in nonpregnant subjects (P < 0.05). Potential elevation in Bax due to PGF-analog gel treatment in pregnancy was only significant in relation to normal diestrus during early pregnancy (P < 0.01). In conclusion, we inferred that the effects of endogenous or exogenous prostaglandin on CL life span in bitches involved increases in apoptotic activity and that increased apoptosis was implicated in normal luteal regression in nonpregnant bitches.     

Radiation-induced apoptosis: a new approach using infrared microspectroscopy

PURPOSE: to characterize radiation-induced apoptosis in human cells using Fourier transform infrared microspectroscopy (FT-IRM) as a new analytical tool. MATERIAL AND METHODS: Normal human circulating lymphocytes were given a gamma ray dose of 6 Gy, or treated with t-butyl hydroperoxide (t-BuOH). HaCaT keratinocytes were given a dose of 20 Gy. Cells were deposited on ZnS windows for infrared spectral acquisition 2 days and 2 h after irradiation and 2 h after t-BuOH treatment. Apoptosis was simultaneously assessed by flow cytometry analysis of cells displaying annexin-V-positive staining. RESULTS: The flow cytometry study showed that about 90% of sham and irradiated cells were annexin-V negative 2 h after irradiation. Two days after irradiation, 68% of lymphocytes and 76% of HaCaT cells were apoptotic, as well as 43% of lymphocytes treated with t-BuOH. In infrared spectra of these apoptotic cells, qualitative and quantitative changes were observed. In the 960-1245 and 1690-1720 cm-1 ranges, mainly attributed to nucleic acids, changes corresponding to conformational changes in DNA were associated with a decrease in the amount of detectable DNA. Conformational changes were also observed in secondary protein structure, in particular an increase in the amount of beta structures. These DNA and protein changes were associated with an increase in the detectable amount of lipids in apoptotic HaCaT cells only. Two hours after irradiation, depending on the dose and (or) the cell type, qualitative and quantitative changes were observed in the IR spectra in the amide I and amide II bands, mainly attributed to proteins. These changes were associated with a significant decrease in the 1700-1750 cm-1 range, mainly attributed to the -C=O ester groups of DNA and phospholipids, in the irradiated HaCaT cells only. CONCLUSION: Our results are in agreement with biochemical published data on radiation-induced apoptosis, and show that DNA is the first cellular target of radiation-induced apoptosis, which, however, also requires conformational changes and synthesis of cell proteins. They also demonstrate that FT-IRM may be useful for assessing the early radiation damage at the molecular level in human cells.     

Critical self-association of bile lipids studied by infrared spectroscopy and viscometry

Fourier transform infrared (FTIR)-attenuated total reflection (ATR) spectroscopy and viscometry were applied to study the micellization of two bile lipids, sodium taurochenodeoxycholate (NaTCDC) and sodium glycocholate (NaGC), in aqueous solutions. The CH2 stretching bands of the bile lipid hydrocarbon region were shifted to higher frequencies suggesting initial critical micellization at 2.5 mM for NaTCDC and 9 mM for NaGC. An abrupt enhancement of the absorption intensity of the CH3 groups of the sterol rings in bile lipids were under conformational strain at 3.5 mM NaTCDC and 9 mM NaGC. Viscometry measurements showed abrupt changes in viscosities in the region of critical micellar concentration (CMC) of both bile lipids. Both infrared and viscometry studies confirmed the onset of conformational strains in tightly packed lipid micelles at their CMC. In addition, FTIR/ATR spectroscopy has defined the specific hydrophobic interactions which bring about critical micellization of bile lipids.     

Changes in activities of superoxide dismutase, nitric oxide synthase, glutathione-dependent enzymes and the incidence of apoptosis in sheep corpus luteum during the estrous cycle

Anti-oxidative enzymes play a role in protecting cells from oxidative stress-induced cell death. The present study was conducted to evaluate whether the anti-oxidant and pro-oxidant enzymatic capacities of the sheep corpus luteum (CL) are correlated with steroidogenic and structural status of the gland during the estrous cycle. Steroidogenic activity, apoptosis and superoxide dismutase (SOD1 and SOD2), nitric oxide synthase (NOS), glutathione peroxidase (GPX), glutathione reductase (GSR) and glutathione S-transferase (GST) activities were determined in the CL at specific developmental stages of the luteal phase. The intensity of apoptotic DNA fragmentation, characteristic of physiological cell death, was much greater in CL at late luteal phase than at early and mid-luteal phase, concomitantly with the diminution in the plasma progesterone concentrations from mid-to late luteal phase. SOD1 and GPX activities increased from early to mid-luteal phase, and increased further at late luteal phase. SOD2 and GST activities were not different between early and mid-luteal phase, but increased at late luteal phase. GSR activity was not different between any luteal phase examined. NOS activity decreased from early to mid- and late luteal phase. These results show that the activities of SOD1, SOD2, NOS, GPX, GSR and GST in the sheep CL are subject to major changes during the estrous cycle, and that the anti-oxidant and pro-oxidant enzymatic capacities of luteal cells are not correlated with cell steroidogenic status and integrity during the late luteal phase.     

