Alterations of bone marrow sinusoidal endothelium in rat and patients with liver cirrhosis.

Whether bone marrow changes occur and potentially contribute to the hematological abnormalities in liver cirrhosis remain unclear. In this study, we established a rat model of liver cirrhosis induced by carbon tetrachloride. Electron microscopy examination showed focal lesions in bone marrow sinusoidal endothelium and hematopoietic cells in animals with cirrhosis. With the persistence of liver cirrhosis, injuries of bone marrow sinusoidal endothelium progressed from mild mitochondrial changes to nuclear pycnosis and cell disruption, and the trilineage hematopoietic cells showed apoptosis and necrosis. Immunohistochemistry revealed increased expression of E-selectin, P-selectin and vWF in bone marrow sinusoidal endothelium of the cirrhotic rats, which was consistent with the data from semiquantitative reverse transcriptase-polymerase chain reaction analysis. Autopsy specimens from patients with liver cirrhosis (in the absence of other disease) showed the same findings as detected by immunohistochemistry in animal models. The results provide evidence of the association between liver cirrhosis and bone marrow alterations by demonstrating the bone marrow sinusoidal endothelium lesions in both a rat model and patients. It also indicates that activation or injury of bone marrow sinusoidal endothelium mediated by E-selectin, P-selectin, and vWF might have a role in pathogenesis of bone marrow changes during liver cirrhosis. The lesions of bone marrow sinusoidal endothelium might contribute to the hematological abnormalities in the end stage of liver disease.

Inhibition of evoked glutamate release by neurosteroid allopregnanolone via inhibition of L-type calcium channels in rat medial prefrontal cortex.

Allopregnanolone is one of the most important neurosteroids in the brain. We studied the effect and mechanism of allopregnanolone on spontaneous and evoked glutamate release in the medial prefrontal cortex using electrophysiological and biochemical methods combined with pharmacological approaches. The results showed that allopregnanolone had no effects on the frequency of miniature excitatory postsynaptic current (mEPSCs), but inhibited the depolarizing agent veratridine-evoked increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) and inhibited the first of the two responses evoked by a pair of electrical pulses more effectively than the second, resulting in increased paired-pulse facilitation (PPF) and thus suggesting a presynaptic inhibitory effect on electrical pulse-evoked glutamate release. A similar effect was also obtained for the effect of allopregnanolone on protein kinase A (PKA) activation, an upstream event of presynaptic glutamate release. Interestingly, allopregnanolone had none of these effects in the striatum. In the study of the upstream mechanism of the PKA inhibition by allopregnanolone, we found that allopregnanolone inhibited extracellular calcium influx-evoked PKA activation, but had no effects on intracellular calcium store release-evoked PKA activation; L-type calcium channel antagonists, but not N- and P/Q-type calcium channel antagonist, blocked the effect of allopregnanolone; allopregnanolone inhibited L-type calcium channel agonist-evoked increase in the PKA activity, intrasynaptosomal calcium concentration and frequency of sEPSCs. These results suggest that allopregnanolone inhibits evoked glutamate release via the inhibition of L-type calcium channels in the medial prefrontal cortex, but does not in the striatum.

Neurosteroid dehydroepiandrosterone sulfate enhances spontaneous glutamate release in rat prelimbic cortex through activation of dopamine D1 and sigma-1 receptor.

This paper studied the effect of neurosteroid dehydroepiandrosterone sulfate on spontaneous glutamate release in the prelimbic cortex by using electrophysiological and biochemical methods combined with a pharmacological approach and made some comparisons with those in the hippocampus. The results showed that dehydroepiandrosterone sulfate increased the frequency of miniature excitatory postsynaptic currents in the prelimbic cortex and hippocampus; sigma-1 receptor antagonist partially blocked the effect of dehydroepiandrosterone sulfate in the prelimbic cortex, but completely blocked it in the hippocampus; D1 receptor antagonist, adenylyl cyclase inhibitor and protein kinase A inhibitor completely blocked the effect of dehydroepiandrosterone sulfate in the prelimbic cortex; dehydroepiandrosterone sulfate increased the activity of protein kinase A in the prelimbic cortex and hippocampus; the effect of dehydroepiandrosterone sulfate on protein kinase A was completely blocked by sigma-1 receptor antagonist in the hippocampus, but was partially blocked in the prelimbic cortex; interestingly, here again, the effect of dehydroepiandrosterone sulfate on protein kinase A was completely blocked by D1 receptor antagonist in the prelimbic cortex. These results suggest that dehydroepiandrosterone sulfate promotes presynaptic glutamate release in the prelimbic cortex via activation of D1 and sigma-1 receptors.

