Acid dissociation constants of diphytanylglycerolphosphorylglycerol-methylphosphate, and diphytanylglycerolphosphorylglycerophosphate and its deoxy analog

Acid dissociation constants of 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-methylphosphate (PGP-Me), the major phospholipid in extreme halophiles (Halobacteriaceae), and of the demethylated 2,3-diphytanyl-sn-glycero-1-phosphoryl-sn-3′-glycero-1′-phosphate (PGP) and its deoxy analog 2,3-diphytanyl-sn-glycero-1-phosphoryl-1′-(1′,3′-propanediol-3′-phosphate) (dPGP), were calculated by an original mathematical procedure from potentiometric titration data in methanol/water (1:1, v/v) and found to be as follows: for PGP-Me (and presumably PGP), pK₁=3.00 and pK₂=3.61; for PGP, pK₃=11.12; and for dPGP, pK₁=2.72, pK₂=4.09, and pK₃=8.43. High-resolution ³¹P NMR spectra of intact PGP-Me in benzene/methanol or in aqueous dispersion showed two resonances corresponding to the two P-OH groups, each of which exhibited a chemical shift change in the pH range 2.0–4.5, corresponding to acid dissociation constants pK₁=pK₂=3.2; no further ionization (pK₃) was detected at higher pH values in the range 5–12. The present results show that PGP-Me titrates as a dibasic acid in the pH range 2–8, but above pH 8, it titrates as a tribasic acid, presumably PGP, formed by hydrolysis of the methyl group during the titration with KOH. Calculation of the concentrations of the ionic molecular species of PGP-Me, PGP and dPGP as a function of pH showed that the dianionic species predominate in the pH range 5–9, covering the optimal pH for growth of Halobacteriaceae. The results are consistent with the concept that the hydroxyl group of the central glycerol moiety in PGP-Me and PGP is involved in the formation of an intramolecular hydrogen-bonded cyclic structure of the polar headgroup, which imparts greater stability to the dianionic form of PGP-Me and PGP in the pH range 5–9 and facilitates lateral proton conduction by a process of diffusion along the membrane surface of halobacterial cells.     

Protein gene product 9.5 is a spermatogonia-specific marker in the pig testis: application to enrichment and culture of porcine spermatogonia

Identification and isolation of spermatogonial stem cells (SSCs) are a prerequisite for culture, genetic manipulation, and/or transplantation research. In this study, we established that expression of PGP 9.5 is a spermatogonia-specific marker in porcine testes. The expression pattern of PGP 9.5 in spermatogonia was compared to cell type-specific protein (GATA-4 or PLZF) expression in seminiferous tubules at different ages, and expression levels of PGP 9.5, Vasa, and Oct-4 were compared in different cell fractions. Enrichment of spermatogonia from 2-week-old (2wo) and 10-week-old (10wo) boars by adhesion to laminin, differential plating, or velocity sedimentation followed by differential plating was assessed by identification of spermatogonia using expression of PGP 9.5 as a marker. Compared to the initial samples, spermatogonia were enriched twofold in laminin-selected cells (P < 0.05), and fivefold either in cells remaining in suspension (fraction I) or in cells slightly attached to the culture dish (fraction II) (P < 0.05) after differential plating. Cells in fraction II appeared to be superior for future experiments due to higher viability (>90%) than in fraction I ( approximately 50%). Velocity sedimentation plus differential plating achieved cell populations containing up to 70% spermatogonia with good viability (>80%). Enriched spermatogonia from 2wo and 10wo testes could be maintained in a simple culture medium without additional growth factors for at least 2 weeks and continued to express PGP 9.5. These data provide the basis for future studies aimed at refining conditions of germ cell culture and manipulation prior to germ cell transplantation in pigs.     

