Levels, distribution characteristics, and sources of heavy metals in indoor dust in Shijiazhuang, China.

Heavy metals in indoor dust are associated with health risks in humans. However, in Shijiazhuang, a city in northern China with severe haze, no research has been published on this topic. To determine the content, distribution characteristics, and sources of heavy metals in indoor dust in the city of Shijiazhuang, indoor dust samples from 33 sampling points in the main urban area of Shijiazhuang were collected and tested. Concentrations of Cu, Ni, Cr, Zn, Cd, and Pb were 87.0, 35.1, 104.4, 568.0, 1.980, and 187.6 mg·kg-1, respectively; their levels have been discussed statistically in comparison with the reported values in other cities in China. The sources of heavy metals were analyzed using enrichment factor, correlation coefficient, and principal component analysis. The results showed that the levels of all six elements in indoor dust in Shijiazhuang exceeded the background values of soil in Hebei Province. Among these, Cd, Pb, and Zn were significantly enriched. The enrichment factors of Cu, Ni, and Cr were below 10, and their levels at different sampling points were similar, indicating their geogenic source. The corresponding pollution levels of Cd, Pb, and Zn were relatively high, and their levels at different points were significantly different and correlated, indicating that they were derived mainly from transportation. Additionally, the level of Zn was significantly affected by the indoor environment. Our findings provide a basis for conducting health risk assessments in the future.

Persistent increased PKMζ in long-term and remote spatial memory.

PKMζ is an autonomously active PKC isoform that is thought to maintain both LTP and long-term memory. Whereas persistent increases in PKMζ protein sustain the kinase's action in LTP, the molecular mechanism for the persistent action of PKMζ during long-term memory has not been characterized. PKMζ inhibitors disrupt spatial memory when introduced into the dorsal hippocampus from 1day to 1month after training. Therefore, if the mechanisms of PKMζ's persistent action in LTP maintenance and long-term memory were similar, persistent increases in PKMζ would last for the duration of the memory, far longer than most other learning-induced gene products. Here we find that spatial conditioning by aversive active place avoidance or appetitive radial arm maze induces PKMζ increases in dorsal hippocampus that persist from 1day to 1month, coinciding with the strength and duration of memory retention. Suppressing the increase by intrahippocampal injections of PKMζ-antisense oligodeoxynucleotides prevents the formation of long-term memory. Thus, similar to LTP maintenance, the persistent increase in the amount of autonomously active PKMζ sustains the kinase's action during long-term and remote spatial memory maintenance.

PKM zeta maintains late long-term potentiation by N-ethylmaleimide-sensitive factor/GluR2-dependent trafficking of postsynaptic AMPA receptors.

Although the maintenance mechanism of late long-term potentiation (LTP) is critical for the storage of long-term memory, the expression mechanism of synaptic enhancement during late-LTP is unknown. The autonomously active protein kinase C isoform, protein kinase Mzeta (PKMzeta), is a core molecule maintaining late-LTP. Here we show that PKMzeta maintains late-LTP through persistent N-ethylmaleimide-sensitive factor (NSF)/glutamate receptor subunit 2 (GluR2)-dependent trafficking of AMPA receptors (AMPARs) to the synapse. Intracellular perfusion of PKMzeta into CA1 pyramidal cells causes potentiation of postsynaptic AMPAR responses; this synaptic enhancement is mediated through NSF/GluR2 interactions but not vesicle-associated membrane protein-dependent exocytosis. PKMzeta may act through NSF to release GluR2-containing receptors from a reserve pool held at extrasynaptic sites by protein interacting with C-kinase 1 (PICK1), because disrupting GluR2/PICK1 interactions mimic and occlude PKMzeta-mediated AMPAR potentiation. During LTP maintenance, PKMzeta directs AMPAR trafficking, as measured by NSF/GluR2-dependent increases of GluR2/3-containing receptors in synaptosomal fractions from tetanized slices. Blocking this trafficking mechanism reverses established late-LTP and persistent potentiation at synapses that have undergone synaptic tagging and capture. Thus, PKMzeta maintains late-LTP by persistently modifying NSF/GluR2-dependent AMPAR trafficking to favor receptor insertion into postsynaptic sites.

