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Background Ixodes scapularis ticks are medically important arthropod vectors that transmit several pathogens to humans. The observations of morphological abnormalities, including nanism, missing leg, extra leg, and gynandromorphism, have been reported in these ticks. In this study, we report the presence of two anuses in a laboratory-reared I. scapularis nymph. Results Larval ticks were allowed to feed on mice and to molt to nymphs. Two anuses were observed in one of the freshly molted nymphs. Stereo and scanning electron microscopy confirmed the presence of two anuses in one nymph within a single anal groove. Conclusions This report confirms the rare occurrence of double anus in I. scapularis. Graphical Abstract more...

When primitive vertebrates evolved from ancestral members of the animal kingdom and acquired complex locomotive and neurological toolsets, a constant supply of energy became necessary for their continued survival. To help fulfill this need, the endocannabinoid (eCB) system transformed drastically with the addition of the cannabinoid-1 receptor (CB1R) to its gene repertoire. This established an eCB/CB1R signaling mechanism responsible for governing the whole organism's energy balance, with its activation triggering a shift toward energy intake and storage in the brain and the peripheral organs (i.e., liver and adipose). Although this function was of primal importance for humans during their pre-historic existence as hunter-gatherers, it became expendable following the successive lifestyle shifts of the Agricultural and Industrial Revolutions. Modernization of the world has further increased food availability and decreased energy expenditure, thus shifting the eCB/CB1R system into a state of hyperactive deregulated signaling that contributes to the 21st century metabolic disease pandemic. Studies from the literature supporting this perspective come from a variety of disciplines, including biochemistry, human medicine, evolutionary/comparative biology, anthropology, and developmental biology. Consideration of both biological and cultural evolution justifies the design of improved pharmacological treatments for obesity and Type 2 diabetes (T2D) that focus on peripheral CB1R antagonism. Blockade of peripheral CB1Rs, which universally promote energy conservation across the vertebrate lineage, represents an evolutionary medicine strategy for clinical management of present-day metabolic disorders. more...

BACKGROUND Acute hemolytic transfusion reactions have a broad clinical presentation from mild and transitory signs and symptoms to shock, disseminated intravascular coagulation, renal failure, and death. We have recently developed a rat model of acute intravascular hemolysis showing that the classical complement pathway mediates antibody-dependent hemolysis. The objective of this study was to evaluate the role of the classical pathway inhibitor peptide inhibitor of complement C1 (PIC1) in this animal model. STUDY DESIGN AND METHODS Male Wistar rats received a 15% transfusion of human red blood cells (RBCs) and blood was isolated from the animals up to 120 minutes. Animals received PIC1 either 2 minutes before or 0.5 minutes after transfusion. Sham-, vehicle-, and cobra venom factor (CVF)-treated animals were used as control groups with a subset of rats also receiving an equivalent dose of intravenous immunoglobulin (IVIG) before transfusion. Blood was analyzed for transfused RBC survival by flow cytometry and free hemoglobin (Hb) in isolated plasma by spectrophotometry. RESULTS Vehicle-treated rats showed decreased human RBC survival and increased free Hb as expected. Rats receiving PIC1 before transfusion showed increased human RBC survival and decreased Hb similar to CVF-treated rats. Notably, rats receiving PIC1 after initiation of transfusion showed similar decreases in hemolysis as animals receiving PIC1 before transfusion. Compared to IVIG and saline controls, PIC1-treated animals demonstrated decreased hemolysis and protection from acute kidney injury. CONCLUSIONS These results demonstrate that PIC1 has efficacy in an animal model of acute intravascular hemolysis in both prevention and rescue scenarios. more...

