[Clinical analysis of 76 cases of sentinel lymph node detection in cervical cancer and endometrial cancer].

Objective: To evaluate the feasibility and clinical value of identifying sentinel lymph node (SLN) and to assess possible factors associated with detection rate in both cervical cancer and endometrial cancer. Methods: Retrospective study of 76 cases (39 with cervical cancer and 37 with endometrial cancer) were conducted in Peking University People's Hospital. All patients underwent SLN biopsy with tracers of indocyanine green (ICG) and (or) carbon nanoparticles. All mapped SLN was resected and followed by procedures that systematic pelvic lymphadenectomy and hysterectomy according to National Comprehensive Cancer Network (NCCN) guidelines. All the lymph nodes were examined postoperatively for the routine paraffin section of hematoxylin and eosin (HE) staining. Detection rate, sensitivity and negative predictive value of SLN were calculated and factors associated with the detection rate were analyzed. Results: The overall detection rate was 95% (72/76), with 74% (56/76) positive bilaterally. The bilateral detection rate of SLN with combined technique was significantly higher than that with single technique (P<0.05). The difference of SLN detection rate between cervical and endometrial cancer patients were not significant (P>0.05). SLN were mostly recognized in obturator (32.1%, 114/355) and external iliac areas (32.4%, 115/355) in cervical cancer, and in external iliac (41.2%, 91/221) and obturator areas (39.4%,87/221) in endometrial cancer. Among 55 patients underwent systematic pelvic lymphadenectomy, the sensitivity of SLN detection was 75% and the negative predictive value was 96%. The sensitivity and negative predictive value were both 100% in patients with successfully bilateral mapped of SLN. Conclusion: s The overall detection rate of SLN in cervical and endometrial cancer is the highest with the combined technique of ICG and carbon nanoparticles. The detection rate and located regions of SLN are similar between cervical and endometrial cancer, and SLN are mostly recognized in the external iliac and obturator areas. The sensitivity and negative predictive value of SLN detection are high, especially when SLN are bilateral mapped.

Isoindoline nitroxide-labeled porphyrins as potential fluorescence-suppressed spin probes.

A series of isoindoline nitroxide-labeled porphyrins were synthesized by the reaction of 5-phenyldipyrromethane and 5-(4'-carboethoxy-methyleneoxyphenyl)dipyrromethane with 5-formyl-1,1,3,3-tetramethylisoindolin-2-yloxyl (FTMIO) using the Lindsey method. The corresponding water-soluble spin-labeled porphyrins were also prepared. Subsequently, these compounds were characterized and their in vitro properties were evaluated. The electrochemical assay demonstrated that these isoindoline nitroxide-labeled porphyrins had similar electrochemical and redox properties to 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl (CTMIO). The electron paramagnetic resonance test showed that these porphyrins exhibited hyperfine splittings and characteristic spectra of CTMIO with typical nitroxide g-values and nitrogen isotropic hyperfine coupling constants. The in vitro cytotoxicity assay indicated that these porphyrins possessed low cytotoxicity to human renal tubular epithelial 293T cells (normal cells) and human hepatoma HepG2 cells (tumor cells). Fluorescence spectroscopy revealed that free base isoindoline nitroxide-labeled porphyrins exhibited fluorescence suppression characteristic of nitroxide-fluorophore systems. In vitro fluorescene imaging demonstrated that the reduced isoindoline nitroxide-labeled porphyrins eliminated fluorescence suppression and displayed strong red fluorescence imaging in HepG2 cells. Thus these isoindoline nitroxide-labeled porphyrins may be considered potentially as biological spin probes for fluorescence imaging and EPR spectroscopy.

Common effects of fat, ethanol, and nicotine on enkephalin in discrete areas of the brain.

