Cell shrinkage is a major characteristic of apoptosis, but the mechanism and role of this process in cell death are poorly understood. The primary factor that controls volume regulation in all cells is ions, and thus we have examined the movement of ions at the single cell level in lymphocytes during apoptosis. Activation of the death program with several stimuli that act through independent pathways to stimulate apoptosis results in a synchronous shift of cells from a normal cell size to a shrunken cell size. Only the shrunken cells exhibit DNA fragmentation and an approximate 4-fold elevation of caspase-3-like activity. Analysis of K ؉ and Na ؉ ion content of individual cells by flow cytometry revealed that the intracellular ionic strength of apoptotic cells decreased substantially from their non-shrunken counterparts. Additionally, we show apoptosis is enhanced under conditions where the intracellular K ؉ concentration is diminished and that apoptosis is inhibited when K ؉ efflux is prevented. These data show that the efflux of ions, primarily potassium, plays a necessary and perhaps a pivotal role in the cell death program.

Little is known about the mechanisms of suppression of apoptosis. We have addressed the novel possibility that the level of intracellular K ؉ regulates the apoptotic process by controlling the activity of death enzymes. We show that K ؉ , at normal intracellular levels, inhibits both apoptotic DNA fragmentation and caspase-3(CPP32)-like protease activation, suggesting that intracellular K ؉ loss must occur early during apoptosis. Direct measurement of K ؉ by inductively coupled plasma/ mass spectrometry and flow cytometry indicates a major decrease in intracellular K ؉ concentration in the apoptotic cell. Flow cytometric analysis revealed that caspase and nuclease activity were restricted to the subpopulation of cells with reduced K ؉ . Disruption of the natural K ؉ electrochemical gradient suppressed the activity of both caspase and nuclease independent of the mode of activation of the apoptotic inducing agent, demonstrating that a decrease in intracellular K ؉ concentration is a necessary, early event in programmed cell death.Apoptosis is a physiological form of cell death that occurs in response to a variety of signals. Although diverse agents induce apoptosis via unique signal transduction pathways, the process is remarkably similar in all systems. Morphologically, the cells in early stages of apoptosis shrink, and chromatin condenses. The nucleus then fragments, and the entire cell blebs into apoptotic bodies that maintain membrane integrity, ensuring encapsulation of the intracellular components. Genetic evidence has shown the requirement of specific gene products for the effective elimination of cells (1-3), although, in mammalian cells, the evidence suggests that these proteins are preformed in the nondying cell and maintained in an inactive state (4 -9). Little is currently known about the mechanisms that provide this chronic suppressive effect on the apoptotic machinery.In response to an apoptotic stimuli, there is a relief of the suppressive influences within a cell that manifests biochemical alterations including the activation of proteases and nucleases. Much attention has focused recently on the role of proteases related to interleukin-1␤ converting enzyme (recently renamed caspases (10)), and increases in caspase activity appear to be an early event in the common component of many apoptotic pathways (11-16). Caspases are synthesized and maintained in the cytoplasm as proenzymes which themselves must undergo a proteolytic activation, perhaps triggering apoptosis. The substrates cleaved by these enzymes are numerous (17) and include both structural proteins (18 -23) and enzymes (22,24,25). In addition to caspase activation, one or more nucleases are activated which destroy the genome through cleavage at specific structures (26 -31). The active apoptotic nuclease(s) cleaves chromatin to produce nucleosomal (180 -200 base pairs) or oligonucleosomal (multiples thereof) DNA fragments (32, 33). When analyzed by electrophoresis these fragments produce the widely recognized "apoptotic ladder." Destruction ...