Immunoassay of the Neuronal and Neuroendocrine Marker PGP 9.5 in Human Tissues

An inhibitory, coated-well immunoassay for the neurone-specific protein PGP 9.5 has been devised and used to measure the concentrations of the protein in human tissues. Concentrations of PGP 9.5 between 40 ng/ml and 10 micrograms/ml could be measured using this assay. In brain PGP 9.5 was present at 100.58 +/- 16.18 micrograms/mg protein. Of the other organs examined only kidney and testis showed significant concentrations of PGP 9.5 (3.97 +/- 0.87 microgram/mg protein and 3.25 +/- 0.36 microgram/mg protein, respectively). All other organs contained less than 2% of the brain level. The tissue levels determined by coated-well immunoassay confirmed the tissue specificity of PGP 9.5 originally determined by high-resolution two-dimensional gel electrophoresis.     

Immunohistochemical Localization of Nerve Fibers in the Pseudocapsule of Fibroids

The pseudocapsule surrounding fibroids consists of compressed myometrium containing nerves and blood vessels that continue into adjacent myometrium. Oxytocin (OXT) is thought to affect wound healing after myomectomy. We determined the presence of OXT and protein gene product 9.5 (PGP9.5) immunoreactive nerve fibers in pseudocapsule compared to adjacent myometrium. Samples (N=106) of pseudocapsule and adjacent myometrium were collected from 57 women with uterine fibroids undergoing myomectomy, and stained with anti-OXT and PGP 9.5 antibodies to demonstrate the presence of nerve fibers. Nerve fibers in the pseudocapsule stained positively with OXT (89/106, 84.0%) and PGP 9.5 (94/106, 88.7%). The densities of nerve fibers staining with PGP 9.5 and OXT in the pseudocapsule were highest in the isthmus (23.68±22.45/mm² and 43.35±40.74/mm², respectively). There were no significant differences in the density of nerve fibers, stained with either OXT or PGP 9.5, between the pseudocapsule, and adjacent normal myometrium regardless of the fibroid location in the uterus (P>0.05). These results suggest that the pseudocapsule should avoid to be damaged during the myomectomy procedure.Key words: fibroid pseudocapsule, nerve fibers, oxytocin, myomectomy, protein gene product 9.5, immunohistochemistry     

Immunohistochemical analysis of protein gene product 9.5, a ubiquitin carboxyl-terminal hydrolase, during placental and embryonic development in the mouse.

Protein gene product 9.5 (PGP9.5) is expressed at high level in the neural and neuroendocrine systems. We investigated the localization and degree of expression of PGP9.5 in the developing mouse placenta and embryo at 6.5, 10.5 and 14 days of gestation using an immunohistochemical technique. At 6.5 days of gestation PGP9.5 was detected at various levels in decidual and primary trophoblast giant cells in the placenta, and in embryonic ectodermal cells in the embryo. At 10.5 and 14 days of gestation PGP9.5 was expressed at moderate to strong levels in neurons in the embryo, but rarely in the placenta. These findings suggest that the protein may play a significant role in implantation and placental development, and differentiation of embryonic ectoderm.     

‘Neuron-specific’ protein gene product 9.5 (PGP 9.5) is also expressed in glioma cell lines and its expression depends on cellular growth state

Protein gene product 9.5 (PGP 9.5), which in the normal nervous system is restricted to certain neurons, has been detected in two glioma cell lines, rat C6 and human GL15, by immunoblotting and immunocytochemistry. Its expression in these cells depends on the cellular growth state, being maximal between the first and second post-plating day. Only a faint PGP 9.5 immunoreactivity can be observed in glioma cells after the eleventh post-plating day, i.e. about one week after confluency has been reached. The present results suggest that PGP 9.5 in cultured glial cells is maximally expressed during the growth phase and that the protein could play a role during brain development in glial cells, in reactive gliosis, or in tumorigenesis of the glial lineage.     

A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter

Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2. The protein contains 160 amino acid residues with a predicted molecular mass of 16.5 kDa. Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms. A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized. Here, we propose a new signature of this family considering the N-terminal: [IV]–X(4)–[AV]–[AP]–X–[AP]–X(3)–Y–X(9)–[LIVF]–X(2)–[SA]–G–[QS], and the C-terminal: [AT]–R–X(2)–[IVFY]–X–[VC]–X(2)–L–P–X(4)–[LIVM]–E–[IVM]–[DE] motifs. Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles. Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.     