Fat, ethanol, and nicotine share a number of properties, including their ability to reinforce behavior and produce overconsumption. To test whether these substances act similarly on the same neuronal populations in specific brain areas mediating these behaviors, we administered the substances short-term, using the same methods and within the same experiment, and measured their effects, in areas of the hypothalamus (HYPO), amygdala (AMYG), and nucleus accumbens (NAc), on mRNA levels of the opioid peptide, enkephalin (ENK), using in situ hybridization and on c-Fos immunoreactivity (ir) to indicate neuronal activity, using immunofluorescence histochemistry. In addition, we examined for comparison another reinforcing substance, sucrose, and also took measurements of stress-related behaviors and circulating corticosterone (CORT) and triglycerides (TG), to determine if they contribute to these substances' behavioral and physiological effects. Adult Sprague-Dawley rats were gavaged three times daily over 5 days with 3.5 mL of water, Intralipid (20% v/v), ethanol (12% v/v), nicotine (0.01% w/v) or sucrose (22% w/v) (approximately 7 kcal/dose), and tail vein blood was collected for measurements of circulating CORT and TG. On day five, animals were sacrificed, brains removed, and the HYPO, AMYG, and NAc processed for single- or double-labeling of ENK mRNA and c-Fos-ir. Fat, ethanol, and nicotine, but not sucrose, increased the single- and double-labeling of ENK and c-Fos-ir in precisely the same brain areas, the middle parvocellular but not lateral area of the paraventricular nucleus, central but not basolateral nucleus of the AMYG, and core but not shell of the NAc. While having little effect on stress-related behaviors or CORT levels, fat, ethanol, and nicotine all increased circulating levels of TG. These findings suggest that the overconsumption of these three substances and their potential for abuse are mediated by the same populations of ENK-expressing neurons in specific nuclei of the hypothalamus and limbic system.

Prenatal ethanol exposure stimulates neurogenesis in hypothalamic and limbic peptide systems: possible mechanism for offspring ethanol overconsumption.

Exposure to ethanol during the prenatal period contributes to increased alcohol consumption and preference in rodents and increased risk for alcoholism in humans. With studies in adult animals showing the orexigenic peptides, enkephalin (ENK), galanin (GAL) and orexin (OX), to stimulate ethanol consumption, the question addressed here is whether prenatal ethanol alters the development in utero of specific neurons that express these peptides. With reports describing suppressive effects of high doses of ethanol, we examined the offspring of dams gavaged from embryonic day 9 to parturition with a control solution or lower ethanol doses, 1 and 3g/kg/day, known to promote ethanol consumption in the offspring. To understand underlying mechanisms, measurements were taken in postnatal offspring of the expression of ENK in the hypothalamic paraventricular nucleus (PVN) and nucleus accumbens (NAc), GAL in the PVN, and OX in the perifornical lateral hypothalamus (PFLH) using real-time qPCR and in situ hybridization, and also of the cell proliferation marker, 5-bromo-2-deoxyuridine (BrdU), and its double-labeling with either neuronal nuclei (NeuN), a marker of mature neurons, or the peptides. On postnatal day 15 (P15), after two weeks without ethanol, the offspring showed increased expression of ENK in the PVN and NAc core but not shell, GAL in the PVN, and OX in the PFLH. In these same areas, prenatal ethanol compared to control increased the density at birth (P0) of neurons expressing these peptides and at P0 and P15 of neurons double-labeling BrdU and NeuN, indicating increased neurogenesis. These BrdU-positive neurons were found to express ENK, GAL and OX, indicating that prenatal ethanol promotes neurogenesis in these specific peptide systems. There were no changes in gliogenesis or apoptosis. This increase in neurogenesis and density of peptide-expressing neurons suggests the involvement of these hypothalamic and accumbal peptide systems in mediating the increased alcohol consumption observed in prenatal ethanol-exposed offspring.

Production and synthetic dyes decolourization capacity of a recombinant laccase from Pichia pastoris.

