Cycle modulation of insulin-like growth factor-binding protein 1 in human endometrium

Endometrium is one of the fastest growing human tissues. Sex hormones, estrogen and progesterone, in interaction with several growth factors, control its growth and differentiation. Insulin-like growth factor 1 (IGF-1) interacts with cell surface receptors and also with specific soluble binding proteins. IGF-binding proteins (IGF-BP) have been shown to modulate IGF-1 action. Of six known isoforms, IGF-BP-1 has been characterized as a marker produced by endometrial stromal cells in the late secretory phase and in the decidua. In the current study, IGF-1-BP concentration and affinity in the proliferative and secretory phase of the menstrual cycle were measured. Endometrial samples were from patients of reproductive age with regular menstrual cycles and taking no steroid hormones. Cytosolic fractions were prepared and binding of 125I-labeled IGF-1 performed. Cross-linking reaction products were analyzed by SDS-polyacrylamide gel electrophoresis (7.5%) followed by autoradiography. 125I-IGF-1 affinity to cytosolic proteins was not statistically different between the proliferative and secretory endometrium. An approximately 35-kDa binding protein was identified when 125I-IGF-1 was cross-linked to cytosol proteins. Secretory endometrium had significantly more IGF-1-BP when compared to proliferative endometrium. The specificity of the cross-linking process was evaluated by the addition of 100 nM unlabeled IGF-1 or insulin. Unlabeled IGF-1 totally abolished the radioactivity from the band, indicating specific binding. Insulin had no apparent effect on the intensity of the labeled band. These results suggest that IGF-BP could modulate the action of IGF-1 throughout the menstrual cycle. It would be interesting to study this binding protein in other pathologic conditions of the endometrium such as adenocarcinomas and hyperplasia.

P-selectin, carcinoma metastasis and heparin: novel mechanistic connections with therapeutic implications

Metastasis is a multistep cascade initiated when malignant cells penetrate the tissue surrounding the primary tumor and enter the bloodstream. Classic studies indicated that blood platelets form complexes around tumor cells in the circulation and facilitate metastases. In other work, the anticoagulant drug heparin diminished metastasis in murine models, as well is in preliminary human studies. However, attempts to follow up the latter observation using vitamin K antagonists failed, indicating that the primary mechanism of heparin action was unrelated to its anticoagulant properties. Other studies showed that the overexpression of sialylated fucosylated glycans in human carcinomas is associated with a poor prognosis. We have now brought all these observations together into one mechanistic explanation, which has therapeutic implications. Carcinoma cells expressing sialylated fucosylated mucins can interact with platelets, leukocytes and endothelium via the selectin family of cell adhesion molecules. The initial organ colonization of intravenously injected carcinoma cells is attenuated in P-selectin-deficient mice, in mice receiving tumor cells pretreated with O-sialoglycoprotease (to selectively remove mucins from cell surfaces), or in mice receiving a single dose of heparin prior to tumor cell injection. In each case, we found that formation of a platelet coating on cancer cells was impeded, allowing increased access of leukocytes to the tumor cells. Several weeks later, all animals showed a decrease in the extent of established metastasis, indicating a long-lasting effect of the short-term intervention. The absence of obvious synergism amongst the three treatments suggests that they all act via a common pathway. Thus, a major mechanism of heparin action in cancer may be inhibition of P-selectin-mediated platelet coating of tumor cells during the initial phase of the metastatic process. We therefore suggest that heparin use in cancer be re-explored, specifically during the time interval between initial visualization of a primary tumor until just after definitive surgical removal.

Effects of terpineol on the compound action potential of the rat sciatic nerve

Terpineol, a volatile terpenoid alcohol of low toxicity, is widely used in the perfumery industry. It is an important chemical constituent of the essential oil of many plants with widespread applications in folk medicine and in aromatherapy. The effects of terpineol on the compound action potential (CAP) of rat sciatic nerve were studied. Terpineol induced a dose-dependent blockade of the CAP. At 100 µM, terpineol had no demonstrable effect. At 300 µM terpineol, peak-to-peak amplitude and conduction velocity of CAP were significantly reduced at the end of 180-min exposure of the nerve to the drug, from 3.28 ± 0.22 mV and 33.5 ± 7.05 m/s, respectively, to 1.91 ± 0.51 mV and 26.2 ± 4.55 m/s. At 600 µM, terpineol significantly reduced peak-to-peak amplitude and conduction velocity from 2.97 ± 0.55 mV and 32.8 ± 3.91 m/s to 0.24 ± 0.23 mV and 2.72 ± 2.72 m/s, respectively (N = 5). All these effects developed slowly and were reversible upon 180-min washout.

