Enhanced activity of the plasma membrane oxidoreductase in circulating lymphocytes from insulin-dependent diabetes mellitus patients

Circulating human lymphocytes contain a transmembrane oxidoreductase (PMOR) capable of reducing dichlorophenol indophenol (DCIP) by endogenous reductants, presumably NADH. Membranes from lymphocytes obtained from buffy coats contain a NADH DCIP reductase having a K(m) of about 1 microM and almost insensible to dicoumarol. The PMOR of lymphocytes from insulin-dependent diabetic patients is higher than that from age-matched controls and, in addition, has a dicoumarol-sensitive component, lacking in most controls, presumably due to membrane association of DT-diaphorase. The increase of PMOR in diabetes is likely due to overexpression of the enzyme, in view of the very low K(m) for NADH indicating that, in intact cells, the enzyme is practically saturated with the reductant substrate.     

Molecular recognition between Azotobacter vinelandii rhodanese and a sulfur acceptor protein

The occurrence of rhodanese-like proteins in the major evolutionary phyla, together with the observed abundance of these proteins also within the same genome, suggests that their function cannot be limited to cyanide scavenging. The aim of the present study was to investigate whether Azotobacter vinelandii RhdA, an enzyme possessing unique biochemical and structural features with respect to other members of rhodanese homology superfamily, could recognize a suitable protein as a potential acceptor of the sulfane sulfur held on its catalytic Cys residue. Both the potential sulfur-delivery RhdA-S and the sulfur-deprived RhdA were found to interact with either holo- or apo-adrenodoxin, the 'substrate' protein used in this work. Interaction of RhdA-S with apo-adrenodoxin led to mobilization of RhdA-S sulfane sulfur. Under appropriate conditions, the sulfur released from RhdA-S was productively used for 2Fe-2S cluster reconstitution to yield holo-adrenodoxin from apo-adrenodoxin in the absence of any other sulfur source. A comparison of the reactivity of RhdA-S with protein and non-protein thiols allowed also some insights into the accessibility of the sulfane sulfur carried by RhdA.     

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo.     

The prediction of membrane protein structure and genome structural annotation

New methods, essentially based on hidden Markov models (HMM) and neural networks (NN), can predict the topography of both beta-barrel and all-alpha membrane proteins with high accuracy and a low rate of false positives and false negatives. These methods have been integrated in a suite of programs to filter proteomes of Gram-negative bacteria, searching for new membrane proteins.     

Insulin resistance,intramyocellular lipid content,and plasma adiponectin in patients with type 1 diabetes

Insulin resistance is a key pathogenic factor of type 2 diabetes (T2DM); in contrast, in type 1 diabetes (T1DM) it is considered a secondary alteration. Increased intramyocellular lipid (IMCL) content accumulation and reduced plasma adiponectin were suggested to be pathogenic events of insulin resistance in T2DM. This study was designed to assess whether IMCL content and plasma adiponectin were also associated with the severity of insulin resistance in T1DM. We studied 18 patients with T1DM, 7 older and overweight/obese patients with T2DM, and 15 nondiabetic, insulin-resistant offspring of T2DM parents (OFF) and 15 healthy individuals (NOR) as appropriate control groups matched for anthropometric features with T1DM patients by means of the euglycemic hyperinsulinemic clamp combined with the infusion of [6,6-2H2]glucose and 1H magnetic resonance spectroscopy of the calf muscles. T1DM and T2DM patients showed reduced insulin-stimulated glucose metabolic clearance rate (MCR: 5.1 +/- 0.6 and 3.2 +/- 0.8 ml x kg(-1) min(-1)) similar to OFF (5.3 +/- 0.4 ml x kg(-1) x min(-1)) compared with NOR (8.5 +/- 0.5 ml x kg(-1) min(-1), P < 0.001). Soleus IMCL content was increased in T1DM (112 +/- 15 AU), T2DM (108 +/- 10 AU) and OFF (82 +/- 13 AU) compared with NOR (52 +/- 7 AU, P < 0.05) and the result was inversely proportional to the MCR (R2 = 0.27, P < 0.001); an association between IMCL content and Hb A1c was found only in T1DM (R2 = 0.57, P < 0.001). Fasting plasma adiponectin was reduced in T2DM (7 +/- 1 microg/ml, P = 0.01) and OFF (11 +/- 1 microg/ml, P = 0.03) but not in T1DM (25 +/- 6 microg/ml), whose plasma level was increased with respect to both OFF (P = 0.03) and NOR (16 +/- 2 microg/ml, P = 0.05). In conclusion, in T1DM, T2DM, and OFF, IMCL content was associated with insulin resistance, demonstrating that IMCL accretion is a marker of insulin resistance common to both primary genetically determined and secondary metabolic (chronic hyperglycemia) alterations. The increased adiponectin levels in insulin-resistant patients with T1DM, in contrast to the reduced levels found in patients with T2DM and in OFF, demonstrated that the relationship of adiponectin to insulin resistance in humans is still unclear.     