Metabolomics using 1H-NMR of apoptosis and Necrosis in HL60 leukemia cells: differences between the two types of cell death and independence from the stimulus of apoptosis used

High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The 1H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH2 and CH3 mobile lipids, principally of CH2, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of 1H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH2 mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in 1H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.     

Early and late responses to oxidative stress in human dermal fibroblasts of healthy donors and rheumatoid arthritis patients. Relationship between the cell death rate and the genomic dosage of active ribosomal genes

A study was made of the effect of the oxidizing agent potassium chromate (K2CrO4, PC) on cultured dermal fibroblasts of a healthy donor and three patients with rheumatoid arthritis (RA). Characteristics of the rRNA gene (RG) complex-RG copy number, active RG (ARG) dosage, and 18S rRNA content--were determined for each cell line. In cells of the healthy donor, oxidative stress caused by low doses of PC (2-4 microM, 1-4 h) induced an early response, including a 50-80% increase in total RNA and rRNA. An appreciable activation of the nucleolus was observed cytochemically, by silver staining and morphometry. The early response grew considerably lower with the increasing passage number and/or PC concentration. Exposure to 6-12 microM PC for 24 h led to a progressive cell death (late response). The existence and intensity of the early response correlated positively with the cell survival during further culturing. Cells of the RA patients displayed almost no early response even at early passages: total RNA did not increase, and rRNA increased by no more than 10%. Cell disruption (apoptosis) during further culturing was more intense than in the line originating from the healthy donor. The apoptosis intensity characterized by the increase in the content of DNA fragments in the culture medium and in the caspase 3 activity, was inversely proportional to the ARG dosage in the genome. The results provide the first quantitative characterization of the early and late responses of cells to PC-induced oxidative stress and suggest a role of the ARG dosage in cell survival in stress.     

Increases in 1H-NMR mobile lipids are not always associated with overt apoptosis: evidence from MG-63 human osteosarcoma three-dimensional spheroids exposed to a low dose (2 Gy) of ionizing radiation

The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.     

Lymphocytes apoptosis: young versus aged and humans versus rats

This paper deals with a comparative study of lymphocyte apoptosis in young versus aged and humans versus rats. Apoptotic rate achieved by the use of different apoptogenic inducers, acting at different cellular levels, and cell surface modifications were analyzed. The results showed that aged human lymphocytes and freshly isolated rat lymphocytes were more prone to undergo apoptosis. Therefore, the same apoptotic signal is recognized by human and rat lymphocytes, but the extent of the answer is related to the species, to the intensity of the apoptotic stimulus and to the metabolic/developmental condition of the cells. Surface modifications (lipids and glycans), typical of apoptosis, were observed. Our data showed that cell surface changes are species and age dependent. They are early events, progressively achieved in the course of the apoptotic process involving lateral membrane movements of molecules.     

脂可平对实验性早期动脉粥样硬化内皮细胞凋亡及Bax、Bcl-2表达的影响

研究脂可平对大鼠血脂、早期动脉粥样硬化血管内皮细胞凋亡及Bax、Bcl-2表达的影响。应用高脂饲料复制SD大鼠早期动脉粥样硬化模型,分别给以脂可平、辛伐他汀片混悬液灌胃,实验10周后,全自动生化分析仪、流式细胞仪定量分别检测各组血脂、主动脉内皮细胞凋亡率及Bax、Bcl-2蛋白的表达;苏木素伊红(HE)染色观察主动脉组织形态学变化。两治疗组均可降低血脂,不同程度的改善了主动脉组织病理损伤,降低内皮细胞凋亡率,调控Bax、Bcl-2蛋白表达。本实验说明脂可平可降低血脂,干预动脉粥样硬化的始动环节及其发生并从蛋白水平调控早期动脉粥样硬化内皮细胞凋亡。     

