Regulation of lewis lung carcinoma invasion and metastasis by protein kinase A

Metastatic Lewis lung carcinoma (LLC-LN7) cells have increased protein kinase A (PKA) activity and are more invasive in vitro than are non-metastatic (LLC-C8) cells. To determine whether PKA mediates the in vitro invasiveness and in vivo metastatic capabilities of these tumor cells, the LLC variants were stably transfected to over-express the C_α subunit of PKA, and thus to have increased PKA activity, or to express a mutant cAMP-resistant PKA R_lα subunit which blocks PKA activation. Wild-type LLC-LN7 tumor cells were invasive in vitro and in vivo, recurred after tumor excision and metastasized to the lungs. However, they lost these properties after transfection to express the mutant R_lα that blocks PKA activation. The non-invasive, non-recurring and non-metastatic LLC-C8 cells gained the capacity to invade, to recur following tumor excision and to metastasize when transfected to express the PKA C_α subunit. © 1995 Wiley-Liss, Inc.     

Cardiac MRI in Congenital Heart Disease – Our Experience

A significant recent advance that has occurred world over in the continuously evolving field of Magnetic Resonance Imaging (MRI) practice is the introduction of Cardiac applications. Cardiac MRI has moved to the centre stage of clinical management strategy by non-invasively imaging the structure as well as function of the heart. It has a wide range of specific applications such as delineation of morphological anatomy, quantification of flow and pressure across cardiac valve dysfunction, evaluation of myocardial function, assessment of infarcts, mapping coronary arteries and so on. Evaluation of congenital heart disease (CHD) is an important application of Cardiac MRI since the morphological details of chambers, septum, defects and anomalous connections are depicted accurately. Besides, flow information across valves, chambers, outflow tracts and shunts are also provided. This article describes our experience in the use of cardiac MRI in congenital heart disease.Key Words: Cardiac MRI, Congenital heart disease, Cyanotic and Acyanotic heart disease     

Comparative study on density gradients and swim-up preparation techniques utilizing neat and cryopreserved spermatozoa

AIM: To 1) compare post-wash and post-thaw parameters of sperm processed with PureSperm density gradient technique and swim-up method; and 2) test the efficacy of two commonly available density gradient media PureSperm and ISolate. METHODS: This prospective study used semen specimens from 22 patients. Specimens from nine patients were processed by both PureSperm density gradient and swim-up method. These specimens were then cryopreserved. Thirteen specimens were processed by both PureSperm (40 % and 80 %) and Isolate (50 % and 90 %) double density gradient techniques. The two fractions processed by both PureSperm and swim-up were analyzed for post-wash sperm characteristics. Post-thaw analysis was done after 24 hours. Sperm fractions obtained after processing with PureSperm and ISolate were compared for post-wash sperm characteristics and ROS levels. Results: Specimens prepared with PureSperm had significantly higher median total motile sperm counts (TMSC) (32.2 x 10(6) vs. 17.6 x 10(6)), recovery rates (69.2 % vs. 50.0 %), and longevity at 4 hours (83.0 % vs. 55.0 %) compared to specimen prepared by swim-up. Post-thaw specimens also had a higher recovery and longevity at 4 hours with PureSperm as compared to the swim-up. Semen specimens processed by PureSperm had significantly higher total sperm count, TMSC, and percentage recovery rates (30.0 % vs. 19.7 %) than ISolate. CONCLUSION: Semen quality is better preserved in fresh and cryopreserved semen prepared with PureSperm density gradient compared to swim-up. A significant enrichment of sperm is observed with PureSperm compared to ISolate. Higher recovery rates of mature motile sperm obtained after PureSperm sperm preparation may be beneficial for successful ART.     

A novel vector for the expression of SCR domains in insect cells

Exploitation of recombinant technology to study proteins containing strings of short consensus repeat (SCR) domains largely depends on expression vectors. In this paper, we describe a vector for cloning and constitutive expression of single or multiple SCR domains. The recombinant vector has unique additive features over commercially available vectors that make it a universal cloning vector for SCR domains as well as a vector suitable for expressing any protein fragment beginning and ending with cysteine residues. As a demonstration of its usefulness, the constitutive extracellular expression of five SCR-containing proteins derived from complement factor H is presented.     

