Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.     

Acute responses of regional vascular conductance to oral ingestion of fructose in healthy young humans

Background

Recently, it was reported in healthy young subjects that fructose containing drinks increased blood pressure acutely, without any apparent change in total vascular conductance (TVC). However, because it is well known that the splanchnic vasculature is dilated by oral fructose ingestion, it is assumed to be the concomitant vasoconstriction in other peripheral region(s) that is responsible for this finding. Thus, the purpose of this study was to determine the acute response of regional VC to oral fructose ingestion in young healthy humans.

Results

In 12 healthy young subjects, mean arterial blood pressure (MAP), heart rate, cardiac output, and blood flow (BF) in the superior mesenteric (SMA), brachial (BA), and popliteal (PA) arteries, in addition to forearm skin BF, were measured continuously for 2 h after ingestion of 400 ml fructose solution (containing 50 g fructose). Regional VC was calculated as BF/MAP. MAP increased for 120 min after fructose ingestion without any change in TVC. While VC in the SMA was elevated after ingestion, VC in BA and PA and forearm skin decreased.

Conclusions

While TVC was apparently unchanged during the 2 h after fructose ingestion, there were coincident changes in regional VCs in the peripheral circulation, but no net change in TVC.     

Epithelial–mesenchymal transition of A549 cells is enhanced by co-cultured with THP-1 macrophages under hypoxic conditions

Epithelial–mesenchymal transition (EMT) is associated with pulmonary fibrosis, including idiopathic pulmonary fibrosis (IPF). In this study, we investigated EMT of human pulmonary epithelial-derived cells (A549). A549 cells was either cultured by itself or co-cultured with THP-1 macrophages under normoxic (21% O₂) and hypoxic (2% O₂) conditions. We evaluated the presence of EMT by determining the expression of EMT markers, E-cadherin, vimentin, and fibronectin. To determine the role of TGF-β1 and IL-1β in EMT of the A549 cells, we analyzed the effects of blocking their activity with TGF-β1 inhibitor or IL-1β neutralizing antibody respectively. The A549 cells presented EMT when they were co-cultured with THP-1 macrophages. The EMT of the A549 cells co-cultured with THP-1 macrophages was exacerbated under hypoxia. In addition, the EMT were prevented by the addition of TGF-β1 type I receptor kinase inhibitor. The hypoxic condition increased the mRNA levels of TGF-β1 in A549 cells and THP-1 macrophages and that of IL-1β in THP-1 macrophages when each cells were co-cultured. Anti-IL-1β neutralizing antibody attenuated TGF-β1 secretion in co-culture media under hypoxic conditions. Thus, the IL-1β from THP-1 macrophages up-regulated the TGF-β1 from A549 cells and THP-1 macrophages, and then the TGF-β1 from both cells induced and promoted the EMT of A549 cells when they were co-cultured under hypoxia. Together, these results demonstrate that the interaction between type II pneumocytes and macrophages under hypoxia is necessary for the development of pulmonary fibrosis.     

Potential role of altered calcium conductance at the onset of intensified spinal reflex after severing the sciatic nerve

The action of substances either increasing or inducing penetration of calcium ions through neuronal membranes on monosynaptic reflex discharges in the ventral horns (MR VH) were investigated in white rats between 5 and 7 days after severing the sciatic nerve. Systemically administered imidazole and 4-aminopyridine were used for the former and verapamil for the latter purposes. Effects of denervation and either imidazole or 4-aminopyridine administration were found to be synergistic; these interventions all led to a considerable increase in MR VH. Verapamil, on the other hand, reduced MR VH amplitude and raised the threshold for triggering these on both the operated and contralateral side. It is suggested that the early intensification of MR VH after severing the nerve is partly due to increased voltage-dependent calcium currents resulting from a reduced cyclic adenosine monophosphate level at presynaptic terminals of the reflex arc investigated.Medical Institute, Ministry of Public Health, Dnepropetrovsk. Translated from Neirofiziologiya, Vol. 22, No. 6, pp. 826–832, November–December, 1990.     

