Emphysematous Pyelonephritis in Type 2 Diabetes - Clinical Profile and Management.

Emphysematous pyelonephritis (EPN) is a rare but life-threatening acute suppurative infection of the kidney among diabetics. There is no current consensus on the management of EPN. A prospective observational study was conducted at the Department of General Surgery, RML Institute of Medical Sciences, Lucknow, as well as at Eras Lucknow Medical College, Lucknow, from 2015 to 2018 to look for clinical, microbial profile and treatment outcome of diabetic patients with EPN. A total of 76 diabetic patients diagnosed with pyelonephritis were identified, of which 15 patients were diagnosed with EPN (26.3%). The mean age of the patients was 58.4 ± 6.5 years. The mean duration of diabetes was 5.3 ± 3.3 years. 12 (82%) of the 15 patients with diabetes mellitus had a glycosylated hemoglobin level higher than 7.5. Renal dysfunction at presentation was seen in 11 (73.3%) patients. Among the unilateral involvement, the left kidney was more affected. Escherichia coli in 11 (73.3%), Klebsiella sp. in one (6.6%), Pseudomonas in one (6.6%), and one each with polymicrobial and fungal urinary tract infection, respectively. Of 15 EPN patients, 13 (86.6 %) survived, and one (6.6 %) expired. Two of them underwent nephrectomy both survived. All patients with Stage I, II, and IIIa EPN (n = 12) were managed with antibiotics with or without percutaneous catheter drainage (PCD). In EPN Stage IIIb/IV (n = 3), all the three (20%) patients were managed with antibiotics and PCD, and later two (13.3%) needed nephrectomy. Only time to diagnosis, altered sensorium, shock at presentation, and thrombocytopenia were associated with poor outcome in EPN patients (P <0.05) Multiple logistic regression tests showed shock (P = 0.04) and disturbance of consciousness (P = 0.05) on (hospital admission as being the independent factors for poor outcome. EPN in diabetics needs a high index of suspicion, timely diagnosis, and good multidisciplinary approach with adequate antibiotics and surgical management for better patient outcomes.

Risk Categorization with Different Grades of Cervical Pre-Neoplastic Lesions - High Risk HPV Associations and Expression of p53 and RARß

Objective: To identify high risk HPV associations by evaluating linked p16 overexpression and also the expression of p53 and RARß together with histopathology for risk categorization of cervical pre-neoplastic lesions. Materials and Methods: Immunohistochemical staining was performed on 100 cases of cervical pre- neoplastic lesions for expression of biomarkers like p16, p53 and RARß for comparison with haematoxylin/eosin (HE) findings. All the experimentally generated data were statistically analyzed. Results: In this study 70% cases showed overexpression of p16INK4A increasing progressively from CIN I to CIN II but reduced in CIN III (p <0.01). p53 oncoprotein expression was seen in 51% cases, again with increments from CIN I to CIN II with slight reduction in CIN III (p<0.01). Some 24% cases showed negative immunoreactivity for the putative tumor suppressor gene RARß (p>0.05). Conclusion: Our study provides support for the idea that p16 can be used to identify associations with HPV , as well as having potential along with p53 and RARß for categorizing cervical pre-neoplastic cases having a higher risk of neoplastic conversion. Thus it may be concluded that accurate risk categorization can be achieved with the help of genetic markers as well as histopathology.

Association of Low Level of High Density Lipoprotein cholesterol with Acute Coronary syndrome.