Protein kinase M zeta regulation of Na/K ATPase: a persistent neuroprotective mechanism of ischemic preconditioning in hippocampal slice cultures.

In ischemic preconditioning, a sublethal ischemic insult protects neurons from subsequent ischemia. In organotypic hippocampal slice cultures a sublethal 5-minute hypoxia-hypoglycemia treatment prevented neuronal loss after a 10-minute experimental ischemic (EI) treatment of hypoxia-hypoglycemia. Whereas preconditioning protected against EI given 24 h later, it did not protect when EI was given 2 h later, suggesting a slow development of neuroprotection. This model identified two regulators of ischemic preconditioning: the atypical protein kinase M zeta (PKMzeta), and the Na/K ATPase. Two hours following preconditioning, when there was no neuroprotection, Na/K ATPase activity was unchanged. In contrast, Na/K ATPase activity significantly increased 24 h after the preconditioning treatment. Elevated Na/K ATPase activity was accompanied by increased surface expression of the alpha1 and alpha2 isoforms of the Na/K ATPase. Similarly, active PKMzeta levels were increased at 24 h, but not 2 h, after preconditioning. PKMzeta overexpression by sindbis virus vectors also increased Na/K ATPase activity. To examine PKMzeta regulation of Na/K ATPase, occlusion experiments were performed using marinobufagenin to inhibit alpha1, dihydroouabain to inhibit alpha2/3 and a zeta-pseudosubstrate peptide to inhibit PKMzeta. These experiments showed that PKMzeta regulated both the activity and surface expression of the alpha1 isoform of the Na/K ATPase. Marinobufagenin, dihydroouabain, and zeta-pseudosubstrate peptide were used to determine if PKMzeta or the alpha1 and alpha2 Na/K ATPase isoforms protected neurons. All three compounds blocked neuroprotection following ischemic preconditioning. PKMzeta levels were elevated 3 days after ischemic preconditioning. These data indicate key roles of PKMzeta and Na/K ATPase in ischemic preconditioning.

A new model of ischemic preconditioning using young adult hippocampal slice cultures.

In ischemic preconditioning (IPC), brief sublethal ischemia protects neurons from a subsequent lethal ischemia. In vivo models faithfully display preconditioning, yet, these models are technically challenging, time-consuming and expensive. In vitro models of preconditioning have also been developed that are technically easier and less expensive. A drawback of pre-existing in vitro models is that since susceptibility to ischemic injury is age-dependent; neuroprotection is being studied in neurons that have intrinsic resistance to oxygen-glucose deprivation (OGD). This study introduces a new in vitro model of ischemic preconditioning in hippocampal slice cultures isolated from 20-30-day-old rats. Slice cultures show a high susceptibility and sharp thresholds toward ischemia that is comparable to that found in vivo. A 5-min OGD treatment was not neurotoxic to young adult slice cultures, while a 10-min OGD treatment was neurotoxic. In addition, the sublethal 5-min OGD treatment protected against a 10-min OGD treatment that was delivered 24 h later. Neuroprotection was seen in preconditioned slice cultures stained with propidium iodide (PI) or with antisera against the neuron-specific antigen NeuN. Energy failure is hypothesized to trigger ischemic preconditioning and a 5-min OGD treatment induced transient energy failure in young adult slice cultures. This model may assist in the search for new therapeutics for the prevention and/or treatment of stroke.

[Construction of pcDNA3.1AM and expression of adrenomedullin in mammalian cells].

