The sodium-activated potassium channel Slack is modulated by hypercapnia and acidosis.

Slack (Slo 2.2), a member of the Slo potassium channel family, is activated by both voltage and cytosolic factors, such as Na(+) ([Na(+)](i)) and Cl(-) ([Cl(-)](i)). Since the Slo family is known to play a role in hypoxia, and since hypoxia/ischemia is associated with an increase in H(+) and CO(2) intracellularly, we hypothesized that the Slack channel may be affected by changes in intracellular concentrations of CO(2) and H(+). To examine this, we expressed the Slack channel in Xenopus oocytes and the Slo 2.2 protein was allowed to be inserted into the plasma membrane. Inside-out patch recordings were performed to examine the response of Slack to different CO(2) concentrations (0.038%, 5%, 12%) and to different pH levels (6.3, 6.8, 7.3, 7.8, 8.3). In the presence of low [Na(+)](i) (5 mM), the Slack channel open probability decreased when exposed to decreased pH or increased CO(2) in a dose-dependent fashion (from 0.28+/-0.03, n=3, at pH 7.3 to 0.006+/-0.005, n=3, P=0.0004, at pH 6.8; and from 0.65+/-0.17, n=3, at 0.038% CO(2) to 0.22+/-0.07, n=3, P=0.04 at 12% CO(2)). In the presence of high [Na(+)](i) (45 mM), Slack open probability increased (from 0.03+/-0.01 at 5 mM [Na(+)](i), n=3, to 0.11+/-0.01, n=3, P=0.01) even in the presence of decreased pH (6.3). Since Slack activity increases significantly when exposed to increased [Na(+)](i), even in presence of increased H(+), we propose that Slack may play an important role in pathological conditions during which there is an increase in the intracellular concentrations of both acid and Na(+), such as in ischemia/hypoxia.

Intracellular pH regulation of CA1 neurons in Na(+)/H(+) isoform 1 mutant mice.

To understand the role of Na(+)/H(+) exchanger 1 (NHE1) in intracellular pH (pH(i)) regulation and neuronal function, we took advantage of natural knockout mice lacking NHE1, the most ubiquitously and densely expressed NHE isoform in the central nervous system (CNS). CA1 neurons from both wild-type (WT) and NHE1 mutant mice were studied by continuous monitoring of pH(i), using the fluorescent indicator carboxy-seminaphthorhodafluor-1 (SNARF-1) and confocal microscopy. In the nominal absence of CO(2)/HCO(3)(-), steady-state pH(i) was higher in WT neurons than in mutant neurons. Using the NH(4)Cl prepulse technique, we also show that H(+) flux in WT neurons was much greater than in mutant neurons. The recovery from acid load was blocked in WT neurons, but not in mutant neurons, by removal of Na(+) from the extracellular solution or by using 100 microM 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate (HOE 694) in HEPES buffer. Surprisingly, in the presence of CO(2)/HCO(3)(-), the difference in H(+) flux between WT and mutant mice was even more exaggerated, with a difference of more than 250 microM/s between them at pH 6.6. H(+) flux in CO(2)/HCO(3)(-) was responsive to diisothiocyanato-stilbene-2, 2'-disulfonate (DIDS) in the WT but not in the mutant. We conclude that (a) the absence of NHE1 in the mutant neurons tended to cause lower steady-state pH(i) and, perhaps more importantly, markedly reduced the rate of recovery from an acid load; and (b) this difference in the rate of recovery between mutant and WT neurons was surprisingly larger in the presence, rather than in the absence, of HCO(3)(-), indicating that the presence of NHE1 is essential for the regulation and/or functional expression of both HCO(3)(-)-dependent and -independent transporters in neurons.

Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster.