Background Prevention of acute hemolytic transfusion reactions is a worldwide concern. The objective of this study was to develop a simple rat model of complement-mediated acute intravascular hemolysis. Study Design and Methods Human AB red blood cells (RBCs) were incubated with complement-sufficient or complement-deficient Wistar rat serum (WRS) in the presence and absence of human RBC antibody in vitro to elucidate the mechanism of hemolysis. To study the role of complement in acute intravascular hemolysis in vivo, Wistar rats were treated either with or without cobra venom factor (CVF) to deplete complement activity. Human AB RBCs were then injected into both groups of rats, followed by serial blood draws up to 2 hours. Venous blood clearance and lysis of transfused RBCs at each time point were measured by flow cytometry and spectrophotometry. RBC sequestration was determined in the liver, spleen, and kidney by immunohistochemistry. Results In vitro incubation of human RBCs with WRS demonstrated that RBC lysis was mediated via the classical complement pathway and that hemolysis was antibody dependent. Transfusion of human RBCs into rats showed significantly less hemolysis in the CVF group versus untreated group. RBC sequestration in the spleen and liver 2 hours posttransfusion were not quantitatively different between the two groups. Conclusions Given the much higher degree of similarity for rat and human complement compared to mice, this simple rat model is ideal for testing novel inhibitors of classical pathway activation for the prevention and treatment of acute intravascular hemolysis. more...

Objective— Prediabetic states are associated with accelerated atherosclerosis, but the availability of mouse models to study connections between these diseases has been limited. The aim of this study was to test the selective role of impaired insulin receptor/insulin receptor substrate-1 signaling on atherogenesis. Methods and Results— To address the effects of impaired insulin signaling associated with hyperinsulinemia on atherosclerosis in the absence of obesity and hyperglycemia, we generated insulin receptor ( Insr )/insulin receptor substrate-1 ( Insr1 ) double heterozygous apolipoprotein ( Apoe )-knockout mice ( Insr +/− Irs1 +/− Apoe −/− ) mice. Insr +/− Irs1 +/− Apoe −/− mice fed a Western diet for 15 weeks showed elevated levels of fasting insulin compared to Insr +/+ Irs1 +/+ Apoe −/− mice. There were no significant differences in glucose, triglyceride, HDL, VLDL, cholesterol levels or free fatty acid in the plasma of Insr +/− Irs1 +/− Apoe −/− and Insr +/+ Irs1 +/+ Apoe −/− mice. Atherosclerotic lesions were increased in male (brachiocephalic artery) and female (aortic tree) Insr +/− Irs1 +/− Apoe −/− compared to Insr +/+ Irs1 +/+ Apoe −/− mice. Bone marrow transfer experiments demonstrated that nonhematopoietic cells have to be Insr +/− Irs1 +/− to accelerate atherosclerosis. Impaired insulin signaling resulted in decreased levels of vascular phospho-eNOS, attenuated endothelium-dependent vasorelaxation and elevated VCAM-1 expression in aortas of Insr +/− Irs1 +/− Apoe −/− mice. In addition, phospho-ERK and vascular smooth muscle cell proliferation were significantly elevated in aortas of Insr +/− Irs1 +/− Apoe −/− mice. Conclusion— These results demonstrate that defective insulin signaling is involved in accelerated atherosclerosis in Insr +/− Irs1 +/− Apoe −/− mice by promoting vascular dysfunction and inflammation. more...

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug with cardiovascular effects that have not been fully described. In the current study, we observed the effects of acute MDMA on rabbit left ventricular function. We also observed the effects of MDMA on nuclear factor-kappa B (NF-kB) activity in cultured rat ventricular myocytes (H9c2). In the rabbit, MDMA (2 mg/kg) alone caused a significant increase in heart rate and a significant decrease in the duration of the cardiac cycle. Inhibition of nitric oxide synthase (NOS) by pretreatment with L-NAME (10 mg/kg) alone caused significant dysfunction in heart rate, systolic pressure, diastolic pressure, duration of relaxation, duration of cardiac cycle, and mean left ventricular pressure. Pretreatment with L-NAME followed by treatment with MDMA caused significant dysfunction in additional parameters that were not abnormal upon exposure to either compound in isolation: duration of contraction, inotropy, and pulse pressure. Exposure to 1.0 mM MDMA for 6 h or 2.0 mM MDMA for 12 h caused increased nuclear localization of NF-kB in cultured H9c2 cells. The current results suggest that MDMA is acutely detrimental to heart function and that an intact cardiovascular NOS system is important to help mitigate early sequelae in some functional parameters. The delayed timing of NF-kB activation suggests that this factor may be relevant to MDMA induced cardiomyopathy of later onset. more...

