Distinct mechanisms regulate cyclooxygenase-1 and -2 in peritoneal macrophages of women with and without endometriosis

Prostaglandin (PG) E2 has been shown to stimulate steroidogenesis in ectopic endometriotic stromal cells and may be involved in the development of endometriosis since this disorder is highly estrogen dependent. The biosynthesis of PGE2 is controlled by the rate-limiting enzyme termed cyclooxygenase (COX). The objective of the current study was to investigate the expression of COX in peritoneal macrophages isolated from women with and without endometriosis, and to explore the effects of pro-inflammatory agents on COX expression in peritoneal macrophages. Using quantitative RT-PCR and Western blot analyses, we found that expression of COX-2 was markedly increased (P < 0.05) in peritoneal macrophages isolated from women with early or severe endometriosis, whereas expression of COX-1 was elevated only in the severe stage (P < 0.05). On the contrary, monocytes/macrophages purified from peripheral blood of patients with endometriosis had minimal or undetectable levels of COX-2, and this was not different from disease-free women. Treatment with interleukin-1beta, tumour necrosis factor-alpha or PGE2 caused a significant increase in COX-2 (P < 0.05) but not COX-1 expression in peritoneal macrophages isolated from disease-free women. In contrast, these agents had no substantial effect on COX-1 and COX-2 expression in peritoneal macrophages from women with endometriosis. In summary, expression of COX in peritoneal macrophages was associated with the severity of endometriosis. Elevated expression of both COX-1 and COX-2 in peritoneal macrophages may contribute to the increased peritoneal fluid PGE2 concentrations and may thus play an important role in the development of endometriosis.     

Increased (CTG/CAG)(n) lengths in myotonic dystrophy type 1 and Machado-Joseph disease genes in idiopathic azoospermia patients

BACKGROUND: An increase in CAG trinucleotide repeat length in the androgen receptor (AR) gene has been linked to idiopathic azoospermia. METHODS: In order to test whether other (CAG/CTG)(n) loci are also affected, the (CAG/CTG)(n) frequency distribution at myotonic dystrophy type 1 (DM1), Machado-Joseph disease (MJD), dentatorubral-pallidoluysian atrophy (DRPLA) and spinocerebellar ataxia type 8 (SCA8) loci, in addition to the AR gene, was investigated in 48 azoospermia patients and 47 controls. RESULTS: The median CAG repeat length in the AR gene was significantly longer in azoospermia patients than in controls (23 versus 21, P < 0.001). Significant differences were also noted in the upper tails of trinucleotide repeat length distributions at both DM1 and MJD loci between the two populations. At the DM1 locus, alleles of more than 18 repeats were observed only in azoospermia patients, and not in controls (P = 0.014). At the MJD locus, the frequency of normal alleles (ANs) with 29 or more CAG repeats was also much higher in azoospermia patients (29.2 versus 7.4%; P = 0.0001). However, the repeat length distribution at DRPLA and SCA8 loci did not differ in the two groups. CONCLUSIONS: These data indicated that, at least in a subset of azoospermia patients, there was an increase in the number of trinucleotide repeats in some disease loci. Thus, it is noteworthy to evaluate whether offspring of these azoospermia patients, if born by assisted reproductive technologies, have an increased risk of trinucleotide repeat diseases.     

Oral preloads of liquid diet and hormonal satiety mechanisms

A hormonal short-term satiety mechanism involving cholecystokinin (CCK) has recently been proposed. We attempted to manipulate endogenous CCK levels in rats by administering oral preloads at varying time intervals before a test meal. Support for the CCK hypothesis was equivocal. In addition, there was little support for other hypothesized satiety mechanisms, such as regulation of volume or calories, or glucose-related mechanisms. The results are discussed in terms of the necessity of considering the total “ecological” situation (diet composition, diet palatability, diet availability, and training) when studying food intake and body weight.     