AIMS: To produce and purify a recombinant laccase from Pichia pastoris and to test its ability in decolourization of synthetic dyes. METHODS AND RESULTS: A cDNA encoding for a laccase was isolated from Pycnoporus sanguineus and was expressed in P. pastoris strain SMD1168H under the control of the alcohol oxidase (AOX1) promoter. The laccase native signal peptide efficiently directed the secretion of the recombinant laccase in an active form. Factors influencing laccase expression, such as cultivation temperature, pH, copper concentration and methanol concentration, were investigated. The recombinant enzyme was purified to electrophoretic homogeneity, and was estimated to have a molecular mass of about 62.8 kDa. The purified enzyme showed a similar behaviour to the native laccase produced by P. sanguineus. Four different synthetic dyes including azo, anthraquinone, triphenylmethane and indigo dyes could be efficiently decolourized by the purified recombinant laccase without the addition of redox mediators. CONCLUSIONS: Heterologous production of P. sanguineus laccase in P. pastoris was successfully achieved. The purified recombinant laccase could efficiently decolourize synthetic dyes in the absence of mediators. SIGNIFICANCE AND IMPACT OF THE STUDY: This study is the first report on the synthetic dye decolourization by the recombinant P. sanguineus laccase. The decolourization capacity of this recombinant enzyme suggested that it could be a useful biocatalyst for the treatment of dye-containing effluents.

Effect of temperature on pharmacokinetics of enrofloxacin in mud crab, Scylla serrata (Forsskål), following oral administration.

The study was conducted to evaluate the pharmacokinetics of enrofloxacin following a single oral gavage (10 mg kg(-1)) in mud crab, Scylla serrata, at water temperatures of 19 and 26 degrees C. Enrofloxacin concentration in haemolymph was determined using high-performance liquid chromatography (HPLC). A multiple and repeated haemolymph sampling from the articular cavity of crab periopods was developed. The haemolymph of an individual crab was successfully sampled up to 11 times from the articular cavity. The profile of haemolymph enrofloxacin concentration of an individual crab versus time was thus achieved. The mean haemolymph enrofloxacin concentration versus time was described by a two-compartment model with first-order absorption at two water temperatures. The peak concentrations of haemolymph enrofloxacin at 19 and 26 degrees C were 7.26 and 11.03 mug mL(-1), at 6 and 2 h, respectively. The absorption and distribution half-life time ( and t(1/2alpha)) at 19 degrees C were 3.7 and 4.5 h, respectively, which were markedly larger than the corresponding values (1.1 and 1.5 h) at 26 degrees C; the elimination half-life time (t(1/2beta)) was 79.1 and 56.5 h at 19 and 26 degrees C, respectively. The area under curve (AUC), total body clearance (Cl) and mean residence time (MRT(0-infinity)) at 19 degrees C were 636.0 mg L(-1) h, 0.016 L h(-1) kg(-1) and 102.5 h, respectively; the corresponding values at 26 degrees C were 583.4 mg L(-1) h, 0.018 L h(-1) kg(-1)and 63.7 h. These results indicate that enrofloxacin is absorbed and eliminated more rapidly in mud crab at 26 degrees C than at 19 degrees C.

A non-biotin polymerized horseradish-peroxidase method for the immunohistochemical diagnosis of canine distemper.

This report describes a modified non-biotin polymerized horseradish peroxidase (HRP) immunohistochemical method for the diagnosis of canine distemper virus (CDV) infection from formalin-fixed, paraffin wax-embedded tissues. This method confirmed infection in seven of eight (87.5%) suspected cases. Labelled CDV antigen was observed in the following sites: cerebrum, cerebellum, meninges, glial cells, neurons, vascular endothelium, periventricular areas and pericytes, and choroid plexus; grey and white matter and central canal of the spinal cord; renal pelvis and tubular epithelium, and urinary bladder epithelium; macrophages and lymphocytes in splenic white pulp and lymph nodes; skin epidermis; bronchiolar epithelium and alveolar macrophages; hepatic Kupffer cells, and gastric and intestinal mucosal epithelium; stratified squamous epithelium of the tongue and oesophagus. With the non-biotin HRP detection system, pretreatment by autoclaving followed by microwave heating gave better labelling results than did microwave pretreatment alone. No obvious difference was noted between the labelling results produced by the non-biotin HRP detection system and the Super Sensitive Link-Label IHC detection system.

Immunohistochemical diagnosis of mouse hepatitis virus and mycoplasma pulmonis infection with murine antiserum.