International audience; The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases. more...

Successful ovulation requires elevated follicular prostaglandin E2 (PGE2) levels. To determine which PGE2 receptors are available to mediate periovulatory events in follicles, granulosa cells and whole ovaries were collected from monkeys before (0 h) and after administration of an ovulatory dose of hCG to span the 40-h periovulatory interval. All PGE2 receptor mRNAs were present in monkey granulosa cells. As assessed by immunofluorescence, PTGER1 (EP1) protein was low/nondetectable in granulosa cells 0, 12, and 24 h after hCG but was abundant 36 h after hCG administration. PTGER2 (EP2) and PTGER3 (EP3) proteins were detected by immunofluorescence in granulosa cells throughout the periovulatory interval, and Western blotting showed an increase in PTGER2 and PTGER3 levels between 0 h and 36 h after hCG. In contrast, PTGER4 (EP4) protein was not detected in monkey granulosa cells. Granulosa cell response to PGE2 receptor agonists was examined 24 h and 36 h after hCG administration, when elevated PGE2 levels present in periovulatory follicles initiate ovulatory events. PGE2 acts via PTGER1 to increase intracellular calcium. PGE2 increased intracellular calcium in granulosa cells obtained 36 h, but not 24 h, after hCG; this effect of PGE2 was blocked by a PTGER1 antagonist. A PTGER2-specific agonist and a PTGER3-specific agonist each elevated cAMP in granulosa cells obtained 36 h, but not 24 h, after hCG. Therefore, the granulosa cells of primate periovulatory follicles express multiple receptors for PGE2. Granulosa cells respond to agonist stimulation of each of these receptors 36 h, but not 24 h, after hCG, supporting the hypothesis that granulosa cells are most sensitive to PGE2 as follicular PGE2 levels peak, leading to maximal PGE2-mediated periovulatory effects just before ovulation. more...

To discourage fibrosis of the filtering bleb, 5 fluorouracil (5-FU) may be injected after trabeculectomy. 5-FU is an antimetabolite that also can damage extraocular tissues at concentrations as low as 0.5%. This study ascertained whether repeated injection of 5-FU has toxic effects on intraocular structures.After unilateral trabeculectomy in anesthetized New Zealand rabbits, 5-FU (5.0 mg/0.1 mL) was injected at the trabeculectomy site every 5 days for 15 days. Evaluation included slit-lamp examination, confocal microscopy, and intraocular pressure (IOP). After sacrifice, aqueous humor (AH) was drawn and eyes excised for scanning electron microscopy (SEM) and light microscopy.The 5-FU injection not decrease IOP beyond trabeculectomy alone. Bleb height remained constant, thickness increased, and vascularity decreased. No changes in cornea or anterior segment were observed. No inflammation was observed in the bleb or surrounding tissues by slit-lamp or histologic examination. Protein in AH increased from 0.6 +/- 0.5 microg/mL at baseline to 19.8 +/- 4.4 microg/mL after trabeculectomy but only to 0.9 +/- 0.6 microg/mL after trabeculectomy plus 5-FU. Both in vivo confocal microscopy and SEM revealed deleterious effects on corneal epithelial and endothelial cells with a minor shift toward smaller cells.In this study 5-FU did not provoke an intraocular inflammatory response and had minimal effect on extraocular structures. Changes in corneal epithelium and endothelium detectable by confocal microscopy suggest a small toxic effect. These in vivo measurements by confocal microscopy were confirmed by SEM. Repeated administration did not cause additional cumulative toxic effects in the anterior segment. Therefore, multiple injections of 5- FU into the filtering bleb pose minimal risk to intraocular structures. more...