Influence of rumen protein degradability on productive and reproductive performance in buffalo cows

The present study aimed to ascertain the influence of crude protein (CP) digestibility in the rumen on the quantity and quality of milk production and reproductive performance, blood (BU) and milk (MU) urea, haematological profile and vaginal mucus urea, ammonia and potassium of buffalo cows. Lactating buffaloes (n = 84), 60 days in milk, were randomly subdivided into Group C (control, n = 42) and Group T (fed a diet supplemented with Aspergillus oryzae, n = 42). In three fistulated buffalo, the diet supplemented with Aspergillus oryzae showed a decrease (P < 0.01) in protein digestibility in the rumen (79.3 vs. 45.9%). No differences were registered in productive performance. Nine buffaloes not in oestrus during the dietary treatment (Groups T1 and C1), 30 days in milk, were used to study the haematological profile and to determine milk urea and ammonia in the vaginal mucus. The animals in Group T1 had higher ammonia values in the blood (P < 0.01) but not in the vaginal mucus than Group C1. A relationship was found between MU and BU. MU was influenced by CP intake and dry matter intake. No differences between the treatments were observed in reproductive performance and the conception rate and calving interval were 37.9% and 41.4% (90 trial-day) and 449 and 419 days respectively in Groups T and C. Reproductive performance was not influenced by high levels of BU nor by blood ammonia levels, although the latter were higher in the group fed the diet supplemented with Aspergillus oryzae.     

Rate limiting step of the allosteric activation of the bacterial adhesin FimH investigated by molecular dynamics simulations

The bacterial adhesin FimH is a model for the study of protein allostery because its structure has been resolved in multiple configurations, including the active and the inactive state. FimH consists of a pilin domain (PD) that anchors it to the rest of the fimbria and an allosterically regulated lectin domain (LD) that binds mannose on the surface of infected cells. Under normal conditions, the two domains are docked to each other and LD binds mannose weakly. However, in the presence of tensile force generated by shear the domains separate and conformational changes propagate across LD resulting in a stronger bond to mannose. Recently, the crystallographic structure of a variant of FimH has been resolved, called ${\text{FimH}}^{\text{FocH}}$, where PD contains 10 mutations near the inter-domain interface. Although the X-ray structures of FimH and ${\text{FimH}}^{\text{FocH}}$ are almost identical, experimental evidence shows that ${\text{FimH}}^{\text{FocH}}$ is activated even in the absence of shear. Here, molecular dynamics simulations combined with the Jarzynski equality were used to investigate the discrepancy between the crystallographic structures and the functional assays. The results indicate that the free energy barrier of the unbinding process between LD and PD is drastically reduced in ${\text{FimH}}^{\text{FocH}}$. Rupture of inter-domain hydrogen bonds involving R166 constitutes a rate limiting step of the domain separation process and occurs more readily in ${\text{FimH}}^{\text{FocH}}$ than FimH. In conclusion, the mutations in ${\text{FimH}}^{\text{FocH}}$ shift the equilibrium toward an equal occupancy of bound and unbound states for LD and PD by reducing a rate limiting step.     

TNFalpha up‐regulates SLUG via the NF‐kappaB/HIF1alpha axis,which imparts breast cancer cells with a stem cell‐like phenotype

Extracellular and intracellular mediators of inflammation, such as tumor necrosis factor alpha (TNFα) and NF‐kappaB (NF‐κB), play major roles in breast cancer pathogenesis, progression and relapse. SLUG, a mediator of the epithelial–mesenchymal transition process, is over‐expressed in CD44⁺/CD24^? tumor initiating breast cancer cells and in basal‐like carcinoma, a subtype of aggressive breast cancer endowed with a stem cell‐like gene expression profile. Cancer stem cells also over‐express members of the pro‐inflammatory NF‐κB network, but their functional relationship with SLUG expression in breast cancer cells remains unclear. Here, we show that TNFα treatment of human breast cancer cells up‐regulates SLUG with a dependency on canonical NF‐κB/HIF1α signaling, which is strongly enhanced by p53 inactivation. Moreover, SLUG up‐regulation engenders breast cancer cells with stem cell‐like properties including enhanced expression of CD44 and Jagged‐1 in conjunction with estrogen receptor alpha down‐regulation, growth as mammospheres, and extracellular matrix invasiveness. Our results reveal a molecular mechanism whereby TNFα, a major pro‐inflammatory cytokine, imparts breast cancer cells with stem cell‐like features, which are connected to increased tumor aggressiveness. J. Cell. Physiol. 225: 682–691, 2010. © 2010 Wiley‐Liss, Inc.