This study established a modified alkaline phosphatase-labelled avidin-biotin-complex (ABC-AP) method for diagnosis of mouse hepatitis virus (MHV) and Mycoplasma pulmonis infection from formalin-fixed, paraffin wax-embedded sections, murine antibody-positive serum being used as the primary reagent. With this method, MHV antigen in cAnNCrj.Cg-Foxn1(nu)/Foxn1(nu) mice and M. pulmonis antigen in Wistar rats were immunolabelled in tissue sections. MHV antigen was clearly detected in samples of liver, stomach, caecal and colonic mucosa, and spleen. M. pulmonis antigen was demonstrated on the luminal surface of bronchiolar epithelial cells. This method may prove useful in diagnosis when commercial antisera are unavailable or when immunosuppression prevents serological diagnosis.

Spectrofluorimetric determination of thiols by use of N[P-(2-benzoxazolyl)phenyl]maleimide.

The weak fluorescence of N-[P-(2-benzoxazolyl)phenyl]maleimide (BOPM) can be greatly enhanced by thiol-containing compounds. A sensitive and simple spectrofluorimetric method based on the use of BOPM has been developed for the determination of thiols such as cysteine (Cys) and reduced glutathione (GSH). Calibration plots were linear in the concentration range from 0 to 1.6 x 10(-7) mol L(-1) for Cys and 0 to 1.7 x 10(-7) mol L(-1) for GSH. The detection limits (3a) were 2.36 x 10(-10) mol L(-1) for Cys and 1.49 x 10(-10) mol L(-1) for GSH. Many other amino acids (present at 100-fold greater concentrations) did not interfere with the determination. The proposed method has been used for the determination of Cys in protein hydrolysate and cystine electrolyte or GSH in serum, with recoveries of 95.4-103.7%.

Incarcerated hernia in a 5-mm cannula wound.

Laparoscopic suturing and repairing of the fascial opening at 10- to 12-mm cannula puncture sites is well established; however, closing a 5-mm cannula wound is not well documented. We often leave the wound open without suture and cover it with gauze after removing the surgical drainage tube. An unusual early postoperative complication of laparoscopic surgery was an incarcerated hernia in a 5-mm cannula site. The 9-year-old girl underwent laparoscopic surgery due to an 8-cm ovarian mature teratoma. After 7 days, she came to our hospital because of a protruding mass in the left cannula wound. The mass was excised, and incarcerated fallopian tube torsion with necrotic change was diagnosed.

A novel water-soluble fluorescent probe for the determination of methylamine.

A novel water-soluble fluorescent probe, monosodium 7-(4,6-dichloro-1,3,5-triazinylamino)-1,3-naphthalenedisulfonic acid (DTND), was synthesized by reacting cyanuric chloride with 7-amino-1,3-naphthalenedisulfonic acid monopotassium salt at 0-5 degrees C. This new reagent was used for the determination of methylamine. The linear range is 3x10(-6)-2x10(-4) mol L(-1) with a detection limit (S/N=3) of 7.2x10(-8) mol L(-1), and the relative S.D. is 1.3% for ten replicate determinations of 1x10(-5) mol L(-1) CH3NH2. Common species in the aqueous environment have no or only slight influence on the determination. The method can be used to determine methylamine in real water samples.

Genotoxic effects of triphenyltin acetate and triphenyltin hydroxide on mammalian cells in vitro and in vivo.

Two organotin pesticides, triphenyltin acetate (TPTA) and triphenyltin hydroxide (TPTH), were evaluated for their ability to induce micronuclei (MN) and sister chromatid exchange (SCE) in vitro using cultured Chinese hamster ovary (CHO) cells and in vivo BALB/c mouse erythrocytes. Both pesticides induced a dose-dependent increase but only TPTH induced a significant increase in MN at the highest dose (150 ng/ml) tested in CHO cells. With adding S9 microsomal fractions, both pesticides induced a meaningful MN induction at 150 ng/ml and a dose-dependent significant increase in SCE. In vivo MN induction in erythrocytes was conducted by treating BALB/c mice orally or intraperitoneally with these pesticides either in a single or triple treatments. Oral gavage (p.o.) of TPTA resulted in a dose-related significant increase of MN induction in peripheral blood and of TPTH induced a significant increase in micronucleated reticulocyte (MNRETs) only in a single treatment. Intraperitoneal administration of TPTA or TPTH, however, resulted in meaningless random increases in MN though these increases might be attributable to toxic effects. The MNRETs levels in the treatment with both pesticides were independent to the sampling time. This study demonstrated that TPTA and TPTH was potential chromosome mutagens.