Murine FGF-4 gene expression is spatially restricted within embryonic skeletal muscle and other tissues

Fibroblast growth factors are believed to play many distinct roles in vertebrate development, owing to their ability to stimulate cell growth, prevent cell death, determine cell fate, and inhibit terminal differentiation in a variety of in vitro culture systems. We have used in situ hybridization to localize fibroblast growth factor-4 (FGF-4, also termed HST and K-FGF) gene expression in 7.5 to 16.5 day gestation mouse embryos. Seven discrete sites of gene expression were detected: (1) primitive streak (E7.5–8.5); (2) paraxial presomitic mesoderm in the trunk (E7.5–11.5); (3) primitive neuroectoderm (E8.0–8.5); (4) pharyngeal pouch endoderm (E8.5–9.5); (5) branchial arch ectoderm (E8.5–9.5); (6) limb apical ectoderm (E10.5–12.5), and (7) skeletal myoblast groups (E9.5–13.5). FGF-4 gene expression is spatially restricted within many of these sites. The profile of FGF-4 gene expression among skeletal muscle groups is overlapping, but distinct, from that of FGF-5, thereby revealing myoblast heterogeneity at the molecular level and suggesting distinct roles for multiple FGFs in muscle development.     

Characterization of growth hormone-releasing hormone (GHRH) binding to cloned porcine GHRH receptor

To study structure-activity relationships of growth hormone-releasing hormone (GHRH), a competitive binding assay was developed using cloned porcine adenopituitary GHRH receptors expressed in human kidney 293 cells. Specific binding of [His¹,¹²⁵I-Tyr¹⁰,Nle²⁷]hGHRH(1–32)-NH₂ increased linearly with protein concentration (10–45 μg protein/tube). Binding reached equilibrium after 90 min at 30°C and remained constant for at least 240 min. Binding was reversible to one class of high-affinity sites (K_d = 1.04 ± 0.19 nM, B_max = 3.9 ± 0.53 pmol/mg protein). Binding was selective with a rank order of affinity (IC₅₀) for porcine GHRH (2.8 ± 0.51 nM), rat GHRH (3.1 ± 0.69 nM), [N-Ac-Tyr¹, -Arg²]hGHRH(3–29)-NH₂ (3.9 ± 0.58 nM), and [ -Thr⁷]GHRH(1–29)-NH₂ (189.7 ± 14.3 nM), consistent with their binding to a GHRH receptor. Nonhydrolyzable guanine nucleotides inhibited binding. These data describe a selective and reliable method for a competitive GHRH binding assay that for the first time utilizes rapid filtration to terminate the binding assay.     

Immunocytochemical evidence of plasticity in the nervous structures of the rat lower lip

In this immunocytochemical study we investigated the distribution of nervous structures in the lower lip of adult rats. The region is characterized by a rich cutaneous and mucosal sensory innervation originating from terminal branches of the trigeminal system. Lower lip innervation was investigated by detection of the general neuronal marker protein gene product 9.5 (PGP 9.5) and the growth-associated protein 43 (GAP-43), a neurochemical marker of neuronal plasticity. The entire neural network of both cutaneous and mucosal aspects was stained by the antibody to PGP 9.5. In particular, nerve fibers were observed in the submucosal and the subepithelial plexuses. Thin immunoreactive fibers were observed within the epithelial layers ending as free fibers or as fibers associated with immunopositive Merkel cells. Well-identified anatomical structures receiving sensory or autonomic innervation were also surrounded by PGP 9.5-ir nerve fibers, in particular, hair follicles, vibrissae, glands, and blood vessels. GAP-43-immunostained nerve fibers were observed in all these structures; however, they were generally less numerous than the PGP 9.5-immunoreactive elements. An equal amount of PGP 9.5 and GAP-43 immunoreactivity occurred, in contrast, in the subepidermal and the submucosal plexuses, or in the epidermis and the mucosal epithelium. The present results show that GAP-43 is normally expressed in the mature trigeminal sensory system of the rat. Skin and oral mucosa are characterized by continuous remodeling that may also involve the sensory nervous apparatus. Continuous neural remodeling, regeneration and sprouting may be the reason for the observed expression of GAP-43.     