Incidence of coronary artery disease (CAD) is increasing in developing countries in Bangladesh with improvement of socioeconomic status, urbanization, changes of dietary habits and lifestyle. Dyslipidaemia is one of the major contributors increase CAD risk. This study was aimed to find out the association of low level HDL-C with acute coronary syndrome. This cross sectional study was conducted in the department of cardiology, Mymensingh Medical College Hospital, Mymensingh, Bangladesh from August 2009 to May 2010. Sociodemographic characteristics, smoking, hypertension, FBS, serum total cholesterol level, HDL-C, LDL-C, Triglyceride level were important variable considered. A total number of 100 respondents consisted of 50 cases (patient) Group I and 50 healthy people (control) Group II. Investigations included ECG, Troponin-I, FBS and Fasting Lipid Profile. The data was analyzed by computer with the help of SPSS. Chi-square test, T-test, ANOVA test used as test of significance. The mean level in cases of HDL-C 39.3±5.1 and in control level HDL-C 34.2±3.4 statistically significant (p<0.0001). In both group low concentration HDL-C (<40mg/dl) risk for CAD. Un-adjusted odds ratio 95% CI determinants of ACS, HDL-C of OR was 0.2. So, HDL-C is not protective factor. In multivariate logistic regression analysis that adjusted for confounders of HDL-C level (age, sex, smoking, hypertension, TC, LDL-C, TG) associated with ACS. HDL-C was strong predictor of ACS (RR in the highest) compared with lowest quarantile = 0.02; (95% CI=0.003-0.173; P for trend <0.0001). The study reflected that low HDL-C level associated with ACS. Categorization of patients with ACS on the basis HDL-C level may be helpful for risk stratification and management.

Immunomodulatory effects of probiotics and prilled fat supplementation on immune genes expression and lymphocyte proliferation of transition stage Karan Fries cows.

BACKGROUND AND AIM: Probiotics are the living microorganism which when administered improves the digestion and health of the animal. Saccharomyces cerevisiae (SC) improves the humoral and innate immunity of the animal. Prilled fat is a hydrogenated palm oil triglyceride which has been reported to promote the release of cytokines from macrophages. The aim of the study was to evaluate the immunomodulatory effect of probiotic and prilled fat during transition stage in Karan Fries (KF) cows. MATERIALS AND METHODS: A total of 12 KF cows at 21 days prepartum were selected and divided into two groups of six animals each. The control group was fed as per the standard feeding practices and the supplemented group cows were supplemented daily with prilled fat at 100 g/cow, SC at 25 g/cow, and sweetener at 1 g/cow in addition to the standard feeding practices from -30 days of prepartum to 21 days of lactation. The sweetener was added to improve the palatability of the feed. The natural sweetener of an African plant leave had 105 times more sweetness than glucose with good aroma. The dry matter intake of the animal was recorded. Plasma samples were collected weekly from all cows for the analysis of blood metabolite beta-hydroxybutyric acid (BHBA). Lymphocytes were isolated from the blood for studying the expression of tumor necrosis factor alpha (TNF-α) and interleukin-1ß (IL-1ß) and for estimating lymphocyte proliferation index (LPI). RESULTS: The upregulated IL-1ß and TNF-α around calving might be possibly associated to the metabolic changes occurring during the transition period and suggest a higher degree of inflammation around parturition. High concentrations of BHBA caused increased expression and synthesis of the pro-inflammatory factors such as TNF-α and IL-1ß in supplemented group in primary calf hepatocytes. The LPI was higher in supplemented group as compared to control which suggests a stimulatory effect of unsaturated fatty acids on mitogen-stimulated T-cell proliferation. CONCLUSION: Dietary supplementation of probiotics, prilled fat, and sweetener alleviated negative energy balance by stimulating feed intake and modulating hepatic lipid metabolism; and both of these additives improved the postpartum health (antioxidant status and immune function) of transition dairy cows.

Novel integrated 3D multidetector computed tomography and fluoroscopy fusion for left atrial appendage occlusion procedures.