Antibody responses after Sendai virus infection and their role in upper and lower respiratory tract disease in rats.

BACKGROUND AND PURPOSE: Sendai virus infection in rats is an excellent model for studying development and role of host defenses throughout the respiratory tract after this infection. Therefore, development of serum antibody responses and disease were studied. METHODS: Forty-two anesthetized pathogen-free 3- to 4- week-old LEW/NCr rats were inoculated intranasally with Sendai virus. At postinoculation days 0, 2, 3, 5, 8, 10, and 14, rats were euthanized by administration of a pentobarbital sodium overdose followed by exsanguination. Serum was obtained from all animals, and nasal wash and bronchoalveolar lavage specimens were collected during selected experiments. An ELISPOT assay was used to measure numbers of Sendai virus-specific antibody-forming cells in respiratory tract lymphoid tissue. RESULTS: Recovery from disease and clearance of virus from respiratory tract tissues coincided with development of serum antibody responses. Upper respiratory tract lymph nodes were the initial and major sites of appearance of antibody-forming cells. Immunoglobulin G was the predominant subtype of these cells during recovery from the infection and in rats resistant to infection. Passive transfer of antisera or specific IgG protected the lower but not the upper respiratory tract. CONCLUSIONS: Circulating components of immunity have a major role in resistance and recovery from disease in the lower respiratory tract, whereas local responses are likely involved in protection of the upper respiratory tract. Local lymphoid tissues are the major production sites of IgG, which contributes to resistance to and recovery from respiratory tract diseases.

Degeneration of the accessory navicular synchondrosis presenting as rupture of the posterior tibial tendon.

Degeneration of the accessory navicular synchondrosis may be associated with decreased function of the posterior tibial tendon in patients who are middle-aged or older. We investigated the role of ultrasonography in differentiating between degeneration of the accessory navicular synchondrosis with separation of the accessory navicular from the navicular, which has not been previously reported to our knowledge, and a rupture of the posterior tibial tendon. We studied fourteen patients (mean age, fifty-five years; range, forty-one to seventy-two years) who had an operatively confirmed injury of the accessory navicular synchondrosis. The mean duration of follow-up was thirty-nine months (range, twenty-seven to fifty-four months). Preoperative radiographs demonstrated a type-II accessory navicular (an accessory navicular with a synchondrosis) in all fourteen patients. Ultrasonography, which was performed for twelve patients, demonstrated a defect in the synchondrosis in eleven patients and a normal posterior tibial tendon in all twelve. The operative findings included incomplete separation of the synchondrosis in four of the fourteen patients, complete separation of the synchondrosis and the periosteum in eight, and avulsion of the accessory navicular in two. On the basis of our findings, we concluded that post-traumatic degeneration of an accessory navicular synchondrosis may present clinically as a variant type of avulsion or rupture of the posterior tibial tendon in this age-group. Ultrasonography is useful for distinguishing between complete or partial separation through the synchondrosis and rupture or attenuation of the posterior tibial tendon.

Interleukin-1 receptor antagonist modulates the progression of a spontaneously occurring IgA nephropathy in mice.