Apoptosis of non-parasitised red blood cells in Plasmodium yoelii malaria

Recently, while studying erythrocytic apoptosis during Plasmodium yoelii infection, we observed an increase in the levels of non-parasitised red blood cell (nRBC) apoptosis, which could be related to malarial anaemia. Therefore, in the present study, we attempted to investigate whether nRBC apoptosis is associated with the peripheral RBC count, parasite load or immune response. To this end, BALB/c mice were infected with P. yoelii 17XL and nRBC apoptosis, number of peripheral RBCs, parasitaemia and plasmatic levels of cytokines, nitric oxide and anti-RBC antibodies were evaluated at the early and late stages of anaemia. The apoptosis of nRBCs increased at the late stage and was associated with parasitaemia, but not with the intensity of the immune response. The increased percentage of nRBC apoptosis that was observed when anaemia was accentuated was not related to a reduction in peripheral RBCs. We conclude that nRBC apoptosis in P. yoelii malaria appears to be induced in response to a high parasite load. Further studies on malaria models in which acute anaemia develops during low parasitaemia are needed to identify the potential pathogenic role of nRBC apoptosis.     

Characterization of Langmuir-Blodgett films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphat idylcholine by FTIR-ATR

Monomolecular films of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphatidylc holine (PPDPC) were transferred from an air/water interface onto a germanium attenuated total reflection crystal by the Langmuir-Blodgett (LB) technique. The assemblies were thereafter investigated by Fourier transform infrared-attenuated total reflection (FTIR-ATR) spectroscopy. To determine the molecular organization in the deposited layers we monitored the CH2 and C = O stretching and the CH2 bending regions of the infrared spectra of these lipids in detail. Using Fourier self-deconvolution technique, the carbonyl stretching mode was resolved into two models corresponding to the conformational differences in the ester linkages of the phospholipid sn-1 and sn-2 acyl chains. By varying the temperature of the subphase and using different surface pressures, we were able to transfer different conformational states of DPPC onto a germanium ATR crystal. Deposition of DPPC at 40 mN m-1 and at 15 degrees C or at 20 mN m-1 and at 35 degrees C results in LB-assemblies in ordered or disordered states, respectively, as judged by the IR spectra. These structures in LB films correspond to the state of DPPC in liposomes below and above the temperature of the order-disorder phase transition. Irrespective of the surface pressure and subphase temperature used during the deposition, an ordering process was found in DPPC films when the number of the transferred layers was increased from one to five. The pyrene-labelled phosphatidylcholine analogue, PPDPC, behaved differently from DPPC. In the case where one to three layers of PPDPC transferred at 35 mN m-1 and at 20 degrees C only conformational structures resembling those in fully hydrated liposomes above the main transition temperature were observed.     

In situ monitoring of cell death using Raman microspectroscopy

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.     

Infrared and time-resolved fluorescence spectroscopic studies of the polymorphic phase behavior of phosphatidylethanolamine/diacylglycerol lipid mixtures

Fourier transform infrared (FTIR) and time-resolved fluorescence spectroscopy have been employed to examine the structural dynamics of lipid fatty acyl chains and lipid/water interfacial region of a binary lipid mixture containing unsaturated phosphatidylethanolamine (PE) and diacylglycerol (DG). Infrared vibrational frequencies of the CH2 symmetric stretching and the C = O stretching bands of the lipids were measured at different lipid compositions and temperatures. For 0% DG, the lamellar gel to lamellar liquid crystalline (L beta-L alpha) and the L alpha to inverted hexagonal (L alpha-HII) phase transitions were observed at approximately 15 degrees and 55 degrees C, respectively. As the DG content increased gradually from 0% to 15%, the L alpha-HII phase transition temperature decreased drastically while the L beta-L alpha phase transition temperature decreased only slightly. At 10% DG, a merge of these two phase transitions was noticed at approximately 10 degrees C. For the composition study at 23 degrees C, the L alpha-HII transition occurred at approximately 6-10% DG as indicated by abrupt increases in both the CH2 and C = O stretching frequencies at those DG contents. Using time-resolved fluorescence spectroscopy, abrupt decreases in both the normalized long time residual and the initial slope of the anisotropy decay function of lipid probes, 1-palmitoyl-2-[[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl]carbonyl]-3-sn-phosphatidylcholine, in these PE/DG mixtures were observed at the L alpha-HII phase transition. These changes in the anisotropy decay parameters suggested that the rotational dynamics and orientational packing of the lipids were altered at the composition-induced L alpha-HII transition, and agreed with a previous temperature-induced L alpha-HII transition study on pure unsaturated PE (Cheng (1989) Biophys. J. 55, 1025-1031). The fluorescence lifetime of water soluble probes, 8,1-anilinonapthalenes sulfonate acid, in PE/DG mixtures increased abruptly at the L alpha-HII phase transition, suggesting that the conformation and hydration of the lipid/water interfacial region also undergo significant changes at the L alpha-HII transition.     