OBJECTIVES: This report demonstrates the application and feasibility of novel 3D-MDCT real-time fusion technology with fluoroscopy, for left atrial appendage (LAA) occlusion procedures. BACKGROUND: A successful LAA occlusion procedure relies on multiple imaging modalities, including TEE or 3D-MDCT, and fluoroscopy. Effectively integrating these imaging modalities may improve implantation safety and success. To our knowledge this technique has not been previously described for LAA occlusions. METHODS: This observational study compared clinical and procedural parameters for procedures performed with or without fusion integration. All patients had a pre-procedural 3D-MDCT for LAA measurements, along with 3D analyses of LAA morphology and surrounding structures. Using the image fusion software (Valve ASSIST 2, GE Healthcare, UK), landmarks were identified on fluoroscopy, and MDCT LAA anatomy outlines were then projected onto the real-time fluoroscopy image during the procedure, to guide all steps of the intervention. RESULTS: A total of 57 patients underwent LAA occlusion, with 16 performed using fusion software. In comparison to the pre-fusion group, reductions in contrast volume (21.0 ± 11.7 vs. 95.9 ± 80.5 ml, P < 0.001), procedure time (63.0 ± 22.0 vs. 87.3 ± 43.0 min, P = 0.01), and fluoroscopy time (6.2 vs. 8.3 min, P = 0.03) were observed. Incomplete sealing (0 vs. 14.6%, P = 0.16) and device deployment success (100 vs. 92.7%, P = 0.17) were not significantly different. CONCLUSIONS: The addition of this novel fusion technology is safe and feasible. To optimize LAA procedural success, fusion integration may offer a promising addition, or alternative, to current imaging modalities. © 2017 Wiley Periodicals, Inc.

Membrane bound pyrophosphatase and P-type adenosine triphosphatase of Leishmania donovani as possible chemotherapeutic targets: similarities and differences in inhibitor sensitivities.

The activities of inorganic pyrophosphatase (PPase) and adenosine triphosphatase (ATPase) were studied in the plasma membrane of Leishmania donovani promastigotes and amastigotes. It was shown that the specific activity of PPase was greater than that of ATPase in the promastigote plasma membrane. We characterized H+-PPase present in the plasma membrane of L. donovani and investigated its possible role in the survival of promastigote and amastigote. PPase activity was stimulated by K+ and sodium orthovanadate and inhibited by pyrophosphate analogs (imidodiphosphate and alendronate), KF, N,N'-dicyclohexylcarbodiimide (DCCD), thiol reagents (p-chloromercuribenzenesulfonate (PCMBS), N-ethylmaleimide (NEM), and phenylarsine oxide (PAO)), the ABC superfamily transport modulator verapamil, and also by the F(1)F(o)-ATPase inhibitor quercetin. ATPase activity was stimulated by K+ and verapamil, inhibited by DCCD, PCMBS, NEM, sodium azide, sodium orthovanadate, and quercetin, and was unaffected by PAO. We conclude that there are significant differences within promastigote, amastigote, and mammalian host in cytosolic pH homeostasis to merit the inclusion of PPase transporter as a putative target for rational drug design.

Enhanced frequency of micronuclei in individuals exposed to arsenic through drinking water in West Bengal, India.

In West Bengal, India arsenic in ground water has been found to be above the maximum permissible limit in seven districts covering an area of 37,493km2. In the present study, evaluation of the micronuclei (MN) formation in oral mucosa cells, urothelial cells and peripheral blood lymphocytes was carried out in the symptomatic individuals exposed to arsenic through drinking water. Forty five individuals with cutaneous signs of arsenicism from four affected districts (368.11 microg/l of As in drinking water) were considered as the exposed group and 21 healthy individuals with no symptoms of arsenic poisoning and residing in two unaffected districts (5.49 microg/l of As) were considered as controls. The exposed and control groups had similar age distribution and socioeconomic status. Standardised questionnaires were utilised and medical examination was conducted to ascertain exposure history, sociodemographic characteristics, diet, health, medication, addiction and chief symptoms in the study participants. Arsenic exposure was confirmed by measuring the arsenic content in the drinking water, nails, hair and urine samples from the volunteers. Arsenic contents in the urine, nail and hair in the exposed group were 24.45 microg/l, 12.58 and 6.97 microg/g, respectively which were significantly high in comparison to corresponding control group values of 4.88 microg/l, 0.51 and 0.34 microg/g, respectively. Exposed individuals showed a statistically significant increase in the frequency of MN in oral mucosa, urothelial cells and lymphocytes (5.15, 5.74 and 6.39/1000 cells, respectively) when compared with the controls (0.77, 0.56 and 0.53/1000 cells, respectively). Thus, the above results indicate that the symptomatic individuals exposed to arsenic through drinking water in this region have significant cytogenetic damage.