O2 deprivation can produce many devastating clinical conditions such as myocardial infarct and stroke. The molecular mechanisms underlying the inherent tissue susceptibility or tolerance to O2 lack are, however, not well defined. Since the fruit fly, Drosophila melanogaster, is extraordinarily tolerant to O2 deprivation, we have performed a genetic screen in the Drosophila to search for loss-of-function mutants that are sensitive to low O2. Here we report on the genetic and molecular characterization of one of the genes identified from this screen, named hypnos-2. This gene encodes a Drosophila pre-mRNA adenosine deaminase (dADAR) and is expressed almost exclusively in the adult central nervous system. Disruption of the dADAR gene results in totally unedited sodium (Para), calcium (Dmca1A), and chloride (DrosGluCl-alpha) channels, a very prolonged recovery from anoxic stupor, a vulnerability to heat shock and increased O2 demands, and neuronal degeneration in aged flies. These data clearly demonstrate that, through the editing of ion channels as targets, dADAR, for which there are mammalian homologues, is essential for adaptation to altered environmental stresses such as O2 deprivation and for the prevention of premature neuronal degeneration.

Circulating DNA for detection of gastric cancer.

OBJECTIVE: Gastric cancer (GC) is one of the most common malignant tumors worldwide, particularly, prevalent in China. Despite the decreasing incidence of GC in China, the 5-year survival rate is still not over 30% yet. Therefore, early diagnosis and therapeutic outcome evaluation of GC remains as the issue to be resolved in a clinical setting. MATERIALS AND METHODS: Recent studies have found the presence of a certain amount of circulating DNA in the peripheral blood of patients with malignant tumor and shown that these free DNA bear tumor-specific genetic information. The circulating DNA detection includes quantitative and qualitative methods and analysis. Combined monitoring of changes in circulating DNA levels and aberrant alteration of relevant tumor genes is likely to provide comprehensive real-time information to patients. RESULTS: Under normal conditions, oncogene presents in the form of proto-oncogene such as K-ras, which is in non-carcinogenic status under the influence of tumor suppressor gene. When tumor suppressor gene is damaged or mutated of oncogene itself is induced for instance P53, oncogene is then activated and induces tumorigenesis. However, compared to gene mutation detection, the detection of DNA methylation is relatively more well-developed and stable. CONCLUSIONS: This article reviews the current status of the research on circulating DNA in the diagnosis, assessment of response to therapy and prognostic evaluation in GC. In addition, the advantage, current issue and prospect of using circulating DNA as tumor marker are also analyzed.

Altered iPSC-derived neurons' sodium channel properties in subjects with Monge's disease.

Monge's disease, also known as chronic mountain sickness (CMS), is a disease that potentially threatens more than 140 million highlanders during extended time living at high altitudes (over 2500m). The prevalence of CMS in Andeans is about 15-20%, suggesting that the majority of highlanders (non-CMS) are rather healthy at high altitudes; however, CMS subjects experience severe hypoxemia, erythrocytosis and many neurologic manifestations including migraine, headache, mental fatigue, confusion, and memory loss. The underlying mechanisms of CMS neuropathology are not well understood and no ideal treatment is available to prevent or cure CMS, except for phlebotomy. In the current study, we reprogrammed fibroblast cells from both CMS and non-CMS subjects' skin biopsies into the induced pluripotent stem cells (iPSCs), then differentiated into neurons and compared their neuronal properties. We discovered that CMS neurons were much less excitable (higher rheobase) than non-CMS neurons. This decreased excitability was not caused by differences in passive neuronal properties, but instead by a significantly lowered Na(+) channel current density and by a shift of the voltage-conductance curve in the depolarization direction. Our findings provide, for the first time, evidence of a neuronal abnormality in CMS subjects as compared to non-CMS subjects, hoping that such studies can pave the way to a better understanding of the neuropathology in CMS.

The role of HCO3(-)-dependent mechanisms in pHi regulation during O2 deprivation.