3,4-Methylenedioxymethamphetamine (MDMA) is an illicit psychoactive drug that has gained immense popularity among teenagers and young adults. The cardiovascular toxicological consequences of abusing this compound have not been fully characterized. The present study utilized a transient transfection/dual luciferase genetic reporter assay, fluorescence confocal microscopy, and gene expression macroarray technology to determine nuclear factor-κB (NF-κB) activity, intracellular calcium balance, mitochondrial depolarization, and gene transcription profiles, respectively, in cultured rat striated cardiac myocytes (H9c2) exposed to MDMA. At concentrations of 1×10−3 M and 1×10−2 M, MDMA significantly enhanced NF-κB reporter activity compared with 0 M (medium only) control. This response was mitigated by cotransfection with IκB for 1×10−3 M but not 1×10−2 M MDMA. MDMA significantly increased intracellular calcium at concentrations of 1×10−3 M and 1×10−2 M and caused mitochondrial depolarization at 1×10−2 M. MDMA increased the transcription of genes that are considered to be biomarkers in cardiovascular disease and genes that respond to toxic indults. Selected gene activation was verified via temperature-gradient RT-PCR conducted with annealing temperatures ranging from 50°C to 65°C. Collectively, these results suggest that MDMA may be toxic to the heart through its ability to activate the myocardial NF-κB response, disrupt cytosolic calcium and mitochondrial homeostasis, and alter gene transcription. more...

CA125 is a mucin commonly employed as a diagnostic marker for epithelial ovarian cancer. Induction of humoral responses to CA125 leads to increased survival times in patients with this form of cancer, suggesting a potential role for this mucin in tumor progression. In this study, oligosaccharides linked to CA125 derived from the human ovarian tumor cell line OVCAR-3 were subjected to rigorous biophysical analysis. Sequencing of the O-glycans indicates the presence of both core type 1 and type 2 glycans. An unusual feature is the expression of branched core 1 antennae in the core type 2 glycans. CA125 is also N-glycosylated, expressing primarily high mannose and complex bisecting type N-linked glycans. High mannose type glycans include Man5-Man9GlcNAc2. The predominant N-glycans are the biantennary, triantennary, and tetraantennary bisecting type oligosaccharides. Remarkably, the N-glycosylation profiles of CA125 and the envelope glycoprotein gp120 (derived from H9 lymphoblastoid cells chronically infected with HIV-1) are very similar. The CA125-associated N-glycans have also recently been implicated in crucial recognition events involved in both the innate and adaptive arms of the cell-mediated immune response. CA125 may therefore induce specific immunomodulatory effects by employing its carbohydrate sequences as functional groups, thereby promoting tumor progression. Immunotherapy directed against CA125 may attenuate these immunosuppressive effects, leading to the prolonged survival of patients with this extremely serious form of cancer. more...

Murine sperm initiate fertilization by binding to specific oligosaccharides linked to the zona pellucida, the specialized matrix coating the egg. Biophysical analyses have revealed the presence of both high mannose and complex-type N-glycans in murine zona pellucida. The predominant high mannose-type glycan had the composition Man(5)GlcNAc(2), but larger oligosaccharides of this type were also detected. Biantennary, triantennary, and tetraantennary complex-type N-glycans were found to be terminated with the following antennae: Galbeta1-4GlcNAc, NeuAcalpha2-3Galbeta1-4GlcNAc, NeuGcalpha2-3Galbeta1-4GlcNAc, the Sd(a) antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc, NeuGcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc), and terminal GlcNAc. Polylactosamine-type sequence was also detected on a subset of the antennae. Analysis of the O-glycans indicated that the majority were core 2-type (Galbeta1-4GlcNAcbeta1-6[Galbeta1-3]GalNAc). The beta1-6-linked branches attached to these O-glycans were terminated with the same sequences as the N-glycans, except for terminal GlcNAc. Glycans bearing Galbeta1-4GlcNAcbeta1-6 branches have previously been suggested to mediate initial murine gamete binding. Oligosaccharides terminated with GalNAcbeta1-4Gal have been implicated in the secondary binding interaction that occurs following the acrosome reaction. The significant implications of these observations are discussed. more...