Age dependence of cellular properties of human septal cartilage: implications for tissue engineering.

BACKGROUND: The persistent need for cartilage replacement material in head and neck surgery has led to novel cell culture methods developed to engineer cartilage. Currently, there is no consensus on an optimal source of cells for these endeavors. OBJECTIVES: To evaluate human nasal cartilage as a potential source of chondrocytes and to determine the effect of donor age on cellular and proliferation characteristics. SUBJECTS: Nasal cartilage specimens were obtained after reconstructive surgery from 46 patients ranging in age from 15 to 60 years. METHODS: Specimens were weighed and chondrocytes were isolated by digestion in 0.2% collagenase type II for 16 hours. Cells were maintained in primary cultures until confluency, then seeded onto polylactic acid-polyglycolic acid scaffolds. Seeding efficiency was determined by quantification of DNA content of seeded constructs by means of Hoechst dye 33258. Specimen weights, cell yields, cell content, and doubling time were also measured and correlated to donor age. RESULTS: Mean (+/-SD) cartilage mass obtained (648 +/- 229 mg) is higher than from typical biopsy specimens of auricular cartilage, and the cellular characteristics show a higher proliferation rate than auricular chondrocytes. Cell yield increased with age, while doubling time decreased with age in samples from patients ranging from 15 to 60 years old. CONCLUSIONS: The use of nasal septal cartilage as a source of cells for tissue engineering may be valid over a wide range of patient ages. The large tissue yield and consequent cell yield make this tissue a potential starting source of chondrocytes for large-volume tissue-engineered implants.

Dehydroepiandrosterone sulfotransferase gene induction by bile acid activated farnesoid X receptor.

Dehydroepiandrosterone sulfotransferase (STD) is a hydroxysteroid sulfo-conjugating enzyme with preferential substrate specificity for C-19 androgenic steroids and C-24 bile acids. STD is primarily expressed in the liver, intestine and adrenal cortex. Earlier studies have shown that androgens inhibit the rat Std promoter function through a negative androgen response region located between -235 and -310 base pair positions (Song, C. S., Jung, M. H., Kim, S. C., Hassan, T., Roy, A. K., and Chatterjee, B. (1998) J. Biol. Chem. 273, 21856-21866). Here we report that the primary bile acid chenodeoxycholic acid (CDCA) also acts as an important regulator of the Std gene promoter. CDCA is a potent inducer of the Std gene, and its inducing effect is mediated through the bile acid-activated farnesoid X receptor (FXR), a recently characterized member of the nuclear receptor superfamily. The ligand-activated FXR acts as a heterodimer with the 9-cis-retinoic acid receptor (RXR) and regulates the Std gene by binding to an upstream region at base pair positions -169 to -193. This specific binding region was initially identified by bile acid responsiveness of the progressively deleted forms of the Std promoter in transfected HepG2 hepatoma and enterocyte-like Caco-2 cells. Subsequently, the precise RXR/FXR binding position was established by protein-DNA interaction using in vitro footprinting and electrophoretic mobility shift analyses. Unlike all other previously characterized FXR target genes, which contain an inverted repeat (IR) of the consensus hexanucleotide half-site (A/G)G(G/T)TCA with a single nucleotide spacer (IR-1), the bile acid response element of the Std promoter does not contain any spacer between the two hexanucleotide repeats (IR-0). A promoter-reporter construct carrying three tandem copies of the IR-0 containing -169/-193 element, linked to a minimal thymidine kinase promoter, can be stimulated more than 70-fold in transfected Caco-2 cells upon CDCA treatment. Autoregulation of the STD gene by its bile acid substrate may provide an important contributing role in the enterohepatic bile acid metabolism and cholesterol homeostasis.

