Intracellular annexin A2 regulates NF-κB signaling by binding to the p50 subunit: implications for gemcitabine resistance in pancreatic cancer.

Annexin A2 (ANXA2) expression is highly upregulated in many types of cancer. Although cell surface localization of ANXA2 has been reported to have a critical role in the progression and metastasis of a variety of tumors, including pancreatic cancer, the biological role of intracellular ANXA2 is not fully understood. Herein the role of intracellular ANXA2 was investigated in a pancreatic cancer cell line. We first determined whether ANXA2 is involved in NF-κB signaling pathways. ANXA2 bound to the p50 subunit of NF-κB in a calcium-independent manner, and the ANXA2-p50 complex translocated into the nucleus. Furthermore, ANXA2 increased the transcriptional activity of NF-κB in both the resting and activated states and upregulated the transcription of several target genes downstream of NF-κB, including that encoding interleukin (IL)-6, which contributes to anti-apoptotic signaling. In Mia-Paca2 cells, we determined the effects of wild-type ANXA2 and an ANXA2 mutant, Y23A, which suppresses the cell surface localization, on upregulation of NF-κB transcriptional activity and secretion of IL-6. Both wild-type and Y23A ANXA2 induced anti-apoptotic effects in response to treatment with tumor necrosis factor-α or gemcitabine. Based on these results, we suggest that ANXA2 mediates resistance to gemcitabine by directly increasing the activity of NF-κB. Collectively, these data may provide additional information about the biological role of ANXA2 in pancreatic cancer and suggest that ANXA2 is a potential biomarker for the drug resistance phenotype and a candidate therapeutic target for the treatment of pancreatic cancer.

Comparison of MR imaging findings between extraligamentous and subligamentous disk herniations in the lumbar spine.

BACKGROUND AND PURPOSE: The method of treating an HIVD in the lumbar spine may depend on the integrity of the PLL. The purpose of this study was to analyze and compare the MR imaging findings of extraligamentous and subligamentous HIVDs in the lumbar spine. MATERIAL AND METHODS: One hundred seventeen patients (M/F = 71:46; mean age, 47 years; age range, 15-79 years) underwent lumbar spine MR imaging and disk surgery (extraligamentous/subligamentous = 66:51) from May 2003 to November 2006. Two radiologists in consensus retrospectively reviewed all MR images, focusing on 10 criteria. RESULTS: The following 5 criteria are suggestive of extraligamentous HIVD in the lumbar spine: 1) spinal canal compromised for more than half its dimension, 2) internal signal difference in the HIVD, 3) an ill-defined margin of the HIVD, 4) disruption of the continuous low-signal-intensity line covering the HIVD, and 5) the presence of an internal dark line in the HIVD (P < .05). When we combined these 5 MR imaging criteria, the sensitivity, specificity, accuracy, and odds ratio were 77.3%, 74.5%, 76.1%, and 9.93 (P < .0001). CONCLUSIONS: Our proposed 5 MR imaging criteria will be helpful in differentiating extraligamentous and subligamentous HIVDs in the lumbar spine.

Predictive value of intra-amniotic and serum markers for inflammatory lesions of preterm placenta.

OBJECTIVE: To compare the relative predictive values of amniotic fluid (AF) matrix metalloproteinase-9 (MMP-9), interleukin-6 (IL-6), and serum C-reactive protein (CRP) for histologic chorioamnionitis and intra-amniotic infection in women with preterm labor or preterm premature rupture of membranes (PROM). STUDY DESIGN: This retrospective cohort study included 99 consecutive women with preterm labor or preterm PROM (21-35 weeks' gestation) who delivered within 72 h of transabdominal amniocentesis. The AF was cultured for aerobic and anaerobic bacteria and for genital mycoplasmas and was assayed for MMP-9 and IL-6 levels. Maternal serum CRP was measured immediately after amniocentesis. The placentas were examined histologically. MAIN OUTCOME MEASURES: histologic chorioamnionitis and intra-amniotic infection. RESULTS: The prevalence of histologic chorioamnionitis and a positive AF culture was 44% (44/99) and 28% (28/99), respectively. In predicting intra-amniotic infection, AF MMP-9 had a significantly higher area under the curve (AUC: 0.94 [95% CI, 0.87-0.98]) than AF IL-6 (0.87 [95% CI, 0.78-0.84]; P < 0.05) and serum CRP (0.76 [95% CI, 0.66-0.84]; P < 0.001) and a higher sensitivity and specificity than serum CRP (P < 0.01, respectively). However, in predicting histologic chorioamnionitis, there were no significant differences in AUCs among the three tests (AF MMP-9: 0.78 [95% CI, 0.68-0.85]; AF IL-6: 0.76 [95% CI, 0.66-0.84]; serum CRP: 0.76 [95% CI, 0.66-0.84]). In a sub-analysis of 71 women without intra-amniotic infection, histologic chorioamnionitis was associated with an elevated serum CRP level (P < 0.05), but not with the level of AF IL-6 or MMP-9 (P = 0.232 and P = 0.402, respectively). CONCLUSIONS: The AF MMP-9 has a better overall diagnostic performance than the AF IL-6 and maternal serum CRP in predicting intra-amniotic infection. However, the serum CRP level obtained up to 72 h before delivery appears to be an important marker for early identification of histologic chorioamnionitis in women without intra-amniotic infection.