The leaves of green plants as well as a cyanobacterium, a red alga, and fungi contain insulin-like antigens

We report the detection of insulin-like antigens in a large range of species utilizing a modified ELISA plate assay and Western blotting. We tested the leaves or aerial parts of species of Rhodophyta (red alga), Bryophyta (mosses), Psilophyta (whisk ferns), Lycopodophyta (club mosses), Sphenopsida (horsetails), gymnosperms, and angiosperms, including monocots and dicots. We also studied species of fungi and a cyanobacterium, Spirulina maxima. The wide distribution of insulin-like antigens, which in some cases present the same electrophoretic mobility as bovine insulin, together with results recently published by us on the amino acid sequence of an insulin isolated from the seed coat of jack bean (Canavalia ensiformis) and from the developing fruits of cowpea (Vigna unguiculata), suggests that pathways depending on this hormone have been conserved through evolution.

Evaluation of the cholinomimetic actions of trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana (Gastropoda, Opisthobranchia)

Trimethylsulfonium, a compound present in the midgut gland of the sea hare Aplysia brasiliana, negatively modulates vagal response, indicating a probable ability to inhibit cholinergic responses. In the present study, the pharmacological profile of trimethylsulfonium was characterized on muscarinic and nicotinic acetylcholine receptors. In rat jejunum the contractile response induced by trimethylsulfonium (pD2 = 2.46 ± 0.12 and maximal response = 2.14 ± 0.32 g) was not antagonized competitively by atropine. The maximal response (Emax) to trimethylsulfonium was diminished in the presence of increasing doses of atropine (P<0.05), suggesting that trimethylsulfonium-induced contraction was not related to muscarinic stimulation, but might be caused by acetylcholine release due to presynaptic stimulation. Trimethylsulfonium displaced [³H]-quinuclidinyl benzilate from rat cortex membranes with a low affinity (Ki = 0.5 mM). Furthermore, it caused contraction of frog rectus abdominis muscles (pD2 = 2.70 ± 0.06 and Emax = 4.16 ± 0.9 g), which was competitively antagonized by d-tubocurarine (1, 3 or 10 µM) with a pA2 of 5.79, suggesting a positive interaction with nicotinic receptors. In fact, trimethylsulfonium displaced [³H]-nicotine from rat diaphragm muscle membranes with a Ki of 27.1 µM. These results suggest that trimethylsulfonium acts as an agonist on nicotinic receptors, and thus contracts frog skeletal rectus abdominis muscle and rat jejunum smooth muscle via stimulation of postjunctional and neuronal prejunctional nicotinic cholinoreceptors, respectively.

Absence-like seizures in adult rats following pilocarpine-induced status epilepticus early in life

Administration of pilocarpine causes epilepsy in rats if status epilepticus (SE) is induced at an early age. To determine in detail the electrophysiological patterns of the epileptogenic activity in these animals, 46 Wistar rats, 7-17 days old, were subjected to SE induced by pilocarpine and electro-oscillograms from the cortex, hippocampus, amygdala, thalamus and hypothalamus, as well as head, rostrum and vibrissa, eye, ear and forelimb movements, were recorded 120 days later. Six control animals of the same age range did not show any signs of epilepsy. In all the rats subjected to SE, iterative spike-wave complexes (8.1 ± 0.5 Hz in frequency, 18.9 ± 9.1 s in duration) were recorded from the frontal cortex during absence fits. However, similar spike-wave discharges were always found also in the hippocampus and, less frequently, in the amygdala and in thalamic nuclei. Repetitive or single spikes were also detected in these same central structures. Clonic movements and single jerks were recorded from all the rats, either concomitantly with or independently of the spike-wave complexes and spikes. We conclude that rats made epileptic with pilocarpine develop absence seizures also occurring during paradoxical sleep, showing the characteristic spike-wave bursts in neocortical areas and also in the hippocampus. This is in contrast to the well-accepted statement that one of the main characteristics of absence-like fits in the rat is that spike-wave discharges are never recorded from the hippocampal fields.