Impact of holmium:YAG and neodymium:YAG lasers on the efficacy of DNA delivery in transitional cell carcinoma

New approaches in the treatment of transitional cell carcinoma (TCC) are using gene therapy to influence the disease at the genetic level. Technical advances in genomics, the availability of tissue-specific gene promoters and other developments have made this approach more realistic. Transporting the gene into the target cell is still the major problem. Several transfection techniques have been introduced. Transfection of naked DNA is one of the simplest to perform but transfection rates have been very poor. We investigated the influence of laser energy on transfection efficacy in urothelial cancer cells in vitro with two types of medical lasers. A suspension of human transitional cancer cells (UM-UC3; 3.5 million cells/ml) was mixed with 200 g of plasmid DNA (pEGFP-N1). Two types of laser energy, neodymium:YAG (Nd:YAG) and holmium:YAG (Ho:YAG), were applied to the cell suspension in different energy settings. Twenty four hours after treatment, transfection rates were measured with FACS analysis. Energy setting parameters that determine the efficacy of laser were investigated. The significance of different transfection rates was estimated with the students t-test. We demonstrated that the Nd:YAG laser was not suitable for achieving significant transfection of the reporter gene to the cells. In contrast, the Ho:YAG laser produced satisfactory transfection rates. There was an increase in transfection with increasing frequency of laser pulses, from 16% with 2 Hz up to 40% with 10 Hz (p<0.0005). Pulse frequency was therefore stabilised at 10 Hz. Pulse energy (mJ) showed the same dependency: a transfection rate of 18.3% was achieved with 1,000 mJ and 53.8% with 2,000 mJ (p>0.0005). Additionally, we investigated the impact of total pulse number (imp) with different pulse energies. At 1,000 mJ, a transfection rate of 18.3% was estimated with 200 imp and 48.56% with 750 imp, (p<0.0005). At 2,000 mJ, a transfection rate of 53.8% was achieved with 200 imp and 58.26% with 500 imp. The optimal laser setting observed in this experiment was 10 Hz, 2,000 mJ and 500 imp. This study indicates that the efficacy of naked DNA delivery into TCC in vitro is improvable by application of Ho:YAG laser energy. The Nd:YAG laser did not increase transfection rates in our model. Our results with the Ho:YAG laser are encouraging for further studies to optimise DNA delivery. As TCC tissue is relatively easy to access, this method could become an effective and minimally invasive procedure in urothelial cancer treatment.     

The influence of oxalate on renal epithelial and interstitial cells

Most renal stones in humans are composed of calcium oxalate. An increase in urinary oxalate levels has been shown to result in renal epithelial cell injury and crystal retention. However, the underlying mechanisms are unclear. Although the localization of primary stone formation and the associated cells playing the pivotal role in stone formation are still unknown, renal epithelial cells and interstitial cells seem to be involved in this process. The aim of this study was to evaluate the effects of oxalate on distinct renal epithelial and endothelial cells as well as fibroblasts. The first part focused on the toxicity of oxalate on the cells and a potential time- and dose-dependency. In the second part, renal epithelial cells were cultured in a two-compartment model to examine the vulnerability of the tubular or basolateral side to oxalate. LLCPK1, MDCK, renal fibroblast and endothelial cell lines were cultured under standard conditions. In part 1, cells were grown in standard culture flasks until confluent layers were achieved. Sodium oxalate was delivered at final concentrations of 1, 2 and 4 mM to either the apical or basolateral side (plain medium was delivered to the contralateral side). Cell survival was assessed microscopically by trypan blue staining after 1, 2 and 4 h. The influence of oxalate on proliferation and apoptosis induction was also investigated. In the second part, MDCK and LLCPK1 cells were grown in 6-well plates until confluent layers were achieved. Sodium oxalate at the above concentrations was applied, to either the apical or basolateral side and plain medium was delivered to the opposite side. The same protocol was then followed as in part 1. Part 1: sodium oxalate led to a time- and concentration-dependent decline in cell survival that was comparable in LLCPK1 and MDCK. Non-tubular cell lines like fibroblasts and endothelial cells were significantly more vulnerable to oxalate. These observations were reflected by significant impairment to cell proliferation. We could not demonstrate an induction of apoptosis in any cell line. Part 2: both cell lines were more vulnerable to oxalate on the basolateral side. This effect was more pronounced in MDCK cells at high oxalate concentrations (4 mM). Cells are apparently more resistant on the apical (tubular) side. Our results show that sodium oxalate has a negative effect on the growth and survival of renal epithelial cells and, to a greater extent, also fibroblasts and endothelial cells. We could not demonstrate any induction of apoptotic processes which implies a direct induction of cell necrosis. The finding of interstitial calcification and the proximity of tubules, vessels and interstitial cells make involvement of non-tubular renal cells in tissue calcification processes possible. Renal epithelial cells are apparently more vulnerable to oxalate on their basolateral side. Therefore, calcification processes within the interstitium may exert pronounced toxic effects to these cells, leading to inflammation and necrosis. These observations further support the idea of the interstitium as a site of primary stone formation.     