International audience; Previous experiments from our laboratories have identified peptides derived from the human astrovirus coat protein (CP) that bind C1q and mannose binding lectin (MBL) inhibiting activation of the classical and lectin pathways of complement, respectively. The purpose of this study was to evaluate the function of these coat protein peptides (CPPs) in an in vitro model of complement-mediated disease (ABO incompatibility), preliminarily assess their in vivo complement suppression profile and develop more highly potent derivatives of these molecules. E23A, a 30 amino acid CPP derivative previously demonstrated to inhibit classical pathway activation was able to dose-dependently inhibit lysis of AB erythrocytes treated with mismatched human O serum. Additionally, when injected into rats, E23A inhibited the animals' serum from lysing antibody-sensitized erythrocytes, providing preliminary in vivo functional evidence that this CPP can cross the species barrier to inhibit serum complement activity in rodents. A rational drug design approach was implemented to identify more potent CPP derivatives, resulting in the identification and characterization of a 15 residue peptide (polar assortant (PA)), which demonstrated both superior inhibition of classical complement pathway activation and robust binding to C1q collagen-like tails. PA also inhibited ABO incompatibility in vitro and demonstrated in vivo complement suppression up to 24h post-injection. CPP's ability to inhibit ABO incompatibility in vitro, proof of concept in vivo inhibitory activity in rats and the development of the highly potent PA derivative set the stage for preclinical testing of this molecule in small animal models of complement-mediated disease. more...

Murine sperm initiate fertilization by binding to the specialized extracellular matrix of their complementary eggs, known as the zona pellucida. On the basis of data reported in this study, mouse sperm also bind to rabbit erythrocytes with higher affinity than they do to murine eggs. This unusual interaction between a germ cell and a somatic cell ("sperm-somatic cell adhesion system") is also carbohydrate dependent based on its sensitivity to mild periodate oxidation. To determine what types of carbohydrate sequences could be involved in this interaction, the protein-linked oligosaccharides of rabbit erythrocytes were sequenced using novel matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry methods that enabled the analysis of individual components up to m/z 9000. The N-glycans are primarily complex biantennary and triantennary types terminated with Galalpha1-3Gal sequences. The majority of these oligosaccharides also possess one antenna consisting of a highly branched polylactosamine-type sequence that is also associated with many glycosphingolipids that coat rabbit erythrocytes. These erythrocytes also express Core 1 and Core 2 O-glycans terminated primarily with Galalpha1-3Gal sequences and to a lesser extent sialic acid. These results confirm that rabbit erythrocytes and mouse eggs present very different types of carbohydrate sequences on their surfaces. However, oligosaccharides terminated with beta1-6-linked N-acetyllactosamine or its alpha1-3 galactosylated analog are expressed on both the mouse zona pellucida and this somatic cell type. The far more abundant presentation of such sequences on rabbit erythrocytes compared with murine eggs could explain why mouse sperm display such exceptional affinity for this somatic cell type. more...