Background Chronic inflammation is believed to be a major mechanism underlying the pathophysiology of type 2 diabetes. Periodontitis is a cause of systemic inflammation. We aimed to assess the effects of periodontal treatment on glycaemic control in people with type 2 diabetes. Methods In this 12 month, single-centre, parallel-group, investigator-masked, randomised trial, we recruited patients with type 2 diabetes, moderate-to-severe periodontitis, and at least 15 teeth from four local hospitals and 15 medical or dental practices in the UK. We randomly assigned patients (1:1) using a computer-generated table to receive intensive periodontal treatment (IPT; whole mouth subgingival scaling, surgical periodontal therapy [if the participants showed good oral hygiene practice; otherwise dental cleaning again], and supportive periodontal therapy every 3 months until completion of the study) or control periodontal treatment (CPT; supra-gingival scaling and polishing at the same timepoints as in the IPT group). Treatment allocation included a process of minimisation in terms of diabetes onset, smoking status, sex, and periodontitis severity. Allocation to treatment was concealed in an opaque envelope and revealed to the clinician on the day of first treatment. With the exception of dental staff who performed the treatment and clinical examinations, all study investigators were masked to group allocation. The primary outcome was between-group difference in HbA,, at 12 months in the intention-to-treat population. This study is registered with the ISRCTN registry, number ISRCTN83229304.

In the present study, we examined the possibility that granulosa cell death during ovarian follicular atresia occurs by apoptosis (programmed cell death). To investigate this possibility, atresia was induced in immature female rats by injecting 15 IU PMSG. Controls received either vehicle or no treatment. PMSG-treated animals were killed on days 1-5 post-injection while controls were killed on days 1 or 5. The onset of atresia was assessed histologically by light microscopic inspection of 5 microns tissue sections and functionally by quantification of serum progesterone and estrogen levels. Apoptosis is characterized by the cleavage of genomic DNA into oligonucleosomal length fragments by a Ca2+/Mg(2+)-dependent endogenous endonuclease. Such fragments form a distinctive ladder pattern when separated electrophoretically. Accordingly, the occurrence of apoptosis in granulosa cells was assessed by examining the pattern of fragmented DNA in cell lysates after agarose gel electrophoresis. Gels were stained with ethidium bromide and DNA visualized by UV transillumination. The earliest morphological signs of atresia were detected 4 days after PMSG injection as evidenced by degeneration and detachment of granulosa cells from the basal lamina. Serum estrogen increased from basal to levels 7-fold over controls by day 3 after PMSG treatment, falling to control values by day 4 and thereafter. In contrast, progesterone remained basal for the first 3 days, rising to levels 3-fold and 8-fold above controls 4 and 5 days after PMSG treatment, respectively. Such shifts in the ratio of estrogen to progesterone production are known to be characteristic of follicular atresia. Finally, electrophoretic analysis of low mol wt DNA in granulosa cell lysates revealed a definitive ladder pattern of oligonucleosomal length DNA fragments (characteristic of apoptosis) on days 4 and 5 after PMSG injection. This pattern was not detectable on days 1 and 2 after treatment. Lysates obtained 3 days after PMSG treatment showed a faint apoptotic-like pattern of DNA fragments; a result consistent with other systems in which DNA cleavage begins before any morphological signs of death. Interestingly, a ladder pattern of DNA fragments was present in control lysates suggesting that granulosa cell death under normal (vs. induced) conditions of atresia in immature rats occurs by apoptosis. These data demonstrate an intimate association between apoptotic-like events and dying granulosa cells and thus support the possibility that apoptosis is involved in the induction of follicular atresia.

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Previous work in our laboratory (Montague, J., Gaido, M., Frye, C., and Cidlowski, J. (1994) J. Biol. Chem. 269, 18877-18880) has shown that human recombinant cyclophilins A, B, and C have sequence homology with the apoptotic nuclease NUC18 and that denatured cyclophilins can degrade DNA. We have now evaluated the nucleolytic activity of recombinant cyclophilins under native conditions. We show that nuclease activity inherent to cyclophilins is distinct from cis-trans-peptidylprolyl isomerase activity and is similar to that described for apoptotic nucleases. Cyclophilin nucleolytic activity is stimulated by Ca 2؉ and/or Mg 2؉, with a combination of the two being optimal for cyclophilins A and B. Mg 2؉ alone is sufficient for cyclophilin C nuclease activity. pH optimums are in the range of pH 7.5-9.5. Cyclophilins can degrade both single-stranded and double-stranded DNA. Additionally, cyclophilins produce 3-OH termini in linear double-stranded substrates, suggesting the cuts produced are similar to those of apoptotic cells. Cyclophilins also display endonucleolytic activity, demonstrated by their ability to degrade supercoiled DNA. In the absence of ions, cyclophilins bind linearized DNA. When added to nuclei from nonapoptotic cells, cyclophilin C induces 50-kilobase pair DNA fragmentation but not internucleosomal fragmentation. Together, these data suggest that cyclophilins are involved in degradation of the genome during apoptosis.

