Regulation of cyclic adenosine 3',5'- monophosphate signaling and pulsatile neurosecretion by Gi-coupled plasma membrane estrogen receptors in immortalized gonadotrophin-releasing hormone neurons

Immortalized GnRH neurons (GT1-7) express receptors for estrogen [estrogen receptor-alpha and-13(ERa and ERI3)] and progesterone (progesterone receptor A) and exhibit positive immunostaining for both intracellular and plasma membrane ERs. Exposure of GT1-7 cells to picomolar estradiol concentrations for 5-60 min caused rapid, sustained,and dose-dependent inhibition of cAMP production. In contrast, treatment with nanomolar estradiol concentrations for 60 min increased cAMP production. The inhibitory and stimulatory actions of estradiol on cAMP formation were abolished by the ER antagonist, ICI 182,780. The estradiol-induced inhibition of cAMP production was prevented by treatment with pertussis toxin, consistent with coupling of the plasma membrane ER to an inhibitory G protein. Coimmunoprecipitation studies demonstrated an estradiol-regulated stimulatory interaction between ERa and G,3 that was prevented by the ER antagonist, ICI 182,780. Exposure of perifused GT1-7 cells and hypothalamic neurons to picomolar estradiol levels increased the GnRH peak interval, shortened peak duration, and increased peak amplitude. These findings indicate that occupancy of the plasma membrane-associated ERs expressed in GT1-7 neurons by physio-logical estradiol levels causes activation of a G, protein and modulates cAMP signaling and neuropeptide secretion.     

Selective toxicity of engineered lentivirus lytic peptides in a CF airway cell model

Lentivirus lytic peptides (LLPs) are derived from HIV-1 and have antibacterial properties. LLP derivatives (eLLPs) were engineered for greater potency against Staphylococcus aureus (SA) and Pseudomonas aeruginosa (PA). Minimum bactericidal concentration (MBC) was determined in low and physiologic salt concentrations. MBC was decreased against SA and equivalent against PA in physiologic salt when compared to the parent compound LLP1. In a novel cystic fibrosis (CF) airway cell model, one derivative, WLSA5, reduced the number of adherent PA and only moderately affected CF cell viability. Overall, eLLPs are selectively toxic to bacteria and may be useful against CF airway infections.     

Deletion of vapA encoding Virulence Associated Protein A attenuates the intracellular actinomycete Rhodococcus equi

Virulent strains of the facultative intracellular bacterium Rhodococcus equi isolated from young horses (foals) with R. equi pneumonia, carry an 80-90 kb virulence plasmid and express a highly immunogenic 15-17 kDa protein of unknown function called VapA (Virulence Associated Protein A). Recent sequencing of the virulence plasmid identified a putative pathogenicity island encoding a novel family of seven Vap proteins including VapA. These proteins exhibit a significant sequence similarity to each other but have no homologues in other organisms. In this study, we describe the construction of an R. equi mutant lacking a 7.9 kb DNA region spanning five vap genes (vapA, -C, -D, -E and -F ). This vap locus mutant was attenuated for virulence in mice as it was unable to replicate in vivo and was rapidly cleared in comparison to the virulent wild-type strain. Complementation analysis of the vap locus mutant showed that expression of vapA alone could restore full virulence, whereas expression of vapC, -D and -E could not. We subsequently constructed an R. equi strain lacking only the vapA gene and found that it was attenuated for growth in vivo to the same degree as the vap locus mutant. Unlike wild-type R. equi which replicates intracellularly, both of the mutant strains exhibited a growth defect in macrophages although their attachment to the macrophages was unaffected. These studies provide the first proof of a role for vapA in the virulence of R. equi, and demonstrate that its presence is essential for intracellular growth in macrophages.     

Function of human intrinsic cardiac neurons in situ

We sought to determine the behavior of intrinsic cardiac neurons in human subjects undergoing cardiac surgery and to correlate their activity with hemodynamics status. A lead II electrocardiogram, pulmonary artery pressure, and systemic arterial pressure were recorded along with extracellular activity generated by right atrial neurons in 10 patients undergoing coronary artery bypass surgery. Identified neurons generated spontaneously activity that was, for the most part, unrelated to the cardiac cycle. Most neurons were activated by gentle mechanical distortion of ventricular epicardial loci. The activity generated by neurons in each patient increased when arterial pressure increased and decreased when arterial pressure fell. Intrinsic cardiac neurons continued to generate activity during cardioplegia and cardiopulmonary bypass, but at reduced levels. Normal neuronal activity was restored postbypass. It is concluded that human intrinsic cardiac neurons generate spontaneous activity and that many receive inputs from ventricular mechanosensory neurites. The latter may account for the fact that their behavior depends, in part, on cardiac dynamics. They are also sensitive to intravenously administered pharmacological agents. These data also indicate that cardiopulmonary bypass and cardioplegia do not induce residual depression of their function.     