Murine sperm initiate fertilization by binding to the zona pellucida (mZP), the specialized extracellular matrix of their homologous eggs. O-Glycans occupying two highly conserved vicinal glycosylation sites (Ser-332 and Ser-334) on the mZP glycoprotein designated mZP3 were previously implicated in this interaction. However, recent biophysical analyses confirm that neither site is occupied, implying that an alternate O-glycosylation domain may be operational in native mZP3. Since human ZP3 (huZP3) can substitute for mZP3 in rescue mice to mediate sperm binding, the site specificity of O-glycosylation in both native mZP3 and huZP3 was analyzed using ultrasensitive mass spectrometric techniques. Two O-glycosylation sites in native mZP3, one at Thr-155 and the other within the glycopeptide at positions 161-168 (ATVSSEEK), are conserved in huZP3 derived from transgenic mice. Thus, there is a specific O-glycosylation domain within native mZP3 expressing two closely spaced O-glycans that is very well conserved in an evolutionarily related glycoprotein. In native mZP3, core 2 O-glycans predominate at both sites. However, in huZP3 derived from rescue mice, the O-glycans associated with Thr-156 (analogous to Thr-155 in mZP3) are exclusively core 1 and related Tn sequences, whereas core 2 O-glycans predominate at the other conserved site. This unique restriction of O-glycan expression suggests that sequence differences in the conserved O-glycosylation domains of mZP3 and huZP3 affect the ability of core 2 N-acetylglucosaminyltransferase(s) to extend the core 1 sequence. However, this difference in O-glycosylation at Thr-156 does not affect the fertility or the sperm binding phenotype of eggs derived from female huZP3 rescue mice. more...

Objectives. CA125 expresses specific oligosaccharides that can inhibit the cytotoxicity of human natural killer (NK) cells. The current study was undertaken to determine the ability of CA125 to modulate NK cell-mediated cytotoxicity. Methods. CA125 was isolated from OVCAR-3 cells and its purity was determined by ELISA and ultra-sensitive mass spectrometric analysis. Peripheral blood-derived NK were treated with CA125 and standard cytotoxicity assays were performed using 51 Cr-labeled K562 cells as targets. The expression of cell surface and intracellular markers on NK cells was determined by either flow cytometry or Western blot analysis. Results. NK cells incubated with CA125 for 72 h exhibited a 50–70% decrease in the lysis of K562 targets. Incubation with CA125 for 4 h and 24 h had no effect on NK-mediated cytolysis. Inhibition of NK function was observed at CA125 concentrations (10,000–100,000 U/ml) that are expected to be significantly lower than those observed in the tumor microenvironment. Co-stimulation with IL-2 did not abrogate the NK inhibitory response of CA125. CA125 did not reduce proliferation or induce apoptosis of NK cells and alter the expression of p56lck, phospholipase Cγ1, ZAP70, or CD3ζ. CA125 did, however, induce major downregulation of CD16 and minor decrease in expression of CD94/NKG2A. Conclusions. Our ongoing research and recent work performed by other laboratories highlights the potential physiologic role of this mucin. Based on the data presented here, it is likely that the tumor-derived CA125 acts as a suppressor of the immune response that is directed against the ovarian tumors. more...

In the present study, human myometrial microvascular endothelial cells (HMMEC) were cocultured with human vascular smooth muscle cells (VSMC) labeled with fluorescent dyes to examine their morphological interaction using confocal laser fluorescent microscopy. HMMEC and VSMC labeled with fluorescent green and red dyes, respectively, attached to opposite sides of polyethyleneterephthalate membranes and remained viable for up to 96 h. In defined medium, 5%+/-3% of the VSMC cytoplasmic processes and 71%+/-17% of the HMMEC processes extended completely across the 13 microm thickness of the transmembrane. However, 41%+/-21% of the VSMC projections and 10%+/-3% of the HMMEC processes that traversed the membrane made contact with the opposing cell type. In cocultures incubated with angiopoietin-1 (Ang-1), although the number of VSMC or HMMEC projections was not significantly increased, the number of VSMC extending across the membrane and making contact with HMMEC was increased (P0.05) to 88%+/-2%. The results of the current study demonstrate that coculture of fluorescent-labeled HMMEC and VSMC on a semipermeable transmembrane coupled with confocal laser fluorescent microscopy provides an in vitro experimental model to study morphological association of microvascular endothelial cells with mural cells. We propose that this system will greatly facilitate study of remodeling of the microvasculature in various organ systems. more...