Postprandial symptoms in dysmotility-like functional dyspepsia are not related to disturbances of gastric myoelectrical activity

Gastric dysrhythmias, such as tachy- or bradygastria, have been reported in patients with functional dyspepsia (FD), but their role in symptom production is uncertain. It is also not known whether gastric dysrhythmias in these patients can be elicited by physiological gastric distension with a meal. We investigated the relationships between symptoms after ingestion of different volumes of water following a test meal and gastric dysrhythmias in FD patients. Fourteen patients with dysmotility-like FD and 13 healthy volunteers underwent paired electrogastrography (EGG) studies. Fasted subjects ingested 150 ml of yoghurt with either 150 ml (low volume) or 300 ml (high volume) water in random order. Fasting and fed EGGs with monitoring of symptoms were performed in both studies. Ten FD patients (71.4%) reported upper abdominal discomfort and bloating after the low volume meal, but only one (7.1%) presented an abnormal EGG (dominant frequency in the 2-4-cpm range: 58%). Following the high volume meal, 7 patients (50%) had symptoms, but none had EGG abnormalities. No significant differences were found between FD patients and controls for any of the EGG variables, in any test. In FD patients with postprandial symptoms, the percentage of the EGG dominant frequency in the normal range (median, 84.6%; range, 76.0-100.0%) was similar (P > 0.20) to that in those without symptoms (88.5%; 75.0-100.0%). We conclude that disturbances of gastric myoelectrical activity are unlikely to play a role in the origin of postprandial upper abdominal discomfort and bloating in dysmotility-like FD.

The compulsive-like aspect of the head dipping emission in rats with chronic electrolytic lesion in the area of the median raphe nucleus

Head dipping (HD) is a behavioral pattern considered to have a risk assessment or an exploratory role and is used as a complementary parameter to evaluate anxiety in experimental animals. Since rats with electrolytic lesion in the area of the median raphe nucleus displayed high frequencies of HD in a previous study, the present investigation was undertaken to confirm this observation and to determine its anxiety-related origin. HD episodes were counted in adult male Wistar rats (270-350 g) with electrolytic lesion (N = 11) and sham-lesioned controls (N = 12). When HD was measured for 60 min on an elevated open platform, lesioned rats emitted 13 times more HD than controls (264.7 ± 93.3 vs 20.3 ± 7.6 episodes), with the difference being statistically significant (P < 0.05). HD counts during 10-min sessions held 7, 14, 21, 27, and 63 days after lesion showed significantly higher means (range: 28.14 ± 5.38 to 62.85 ± 9.48) compared to sham-lesioned controls (range: 7.37 ± 1.13 to 8.5 ± 1.45). Normal rats stepped down into their home cages when the vertical distance between them and the cage was short (16 cm), and the step-down latencies increased with increasing depths (36.7 ± 7.92 to 185.87 ± 35.44 s). Lesioned rats showed a similar behavior when facing the shortest depth, but had a significantly increased number (23.28 ± 2.35 episodes) and latency (300 ± 0.00 s) of HD compared to normal rats (9.25 ± 1.37 episodes and 185.87 ± 35.44 s) when facing the greatest depth (30 cm). This suggests that HD may be a depth-measuring behavior related to risk assessment.

Nocardia otitidiscaviarum (GAM-5) induces parkinsonian-like alterations in mouse

Parkinson's disease, a major neurodegenerative disorder in humans whose etiology is unknown, may be associated with some environmental factors. Nocardia otitidiscaviarum (GAM-5) isolated from a patient with an actinomycetoma produced signs similar to Parkinson's disease following iv injection into NMRI mice. NMRI mice were infected intravenously with a non-lethal dose of 5 x 10(6) colony forming units of N. otitidiscaviarum (GAM-5). Fourteen days after bacterial infection, most of the 60 mice injected exhibited parkinsonian features characterized by vertical head tremor, akinesia/bradykinesia, flexed posture and postural instability. There was a peak of nocardial growth in the brain during the first 24 h followed by a decrease, so that by 14 days nocardiae could no longer be cultured. At 24 h after infection, Gram staining showed nocardiae in neurons in the substantia nigra and occasionally in the brain parenchyma in the frontal and parietal cortex. At 21 days post-infection, tyrosine hydroxylase immunolabeling showed a 58% reduction of tyrosine hydroxylase in the substantia nigra, and a 35% reduction of tyrosine hydroxylase in the ventral tegmental region. Dopamine levels were reduced from 110 ± 32.5 to 58 ± 16.5 ng/mg protein (47.2% reduction) in brain from infected mice exhibiting impaired movements, whereas serotonin levels were unchanged (191 ± 44 protein in control and 175 ± 39 ng/mg protein in injected mice). At later times, intraneuronal inclusion bodies were observed in the substantia nigra. Our observations emphasize the need for further studies of the potential association between Parkinson's disease or parkinsonism-like disease and exposure to various nocardial species.