A method for large-scale multiplication of Curculigo orchioides through bulbil formation from leaf explant in shake flask culture

An efficient method has been developed for large-scale multiplication of Curculigo orchioides (Hypoxidaceae), an endangered medicinal plant, through direct bulbil formation from leaf explants in shake flask cultures. Leaf-segments (7 x 10 mm) were cultured in B5 liquid medium containing KNO3 (200 mgNL-1), (NH4)2SO4 (50 mgNL-1), benzyl adenosine (2.2 microM), adenine (0.11 mM), indole butyric acid (1.0 microM) and polyvinyl pyrrolidone (250 mgL-1). About 95% of explants produced maximum number of bulbils (546/flask at 6 weeks growth) in the medium. Shake flask cultures yielded 2737 bulbils/L medium whereas static cultures yielded 624 bulbils/L medium. Germination of bulbils was maximum (90.62%) on agar-gelled B5 medium containing benzyl adenosine (2.2 microM) and gibberellic acid (3.5 microM). Plantlets developed in vitro were successfully transferred to soil with a high rate of survivability (90%) and were comparable to natural population in growth and vigour.     

Testicular fine needle aspiration cytology for the diagnosis of azoospermia and oligospermia

OBJECTIVE: To evaluate qualitative and quantitative cytologic features on testicular fine needle aspiration biopsy in the diagnosis of azoospermia and oligospermia and to correlate cytologic and histologic diagnoses. STUDY DESIGN: In this prospective study, 50 infertile males selected from the infertility clinic of Guru Tegh Bahadur Hospital were studied. Fine needle aspiration cytology (FNAC) smears from both testes of 27 azoospermic and 23 oligospermic patients (sperm count < 10 million per milliliter) were stained with May-Grünwald-Giemsa and Papanicolaou stain. Differential counting of 500 spermatogenic cells was done, and the number of Sertoli cells per 500 germ cells was determined for calculating the spermatic index and Sertoli cell index, respectively. FNAC and testicular biopsy were performed under local anesthesia as a minor surgical procedure. RESULTS: Six groups were identified on FNAC smears from azoospermic patients: I. normal spermatogenesis (8), II. hypospermatogenesis (2), III. maturation arrest (2), IV. Sertoli cells only (6), V. atrophic pattern (7), and VI. Leydig cell predominance (2). In oligospermic patients two groups were identified: I. those with normal spermatogenesis (4), and II. those with subnormal spermatogenesis (19). Correlation with histopathologic examination was seen in 81.5% azoospermic and 65.2% oligospermic patients. CONCLUSION: Qualitative and quantitative evaluation of testicular FNAC provides useful information on both azoospermic and oligospermic patients. FNAC performed under local anesthesia is an acceptable outpatient procedure that consistently yields sufficient diagnostic material in all patients.     

Intracellular osteopontin is an integral component of the CD44-ERM complex involved in cell migration

Osteopontin (OPN) is a secreted glycoprotein with mineral- and cell-binding properties that can regulate cell activities through integrin receptors. Previously, we identified an intracellular form of osteopontin with a perimembranous distribution in migrating fetal fibroblasts (Zohar et al., J Cell Physiol 170:88-98, 1997). Since OPN and CD44 expression are increased in migrating cells, we analyzed the relationship of these proteins with immunofluorescence and confocal microscopy. A distinct co-localization of perimembranous OPN and cell-surface CD44 was observed in fetal fibroblasts, periodontal ligament cells, activated macrophages, and metastatic breast cancer cells. The co-localization of OPN and CD44 was prominent at the leading edge of migrating fibroblasts, where OPN also co-localized with the ezrin/radixin/moesin (ERM) protein ezrin, as well as in cell processes and at attachment sites of hyaluronan-coated beads. The subcortical location of OPN in these cells was verified by cell-surface biotinylation experiments in which biotinylated CD44 and non-biotinylated OPN were isolated from complexes formed with hyaluronan-coated beads and identified with immunoblotting. That perimembranous OPN represents secreted protein internalized by endocytosis or phagocytosis appeared to be unlikely since exogenous OPN that was added to cell cultures could not be detected inside the cells. A physical association with OPN, CD44, and ERM, but not with vinculin or alpha-actin, was indicated by immunoadsorption and immunoblotting of cell proteins in complexes extracted from hyaluronan-coated beads. The functional significance of OPN in this complex was demonstrated using OPN-/- and CD-/- mouse fibroblasts which displayed impaired migration and a reduced attachment to hyaluronan-coated beads. These studies indicate that OPN exists as an integral component of a hyaluronan-CD44-ERM attachment complex that is involved in the migration of embryonic fibroblasts, activated macrophages, and metastatic cells.