[Effects of Astragalus polysaccharides and Cordyceps sinensis mycelium mixture on the transforming growth factor-beta1 expression in chronic rejection of artery transplantation: experiment with rats].

OBJECTIVE: To explore the effect of Astragalus polysaccharides (APS) and Cordyceps sinensis (CP) mycelium mixture on the chronic rejection of artery transplantation. METHODS: The abdominal arteries of 180 Wistar rats were isolated and transplanted to 180 SD rats whose with their abdominal arteries resected. The 180 recipient SD rats were randomly divided into blank control group (n = 20), APS group (n = 40, receiving gastric perfusion of APS 1 mg/kg bid), APS + low-dose CP group (n = 40, receiving APS and CP mycelium powder 1.25 g/d), APS + medium-dose CP group (n = 40, receiving APS and CP mycelium powder 2.5 g/d), and APS + high-dose CP group (n = 40, receiving APS and CP mycelium powder 5 g/d). Thirty days later the SD rats were killed with the transplanted abdominal arteries taken out to undergo microscopy. The expression of transforming growth factor (TGF)-beta(1) in the transplanted artery was examined by immunohistochemistry. RESULTS: In the control group, overexpression of TGF-beta(1) was observed in the intimal smooth muscle cells, monocytes, arterial endothelial cells, and arteriole, venule, and blood capillary endothelial cells in extima. However, the expression levels of TGF-beta(1) in the APS group and the 3 mixture administration groups were all significantly lower, especially in the medium- and high-dose groups (all P < 0.01). Severe edema of arterial endothelial cells and proliferation of monocytes were observed microscopically in the control group. Under electron microscope, rough endoplasmic reticulum and Golgi body were abundant in the control group. While in the mixture administration groups these changes were either weakened or absent. CONCLUSION: Astragalus polysaccharides and Cordyceps sinensis mycelium polysaccharides mixture can decrease the expression of TGF-beta(1) and inhibit the synthesis of collagen in the transplanted arteries.

Association between the expression of inhibitor of growth family member 4 and the progression of clear cell renal carcinoma.

Inhibitor of growth family member 4 (ING4) is a candidate tumor suppressor that serves important roles in tumor growth and angiogenesis. In the present study ING4 expression was assessed in clear cell renal carcinoma (CCRC) tissues and its association with the progression of CCRC was determined. The expression of ING4 in 125 patients with CCRC was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot analysis and immunohistochemical methods. A total of 40 adjacent normal renal tissues were used as control samples. The results identified that ING4 expression was positive in 100% of normal renal tissues, but in only 82.3% CCRC samples. RT-qPCR and western blotting results demonstrated that expression levels of ING4 mRNA and protein were significantly decreased in CCRC compared with in normal tissues (P<0.0001). ING4 expression was not associated with sex, age or tumor volume (P>0.05), but was associated with the nuclear grade of renal cancer (P<0.0001), the clinical stage of CCRC (P<0.0001) and lymphatic metastasis (P<0.0001). The results of the present study indicated that dysregulation of ING4 expression may be involved in the initiation and progression of CCRC.

Neurosteroid pregnenolone sulfate inhibits stimulus-evoked EPSC via presynaptic inhibition of protein kinase A in rat prelimbic cortical neurons.