To determine the cardiovascular molecular events associated with acute exposure to cocaine, the present study utilized in vivo analysis of left-ventricular heart function in adult rabbits, fluorescence confocal microscopy of fluo-2, rhod-2, (5-(and-6) carboxy 2',7' dichlorodihydrofluores-cein diacetate (carboxy-H2DCFDA), and JC-1 in H9C2 cells and gene expression microarray technology for analysis of gene activation in both rabbit ventricular tissue and H9C2 cells. In the rabbit, acute cocaine exposure (2 mg/kg) caused left-ventricular dysfunction and 0.1-10 mM cocaine increased cytosolic and mitochondrial calcium activity and mitochondrial membrane depolarization in H9C2 cells. A 3-min pretreatment of H9C2 cells by 10 microM verapamil, nifedipine, or nadolol inhibited calcium increases, but only 1 mM N-acetylcysteine (NAC) or 1 mM glutathione blocked mitochondrial membrane depolarization. Cocaine induced activation of genes in the rabbit heart and H9C2 cells including angiotensinogen, ADRB1, and c-reactive protein (CRP). In H9C2 cells, NAC pretreatment blocked cocaine-mediated increases in CRP, FAS, FAS ligand, and cytokine receptor-like factor1 (CRLF1) expression. Collectively, these data suggest that acute cocaine administration initiates cellular and genetic changes that, if chronically manifested, could cause cardiac deficits similar to those seen in heart failure and ischemia, such as ventricular dysfunction, cardiac arrhythmias, and cardiac remodeling. more...

Murine sperm initiate fertilization by binding to the outer covering of the egg known as the murine zona pellucida (mZP). This binding is thought to require the interaction of O-glycans linked to a specific mZP glycoprotein (mZP3) with egg-binding proteins coating the sperm plasma membrane. The precise molecular basis of this interaction remains to be resolved. In this study, we analyzed the O-glycosylation of the individual mZP glycoproteins by using ultrasensitive MS methods. We found that the majority of the O-glycans that are linked to mZP3 are core type 2 sequences terminated with sialic acid, lacNAc (Galβ1-4GlcNAc), lacdiNAc (Gal-NAcβ1-4GlcNAc), Galα1-3Gal, and NeuAcα2-3[GalNAcβ1-4]Galβ1-4 (Sd a antigen). Many of these terminal sequences have been implicated previously in murine sperm–egg binding. Core type 1 O-glycans are also present and are generally unmodified, although some are terminated with sialic acid, β-linked N -acetylhexosamine, or NeuAcα2-3[GalNAcβ1-4]Galβ1-4. Eggs expressing human ZP (huZP) glycoprotein huZP3, derived from transgenic mice, bind murine but not human sperm, implying that huZP3 acquires the same O-glycans as native mZP3. Sequencing of huZP3-associated O-glycans confirms that this implication is correct. The data obtained in this investigation may prove to be very useful for studies to determine the precise molecular basis of initial murine sperm–egg binding. more...

Human seminal plasma is a complex mixture of proteins, glycoproteins, peptides, glycopeptides, and prostaglandins secreted by organs of the male reproductive tract. The components of this fluid have been implicated in the suppression of immune response, agonistic effects on sperm-egg binding, and promotion of successful implantation of the human embryo. Fractionation followed by biophysical analyses revealed that free oligosaccharides constitute a major component of the total glycoconjugates within seminal plasma. Significant findings of our analyses include the following: (i) the concentration of free oligosaccharides is 0.3-0.4 mg/ml; (ii) mono- and difucosylated forms of the disaccharide lactose are major components; (iii) many of the remaining oligosaccharides are also rich in fucose and carry Lewis(x) and/or Lewis(y) epitopes; (iv) a subset of the oligosaccharides express the reducing end sequence (GlcNAcbeta1-3/4Glc) not reported in human milk oligosaccharides; (v) oligosaccharides in seminal plasma exclusively express type 2 (Galbeta1-4GlcNAc) but not the type 1 sequences (Galbeta1-3GlcNAc) that predominate in human milk glycans; and (vi) the structural diversity of seminal plasma oligosaccharides is far less than human milk oligosaccharides. The agonistic effect of both fucose and fucosylated glycoconjugates on human sperm-egg binding in vitro suggests that fucosylated oligosaccharides may also promote fertilization in the female reproductive tract. more...