We have reported in our previous work that, in the absence of HCO(3)(-), Na(+)/H(+) exchanger is responsible for an anoxia-induced alkalinization in hippocampal CA1 neurons. HCO(3)(-)-dependent mechanisms have been reported to play a key role in pH(i) regulation in nerve cells, but how their function is affected by O(2) deprivation has not been well studied. In this work, pH(i) measurements (obtained from dissociated neurons loaded with carboxy-seminaphthorhodafluor-1 and using confocal microscopy) and whole-cell patch clamp recording techniques were used to investigate the role of HCO(3)(-)-dependent membrane exchangers on CA1 neurons during O(2) deprivation. Anoxia (5 min) induced a small acidification in neurons in the presence of HCO(3)(-) and this acidification was changed to a significant alkalinization when neurons were bathed with Hepes buffer or when 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid was applied in a HCO(3)(-) solution, indicating that HCO(3)(-)-dependent mechanisms were involved. A marked anoxia-induced acidification (0.33+/-0.11 pH unit) was seen when the Na(+)/H(+) exchange was blocked with 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate in the presence of HCO(3)(-), but the same anoxia did not cause a significant pH(i) change in a Na(+) free, HCO(3)(-) solution, suggesting that the anoxia-induced acidification in the presence of 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate is dependent on both Na(+) and HCO(3)(-). Furthermore, anoxia did not cause a significant pH(i) change when both 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid and 3-(methylsulfonyl-4-piperidino-benzoyl)-guanidine methanesulfonate were present. Current clamp recordings showed a significant membrane depolarization following anoxia in HCO(3)(-) solution but not in Hepes buffer. Our data suggest that, in hippocampal neurons: a) pH(i) regulation during O(2) deprivation is affected not only by metabolism but also by membrane exchangers, and b) besides the activation of Na(+)/H(+) exchange, anoxia activates a 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid-sensitive, Na(+)-dependent acid loader (possibly electrogenic).

Effects of lectins on quantal release at the frog neuromuscular junction.

This work was initiated because pretreatment with concanavalin A was reported to abolish the increase in spontaneous quantal release produced by hypertonic solutions [Gorio A. and Mauro A. (1979) J. gen. Physiol. 73, 245-263]. This suggested that lectins might be valuable tools for investigating the role of glycoproteins in the response to tonicity. We compared muscles soaked for 2 h in hypertonic solution containing concanavalin A with paired muscles soaked in hypertonic solution without lectin. The lectin treatment decreased miniature end-plate potential frequencies in Ringer and in hypertonic solutions compared with the controls. Even after lectin treatment hypertonic solutions and elevated K+ solutions increased miniature end-plate potential frequencies, and the proportional increases were the same as in controls. The lectin treatment lowered baseline frequency, but the preparation still responded to hypertonic solutions. Concanavalin A effects appeared after treatment for more than 1 h and required concentrations of 10 micrograms/ml or higher. Higher concentrations did not produce more effect. Similar results were obtained with four other lectins with different sugar specificities. Treatment in hypertonic solution without lectin produces a similar, but smaller, decrease in baseline frequency. Concanavalin A pretreatment had no detectable effects on evoked release or facilitation. We conclude that the effects of lectins on quantal release are not mediated by binding to a single sugar group. The lectins do not produce a unique effect; they exaggerate the changes produced by hypertonic pretreatment. All of the effects could be accounted for by a reduction in baseline [Ca2+] in the nerve terminal. Such reductions are produced by lectins in many cell types.

Hematoporphyrin photoradiation therapy for iris neovascularization. A preliminary report.

Photoradiation therapy is a new technique currently under clinical investigation for the treatment of a variety of solid malignant tumors. The technique involves intravenous (IV) administration of hematoporphyrin derivative, a photosensitizing compound that is preferentially retained by malignant cells, and photoactivation of a neoplasm with red light (630 nm) to obtain selective destruction of cancer cells. A modification of this technique was used to treat iris neovascularization produced in a primate model of retinal vein occlusion. Following IV injection of hematoporphyrin derivative, the anterior surface of the iris was exposed to red light for 30 to 45 minutes. In the four eyes treated, there was a marked reduction of fluorescein leakage from the neovascular tissue within 24 hours following treatment. Histologic examination of two eyes demonstrated a selective response of the iris neovascularization to this therapy, with apparent preservation of surrounding tissue.