Localization and structural characterization of an oligosaccharide O-linked to bovine PDC-109 Quantitation of the glycoprotein in seminal plasma and on the surface of ejaculated and capacitated spermatozoa

PDC-109 (13 kDa) is the most abundant component, and the major heparin-binding protein, of bovine (Bos taurus) seminal plasma. Here, we show that PDC-109 contains a single O-linked oligosaccharide (NeuNAc(2–6)-Galβ(1–3)-GalNAc-) attached to Thr¹¹. Immunoquantitation of PDC-109 indicates that its concentration in seminal plasma is 15–20 mg/ml. Though PDC-109 is not present on epididymal sperm, ejaculated spermatozoa on average are coated with (9.5 ± 0.3) × 10⁶ molecules of PDC-109/cell. This value remained constant in swim-up sperm and decreased to (7.7 ± 0.4) × 10⁶/spermatozoon after incubation for 24 h in capacitation medium at 39°C. These data substantiate the hypothesis that PDC-109 may be one of the seminal plasma components that enhance the fertilizing capacity of bull spermatozoa upon interaction with heparin-like glycosaminoglycans present in the female genital tract.     

Phenotypic variations and dynamic topography of transformed cells in an experimental model of diethylstilbestrol-induced renal tumour in male Syrian hamster

This work explores the phenotypic changes affecting transformed cells in an experimental model of diethylstilbestrol (DES)-induced renal tumours in male Syrian hamster. This estrogen-induced neoplasm presents an important cytological pleomorphism and its origin remains largely controversial. In order to characterize phenotypic variations during tumour progression, the occurrence of seven lineage markers was analysed by a morphometric approach in kidney sections of DES-exposed hamsters (6–;11 months). S100 protein, neuron-specific enolase (NSE) and vimentin are expressed by a large percentage of malignant cells during tumour development. Glial fibrillary acidic protein (GFAP), protein gene product 9.5 (PGP 9.5) and desmin are mostly evidenced in advanced neoplasm whereas Leu 7 always presents a focal expression. As evidenced by double-label immunofluorescence, the coexpression of three important neuroectodermal lineage markers (S100, NSE and PGP 9.5) in earliest tumour buds points to a peripheral nerve sheath origin for this neoplasm thus confirming previously published data. For each marker, the fluctuations of expression levels during tumour progression as well as the spatial heterogeneity of distribution suggest variable phenotypic differentiation of transformed cell populations. This observation is largely corroborated by double-label immunofluorescence showing coexpression modification of several markers during tumour progression. This points to a complex dynamic and spatial self-organization of different phenotypes within neoplasms. Altogether, these results support the concept that DES-induced kidney tumours are not made of unstructured cell populations but represent adaptive complex dynamic biosystems.     

Evidence for neural progenitor cells in the human adult enteric nervous system

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.     

Morphological and morphometrical characteristics of the esophageal intrinsic nervous system in the golden hamster

Very little is known about esophageal innervation in the hamster. In the present study, we used protein gene product 9.5 (PGP 9.5) to determine immunohistochemically the architectural features of the enteric nervous system in the hamster esophagus. The myenteric plexus consisted of a loose and irregular network of ganglia and interganglionic nerve bundles. The density of the neurons in the myenteric plexus was relatively low (479 +/- 75/cm(2), n = 5), with a preferentially higher density in the upper cervical portion than other parts of the esophagus. Regional differences in the number of PGP 9.5-positive neurons and ganglia were observed. PGP 9.5-immunoreactive fibers in the ganglia often branched, giving rise to expanding nerve endings of laminar morphology resembling intraganglionic laminar endings described in rats and cats. Fine varicose fibers originating from the secondary plexus were occasionally observed near the motor endplates, suggested a dual innervation of the striated muscle. The submucosal plexus was free from ganglionated plexus. A regional difference in the submucosal nervous network was observed. The number of motor endplates in the inner muscle layer was higher than that in the outer muscle layer.     