Evoked activity of spinal neurons in the early period after sciatic nerve division

The character of dorsal horn motoneurons and interneurons evoked by stimulation of the dorsal root, and activity of Renshaw cells in response to stimulation of the ventral root were studied in albino rats in the lower lumbar segments of the spinal cord 5 days after sciatic nerve division. A significant increase in the mean amplitude of excitatory postsynaptic potentials of motoneurons was observed on the side of division of the nerve. No significant change in membrane potential and in the threshold of appearance of the action potential of these motoneurons took place. The mean number of action potentials and the duration of discharge of the Renshaw cells and dorsal horn interneurons likewise were not significantly changed.Dnepropetrovsk Medical Institute, Ukrainian Ministry of Health. Translated from Neirofiziologiya, Vol. 24, No. 3, pp. 306–314, May–June, 1992.     

In vitro inhibition of glycogen-degrading enzymes and glycosidases by six-membered sugar mimics and their evaluation in cell cultures

We investigated in vitro inhibition of mammalian carbohydrate-degrading enzymes by six-membered sugar mimics and their evaluation in cell cultures. 1-Deoxynojirimycin (DNJ) showed no significant inhibition toward glycogen phosphorylase (GP) but was a potent inhibitor of another glycogen-degrading enzyme, amylo-1,6-glucosidase (1,6-GL), with an IC(50) value of 0.16 microM. In primary rat hepatocytes, the inhibition of glycogen breakdown by DNJ reached plateau at 100 microM with 25% inhibition and then remained unchanged. The potent GP inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (D-AB1) inhibited hepatic glucose production with an IC(50) value of about 9 microM and the inhibition by D-AB1 was further enhanced in the presence of DNJ. DNJ and alpha-homonojirimycin (HNJ) are very potent inhibitors of rat intestinal maltase, with IC(50) values of 0.13 and 0.08 microM, respectively, and also showed a similar strong inhibition toward maltase in Caco-2 cell model system, with IC(50) value of 0.05 and 0.10 microM, respectively. D-Isofagomine (D-IFG) and L-IFG are competitive and noncompetitive inhibitors of human lysosomal beta-glucosidase (beta-GL), respectively, with K(i) values of 8.4 nM and 6.9 microM. D-IFG increased intracellular beta-GL activity by twofold at 10 microM in Gaucher N370S cell line as an 'active-site-specific' chaperone, and surprisingly a noncompetitive inhibitor L-IFG also increased intracellular beta-GL activity by 1.6-fold at 500 microM.     

Dynamics of post-denervational modifications of spinal reflex activity in albino rats

In experiments on rats, we studied the characteristics of reflex discharges in the ventral root (VR) L ₅; the discharges were evoked by stimulation of segmental (peripheral nerve or dorsal root, DR) and suprasegmental vestibular (stimulation of the round window of the labyrinth) inputs. Potentials were recorded within different time intervals (from 1 to 150 days) after transection of the sciatic nerve (SN); measures preventing regeneration of its fibers were used. Modifications of the segmental responses related to post-denervational changes included four phases: (i) latent period, (ii) post-denervational spinal hyperreflexia (PDSH), (iii) partial suppression of monosynaptic discharges (MDs) in the VR, and (iv) complete disappearance of VR MDs resulting from late post-denervational changes. The latency of post-denervational modifications was about 18–48 h after the moment of transection of the SN. Within the PDSH phase, modifications were the greatest 3 to 5 days after transection; these changes could be more adequately estimated in the case of stimulation of the DR on the side of transection and not under conditions of stimulation of the central segment of the transected SN per se. Within this phase, the amplitudes of VR MDs and responses to vestibular stimulation were augmented two to three and four to five times, as compared with the respective indices in intact animals. From the 7th to 10th day after the nerve transection, the amplitude of VR MDs progressively dropped, and on about the 20th day these discharges practically disappeared, while polysynaptic components of segmental responses were preserved. Vestibular responses within this period were, as earlier, considerably facilitated. On the 60th and 150th days (within the phase of late post-denervational modifications) there were no VR MDs after stimulation of segmental inputs, and polysynaptic responses were exclusively observed. The amplitude of discharges evoked by vestibular stimulation became lower than in the PDSH state but remained significantly higher than the control values of this parameter. Probable mechanisms of post-denervational modifications of the evoked spinal activity within different time intervals after transection of the SN are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 37–46, January–February, 2007.     