Ocular neovascularization. Tissue culture studies.

The proliferative activity of a number of intraocular fluids, bovine retinal extract, and normal serum (from humans and cynomolgus monkeys) was investigated by in vitro tissue culture studies, with the use of tritiated thymidine incorporation by the cultured endothelial cells of human umbilical veins. There was increased tritiated thymidine incorporation by (1) the aqueous, vitreous, and intraocular fluid (IOF) (which filled the eye after lensectomy and vitrectomy) removed from cynomolgus monkey eyes with iris neovascularization or with neovascular glaucoma (NVG) that developed after experimental retinal vein occlusion, (2) by aqueous and vitreous removed from human eyes with NVG or proliferative diabetic retinopathy; (3) by the serum, and (4) by the bovine retinal extract. However, tritiated thymidine incorporation was not increased by the normal aqueous, vitreous, or IOF.

Decreased neuronal excitability in hippocampal neurons of mice exposed to cyclic hypoxia.

To study the physiological effects of chronic intermittent hypoxia on neuronal excitability and function in mice, we exposed animals to cyclic hypoxia for 8 h daily (12 cycles/h) for approximately 4 wk, starting at 2-3 days of age, and examined the properties of freshly dissociated hippocampal neurons in vitro. Compared with control (Con) hippocampal CA1 neurons, exposed (Cyc) neurons showed action potentials (AP) with a smaller amplitude and a longer duration and a more depolarized resting membrane potential. They also have a lower rate of spontaneous firing of AP and a higher rheobase. Furthermore, there was downregulation of the Na(+) current density in Cyc compared with Con neurons (356.09 +/- 54.03 pA/pF in Cyc neurons vs. 508.48 +/- 67.30 pA/pF in Con, P < 0.04). Na(+) channel characteristics, including activation, steady-state inactivation, and recovery from inactivation, were similar in both groups. The deactivation rate, however, was much larger in Cyc than in Con (at -100 mV, time constant for deactivation = 0.37 +/- 0.04 ms in Cyc neurons and 0.18 +/- 0.01 ms in Con neurons). We conclude that the decreased neuronal excitability in mice neurons treated with cyclic hypoxia is due, at least in part, to differences in passive properties (e.g., resting membrane potential) and in Na(+) channel expression and/or regulation. We hypothesize that this decreased excitability is an adaptive response that attempts to decrease the energy expenditure that is used for adjusting disturbances in ionic homeostasis in low-O(2) conditions.

Action potential-like responses in B104 cells with low Na+ channel densities.

Action potential generation and Na+ currents were studied in B104 neuroblastoma cells in vitro using the whole-cell patch-clamp method in voltage-clamp and current-clamp mode. Action potential-like responses were elicited in 38 of 42 cells, with a threshold close to -55 mV for depolarizing stimuli, and -56 mV for anode-break stimuli. Response amplitudes were larger when cells were held at more negative prepulse potentials, and were well fit by a Boltzmann distribution with a midpoint of approx. -75 mV, close to the V1/2 for Na+ current steady-state inactivation in these cells. Cells displaying action potential-like responses exhibited a peak Na+ current density of 133 +/- 0.14 pA/pF (range, 10.2-296.2 pA/pF) and a low gK:gNa ratio (0.0067 +/- 0.0023). Exposure to 0.1 mM Cd2+ did not block the generation of action potential-like responses in B104 cells, while 1 microM TTX abolished the responses. We conclude that low densities of Na+ channels (< 3/microns2, and < 1/micron2 in some cells) can support the generation of action potential-like responses in B104 cells if they are held at hyperpolarized levels to remove inactivation. The low leak and K+ conductance of these cells may contribute to their ability to generate action potential-like responses under these circumstances.

Drosophila neurons respond differently to hypoxia and cyanide than rat neurons.