Chasing hyponatraemia: unusual presentation.

Two females, in their sixth decade, presented with recurrent episodes of headache, vertigo, vomiting and altered sensorium. Both patients had persistent hyponatraemia as the only clue. Detailed investigations revealed a pituitary aetiology in both. One patient had a pituitary microadenoma while the other had an empty sella syndrome. The diagnosis and management is discussed and the relevant literature reviewed.

Effects of tomato extract on human platelet aggregation in vitro.

Among all fruits tested in vitro for their anti-platelet property, tomato had the highest activity followed by grapefruit, melon, and strawberry, whereas pear and apple had little or no activity. Tomato extract (20-50 microl of 100% juice) inhibited both ADP- and collagen-induced aggregation by up to 70% but could not inhibit arachidonic acid-induced platelet aggregation and concomitant thromboxane synthesis under similar experimental conditions. The anti-platelet components (MW <1000 Da) in tomatoes are water soluble, heat stable and are concentrated in the yellow fluid around the seeds. The active fractions were separated using gel filtration and HPLC. The aqueous fraction (110 000 xg supernatant) of tomatoes containing anti-platelet activity was subjected to gel filtration column chromatography (Biogel P2 column). The activity was fractionated into two peaks, peak-3 and peak-4 (major peak). Subsequently, peak-4 was further purified by HPLC using a reversed-phase column. NMR and mass spectroscopy studies indicated that peak F2 (obtained from peak 4) contained adenosine and cytidine. Deamination of peak F2 with adenosine deaminase almost completely abolished its anti-platelet activity, confirming the presence of adenosine in this fraction. In comparison, deamination of peak-4 resulted in only partial loss of inhibitory activity while the activity of peak-3 remained unaffected. These results indicate that tomatoes contain anti-platelet compounds in addition to adenosine. Unlike aspirin, the tomato-derived compounds inhibit thrombin-induced platelet aggregation. All these data indicate that tomato contains very potent anti-platelet components, and consuming tomatoes might be beneficial both as a preventive and therapeutic regime for cardiovascular disease.

Further characterization of a novel triacylglycerol hydrolase activity (pH 6.0 optimum) from microvillous membranes from human term placenta.

We recently identified the presence of two distinct triacylglycerol hydrolases with pH optima of 6.0 and 8.0 in human placental microvillous membranes (MVM). The TAG hydrolase with a pH optimum of 8.0 has properties similar to lipoprotein lipase, whereas TAG hydrolase with a pH optimum of 6.0 still to be fully characterized. In order to understand the functional and structural relationships between these two TAG hydrolases of MVM we have further investigated their biochemical and molecular properties. The presence of oleic acid inhibited TAG hydrolase activity with a pH optimum of 8.0 by 60 per cent whilst it had very little effect on the pH 6.0 TAG hydrolase activity. K(m)values for TAG hydrolases at pH 6.0 and pH 8. 0 optima were 170.6 and 9.83 nmol triolein, respectively, whereas the corresponding V(max)values were 0.32 and 0.037 nmol oleic acid/min mg/protein. Treatment of MVM with phenylmethylsulphonofluoride or protamine had no effect on TAG hydrolase at pH 6.0 whereas both decreased activity at pH 8.0, by 70 per cent and 52 per cent, respectively (P< 0.05), compared with control. p-Chloromercuribenzoate inhibited both TAG hydrolase activities by 25-30 per cent whereas iodoacetate inhibited TAG hydrolase activity with optimum pH 8.0 by 74 per cent and the activity at pH 6.0 by 28 per cent. Unlike the TAG hydrolase activity at pH 8.0, the activity at pH 6.0 was not affected by heparin. TAG hydrolase activity at pH 6.0 was significantly decreased compared with that of pH 8.0 optimum TAG hydrolase activity in smokers placenta. A threefold increase in pH 6.0 TAG hydrolase activity was observed following differentiation, whereas membrane associated TAG hydrolase activity with optimum pH 8.0 did not change. The TAG hydrolase with optimum pH 6.0 was subsequently purified from MVM to almost 1000-fold enrichment of the activity over the starting material. The final preparation however, still contained three distinct protein bands (90, 70 and 45 kDa). When extracted from non-denaturing polyacrylamide gels, the 70 kDa protein was the only protein to have TAG hydrolysing activity and had a pH optimum of 6.0. Labelling of samples with [(14)C]tetrahydrolipstatin also confirmed that the TAG hydrolase active protein was a 70 kDa protein. In conclusion, we report that there is a 70 kDa TAG hydrolase with optimum pH 6.0 in human placental MVM which is quite distinct from placental lipoprotein lipase.

