Culture and differentiation of preadipocytes in two-dimensional and three-dimensional in vitro systems

Adipogenesis is a complex process that involves the differentiation of preadipocytes into mature adipocytes. We have developed two-dimensional (2D) and three-dimensional (3D) cell culture systems for the purpose of culturing and differentiating primary preadipocytes in vitro. Differentiating preadipocytes show multiple lipid droplet accumulation and comparable protein expression patterns to mature adipocytes in vivo. We report that in both in vitro systems terminally differentiated adipocytes show characteristics similar to those of mature adipocytes in vivo, assessed by the expression of the S100alpha/beta protein, insulin receptor and caveolin-1, and receptors for inflammatory mediators, namely tumor necrosis factor-alpha receptors I and II (TNFRI and TNFRII) and chemokine receptor 5 (CCR5). Our results demonstrate that the S100 protein, caveolin-1, and insulin receptor are expressed and up-regulated in differentiating and terminally differentiated cells. In addition, the receptors for TNFalpha are not present in preadipocytes but are expressed in differentiating preadipocytes and in differentiated adipocytes. Similarly, CCR5 was exclusively expressed in differentiating preadipocytes and terminally differentiated adipocytes, but not in preadipocytes. Both 2D and 3D culture models are highly robust and reproducible and offer the potential to study adipogenesis and cellular interactions closely resembling and comparable to those in vivo. Our 3D collagen system offers a distinct advantage over the 2D system in that the adipocytes remain confined within the matrix and remain intact during biochemical analysis. Moreover, the collagen matrix allows adipocytes to closely simulate morphological characteristics and behavior as in vivo whilst permitting manipulation of the microenvironment in vitro to study adipogenesis.     

Oncocytic carcinoid tumor of the lung: a case report of diagnostic pitfall in filter membrane preparation of bronchial washings

BACKGROUND: Oncocytic carcinoid tumor of the lung is a rare variant of pulmonary carcinoid. This report describes the morphologic appearance of this rare tumor on filter membrane preparation along with potential pitfalls. CASE: A 49-year-old woman presented with cough and expectoration. On chest radiograph a mass lesion was seen in the upper zone of the right lung. Bronchial washings were sent for evaluation. On filter membrane (Millipore) preparation of bronchial washing the possibility of a non-small cell carcinoma, possibly squamous, was suggested. Right upper lobectomy was subsequently performed and a histologic diagnosis of oncocytic carcinoid given. The cytomorphologic features of this tumor on the Millipore preparation were reviewed. CONCLUSION: Differential diagnosis of oncocytic carcinoid should be kept in mind while assessing cytologic material when tumor cells show abundant granular cytoplasm and prominent nucleoli. Oncocytic carcinoid also must be differentiated from oncocytoma and granular cell tumor. Immunocytochemistry and electron microscopy are useful in confirming the diagnosis.     

Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM*

The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD⁺. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O₂-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.     

Catheter ablation of recurrent polymorphic tachycardia: Use of sodium channel blockade to organize the tachycardia: A case report

A 55 year old male presented with recurrent implantable cardioverter defibrillator (ICD) shocks due to polymorphic ventricular tachycardia (PMVT). He had undergone prior catheter ablation for VT three years ago. During the prior attempt he underwent voltage guided substrate ablation. With programmed ventricular extrastimulation (PVES), PMVT was repeatedly induced requiring DC shock. Intravenous procainamide was administered and PVES was repeated which induced sustained monomorphic ventricular tachycardia (MMVT). This VT had pseudo delta waves with maximum deflection index of 0.68, suggestive of epicardial origin. Activation mapping was performed epicardially. Presystolic potentials were recorded in mid anterolateral wall of left ventricular epicardial region. Radiofrequency (RF) ablation at this site terminated the VT. Post ablation there was no inducible tachycardia and patient is free of arrhythmias during 2 years of follow-up.     

Reprogramming events of mammalian somatic cells induced by Xenopus laevis egg extracts

It is known that differentiated cells can be reprogrammed to an undifferentiated state in oocyte cytoplasm after nuclear transfer. Recently, some reports suggested that Xenopus egg extracts have the ability to reprogram mammalian somatic cells. Reprogramming events of mammalian cells after Xenopus egg extract treatment and after cell culture of extract-treated cells have not been elucidated. In this experiment, we examined reprogramming events in reversibly permeabilized or nonpermeabilized porcine fibroblast cells after Xenopus egg extract treatment. The Xenopus egg-specific histone B4 was assembled on porcine chromatin and nuclear lamin LIII was incorporated into nuclei. Deacetylation of histone H3 at lysine 9 in extract-treated cells was detected in nonpermeabilized cells, suggesting that a part of reprogramming may be induced even in nonpermeabilized cells. Following culture of extract-treated cells, the cells began to express the pluripotent marker genes such as POU5F1 (OCT4) and SOX2 and to form colonies. Reactivation of the OCT4 gene in extract-treated cells was also confirmed in bovine fibroblasts transformed with an OCT4-EGFP construct. These results suggest that nuclei of mammalian cells can be partially reprogrammed to an embryonic state by Xenopus egg extracts and the remodeled cells partly dedifferentiate after cell culture. A system using egg extracts may be useful for understanding the mechanisms and processes of dedifferentiation and reprogramming of mammalian somatic cells after nuclear transfer.     

Chyluria with proteinuria or filarial nephropathy? An enigma

Lymphatic filariasis is endemic in India. Out of 128 million infected individuals worldwide, India accounts for 48 million cases [Manson's Tropical Diseases, 21st Ed. p 1488]. Filariasis can have protean manifestations, but Tropical pulmonary eosinophilia and chyluria are unusual manifestations reported mainly from South Asian countries [Manson's Tropical Diseases, 21st Ed. p 1494]. Chyluria occurs only in 2% of filarial afflicted patients in the filarial belt [Diamond E, Schapira HE. Chyluria--a review of literature. Urology 1985;26(5): 427-31]. Lymphatic filariasis presenting as chyluria may be equally rare. Predominant chyluria with no overt lymphatic filariasis remains an enigma.     

Increased expression of mitochondrial glycerophosphate dehydrogenase and antioxidant enzymes in prostate cancer cell lines/cancer

The involvement of mitochondrial glycerophosphate dehydrogenase (mGPDH) has previously been established in the production of ROS in prostate cancer cell lines (LNCaP, DU145, PC3 and CL1). The current study demonstrates that the mRNA level of mGPDH in prostate cancer cells is 3.3-8.9-fold higher compared to the normal prostate epithelial cell line, PNT1A. This is consistent with the enzymatic activity and protein level of mGPDH. However, cytochrome c oxidase (COX) activity is 2.9-3.2-fold down-regulated in androgen-independent prostate cancer cell lines. The level of antioxidant enzymes, catalase, MnSOD and CuZnSOD are up-regulated in prostate cancer cell lines. Furthermore, it was observed that the activity of mGPDH is significantly higher in liver tissues from all mice with cancer compared to liver tissues from control mice. These data suggest that the up-regulation of mGPDH, due to a highly glycolytic environment, contributes to the overall increase in ROS generation and may result in the progression of the cancer.