Compared with mammalian species, Drosophila melanogaster exhibits marked tolerance to hypoxia or anoxia. However, the underlying cellular mechanisms of tolerance are still largely unknown. In order to assess the electrophysiologic response to O(2) lack in Drosophila neurons and compare them to those in mammals, we used neurons from embryonic cultures of both Drosophila and rat. We studied the effects of hypoxia on membrane potential V(m), input resistance R(m), rheobase, and action potential characteristics before, during and after 3 to 5 min of hypoxia (measured PO(2)<20 Torr). In Drosophila neurons, on the one hand, V(m) reversibly hyperpolarized with hypoxia by an average of about 20 mV and input resistance decreased by 71% from control. In most cells studied, action potential (AP) amplitude decreased, its duration increased, and its threshold shifted in a hyperpolarized direction before AP generation was attenuated. On the other hand, V(m) in rat cortical neurons reversibly depolarized by an average of 10 mV with hypoxia. Input resistance was reversibly reduced by 58% and, in most cells studied, AP amplitude also decreased and its duration increased. In contrast to the effects of hypoxia on V(m), CN caused a depolarization by 22 mV and a slight increase in R(m) in Drosophila. In the rat, CN was similar to hypoxia in its effect on R(m). We conclude that (1) rat and Drosophila neurons decrease their excitability in hypoxia by activating different mechanisms; (2) the most likely explanation for the hyperpolarization and the decrease in R(m) in Drosophila neurons is the activation of a K(+) conductance; this activation, by itself, cannot explain the results in rat neurons and (3) hypoxia and cyanide have similar effects in rat neurons but are divergent in their effects in Drosophila neurons.

Experience in controlling lead poisoning in the People's Republic of China.

This paper summarizes the work done to control lead poisoning in the People's Republic of China. The work can be divided into the following four phases: (i) health surveillance of lead workers, (ii) health standard setting, (iii) research for early detection indicators, and (iv) improvement of the work conditions and health surveillance of "undeserved" groups. In 1949 prevention started with the initiation of the health surveillance of lead workers. Since then health surveillance has been used to supplement environmental monitoring in the early detection of lead poisoning, and the prevalence of lead poisoning has decreased. This activity has also helped to identify the needs for improvement in technological control. The next step has been the promulgation of health standards. The current standard for lead gives the maximum allowable concentration in air as 0.03 mg/m3 for lead fume and 0.05 mg/m3 for lead dust. Research on indicators for the early detection of lead has improved the quality of prevention. It has recently led to the addition of zinc protoporphyrin to the list of indicators in use. Finally the work conditions and surveillance of workers that had earlier been considered "underserved," particularly workers in agriculture and small industries in rural areas, are being improved.

The role of early detection in the prevention of occupational disease--a review of work in the People's Republic of China.

The early detection of occupational diseases is the main purpose of the health surveillance of workers exposed to occupational hazards. While health surveillance is one of the important approaches to occupational disease prevention (in addition to environmental monitoring and improved technology), promulgating legislation and organizing and training professional personnel could guarantee the implementation. Chinese experience in industrial development is similar to that of other countries. Research determining indicators for the early detection of occupational diseases could improve the quality of preventive measures.

Treatment of yellow phosphorus skin burns with silver nitrate instead of copper sulfate.

The authors recommend the application of silver nitrate solution for the treatment of yellow phosphorus skin burns instead of the traditionally used copper sulfate solution. The latter may cause copper poisoning if absorbed through damaged skin. Silver nitrate treatment has been successfully applied in 13 cases of phosphorus burns.

Occupational health services in "small plants" in Shanghai County.

With the reorganization of the grassroots structure and economic readjustment, the commune- and brigade-run plants have now been changed to district (town)- and village-run plants, respectively, and the role of industrial production has become more important to the total income of the people. The Qi-yi Commune (District) is a good example. The number of plants showed an increase of 80% from 1975 to 1983, whereas the number of workers increased by 149%. The number of workers as a percentage of the total population of the district grew from 10% to about 25%. The rapid progress of industry led to a need to promote occupational health services as part of primary health care. This article reviews the implementation of such services with the objective of attaining the goal "Health for all by the year 2000."