Carotid artery injury during transsphenoidal resection of pituitary tumor: anesthesia perspective

The authors describe two patients who suffered carotid artery injury during transsphenoidal resection of a pituitary tumor. Anesthesiologists were involved in resuscitation after initial hemorrhage, in securing the airway, in initiating cerebral protection strategies, and in transporting these patients. Anesthesia was provided for resection of the tumors, removal of packs from the pituitary fossae, and diagnostic and therapeutic radiologic procedures. In each case hemostasis was achieved by packing the sella turcica, sphenoid sinus, and nostrils. Both patients were electively ventilated. In one patient, a pseudoaneurysm of the carotid artery was detected by angiography in the operating room. It was treated by trapping the internal carotid artery. The other patient developed a carotid-cavernous fistula, which was treated by balloon embolization. Both patients were discharged after dealing with these complications.     

Cerebral hyperperfusion syndrome after carotid endarterectomy: predictive factors and hemodynamic changes

PURPOSE: It is believed that cerebral hyperperfusion syndrome (CHS) is caused by loss of cerebral autoregulation resulting from chronic cerebral ischemia and that factors including increased intraoperative cerebral blood flow, ipsilateral or contralateral carotid disease, and postoperative hypertension may cause CHS. We describe our experience with CHS, which diverges from published reports. MATERIALS AND METHODS: From March 2000 to February 2002 we performed 455 carotid endarterectomy (CEA) procedures in 404 patients at our institution. CHS developed 1 to 8 days (mean, 3.2 +/- 2.5 days) postoperatively in 9 patients (2%), 6 women and 3 men, whose age ranged from 52 to 84 years (mean, 69 +/- 8 years). Indications for surgery in 8 patients without neurologic symptoms were ipsilateral internal carotid artery (ICA) stenoses ranging from 70% to 99% (mean, 80% +/- 7%); the remaining patient had an ipsilateral stroke, with good clinical recovery, 7 weeks before CEA. Only 1 patient had significant contralateral ICA stenosis (70%). However, 5 patients had undergone contralateral CEA within the previous 3 months. CHS symptoms were severe headache in 5 patients, seizures in 3 patients (1 stroke), and visual disturbance and ataxia in 1 patient. All 404 patients (455 cases) underwent intraoperative and early (2 weeks) postoperative carotid artery duplex scanning. The 9 patients with CHS also underwent carotid artery duplex scanning at the time of the neurologic event. RESULTS: Mean intraoperative ICA volume flow (MICAVF) in the 9 CHS cases was not significantly different from that in the other 446 cases (170 +/- 47 mL/min and 182 +/- 81 mL/min, respectively). However, mean ICA volume flow (481 +/- 106 mL/min) and peak systolic velocity (PSV) (108 +/- 33 cm/s) for the 9 CHS cases measured at onset of symptoms were higher than those for the remaining 446 cases (267 +/- 87 mL/min and 80 +/- 26 cm/s, respectively) (P <.01). Of the 9 patients with CHS, only 3 had systolic blood pressures more than 160 mm Hg at onset of symptoms. Severity of ipsilateral and contralateral ICA stenoses was not significantly different between the 9 CHS cases and the remaining 446 cases. CONCLUSIONS: These data do not corroborate the common belief that CHS occurs preferentially in patients with severe ipsilateral or contralateral carotid disease, increased intraoperative cerebral perfusion, or severe hypertension. Recently performed contralateral CEA (<3 months) appears to be predictive of CHS.     

The blighted hills of Roro,Jharkhand, India: a tale of corporate greed and abandonment

In the Chaibasa region of the West Singhbhum district of Jharkhand, India, an abandoned chrysotile asbestos mine is a health scourge for villagers and former mine workers. A massive pile of asbestos waste mixed with chromite has lain atop the hilltops of Roro village for two decades, gradually seeping into the land, water, homes, and bodies of the tribal communities living at the foothills of Roro. To investigate the status of the asbestos waste and its impact on the community and the environment, a fact-finding team made a preliminary assessment. Its findings suggest that the careless closure of the mines and the unscientific disposal of toxic asbestos and chromite waste by the mining company pose a serious threat to the health of the local community and the environment. The preliminary health survey of 14 villages around the Roro hills, with 45% of the respondents being former workers of the Roro asbestos mines, indicates a highly probable link between the asbestos exposures and several adverse health effects such as low back pain, dyspnea, hemoptysis, and blindness.