Identification of medically important molds by an oligonucleotide array

Infections caused by fungi have increased in recent years. Accurate and rapid identification of fungal pathogens is important for appropriate treatment with antifungal agents. On the basis of the internal transcribed spacer 1 (ITS 1) and ITS 2 sequences of the rRNA genes, an oligonucleotide array was developed to identify 64 species (32 genera) of clinically important filamentous (or dimorphic) fungi. These 64 species included fungi causing superficial, cutaneous, subcutaneous, and invasive infections. The method consisted of PCR amplification of the ITS regions using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species- or group-specific oligonucleotides immobilized on a nylon membrane. Of 397 fungal strains (290 target and 107 nontarget strains) tested, the sensitivity and specificity of the array was 98.3% (285/290) and 98.1% (105/107), respectively. Misidentified strains were usually those belonging to the same genus of the target species or having partial homology with oligonucleotide probes on the membrane. The whole procedure can be finished within 24 h starting from isolated colonies; reproductive structures, which are essential for the conventional identification methods, are not needed. In conclusion, the present array is a powerful tool for identification of clinically important filamentous fungi and may have the potential to be continually extended by adding further oligonucleotides to the array without significantly increasing the cost or complexity.     

Signal transduction modulation by lithium: cell culture, cerebral microdialysis and human studies.

Considerable evidence suggests that signal transduction pathways are targets of lithium (Li) action. A number of investigators have reported that Li attenuates both adenylate cyclase (AC) activity and phosphoinositide (PI) turnover in rodents and in humans, thus "dampening" these systems. We have studied selected components of these second-messenger systems in a series of clinical and preclinical investigations. To overcome confounding effects of alterations in mood state, we examined AC activity and G-protein ribosylation in peripheral blood cells from 10 healthy volunteers, prior to and following 14 days of Li administration. Basal and postreceptor [cesium fluoride (CsF) or Gpp(NH)p] stimulated AC activity were unaffected in lymphocytes. In contrast, both basal and stimulated AC activity in platelets were significantly augmented, compatible with an attenuation of Gi function. Ribosylation of platelet Gs by cholera toxin was unchanged, whereas that of Gi by pertussis toxin (PT) was increased. Given that undissociated G protein is the preferred substrate for PT, our results suggest that Li interferes with subunit dissociation and the subsequent activation of Gi. To determine if Li has similar effects on Gi in the central nervous system, we measured extracellular (EC) cyclic adenosine monophosphate (cAMP) in rat brain by in vivo microdialysis, revealing a dose-dependent increase in cAMP by norepinephrine (NE) antagonized by propranolol. Chronic (4-week) Li doubled basal EC cAMP, while decreasing the fractional response to 100 microM NE. Thus, using in vivo microdialysis, we observed the reported reduction in NE-stimulated AC activity, but only as a function of elevated basal cAMP. Increased basal AC activity has been observed following chronic Li in both humans and rat tissues but generally has not been considered relevant. The PI generating system is another proposed major target for Li that we have studied using an in vitro cell culture model of peripheral blood cells. Chronic (6-day) exposure of neutrophil-like HL60 cells to 1 mM LiCl did not affect agonist fMet-Leu-Phe (fMLP) induced PI turnover. In contrast, Li attenuated both agonist and phorbol ester stimulated Na+/H+ exchange, suggesting reduced protein kinase C (PKC) function. Western blot analysis revealed altered levels of PKC in both membrane and cytosolic fractions. The functional consequences of these complex effects on the two major signal transduction pathways and their interactions in the intact living organism remain to be elucidated.     

Outward currents influencing bursting dynamics in guinea pig trigeminal motoneurons

To initiate and maintain bursts (and plateau potentials) in the presence of serotonin, guinea pig trigeminal motoneurons utilize L-type Ca2+ and persistent Na+ inward currents. However, the intrinsic currents that contribute to burst termination and determine the duration of the interburst interval are unknown. Therefore we investigated the roles of outward currents, whose slow activation is coupled to cytosolic cation (Ca2+ and Na+) accumulation. First we examined a Ca2+-dependent K+ current (IK-Ca) with apamin and Ba2+-substituted, low-Ca2+ solution. Blockade of IK-Ca lengthened burst duration and cycle time but did not abolish bursting. Next we studied the Na+/K+-ATPase pump current (Ip) with cardiac glycosides. In the presence of apamin or low-Ca2+/Ba2+ solution, blocking Ip (with ouabain or strophanthidin) decreased both burst duration and cycle time and ultimately transformed bursting into tonic spiking. We conclude that IK-Ca and Ip contribute to burst termination in trigeminal motoneurons. These currents influence temporal bursting properties such as burst duration and cycle time and may help determine the phasic activity of motoneurons during rhythmic oral-motor behaviors.     