Prevention of acute parathion and demeton poisoning in farmers around Shanghai.

After the occurrence of poisoning episodes among commune members who handled the insecticides parathion and demeton during the first few years of application in the early 1960s, a series of surveys was conducted and comprehensive regulatory actions were adopted. The surveys showed that the cause of most of the poisoning cases was percutaneous absorption of toxicant as a consequence of skin contamination during careless operating. As a result of a comprehensive program carried out by large numbers of administrators, health workers, and commune members, the incidence of intoxication quickly declined, starting in 1965, to a negligible level and has remained so to the present, even though parathion and demeton use has increased greatly. It is suggested that the experience obtained might be helpful to other areas in the People's Republic of China and in some developing countries.

Epidemiologic study on the skin lesions of workers exposed to N,N'-methylene-bis-(2-amino-1,3,4-thiadiazole) and of children in the neighborhood of the factory.

The air in a factory producing N,N' methylene-bis-(2-amino-1,3,4-thiadiazole) (MATDA) and water in a nearby river were often polluted by the intermediate and finished product. The levels of MATDA in the air, soil, and river water were 0.1-0.8 mg/m3, 6-2 650 mg/kg, and 0.5-2 mg/kg, respectively. Seventy-nine workers engaged in MATDA production were observed; 44% of them suffered from contact dermatitis; and 72% showed changes of pigment in the skin (versus 2 and 16%, respectively, for the referents). All of the exposed workers had experienced dermatitis at some time. For 218 pupils of a primary school in the neighborhood of the factory, the incidence of hypopigmentation and pigmentation of the skin was 14 and 72%, respectively, but in the reference group the corresponding incidence rates were 0 and 20%. Pigment changes of workers' children increased with the duration of residence in the neighborhood of the factory. The results of the survey led to a change in the formulation of the product from a powder to an emulsion. Improved technology was also initiated which allowed most of the waste to be reused in the processing. After these control measures the dermatitis of the workers was greatly reduced.

The current status of hygiene standard setting for chemical substances in workplaces in China.

The hygiene standard setting for occupational hazards was started in the mid 1950s in China. Three documents on exposure limits for chemical substances in the work environment and some other documents related to exposure limits for physical agents have been published since then. The latest documentation on "Hygiene Standards for the Design of Industrial Premises" (Standard TJ 36-79) was promulgated in 1979. It contains a MAC list of 134 toxin agents and dusts which are most frequently used and encountered in China. However, a more sophisticated system of scientific research on hygiene standard setting has been created since the establishment of the National Scientific Commission of Hygiene Standard Setting in 1980. This system emphasizes health as a basic criterion, it strengthens epidemiological study of the human population, it integrates domestic and international information sources and it periodically reappraises the recommended standards. Based on these principles, more than forty new standards have been set for both chemicals and dusts since the founding of the committee. These new MACs are now in the process of being promulgated and are expected to take effect as the additional part of the MAC list published in 1979. In addition, further considerations of hygiene standard setting related to the conceptual renewal, selection of safety factor, legislation and enforcement of the hygiene standard, recommendations of exposure limits for occupational carcinogens, and speeding up the pace of hygiene standard setting are proposed in this paper.

New challenges for occupational health services facing economic reform in China.

In China, which is facing economic reform and the rapid development of small-scale industries, various occupational hazards are reappearing. The problems are related to the ignorance of the managers and the workers, a lack of capital, the insufficiency of occupational health services, and the transfer of some hazardous work from large factories in the cities to small plants in the towns and villages. The change in life-style, especially in smoking habits, has aggravated occupational hazards. The problems may be solved by renewing the concept of health among the managers and workers, enforcing legislation related to occupational hazards, strengthening health personnel training, and reinforcing health education and self-help.