Cytokines, such as interleukin-1 (IL-1), may play a key role in the pathogenesis of IgA nephropathy (IgAN). This study was conducted to evaluate the effects of IL-1 receptor antagonist (IL-1ra) in the treatment of a spontaneously occurring experimental IgAN in established phase. ddY mice (12/group) were injected twice daily with 3 mg/kg of IL-1ra, intraperitoneally, for 8 consecutive weeks. The placebo mice were injected with saline only. As normal controls, ddY mice, which were not treated with IL-1ra or saline, were killed at 6 weeks of age. Results showed a significant reduction of proteinuria in the IL-1ra-treated mice, compared with saline-treated mice (urinary albumin/creatinine, 0.24 +/- 0.04 v 0.39 +/- 0.03, P < 0.001). A significant improvement of renal 51Cr-EDTA (ethylenediaminetetra-acetic acid) clearance was observed in the IL-1ra-treated mice (t1/2, 12 +/- 2.7 minutes, compared with saline-treated mice 25 +/- 2.0 minutes, P < 0.001). Similarly, serum levels of creatinine (1.0 +/- 0.4 v 2.4 +/- 0.3 mg/dL, P < 0.001) and urea nitrogen (46 +/- 6 v 58 +/- 2 mg/dL, P < 0.01) were significantly lower in IL-1ra-treated mice than in saline-treated mice. In renal tissue studies, the IL-1ra-treated mice exhibited significantly decreased mesangial cell proliferation, compared with saline-treated mice (P < 0.001), as shown by light and electron microscopy. In addition, the IL-1ra-treated mice showed significantly lower glomerular expression of collagen type IV, fibronectin, laminin, and IL-6 (P < 0.001) than saline-treated mice, although they still showed higher glomerular expression of collagen type IV (P < 0.01), fibronectin (P < 0.01), laminin (P < 0.001), IL-1 (P < 0.001), and IL-6 (P < 0.01) than did normal control mice. Meanwhile, glomerular C3 deposition was significantly lower in IL-1ra-treated mice than in saline-treated mice (P < 0.001). These findings indicate that IL-1ra partially prevented the progression of spontaneously occurring IgAN in this experimental model. Data from these experiments also confirm the pathogenic effects of IL-1 in the established phase of IgAN in ddY mice.

Diagnostic efficacy of ultrasonography in stage I posterior tibial tendon dysfunction: sonographic-surgical correlation.

The purpose of this study is to evaluate the diagnostic efficacy of ultrasonography in stage I posterior tibial tendon dysfunction. Fourteen of the 17 consecutive patients who underwent tenosynovectomy for stage I posterior tibial tendon dysfunction were included in this study. The preoperative diagnosis was based primarily on the clinical suspicion of dysfunction, which was confirmed by ultrasonography. Two measurements were obtained at the midpoint between the insertion site and the medial malleolus in both feet of the 14 patients: the diameter of the posterior tibial tendon and the diameter of the tendon sheath measured from its inner walls. The mean diameter of the tendon was 4.61 +/- 0.50 mm and that of the tendon sheath in the symptomatic foot was 7.24 +/- 0.75. In the unaffected foot, the mean diameter of the tendon was 3.30 +/- 0.34 mm and that of the tendon sheath was 3.64 +/- 0.35 mm, respectively. In the symptomatic tendon, the increase of peritendinous space was significantly higher than the increase in the tendinous portion (P < 0.0001). Surgical findings proved the accuracy of diagnosis in all patients. Although many cases of stage I posterior tibial tendon dysfunction remain undiagnosed owing to the mild clinical symptoms, this series indicates that ultrasonography is a valuable adjunctive diagnostic tool in the clinical examination and assists in achieving an accurate diagnosis of stage I posterior tibial tendon dysfunction, allowing early treatment.

Synthesis and evaluation of coumarin alpha-methylene-gamma-butyrolactones: a new class of platelet aggregation inhibitors.

In a search for new inhibitors of platelet aggregation, certain coumarin-bearing alpha-methylene-gamma-butyrolactones were synthesized and evaluated for inhibitory activity against thrombin (Thr)-, arachidonic acid (AA)-, collagen (Col)-, and platelet-activating factor (PAF)-induced aggregation in washed rabbit platelets. These compounds were efficiently synthesized from commercially available 7-hydroxycoumarin or its derivatives. Among them, 7-[(2,3,4,5-tetrahydro-4-methylene-5-oxo-2-phenyl-2-furanyl) methoxy]-2H-1-benzopyran-2-one (3d) showed the most potent inhibition of AA- and PAF- induced aggregation, with IC50 values of 3.65 and 16.36 microM respectively.