Progesterone metabolism in human fibroblasts is independent of P-glycoprotein levels and Niemann-Pick type C disease

Progesterone inhibits intracellular transport of lysosomal cholesterol in cultured cells, and thus at least in part mimics the biochemical phenotype of Niemann–Pick type C disease (NPC) in human fibroblasts. The goal of this study was to determine whether metabolism of progesterone to other steroids is affected by the NPC mutation or by P-glycoprotein (a known progesterone target). We found that human fibroblasts metabolize progesterone in three steps: rapid conversion to 5-pregnane-3,20-dione, which is then reduced to 5-pregnane-3β()-ol-20-one with subsequent 6-hydroxylation. The pattern and rates of progesterone metabolism were not significantly different in a variety of fibroblasts from normal individuals, NPC patients, and obligate heterozygotes. Inhibition of steroid 5-reductase with finasteride completely blocked metabolism of progesterone but had no effect on inhibition of LDL-stimulated cholesterol esterification (IC₅₀=10 μM). Progesterone also partially inhibited 25-hydroxycholesterol-induced cholesterol esterification, with similar dose-dependence in normal and NPC fibroblasts. P-glycoprotein levels varied significantly among the various fibroblasts tested, but no correlation with NPC phenotype or rate of progesterone metabolism was noted, and P-glycoprotein inhibitors did not affect conversion of progesterone to products. These results indicate that metabolism of progesterone in human fibroblasts is largely independent of its ability to interfere with cholesterol traffic and P-glycoprotein function.     

Isolation of PGP 9.5, a New Human Neurone-Specific Protein Detected by High-Resolution Two-Dimensional Electrophoresis

Protein gene product (PGP) 9.5 is a new brain-specific protein originally detected by high-resolution two-dimensional electrophoresis of the soluble proteins of human brain and other organs. We have purified this protein from human brain and raised a rabbit antihuman PGP 9.5 antiserum. The protein has a monomer molecular weight of approximately 27,000 and is present in brain at concentrations at least 50 times greater than in other organs. Immunoperoxidase labelling has localised PGP 9.5 to neurones in the human cerebral cortex with no evidence of staining of glial elements. PGP 9.5 is estimated to be present in brain at concentrations of 200-500 micrograms/g wet weight and represents a major protein component of neuronal cytoplasm. This new neurone-specific cytoplasmic marker may prove useful in studies of neuronal development and in the detection of neuronal damage in disease of the nervous system.     

Protein gene product (PGP) 9.5 immunoreactivity in nerve fibres and pinealocytes of guinea-pig pineal gland: interrelationship with tyrosine-hydroxylase- and neuropeptide-Y-immunoreactive nerve fibres

This light-microscopic (LM) immunohistochemical study has evaluated the presence and distribution of the pan-neural and neuroendocrine marker protein gene product (PGP) 9.5 in pinealocytes and nerve fibres of guinea-pig pineal gland. The pattern of PGP 9.5-immunoreactive (ir) nerve fibres has been compared with that of fibres staining for tyrosine hydroxylase (TH) or neuropeptide Y (NPY). The vast majority of pinealocytes stained for PGP 9.5, although with variable intensity. PGP 9.5 immunoreactivity was localized in pinealocytic cell bodies and processes. Double-immunofluorescence revealed that PGP 9.5 immunoreactivity was absent from glial cells identified with a monoclonal antibody against glial fibrillary acidic protein (GFAP), PGP 9.5 immunoreactivity was also present in a large number of nerve fibres and varicosities distributed throughout the pineal gland. The number of TH-ir and NPY-ir nerve fibres was lower compared with those containing PGP 9.5 immunoreactivity. All fibres staining for NPY also stained for TH. NPY-ir nerve fibres were found to be much more numerous than previously reported for this species. The double-immunofluorescence analysis indicated that almost all TH-ir nerve fibres of the pineal gland contained PGP 9.5 immunoreactivity. However, few PGP 9.5-ir nerve fibres, located in the periphery and the central part of the gland, were TH-negative. A large number of PGP 9.5-ir fibres was concentrated in the pineal stalk. In contrast, TH-ir and NPY-ir nerve fibres were rare in this part of the pineal gland. Our data provide evidence that immunohistochemistry for PGP 9.5 may be a useful tool further to differentiate central and peripheral origins of pineal innervation. Furthermore, the staining of pinealocytes for PGP 9.5 may be exploited to study the three-dimensional morphology and the architecture of pinealocytes and their processes under various experimental conditions.