Refolding and purification of human papillomavirus type 16 E7-lacZ fusion protein expressed in Escherichia coli.

Human papillomavirus (HPV) type 16 E7-lacZ fusion protein was produced in Escherichia coli, extracted as inclusion bodies, refolded with reducing reagents, and subjected to gel filtration. The refolded protein was purified by ion-exchange column chromatography, resulting in a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 1H nuclear magnetic resonance spectral changes were observed in the high field methyl region in the presence of Zn2+ ion, suggesting that the refolded form of the fusion protein is possibly renaturated into the putative zinc finger motif (C. Edmond and K. H. Vousden, 1989, J. Virol. 63, 2650-2656) and supporting the data of J. A. Rawls, R. Pusztai, and M. Green (1990, J. Virol. 64, 6121-6129) on zinc binding to E7 protein using radioisotopically labeled zinc ion.     

Effects of Long-Lasting Afferent Activation on Segmental Reflex Responses in Albino Rats,and Modifications of These Effects by Thyroxine and 4-Aminopyridine

In albino rats, we studied the effects of long-lasting tetanization of the dorsal roots of the L ₅ (homosynaptic activation) and L ₄ (heterosynaptic activation) segments on reflex discharges in the L ₅ ventral root evoked by single stimulation of the dorsal root of the same segment. Tetanization trains consisted of 5,000 stimuli applied with frequencies of 10, 50, 100, or 300 sec^–1, and their effects were tested during 10 min. There were no long-term post-tetanic potentiation (PTP) of monosynaptic responses when low frequencies of homosynaptic tetanization (10 and 50 sec^–1) were used. In the case of higher frequencies, PTP was rather clear and long-lasting. Under conditions of heterosynaptic activation, there was no PTP. Facilitation of polysynaptic responses developed at all the frequencies of homosynaptic tetanization used; when heterosynaptic tetanization was applied, such facilitation (although weaker) was also observed. In rats treated with agents increasing the excitability of spinal neuronal systems, such as thyroxine and 4-aminopyridine, tetanization of the studied inputs evoked long-term depression (LTD) of both mono- and polysynaptic components of the reflex discharges instead of PTP. Probable mechanisms of postsynaptic changes in the segmental reflex responses are discussed.     

Evoked Activity of Afferent and Efferent Fibers of the Sciatic Nerve in Rats under Conditions of Experimental Hyperthyroidism

In Wistar albino rats with experimental hyperthyroidism (HTh) and control animals, we measured parameters of the responses evoked in peripheral segments of the ventral and dorsal roots (VR and DR, respectively) by stimulation of the sciatic nerve. We found that the chronaxia of the afferent fibers of the sciatic nerve in HTh animals is shorter, while the duration of the mass action potential (AP) in the DR is somewhat longer than in the control. Under conditions of HTh, the excitation threshold of the efferent fibers became higher, the chronaxia decreased, and the second high-amplitude component could appear in the AP recorded from the VR. Possible mechanisms of changes in the excitability of afferent and efferent fibers of the sciatic nerve and specific features of the AP recorded from the VR under HTh conditions are discussed. In particular, we consider the possibility of ephaptic spreading of excitation in VR fibers under HTh conditions.