Pregnenolone sulfate is one of the most abundantly produced neurosteroids in the brain. The present paper studies the effect of pregnenolone sulfate on excitatory synaptic transmission in the pyramidal cells of the layer V-VI of the prelimbic cortex using whole-cell patch-clamp in slices. We found that pregnenolone sulfate inhibited stimulus-evoked excitatory postsynaptic currents (EPSC); the effect of pregnenolone sulfate was significant at concentration of 1 microM and increased with an increase in concentrations; pregnenolone sulfate had no effect on the amplitude and frequency of miniature excitatory spontaneous postsynaptic currents; pregnenolone sulfate significantly enhanced the paired-pulse facilitation (PPF) and inhibited dopamine and 5-HT-evoked increase in the frequency of spontaneous excitatory postsynaptic currents; the protein kinase A inhibitor H89 and Rp-cAMPS enhanced PPF and canceled the effect of pregnenolone sulfate on PPF; pregnenolone sulfate inhibited the protein kinase A agonist forskolin-evoked increase in the frequency of spontaneous excitatory postsynaptic currents; the G(i) protein inhibitor N-ethylmaleimide canceled the effect of pregnenolone sulfate on PPF. These results suggest that pregnenolone sulfate inhibits stimulus-evoked EPSC via presynaptic inhibition of protein kinase A in rat prelimbic cortical neurons.

Progesterone inhibition of dopamine-induced increase in frequency of spontaneous excitatory postsynaptic currents in rat prelimbic cortical neurons.

We examined the effects of progesterone on frequency of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs), and dopamine-induced increase in the frequency of sEPSCs in pyramidal cells of layers V-VI of the rat prelimbic cortex using whole-cell patch-clamp techniques in slices. The results showed that progesterone 100 microM had no effects on the frequency of mEPSCs and sEPSCs, but significantly inhibited dopamine-induced increase in frequency of sEPSCs. This was in contrast to the effect of progesterone on the effect of 5-HT, which showed no changes after progesterone. When studying the mechanism of the progesterone effect, we observed that GABA(A) receptor antagonist and progesterone receptor antagonist did not influence the effect of progesterone; progesterone had no effects on D1 receptor agonist, protein kinase A and protein kinase C activator-induced increase in the frequency of sEPSCs. Interestingly, sigma(1) receptor antagonist could inhibit the effect of dopamine and sigma(1) receptor agonist had a synergistic effect on the effect of D1 receptor agonist. These results suggest that progesterone may inhibit dopamine-induced increase in frequency of sEPSCs in rat prelimbic cortical neurons via inhibition of sigma(1)/D1 receptor synergism because progesterone has been known to be an antagonist of sigma(1) receptor.

Genomic diversification among archival strains of Salmonella enterica serovar typhimurium LT7.

To document genomic changes during long periods of storage, we analyzed Salmonella enterica serovar Typhimurium LT7, a mutator strain that was previously reported to have higher rates of mutations compared to other serovar Typhimurium strains such as LT2. Upon plating directly from sealed agar stabs that had been stocked at room temperature for up to four decades, many auxotrophic mutants derived from LT7 gave rise to colonies of different sizes. Restreaking from single colonies consistently yielded colonies of diverse sizes even when we repeated single-colony isolation nine times. Colonies from the first plating had diverse genomic changes among and even within individual vials, including translocations, inversions, duplications, and point mutations, which were detected by rare-cutting endonuclease analysis with pulsed-field gel electrophoresis. Interestingly, even though the colony size kept diversifying, all descendents of the same single colonies from the first plating had the same sets of detected genomic changes. We did not detect any colony size or genome structure diversification in serovar Typhimurium LT7 stocked at -70 degrees C or in serovar Typhimurium LT2 stocked either at -70 degrees C or at room temperature. These results suggest that, although colony size diversification occurred during rapid growth, all detected genomic changes took place during the storage at room temperature and were carried over to their descendents without further changes during rapid growth in rich medium. We constructed a genomic cleavage map on the LT7 strain that had been stocked at -70 degrees C and located all of the detected genomic changes on the map. We speculated on the significance of mutators for survival and evolution under environmentally stressed conditions.