Hypercaloric cafeteria-like diet induced UCP3 gene expression in skeletal muscle is impaired by hypothyroidism

The uncoupling protein UCP3 belongs to a family of mitochondrial carriers located in the inner mitochondrial membrane of certain cell types. It is expressed almost exclusively at high levels in skeletal muscle and its physiological role has not been fully determined in this tissue. In the present study we have addressed the possible interaction between a hypercaloric diet and thyroid hormone (T3), which are strong stimulators of UCP3 gene expression in skeletal muscle. Male Wistar rats weighing 180 ± 20 g were rendered hypothyroid by thyroidectomy and the addition of methimazole (0.05%; w/v) to drinking water after surgery. The rats were fed a hypercaloric cafeteria diet (68% carbohydrates, 13% protein and 18% lipids) for 10 days and sacrificed by decapitation. Subsequently, the gastrocnemius muscle was dissected, total RNA was isolated with Trizol™ and UCP3 gene expression was determined by Northern blotting using a specific probe. Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post-test. Skeletal muscle UCP3 gene expression was decreased by 60% in hypothyroid rats and UCP3 mRNA expression was increased 70% in euthyroid cafeteria-fed rats compared to euthyroid chow-fed animals, confirming previous studies. Interestingly, the cafeteria diet was unable to stimulate UCP3 gene expression in hypothyroid animals (40% lower as compared to euthyroid cafeteria-fed animals). The results show that a hypercaloric diet is a strong stimulator of UCP3 gene expression in skeletal muscle and requires T3 for an adequate action.

Colocalization of coilin and nucleolar proteins in Cajal body-like structures of micronucleated PtK2 cells

Cajal bodies (CB) are ubiquitous nuclear structures involved in the biogenesis of small nuclear ribonucleoproteins and show narrow association with the nucleolus. To identify possible relationships between CB and the nucleolus, the localization of coilin, a marker of CB, and of a set of nucleolar proteins was investigated in cultured PtK2 cells undergoing micronucleation. Nocodazol-induced micronucleated cells were examined by double indirect immunofluorescence with antibodies against coilin, fibrillarin, NOR-90/hUBF, RNA polymerase I, PM/Scl, and To/Th. Cells were imaged on a BioRad 1024-UV confocal system attached to a Zeiss Axiovert 100 microscope. Since PtK2 cells possess only one nucleolus organizer region, micronucleated cells presented only one or two micronuclei containing nucleolus. By confocal microscopy we showed that in most micronuclei lacking a typical nucleolus a variable number of round structures were stained by antibodies against fibrillarin, NOR-90/hUBF protein, and coilin. These bodies were regarded as CB-like structures and were not stained by anti-PM/Scl and anti-To/Th antibodies. Anti-RNA polymerase I antibodies also reacted with CB-like structures in some micronuclei lacking nucleolus. The demonstration that a set of proteins involved in RNA/RNP biogenesis, namely coilin, fibrillarin, NOR-90/hUBF, and RNA polymerase I gather in CB-like structures present in nucleoli-devoid micronuclei may contribute to shed some light into the understanding of CB function.

Effects of estragole on the compound action potential of the rat sciatic nerve

Estragole, a relatively nontoxic terpenoid ether, is an important constituent of many essential oils with widespread applications in folk medicine and aromatherapy and known to have potent local anesthetic activity. We investigated the effects of estragole on the compound action potential (CAP) of the rat sciatic nerve. The experiments were carried out on sciatic nerves dissected from Wistar rats. Nerves, mounted in a moist chamber, were stimulated at a frequency of 0.2 Hz, with electric pulses of 50-100-µs duration at 10-20 V, and evoked CAP were monitored on an oscilloscope and recorded on a computer. CAP control parameters were: peak-to-peak amplitude (PPA), 9.9 ± 0.55 mV (N = 15), conduction velocity, 92.2 ± 4.36 m/s (N = 15), chronaxy, 45.6 ± 3.74 µs (N = 5), and rheobase, 3.9 ± 0.78 V (N = 5). Estragole induced a dose-dependent blockade of the CAP. At 0.6 mM, estragole had no demonstrable effect. At 2.0 and 6.0 mM estragole, PPA was significantly reduced at the end of 180-min exposure of the nerve to the drug to 85.6 ± 3.96 and 13.04 ± 1.80% of control, respectively. At 4.0 mM, estragole significantly altered PPA, conduction velocity, chronaxy, and rheobase (P <= 0.05, ANOVA; N = 5) to 49.3 ± 6.21 and 77.7 ± 3.84, 125.9 ± 10.43 and 116.7 ± 4.59%, of control, respectively. All of these effects developed slowly and were reversible upon a 300-min wash-out. The data show that estragole dose-dependently blocks nerve excitability.