A co-twin study of the relative effect of birth weight and gestational age on retinopathy of prematurity.

PURPOSE: To determine the relative effect of birth weight and gestational age on retinopathy of prematurity (ROP) using preterm twin pairs discordant for birth weight. METHODS: This study was a retrospective cohort study including 55 consecutive twin pairs of 110 preterm infants (gestational age ≤33 weeks). The outcomes of ROP including occurrence (any stage), severe ROP (stage 3 or more), and clinically significant ROP requiring laser treatment were compared between twins with the lower birth weight from each pair and their co-twins with the higher birth weight. Using twin pairs having different birth weight and identical gestational age, the independent effects of prematurity and intrauterine growth on ROP could be evaluated. Other perinatal morbidities related to prematurity were also compared between twin pairs. RESULTS: No significant differences in ROP between larger and smaller infants were observed in the twin-paired analysis while analysis on individual infants showed strong association between small birth weight and ROP outcomes. However, in both the larger and smaller infant groups, gestational age of <28 weeks was significantly associated with ROP outcomes. No differences were found between twin pairs regarding other perinatal morbidities including bronchopulmonary dysplasia, respiratory distress syndrome, patent ductus arteriosus, intraventricular hemorrhage, and periventricular leukomalacia. CONCLUSIONS: Birth weight is not associated with ROP, while gestational age is in the twin-paired study, suggesting that gestational age is a better predictor of ROP than birth weight. This indicates that maturity is more important in the pathogenesis of ROP than intrauterine growth.

Comparison between sonographic cervical length and Bishop score in preinduction cervical assessment: a randomized trial.

OBJECTIVE: To compare sonographically measured cervical length with the Bishop score in determining the requirement for prostaglandin administration for preinduction cervical ripening in nulliparae at term. METHODS: One hundred and fifty-four women with singleton pregnancies at term who were scheduled for induction of labor were randomly assigned to receive prostaglandin for preinduction cervical ripening based on the Bishop score or sonographic cervical length. A cervix unfavorable for treatment with prostaglandin for preinduction cervical ripening was defined as having either a Bishop score of ≤ 4 or a cervical length of ≥ 28 mm. The primary outcome measures were induction success (defined as an ability to achieve the active phase of labor) and the percentage of patients treated with prostaglandin for preinduction cervical ripening. RESULTS: The two groups were similar with respect to maternal demographics, gestational age, cervical length, and Bishop score. The rates of induction success and Cesarean delivery, the interval to active phase of labor, and the interval to delivery were also similar in the two groups. However, in the transvaginal ultrasound group (n = 77), prostaglandin was administered to only 36% of the nulliparae compared with 75% of those in the Bishop score group (n = 77) (P < 0.0001). CONCLUSION: In comparison with the Bishop score, the use of sonographic cervical length for assessing the cervix prior to induction of labor can reduce the need for prostaglandin administration by approximately 50% without adversely affecting the outcome of induction in nulliparae at term if the cut-off values used are a Bishop score of ≤ 4 and a cervical length of ≥ 28 mm.

The presence of funisitis is associated with a decreased risk for the development of neonatal respiratory distress syndrome.