Estrogen stimulates water imbibition in the uterine endometrium. This water then crosses the epithelial cells into the lumen, leading to a decrease in viscosity of uterine luminal fluid. To gain insight into the mechanisms underlying this estrogen-stimulated water transport, we have explored the expression profile and functionality of water channels termed aquaporins (AQPs) in the ovariectomized mouse uterus treated with ovarian steroid hormones. Using immunocytochemical analysis and immunoprecipitation techniques, we have found that AQP-1, -3, and -8 were constitutively expressed. AQP-1 expression was restricted to the myometrium and may be slightly regulated by ovarian steroid hormones. AQP-3 was expressed at low levels in the epithelial cells and myometrium, whereas AQP-8 was found in both the stromal cells and myometrium. AQP-2 was absent in vehicle controls but strongly up-regulated by estrogen in the epithelial cells and myometrium of the uterus. This localization implicates all four isotypes in movement of water during uterine imbibition and, based on their localization to the luminal epithelial cells, AQP-2 and -3 in facilitating water movement into the lumen of the uterus. The analysis of the plasma membrane permeability of luminal epithelial cells by two separate cell swelling assays confirmed a highly increased water permeability of these cells in response to estrogen treatment. This finding suggests that estrogen decreases the luminal fluid viscosity, in part, by enhancing the water permeability of the epithelial layer, most likely by increasing the expression of AQP-2 and/or the availability of AQP-3. Together these results provide novel information concerning the mechanism by which estrogen controls water imbibition and luminal fluid viscosity in the mouse uterus.

SUMMARY Exposure to environmentally prevalent heavy metals such as cadmium can have detrimental effects on a variety of commercially and ecologically important species such as oysters. Since Cd2+ is known to induce apoptosis in immune cells of vertebrates, we have investigated the effects of this metal on isolated oyster hemocytes, the main cellular immune defense in mollusks. Enhanced apoptosis of these cells could conceivably create immunosuppressed conditions in these organisms and result in reduced disease resistance and increased opportunistic infection, resulting in decline of their populations. Cd2+ exposure induced apoptosis in oyster hemocytes in a dose-dependent manner in the range of 10-100 μmol l-1, as indicated by the translocation of phosphatidylserine to the outer leaflet of the plasma membrane. At higher concentrations (200-1000 μmol l-1), there was no further increase in apoptosis but a significant increase in the level of necrosis. In stark contrast to vertebrate immune cells, there was no decrease in the mitochondrial membrane potential or activation of caspases in response to Cd2+ in the apoptotic range. Surprisingly, Cd2+ exposure in this range did cause a significant decrease in intracellular ATP levels, indicating a severe disturbance of energy metabolism. Similarly, Cd2+ exposure of isolated mitochondria resulted in partial uncoupling of mitochondria but no difference in mitochondrial membrane potential. The results demonstrate that the important environmental pollutant Cd2+ induces apoptosis in oyster immune cells and does so through a mitochondria/caspase-independent pathway,suggesting that a novel, perhaps ancient, apoptotic pathway is active in these cells. Furthermore, it appears that the observed decrease in ATP production during apoptosis is not due to the loss of the mitochondrial proton-motive force but is more likely to be due to inhibition of the F0/F1-ATPase and/or mitochondrial ADP/ATP or substrate transport.