Control of the rat angiotensin I converting enzyme gene by CRE-like sequences

We characterized the role of potential cAMP-responsive elements (CRE) in basal and in induced angiotensin converting enzyme (ACE) gene promoter activity in order to shed light on the regulation of somatic ACE expression. We identified stimulators and repressors of basal expression between 122 and 288 bp and between 415 and 1303 bp upstream from the transcription start site, respectively, using a rabbit endothelial cell (REC) line. These regions also contained elements associated with the response to 8BrcAMP. When screening for CRE motifs we found pCRE, a proximal sequence between 209 and 222 bp. dCRE, a distal tandem of two CRE-like sequences conserved between rats, mice and humans, was detected between 834 and 846 bp. Gel retardation analysis of nuclear extracts of REC indicated that pCRE and dCRE bind to the same protein complexes as bound by a canonical CRE. Mutation of pCRE and dCRE in REC established the former as a positive element and the latter as a negative element. In 293 cells, a renal cell line, pCRE and dCRE are negative regulators. Co-transfection of ATF-2 or ATF-2 plus c-Jun repressed ACE promoter activity, suggesting that the ACE gene is controlled by cellular stress. Although mapping of cAMP responsiveness was consistent with roles for pCRE and dCRE, mutation analysis indicated that they were not required for cAMP responsiveness. We conclude that the basal activity of the somatic ACE promoter is controlled by proximal and distal CREs that can act as enhancers or repressors depending on the cell context.

A role for histone-like protein H1 (H-NS) in the regulation of hemolysin expression by Serratia marcescens

The histone-like protein H1 (H-NS) is an abundant structural component of the bacterial nucleoid and influences many cellular processes including recombination, transcription and transposition. Mutations in the hns gene encoding H-NS are highly pleiotropic, affecting the expression of many unrelated genes. We have studied the role of H-NS on the regulation of hemolysin gene expression in Serratia marcescens. The Escherichia coli hns mutant carrying S. marcescens hemolysin genes on a plasmid constructed by ligation of the 3.2-kb HindIII-SacI fragment of pR02 into pBluescriptIIKS, showed a high level of expression of this hemolytic factor. To determine the osmoregulation of wild-type and hns defective mutants the cells were grown to mid-logarithmic phase in LB medium with 0.06 or 0.3 M NaCl containing ampicillin and kanamycin, whereas to analyze the effect of pH on hemolysin expression, the cells were grown to late-logarithmic phase in LB medium buffered with 0.1 M Tris-HCl, pH 4.5 to 8.0. To assay growth phase-related hemolysin production, bacterial cells were grown in LB medium supplemented with ampicillin and kanamycin. The expression of S. marcescens hemolysin genes in wild-type E. coli and in an hns-defective derivative at different pH and during different growth phases indicated that, in the absence of H-NS, the expression of hemolysin did not vary with pH changes or growth phases. Furthermore, the data suggest that H-NS may play an important role in the regulation of hemolysin expression in S. marcescens and its effect may be due to changes in DNA topology influencing transcription and thus the amount of hemolysin expression. Implications for the mechanism by which H-NS influences gene expression are discussed.

Activation of a P2Y4-like purinoceptor triggers an increase in cytosolic [Ca2+] in the red blood cells of the lizard Ameiva ameiva (Squamata, Teiidae)

An increasing number of pathophysiological roles for purinoceptors are emerging, some of which have therapeutic potential. Erythrocytes are an important source of purines, which can be released under physiological and physiopathological conditions, acting on purinergic receptors associated with the same cell or with neighboring cells. Few studies have been conducted on lizards, and have been limited to ATP agonist itself. We have previously shown that the red blood cells (RBCs) of the lizard Ameiva ameiva store Ca2+ in the endoplasmic reticulum (ER) and that the purinergic agonist ATP triggers a rapid and transient increase of [Ca2+]c by mobilization of the cation from internal stores. We also reported the ability of the second messenger IP3 to discharge the ER calcium pool of the ER. Here we characterize the purinoceptor present in the cytoplasmic membrane of the RBCs of the lizard Ameiva ameiva by the selective use of ATP analogues and pyrimidine nucleotides. The nucleotides UTP, UDP, GTP, and ATPgammaS triggered a dose-dependent response, while interestingly 2MeSATP, 2ClATP, alpha, ß-ATP, and ADP failed to do so in a 1- to 200-µm con- centration. The EC50 obtained for the compounds tested was 41.77 µM for UTP, 48.11 µM for GTP, 53.11 µM for UDP, and 30.78 µM for ATPgammaS. The present data indicate that the receptor within the RBCs of Ameiva ameiva is a P2Y4-like receptor due to its pharmacological similarity to the mammalian P2Y4 receptor.