OBJECTIVES: Fetal lung maturation and respiratory outcomes are influenced by the exposure to intrauterine inflammation. Funisitis is considered as the histologic hallmark of fetal inflammatory response. This study was performed to determine if there is a difference in the rate of neonatal respiratory distress syndrome (RDS) according to the presence or absence of funisitis in preterm gestations. STUDY DESIGN: The relationship between the presence of funisitis and the development of neonatal RDS was examined in 301 consecutive singleton preterm births (24-32 weeks' gestation). Cases without placental histological examination and those with major congenital anomalies were excluded. Funisitis was diagnosed in the presence of neutrophil infiltration into the umbilical vessel walls or Wharton's jelly on the placental histological examination. RESULTS: Funisitis was diagnosed in 25% and RDS was diagnosed in 46% of cases. The rate of RDS in babies with funisitis was lower than in those without funisitis (28.4% vs. 51.1%, p = 0.001). Logistic regression analysis demonstrated that the presence of funisitis was associated with a decreased risk for RDS after adjusting for confounding variables (Odds ratio = 0.44, 95% CI 0.22-0.90). The downward trend of the frequency of RDS was related to the presence of histologic chorioamnionitis and funisitis (p < 0.001). CONCLUSIONS: The presence of funisitis is associated with a decreased risk for the development of neonatal RDS in preterm gestations. Furthermore, this observation suggests that the fetal involvement of placental inflammation may be beneficial to the maturation of the fetal lung.

Non-invasive prediction of intra-amniotic inflammation in women with preterm labor.

OBJECTIVE: To develop a model based on non-invasive variables to predict the probability of intra-amniotic inflammation in women with preterm labor and intact membranes. METHODS: Transvaginal ultrasonography and digital examination for the assessment of cervical length and cervical dilatation were performed, and maternal blood was collected for the determination of C-reactive protein and white blood cell (WBC) count immediately after amniocentesis in 153 consecutive women with preterm labor. Amniotic fluid obtained by amniocentesis was cultured for aerobic and anaerobic bacteria and mycoplasmas, and the WBC was determined. Intra-amniotic inflammation was defined as an elevated amniotic fluid interleukin-6 concentration (> 2.6 ng/mL). Receiver-operating characteristics (ROC) curves and logistic regression analysis were used for statistical analysis. RESULTS: The prevalence of a positive amniotic fluid culture was 7.2% (11/153) and the prevalence of intra-amniotic inflammation was 19.6% (30/153). The final logistic regression model was based on non-invasive clinical variables, including gestational age at assessment, cervical length and maternal blood WBC count, which were the best predictors of intra-amniotic inflammation. The model was shown to have an adequate goodness of fit (P = 0.754), and the area under the ROC curve was 0.724, indicating reasonably good discrimination. CONCLUSION: In women with preterm labor and intact membranes, the risk for intra-amniotic inflammation can be predicted non-invasively with a risk score based on gestational age, cervical length and maternal blood WBC count.

Expression of extracellular signal-regulated kinase1/2 and p38 mitogen-activated protein kinase in the invasive trophoblasts at the human placental bed.

BACKGROUND: Mitogen-activated protein kinases (MAP kinases) participate in signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. The roles of extracellular signal-regulated kinase1/2 (ERK1/2) and p38 MAP kinase in the differentiation and invasion of human trophoblasts have been studied. However, the in vivo expression and activation of ERK1/2 and p38 at the placental bed have not been elucidated. METHODS: The study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n=24) and with preeclampsia (n=8) between 31 and 40 weeks of gestation. We evaluated the expressions and phosphorylations of ERK1/2 and p38 MAP kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. RESULTS: p38 and phospho-p38 MAP kinase were not detected in invasive trophoblasts in cases or controls. ERK1/2 and phospho-ERK1/2 were positive in invasive trophoblasts albeit with variable staining. Phosphorylation of ERK1/2 was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. CONCLUSION: These findings suggest that preeclampsia is associated with decreased activation of ERK1/2 in invasive trophoblasts in vivo.

Arthroscopic management of acute painful hip following occult subluxation: evidence-based case report.

Arthroscopy of the hip joint has gained popularity in the recent past leading to an explosive increase in our knowledge of intra-articular hip pathologies. However, a spectrum of intra-articular hip lesions still needs to be explored to further advance the understanding, diagnosis and treatment of hip pathologies. The orthopedic surgeon occasionally affronts a situation when etiology of traumatic painful hip joint is not vivid and lack of definitive diagnosis prolongs the patient's suffering; however, an elaborate history taking and pragmatic apt arthroscopic intervention can curtail the illness span. Radiological examination generally fails to provide complete diagnosis in hip joints due to compact anatomy of the joint, and a negative report should not be considered as a deterrent for arthroscopic intervention. We report two evidence-based cases to highlight the significance of arthroscopic evaluation and management for occult subluxation of the hip. In both the cases, there was significant and prompt relief of symptoms after arthroscopic debridement.