Cellular uptake of long-chain fatty acids: role of membrane-associated fatty-acid-binding/transport proteins.

The critical importance of long-chain fatty acids in cellular homeostasis demands an efficient uptake system for these fatty acids and their metabolism in tissues. Increasing evidence suggests that the plasma-membrane-associated and cytoplasmic fatty-acid-binding proteins are involved in cellular fatty acid uptake, transport and metabolism in tissues. These binding proteins may also function in the fine tuning of cellular events by modulating the metabolism of long-chain fatty acids implicated in the regulation of cell growth and various cellular functions. Several membrane-associated fatty-acid-binding/transport proteins such as plasma membrane fatty-acid-binding protein (FABPpm, 43 kDa), fatty acid translocase (FAT, 88 kDa) and fatty acid transporter protein (FATP, 63 kDa) have been identified. In the feto-placental unit, preferential transport of maternal plasma arachidonic and docosahexaenoic acids across the placenta is of critical importance for fetal growth and development. Our studies have shown that arachidonic and docosahexaenoic acids are preferentially taken up by placental trophoblasts for fetal transport. The existence of a fatty-acid-transport system comprising multiple membrane-binding proteins (FAT, FATP and FABPpm) in human placenta may be essential to facilitate the preferential transport of maternal plasma fatty acids in order to meet the requirements of the growing fetus. The preferential uptake of arachidonic and docosahexaenoic acids by the human placenta has the net effect of shunting these maternal plasma fatty acids towards the fetus. The roles of plasma membrane-associated binding/transport proteins (FABPpm, FAT and FATP) in tissue-specific fatty acid uptake and metabolism are discussed.

Infantile neuroaxonal dystrophy and giant axonal neuropathy--overlap diseases of neuronal cytoskeletal elements in childhood?

Giant axonal neuropathy (GAN) and infantile neuroaxonal dystrophy (INAD) are two progressive neurodegenerative disorders of childhood that have considerable clinical as well as histological overlap but are believed to be ultrastructurally distinct. The clinicopathological and ultrastructural features of three cases of INAD, two of whom are siblings and one case of GAN are described. The sural nerve biopsies in all four cases were essentially similar on light microscopy revealing giant axons. On electron microscopy, the findings in the case of GAN were typical with dense accumulation of neurofilaments within the giant axons. In the three cases of INAD, too, in addition to accumulation of mitochondria and organelles with vesiculotubular profiles, a similar increase in neurofilaments was evident. We, therefore, believe that these two disorders may represent a spectrum in evolution of intermediate filament pathology with various organelles participating in the temporal evolution of the disease process.

Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells.

An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.

Preferential distribution of long chain polyunsaturated fatty acids in phospatidyl ethanolamine fraction of guinea pig alveolar apical membranes.