Lacritin is a novel human tear glycoprotein that promotes basal tear peroxidase secretion by rat lacrimal acinar cells in vitro. This study investigates whether lacritin is prosecretory when added topically to the ocular surface of normal living rabbits, and if so, what is its efficacy and tolerability versus cyclosporine and artificial tears.Purified recombinant human lacritin (1, 10, 50, or 100 μg/mL), inactive lacritin truncation mutant C-25 (10 μg/mL), cyclosporine (0.05%), or artificial tears were topically administered to eyes of normal New Zealand White rabbits either as a single dose or three times daily for 14 days with monitoring of basal tear production. Basal tearing under proparacaine anesthesia was repeatedly assessed throughout and 1 week after chronic treatment ceased. Eyes were examined weekly by slit-lamp biomicroscopy.Lacritin acutely increased basal tearing to 30% over vehicle at 240 minutes. Three times daily treatment with 10-100 μg/mL lacritin was well tolerated. Basal tearing became progressively elevated 4, 7, and 14 days later and was 50% over baseline (50 μg/mL lacritin) 1 week after treatment had ceased. Cyclosporine elevated tearing to a similar level on days 4 and 7 but had little or no effect on day 14 and had returned to baseline 1 week after ending treatment. C-25 and artificial tears had no effect.Lacritin acutely stimulates basal tear flow that is sustained for at least 240 minutes. Two weeks of lacritin treatment three times daily was well tolerated and progressively elevated the basal tear flow. One week after treatment ended, basal tearing was still 50% over baseline. In contrast, cyclosporine triggered mild to moderate corneal irritation and a temporary elevation in tearing. more...

Summary Cell surface receptors for progesterone were visualized in human sperm using fluorescein isothiocyanate-progesterone 3-(0-carboxymethyl) oxime-bovine serum albumin (FITC prog CMO BSA). The receptors were confined to the head and not the midpiece or tail. FITC prog CMO BSA was also an effective stimulus to elevate intracellular free calcium in human sperm as detected by fura-2 fluorescence. The elevation of intracellular free calcium is a stimulus for the acrosome reaction, a process which is necessary to occur for sperm to fertilize the egg. It is proposed that progesterone, which is present in the female reproductive tract, can bind to progesterone receptors located in the plasma membrane of the sperm head and elicit an influx of Ca 2+ into the underlying cytoplasm and or acrosome and induce the acrosome reaction and facilitate fertilization. more...

Subfractions of adrenal medullary homogenates were analyzed in two-dimensional polyacrylamide gels to assess the extent of protein homology. Chromaffin granule proteins were highly acidic, with the exception of the soluble form of the enzyme dopamine β-hydroxylase (EC 1.14.17.1). The purified granule membrane proteins were more heterogeneous, but still predominantly acidic. The soluble and membrane forms of dopamine β-hydroxylase behaved identically in this gel system. Lactoperoxidase-catalyzed iodination of intact granules revealed that most, but not all, granule membrane proteins are accessible at the cytoplasmic face. Prominent proteins of the purified adrenal medullary mitochondria showed little if any homolog with purified granule membranes. The crude microsome fraction showed significant homology with purified granule membranes despite low levels of cross-contamination between the two fractions in marker enzyme analysis. Among proteins that could be identified, dopamine β-hydroxylase was at a low level in the microsomes, while the granule membrane protein cytochrome b -561 appeared to be in both fractions. The pattern obtained from primary cultures of adrenal chromaffin cells was very complex, but prominent proteins from the subcellular fractions were seen without difficulty. Actin and tubulin were very prominent in the whole cell pattern. Radioiodination of the whole cells resulted in a number of spots being labelled, although the majority of the label appeared to be in only two proteins of molecular weight 70000 and isoelectric point 5.7. more...