Reversible renal vasoconstriction in a patient with acute renal failure after exercise.

Acute renal failure (ARF) with severe loin pain induced by anaerobic exercise is a rare condition that is accompanied by wedge-shaped contrast enhancement seen on computerized tomographic (CT) scan without evidence of rhabdomyolysis. An 18-year-old Korean male was transferred to our hospital for evaluation of mild azotemia, that developed after anaerobic exercise. The laboratory tests revealed that the serum creatinine was 2.1 mg/dl and the serum uric acid level was 1.6 mg/dl without any elevation of the serum myoglobin or creatine phosphokinase. Under the impression of exercise-induced ARF, we tried to determine the relationship between the occurrence of clinical symptoms, renal dysfunction and the characteristic CT findings by observing those changes prospectively before and after anaerobic exercise. After obtaining a written consent, the patient underwent a strenuous period of anaerobic exercise to induce the clinical symptoms. Before exercise, he was completely asymptomatic; his serum creatinine level was 0.9 mg/dl and CT scan of the kidneys showed no abnormalities. Loin pain developed 2 hours after exercise, and the serum creatinine level increased to 1.2 mg/dl 18 hours after the exercise. CT scan 18 hours after exercise showed multiple perfusion defects, and a 24-hour delayed CT scan showed multiple areas of wedge-shaped enhancement on both kidneys. These changes were completely resolved on the follow-up CT scan obtained 13 days after exercise with the return of a normal serum creatinine level. We conclude that reversible renal vasoconstriction is probably the main pathophysiologic mechanism of acute renal failure induced by anaerobic exercise.

Effects of local infusion of OP-1 on particle-induced and NSAID-induced inhibition of bone ingrowth in vivo.

Excessive polyethylene wear particles from joint replacements may lead to periprosthetic osteolysis and loosening. Nonsteroidal anti-inflammatory drugs (NSAIDs) decrease fracture healing and bone ingrowth. We hypothesized that continuous local infusion of OP-1 (BMP-7) would increase local bone formation in the presence of two different adverse stimuli, polyethylene particles, and an oral NSAID. The Drug Test Chamber (DTC) was implanted in the proximal tibia of mature rabbits. The tissue growing into the chamber was exposed to OP-1 solution (110 ng/day), which was infused via an osmotic pump. Infusion of OP-1 alone for 6 weeks enhanced local bone formation in the chamber by 80% (p < 0.05) over infusion of carrier alone. In the presence of polyethylene particles, infusion of OP-1 increased local bone formation by 38% (p < 0.05) over treatment with particles and carrier. Oral administration of NSAID reduced local bone formation by 58% (p < 0.05); this suppressive effect caused by NSAIDS was completely reversed by the infusion of OP-1 (p < 0.05). These findings underline a potential role for local treatment with OP-1 to increase bone formation in the presence of potentially adverse stimuli such as polyethylene wear particles or NSAID use.

Characterization of salicylic acid-induced genes in Chinese cabbage.

Salicylic acid is a messenger molecule in the activation of defense responses in plants. In this study, we isolated four cDNA clones representing salicylic acid-induced genes in Chinese cabbage (Brassica rapa subsp. pekinensis) by subtractive hybridization. Of the four clones, the BC5-2 clone encodes a putative glucosyltransferase protein. The BC5-3 clone is highly similar to an Arabidopsis gene encoding a putative metal-binding farnesylated protein. The BC6-1 clone is a chitinase gene with similarities to a rapeseed class IV chitinase. Class IV chitinases have deletions in the chitin-binding and catalytic domains and the BC6-1 chitinase has an additional deletion in the catalytic domain. The BCP8-1 clone is most homologous to an Arabidopsis gene that contains a tandem array of two thiJ-like sequences. These four cabbage genes were barely expressed in healthy leaves, but were strongly induced by salicylic acid and benzothiadiazole. Expression of the three genes represented by the BC5-2, BC5-3 and BCP8-1 clones were also induced by Pseudomonas syringae pv. tomato, a nonhost pathogen that elicits a hypersensitive response in Chinese cabbage. None of these four genes, however, was strongly induced by methyl jasmonate or by ethylene.

Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations.

Magnetic dipolar interactions between pairs of solvent-exposed nitroxide side chains separated by approximately one to four turns along an alpha-helix in T4 lysozyme are investigated. The interactions are analyzed both in frozen solution (rigid lattice conditions) and at room temperature as a function of solvent viscosity. At room temperature, a novel side chain with hindered internal motion is used, along with a more commonly employed nitroxide side chain. The results suggest that methods developed for rigid lattice conditions can be used to analyze dipolar interactions between nitroxides even in the presence of motion of the individual spins, provided the rotational correlation time of the interspin vector is sufficiently long. The distribution of distances observed for the various spin pairs is consistent with rotameric equilibria in the nitroxide side chain, as observed in crystal structures. The existence of such distance distributions places important constraints on the interpretation of internitroxide distances in terms of protein structure and structural changes.

Nucleation of water and methanol droplets on cations and anions: the sign preference.

The barrier height to cation- and anion-induced nucleation to produce water and methanol droplets are calculated by means of an umbrella-sampling Monte Carlo method. The computer simulation corroborates the century-old finding of Wilson that the anion is a better nucleator to produce water droplets than the cation having the same magnitude of charge, even without the presence of external electric field. The simulation also shows that the cation is a better nucleator to produce methanol droplets than the anion.

Roles of histidine residues in tobacco acetolactate synthase.

Acetolactate synthase (ALS) catalyzes the first common step in the biosynthesis of valine, leucine, and isoleucine in plants and microorganisms. ALS is the target of several structurally diverse classes of herbicides, including sulfonylureas, imidazolinones, and triazolopyrimidines. The roles of three well-conserved histidine residues (H351, H392, and H487) in tobacco ALS were determined using site-directed mutagenesis. Both H487F and H487L mutations abolished the enzymatic activity as well as the binding affinity for the cofactor FAD. Nevertheless, the mutation of H487F did not affect the secondary structure of the ALS. The K(m) values of H351M, H351Q, and H351F are approximately 18-, 60-, and fivefold higher than that of the wild-type ALS, respectively. Moreover, the K(c) value of H351Q for FAD is about 137-fold higher than that of wALS. Mutants H351M and H351Q showed very strong resistance to Londax (a sulfonylurea) and Cadre (an imidazolinone), whereas mutant H351F was weakly resistant to them. However, the secondary structures of mutants H351M and H351Q appeared to be different from that of wALS. The mutation of H392M did not have any significant effect on the kinetic parameters nor the resistance to ALS-inhibiting herbicides. These results suggest that the His487 residue is located at the active site of the enzyme and is likely involved in the binding of cofactor FAD in tobacco ALS. Mutational analyses of the His351 residue imply that the active site of the ALS is probably close to its binding site of the herbicides, Londax and Cadre.

The diphtheria toxin channel-forming T-domain translocates its own NH2-terminal region and the catalytic domain across planar phospholipid bilayers.

The T-domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy-terminal 57-amino acid segment (residues 322-378) in the T-domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T-domain upstream from this. Here we show that in association with channel formation by the T-domain, its hydrophilic 63-amino acid NH2-terminal region (residues 202-264) as well as the entire catalytic A fragment (residues 1-201) cross the lipid bilayer. The phenomenon that enabled us to demonstrate this was the rapid closure of channels at cis negative voltages when a histidine tag was placed at various positions in the NH2-terminal region of the T-domain or in the A fragment; the inhibition of this effect by trans nickel established that the histidine tag was present on the trans side of the membrane. Thus, all of the machinery necessary to translocate the A fragment across membranes is built into the 114 residues at the carboxy-terminal end of the T-domain (residues 265-378), without the requirement of any proteins in the plasma membrane (e.g., toxin receptor) or of any other cellular components.

Posttranslational N-myristoylation of BID as a molecular switch for targeting mitochondria and apoptosis.

Many apoptotic molecules relocate subcellularly in cells undergoing apoptosis. The pro-apoptotic protein BID underwent posttranslational (rather than classic cotranslational) N-myristoylation when cleavage by caspase 8 caused exposure of a glycine residue. N-myristoylation enabled the targeting of a complex of p7 and myristoylated p15 fragments of BID to artificial membranes bearing the lipid composition of mitochondria, as well as to intact mitochondria. This post-proteolytic N-myristoylation serves as an activating switch, enhancing BID-induced release of cytochrome c and cell death.