We investigated the fatty acid distribution in guinea pig alveolar apical membranes at different developmental stages. Fatty acid composition of the purified membranes isolated from guinea pig fetuses (at 65 day, term=68 day), neonates (day 1) and adult males was determined. The levels of arachidonic acid (AA) and docosahexaenoic acid (DHA) were higher in the adult guinea pig alveolar apical membrane phosphatidylethanolamine (PE) fraction (9. 3+/-2.2 and 2.9+/-1.0%, respectively) while in other phospholipids (PL) fractions their levels were low or absent (P<0.01). Furthermore, levels of AA and DHA in the PE fraction of apical membrane increased significantly from fetal (6.6+/-3.0 and 0.8+/-0.4%, respectively) to neonatal life (10.3+/-1.5 and 3.0+/-0.8%, respectively). Increase in the level of DHA (almost four-fold) was much more pronounced than that of AA (P<0.05). As for guinea pig alveolar membranes, EPA and AA were mostly present in the PE fraction in pulmonary adenocarcinoma derived cells (A549 cells), a parallel model of type II pneumocytes, with the levels of AA around three-fold greater than that of EPA, Binding of radiolabelled fatty acids to A549 cells showed no significant differences between the maximum uptake achieved for different fatty acids (AA, 1.7+/-0.2, EPA, 2.3+/-0.3, LA, 1.7+/-0.2, OA, 2.0+/-0.2nmol/mg protein, P>0.5). Once the fatty acids were taken up by these cells AA was mostly identifiable in the monoacylglycerol (MAG) fraction, whereas EPA was equally distributed between the MAG and PL fractions. Oleic acid was mainly present in the triglyceride (TAG) fraction whereas LA was evenly distributed between the TAG, MAG, and PL fractions. Our data demonstrate a preferential distribution of AA and DHA in PE fractions of alveolar apical membranes during development.

Age-dependent increase of heme oxygenase-1 gene expression in the liver mediated by NFkappaB.

Heme, the iron-porphyrin coordination complex, released from the degradation of hemoproteins, is a strong prooxidant. It is enzymatically degraded by heme oxygenase to free iron, carbon monoxide and biliverdin. Biliverdin and its reduced metabolite bilirubin are two potent physiological antioxidants. Here we show a progressive increase of steady-state levels of the mRNA encoding the inducible isoform of this enzyme (heme oxygenase-1) in the rat liver during aging. We had previously reported that aging is associated with increased activation of the nuclear factor kappaB (NFkappaB). We now provide evidence to establish that overexpression of NFkappaB in transfected liver-derived HepG2 cells can cause a marked induction of the endogenous heme oxygenase-1 (HO-1) mRNA and activation of the cotransfected HO-1 gene promoter. Taken together, these results support the conclusion that enhanced oxidative stress during aging is accompanied by compensatory induction of the antioxidant enzyme HO-1 through activation of the NFkappaB pathway.

Transport mechanisms for long-chain polyunsaturated fatty acids in the human placenta.

To understand the placental role in the processes responsible for the preferential accumulation of maternal long-chain polyunsaturated fatty acids (LCPUFAs) in the fetus, we investigated fatty acid uptake and metabolism in the human placenta. A preference for LCPUFAs over nonessential fatty acids has been observed in isolated human placental membranes as well as in BeWo cells, a human placental choriocarcinoma cell line. A placental plasma membrane fatty acid binding protein (p-FABP(pm)) with a molecular mass of approximately 40 kDa was identified. The purified p-FABP(pm) preferentially bound with essential fatty acids (EFAs) and LCPUFAs over nonessential fatty acids. Oleic acid was taken up least and docosahexaenoic acid (DHA) most by BeWo cells, whereas no such discrimination was observed in HepG2 liver cells. Studies on the distribution of radiolabeled fatty acids in the cellular lipids of BeWo cells showed that DHA is incorporated mainly into the triacylglycerol fraction, followed by the phospholipid fraction; the reverse is true for arachidonic acid (AA). The greater cellular uptake of DHA and its preferential incorporation into the triacylglycerol fraction suggests that both uptake and transport modes of DHA by the placenta to the fetus are different from those of AA. p-FABP(pm) antiserum preferentially decreased the uptake of LCPUFAs and EFAs by BeWo cells compared with preimmune serum. Together, these results show the preferential uptake of LCPUFAs by the placenta that is most probably mediated via the p-FABP(pm).