Proliferation-associated expression of DNA methyltransferase in human embryonic lung cells

The cell cycle-dependent and proliferation-associated expression of the enzyme DNA methyltransferase has been evaluated immunocytochemically in synchronized L-132 human embryonic lung cells, using the anti-DNA methyltransferase monoclonal antibody M1F6D7/5C10. DNA methyltransferase-reactivity was firstly seen in mid-G1 cells. An intense and granular reaction in the cell nuclei with a sparing of the nucleoli was observed in addition to a homogenous and faint cytoplasmic staining. The staining intensity in the cell nuclei increased progressively up to mitosis. In early mitotic cells an intense perichromosomal staining was observed in addition to a homogenous staining of cyto- and karyoplasm after the resolving of the core membrane. In late mitosis the staining intensity decreased rapidly. Early G1 cells and density inhibited, resting G0 cells showed no DNA methyltransferase reactivity at all. Our results indicate that anti-DNA methyltransferase monoclonal antibodies could become valuable tools to detect proliferating cells in cell cultures and tissues.     

Inter- and intramolecular disulfide bond formation and related structural changes in the lens proteins. A Raman spectroscopic study in vivo of lens aging

Raman spectra have been measured for intact rat lens nuclei at various stages of aging in an attempt to gain further insight into age-related structural changes in the lens proteins, especially changes concerning protein sulfhydryl groups. Two Raman bands at 2579 and 2561 cm-1 were observed to be assignable to SH stretching modes of the cysteine residues. These bands have been attributed to "exposed" and "buried" sulfhydryl groups of the lens proteins, respectively, on the basis of a model compound study. The relative intensities of both SH stretching modes decreased with lens aging, and concurrently the intensity of a S-S stretching mode at 509 cm-1 due to disulfide bridges increased, suggesting that not only exposed but also buried protein sulfhydryl groups are converted to disulfide groups as a result of aging. The rate of the intensity decrease in the 2561 cm-1 band was similar to that in the 2579 cm-1 band. Therefore, it seems likely that the sulfhydryl groups in the two distinct environments are nearly equally subjected to the oxidation. Cysteine and cystine residues of the lens proteins gave their C-S stretching modes at 708 cm-1, indicating that they predominantly assume PC and/or PN conformers. The intensity ratio of a tyrosine doublet near 840 cm-1 (I832/I855) changed from approximately 0.86 to approximately 0.81 with the aging of the rat lens. This result implies that some tyrosine residues undergo a change in their hydrogen bonding environments during the course of aging. Of particular importance is that the relative intensity change of the tyrosine doublet with normal aging and that with cataract formation are in opposite directions.     

Apoptotic cell nuclei favour aggregation and fluorescence quenching of DNA dyes

 Apoptotic cell nuclei are known to stain hyperchromatically with absorption dyes and dimly with many DNA fluorochromes. We hypothesised that both optical phenomena have the same cause - the ability of apoptotic chromatin to aggregate cationic dyes. This hypothesis was tested using prednisolone-primed rat thymus, which is known to contain apoptotic cells. The apoptotic cells were classified as early and late, based on their morphology, in thin and semithin sections and in thymus imprints on slides. Direct reaction for DNA strand breaks (TUNEL) indicated the presence of breaks in both categories of cells, with more intense labelling in late apoptosis. The chromatin ultrastructure of early apoptotic cells initially retained the supranucleosomal order of packaging which characterises control cells, whereas the dense chromatin of late apoptotic cells possessed the degraded structure. Absorption spectra of the toluidine blue-stained early apoptotic cell chromatin revealed a metachromatic shift, indicating a change of DNA conformation and polymerisation of the dye. When the staining was performed by acridine orange (preceded by a short acid treatment), a paradoxical several-fold increase of fluorescence intensity at a several-fold dilution of the dye was found. The simultaneous reduction of the ratio of red to green components of fluorescence confirmed that the concentration-dependent fluorescence quenching was due to aggregation of the dye. The results suggest that the enhanced affinity of the chromatin of early apoptotic cells for cationic dyes is associated with conformational relaxation rather than degradation of DNA. In late apoptotic cells, the very dense packaging of degraded DNA promotes further aggregation of dyes. The results suggest alternative methods for detection and discrimination of early and late apoptotic cells. Accepted: 12 February 1997