Synthesis and properties of poly(ether imide)s derived from 4,4′-(1,5-naphthylenedioxy)diphthalic anhydride and various aromatic diamines

A naphthalene unit-containing bis(ether anhydride), 4,4′-(1,5-naphthylenedioxy)-diphthalic anhydride, was prepared in three steps starting from the nucleophilic nitro-displacement reaction of 1,5-dihydroxynaphthalene and 4-nitrophthalonitrile in N,N-dimethylformamide (DMF) solution in the presence of potassium carbonate. High-molarmass aromatic poly(ether imide)s were synthesized using a two-stage polymerization process from the bis(ether anhydride) and ten aromatic diamines. The intermediate poly(ether amic acid)s had inherent viscosities of 0,66–1,27 dL/g. The films of poly(ether imide)s derived from some diamines, such as p-phenylenediamine, benzidine, and bis[4-(4-aminophenoxy)phenyl] ether, crystallized and embrittled during the thermal imidization process. The other poly(ether imide)s were amorphous materials and could be fabricated into transparent, flexible, and tough films. These poly(ether imide) films had yield strengths of 111–125 MPa, tensile strengths of 96–150 MPa, elongations to break of 10–38%, and initial moduli of 1,6–2,4 GPa. All of these polymers were insoluble in organic solvents, except for that derived from 2,2-bis[(4-aminophenoxy)phenyl]propane. Their T_g's were recorded in the range of 226–265°C by DSC. Thermogravimetric analysis (TG) showed that all the polymers were stable up to 535°C in both air and nitrogen atmosphere.     

Synthesis and properties of polyamides and poly(amideimide)s based on 4,4′-[1,4(or 1,3)-phenylenebis(isopropylidene-1,4-phenyleneoxy)]dianiline

The diamines 4,4′-[1,4(or 1,3)-phenylenebis(isopropylidene-1,4-phenyleneoxy)]dianiline (p- 3 and m- 3 ) were synthesized in two steps from the condensation of 4,4′-[1,4(or 1,3)-phenylenediisopropylidene]diphenol (p- 1 and m- 1 ) and p-chloronitrobenzene in the presence of K₂CO₃ in N,N-dimethylformamide (DMF), giving the corresponding bis(4-nitrophenoxy) compounds, followed by reduction with the hydrazine/Pd-C system. A series of aromatic polyamides, aliphatic-aromatic polyamides, and poly(amide-imide)s were prepared by the direct polycondensation of the diamines with aromatic or aliphatic dicarboxylic acids and phthalimide unit-bearing dicarboxylic acids in N-methyl-2-pyrrolidone (NMP) using triphenyl phosphite and pyridine as condensing agents. In addition, two series of polyamides were prepared from terephthalic acid or isophthalic acid and aromatic diamines with isopropylidene and/or ether linking groups between the phenylene units, and the structure-property relationships of these polyamides were studied. Almost all the resultant polymers were amorphous in nature and could be solution-cast into transparent, tough, and flexible films. Most of the polymers exhibited moderately high thermal stability. Thus, they are considered as new candidates for processable high-performance polymeric materials.     

New poly(amide-imide) syntheses, 4 Poly(amide-imide)s derived from 2,5-bis(4-aminophenyl)-3,4-diphenylthiophene and various bis(trimellitimide)s

Fifteen bis(trimellitimide)s 2a–o were prepared by condensation of the corresponding aliphatic or aromatic diamines with trimellitic anhydride. A series of structurally new poly(amideimide)s were synthesized by the direct polycondensation of these bis(strimellitimide)s with 2,5-bis(4-aminophenyl)-3,4-diphenylthiophene using triphenyl phosphite and pyridine as condensing agents in N-methyl-2-pyrrolidone (NMP). The resultant poly(amide-imide)s have inherent viscosities in the range between 0,72 and 2,73 dL/g at 30°C in N,N-dimethylacetamide (cf. Tab. 2). The polymers are amorphous and readily soluble in polar aprotic solvents such as N,N-dimethylacetamide (DMAc) and NMP. Transparent and tough films can be cast from their solutions. Most aliphatic-aromatic poly(amide-imide)s show a glass transition in the range between 203 and 242°C in their differential scanning calorimetry (DSC) traces, whereas the wholly aromatic poly(amide-imide)s show no discernible transition before decomposition. The thermal stability of the polymers was evaluated by thermogravimetry which showed the 10% weight-loss temperatures in the range between 414 and 459°C in air and between 451 and 578°C in a nitrogen atmosphere for the aliphatic-aromatic poly(amide-imide)s and above 500°C in both air and nitrogen atmosphere for the wholly aromatic poly(amide-imide)s.