Molecular mechanism of cellular uptake and intracellular translocation of alpha-tocopherol: role of tocopherol-binding proteins.

Vitamin E (alpha-tocopherol) is a lipid-soluble antioxidant which is present in cellular membranes where it plays an important role in the suppression of free radical-induced lipid peroxidation. There are eight naturally occurring homologues of vitamin E which differ in their structure and in biological activity in vivo and in vitro. Various studies have suggested that the tocopherol distribution system favours the accumulation of alpha-tocopherol both in the plasma and different tissues. Mechanisms involved in the preferential accumulation of alpha-tocopherol are not yet well established; however, recent data indicate that both intracellular and membrane alpha-tocopherol-binding proteins may be involved in these processes. A 30 kDa alpha-tocopherol-binding protein (TBP) in the liver cytoplasm is now known to regulate plasma vitamin E concentrations by preferentially incorporating alpha-tocopherol into nascent very low density (VLDL) whereas the 15 kDa TBP may be responsible for intracellular distribution of alpha-tocopherol. The 30 kDa TBP is unique to the hepatocyte whereas the 15 kDa TBP is present in all major tissues. The 15 kDa TBP specifically binds alpha-tocopherol in preference to the delta- and gamma-tocopherol and may exclusively transport alpha-tocopherol to these intracellular sites. In addition, the presence of a membrane TBP (TBPpm) in tissues may regulate their alpha-tocopherol levels. Activity of erythrocyte TBPpm appears to be reduced in smokers, which may lead to reduced levels of alpha-tocopherol in these cells despite smokers have similar plasma levels of vitamin E as in non-smokers. The current status of the evidence for this directed flow of alpha-tocopherol through interactions with these proteins (TBP and TBPpm) is discussed.

Negative regulation of the androgen receptor gene promoter by NFI and an adjacently located multiprotein-binding site.

The upstream promoter of the rat androgen receptor (AR) gene contains a strong negative regulatory region located at the -388 to -340 nucleotide position. The distal part (-388/-373) of this regulatory region binds NFI, a ubiquitous transcription factor, while the proximal portion (-372/-340) contains an overlapping binding site for two nuclear proteins. This composite regulatory region (-388/-340) was initially defined by deoxyribonuclease I footprinting as the continuous stretch of a nuclease-protected site. NFI specificity of the distal portion (-388/-373) of the footprint was established through cross-competition in electrophoretic mobility shift assay (EMSA) using the well characterized NFI element of the adenovirus major late promoter and by immunoreactivity to the NFI antibody. EMSA with oligonucleotide duplexes corresponding to the proximal domain (-372/-340) indicated multiple retarded bands with at least two major DNA-protein complexes. Further analysis with truncated oligonucleotide duplexes showed that these two major proteins bind to this domain in an overlapping manner. Within this overlapping area, the position spanning -359 to -347 is essential for the formation of either of these two complexes. Substitution of four G with T residues in the overlapping area totally abolished all protein binding at the downstream -372/-340 site. Point mutations that abolish specific binding at either the NFI or immediately downstream multiprotein-binding site caused about a 10-fold increase in AR promoter activity in transfected HepG2 cells. Double mutation involving both the NFI and proximal overlapping protein-binding sites failed to cause any additional increase in promoter function. From these results we conclude that the AR promoter contains a composite negative regulatory region at -388/-340, and the repressor function may involve a coordinate interaction between NFI and at least two other nuclear factors.