The newly discovered endogenous vasodilating and diuretic peptide adrenomedullin (AM) was considered to be of attractive value in clinical treatment of hypertension and congestive heart failure. In order to explore the treatment of cardiovascular diseases by expressing AM in vivo, AM cDNA was inserted into mammalian expressing vector pcDNA3.1, and in vitro expression of AM was carried out in cultured K(562) cell line. AM mRNA was amplified by RT-PCR from the total RNA isolated from the adrenal glands of rats and was inserted into pcDNA3.1 vector to form pcDNA3.1AM, the recombinant pcDNA3.1AM was then transferred into cultured K(562) cell line by liposome. The expression of AM in pcDNA3.1AM transferred cell was identified by RT-PCR and dot immunoblot assay. The results demonstrated that there were AM mRNA in the pcDNA3.1AM-transferred K(562) cell line and AM peptides in the culturing medium, indicating that the recombinant pcDNA3.1AM vector can express AM in mammalian cell line.

Low-dose cardiotonic steroids increase sodium-potassium ATPase activity that protects hippocampal slice cultures from experimental ischemia.

The sodium-potassium ATPase (Na/K ATPase) is a major ionic transporter in the brain and is responsible for the maintenance of the Na(+) and K(+) gradients across the cell membrane. Cardiotonic steroids such as ouabain, digoxin and marinobufagenin are well-characterized inhibitors of the Na/K ATPase. Recently, cardiotonic steroids have been shown to have additional effects at concentrations below their IC(50) for pumping. The cardiotonic steroids ouabain, digoxin, and marinobufagenin all show an inverted U-shaped dose-response curve with inhibition of pumping at concentrations near their IC(50), while increasing Na/K ATPase activity at doses below their IC(50). This stimulatory effect of cardiotonic steroids was observed in vitro in hippocampal slice cultures as well as in the hippocampus in vivo. Increased Na/K ATPase activity has been shown to protect slice culture neurons from hypoxia-hypoglycemia. Ouabain protected slice culture neurons from experimental ischemia at concentrations that increased Na/K ATPase. This protective effect was observed when ouabain was dosed 30min before, or 2h following experimental ischemia. Ouabain no longer protected against experimental ischemia if the increase of Na/K ATPase was blocked. These data suggest that the protective effect of ouabain was due to increased Na/K ATPase activity. The demonstration of a neuroprotective effect of cardiotonic steroids could potentially assist in the treatment of stroke since digoxin, one of the cardiotonic steroids examined in this study, has approval by the Food and Drug Administration and can be safely administered at the concentrations that increase Na/K ATPase activity.

Amelioration of cardiac function in chronic myocardial infarcted rats following administration of vector pcDNA3.1AM.

1. The present study was designed to examine the cardiovascular effects of intravenously administered pcDNA3.1AM, a recombinant non-virus vector carrying a rat adrenomedullin (AM) gene translation fragment, in rats with chronic cardiac dysfunction induced by ligation of the left descending coronary artery. 2. Haemodynamic parameters were recorded by intraventricular catheterization. In situ hybridization and polymerase chain reaction (PCR) were performed to identify the distribution of the introduced vector. The concentration of AM was determined by radioimmunoassay. 3. Progressive cardiac dysfunction was observed following coronary artery ligation, as indicated by a significant reduction in mean arterial pressure (MAP) and increases in both central venous pressure (CVP) and end-diastolic pressure of the left ventricle (LVEDP; P < 0.01). Administration of pcDNA3.1AM significantly attenuated the progressive cardiac dysfunction and lowered the elevated CVP and LVEDP. The introduced vector was widely distributed in different organs, including the lungs, kidney, heart, liver, spleen and brain. However, intense staining of pcDNA3.1 AM was observed in the lungs and kidneys. The introduced vector was localized mainly in the endothelial cells of blood vessels. Radioimmunoassay showed elevated levels of AM in the plasma and lung and heart after surgery, but there was no significant further increase in the concentration of AM after pcDNA3.1AM delivery. 4. The present study has provided some novel findings on the potential beneficial effects of AM gene delivery on chronic cardiac function in rats. Expression of AM by a non-virus vector may also have therapeutic value against cardiac dysfunction in vivo.