Lactoferrin (Lf): Retinoid interactions in the mammary glands of transgenic mice overexpressing human Lf

Induction of protein expression in a tissue-specific manner by gene transfer over-expression techniques has been one means to define the function of a protein in a biological paradigm. Studies with retinoid reporter constructs transfected in mammary cell lines suggests that lactoferrin (Lf) affects retinoid signaling pathways and alters apoptosis. We tested the effects and interactions of over-expressed mammary-specific human lactoferrin (hLf) and dietary retinol palmitate on lactation and mammary gland development in mice. Increased retinol palmitate in the diet increased daily retinol equivalents (RE) to 2.6-fold over the normal mouse control diet. Transgene (Tg) expression in the dam fed control diet depressed pup weight gain. Severe depression of pup weight gain was observed when homozygote TgTg dams were fed the RE diet. Normal weight gain was restored when pups were placed with a wild type dam fed the RE diet; conversely, normal growing pups from the wild type dams showed declining weight gains when fostered to the TgTg RE-fed dams. Northern analysis of mammary tissue extracts showed a reduction in WAP and an increase in IGFBP-3 mRNA that was associated with the presence of the transgene. Histological evaluation of 3 days lactating mammary tissue showed mammary epithelial cells from TgTg animals contained excessive secretory products, suggesting a block in cellular secretion mechanisms. In addition, the mammary cells displayed a cellular apical membrane puckering that extended into the alveoli lumens. These studies demonstrate an in vivo interaction of Tg-hLf expression and dietary retinoids in mouse mammary glands. While normal mammary gland physiology may not be representative by these experiments because high Lf concentrations during early lactation are abnormal, the demonstrated biological interaction suggests that typical periods of high Lf concentrations may have impact upon developing and involuting mammary glands.

Why is 11beta-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11beta-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

Silhouettella loricatula (Arachnida, Araneae, Oonopidae): a Haplogyne spider with complex female genitalia

The female genital system of the oonopid Silhouettella loricatula is astonishingly complex. The genital opening is situated medially and leads into an oval receptaculum that is heavily sclerotized except for the ventral half of the posterior wall that appears chitinized only. A large striking sclerite lying in the posterior wall of the uterus externus is attached anteriorly to the receptaculum and continues dorsally into a globular appendix that bears a furrow. The uterus externus shows a peculiar modification in its anterior wall: a paddle-like sclerite with a nail-like posterior process. This sclerite lies opposite to the furrow proceeding in the globular appendix and may serve females to lock the uterus externus by muscle contractions. Massive muscles connect the sclerite with the anterior scutum of the opisthosoma and with two other sclerites that are attached to the receptaculum and serve as attachments for further muscles. Gland cells extend around a pore field of the receptaculum. They produce secretion that encloses spermatozoa in a discrete package (secretory sac) inside the receptaculum. In this way, the mixing of sperm from different males and thus sperm competition may be severely limited or completely prevented. During a copulation in the laboratory the ejection of a secretory sac that most probably contained spermatozoa was observed, indicating sperm dumping in S. loricatula. The ejection of the secretory sac may be caused by female muscle contractions or by male pedipalp movements. The majority of the investigated females have microorganisms in the receptacula that could represent symbionts or infectious agents. The microorganisms can be identified partly as bacteria. They are enclosed in secretion and are always found in the same position inside the receptaculum.

Complex genital system of a haplogyne spider (Arachnida, Araneae, Tetrablemmidae) indicates internal fertilization and full female control over transferred sperm

The female genital organs of the tetrablemmid Indicoblemma lannaianum are astonishingly complex. The copulatory orifice lies anterior to the opening of the uterus externus and leads into a narrow insertion duct that ends in a genital cavity. The genital cavity continues laterally in paired tube-like copulatory ducts, which lead into paired, large, sac-like receptacula. Each receptaculum has a sclerotized pore plate with associated gland cells. Paired small fertilization ducts originate in the receptacula and take their curved course inside the copulatory ducts. The fertilization ducts end in slit-like openings in the sclerotized posterior walls of the copulatory ducts. Huge masses of secretions forming large balls are detectable in the female receptacula. An important function of these secretory balls seems to be the encapsulation of spermatozoa in discrete packages in order to avoid the mixing of sperm from different males. In this way, sperm competition may be completely prevented or at least severely limited. Females seem to have full control over transferred sperm and be able to express preference for spermatozoa of certain males. The lumen of the sperm containing secretory balls is connected with the fertilization duct. Activated spermatozoa are only found in the uterus internus of females, which is an indication of internal fertilization. The sperm cells in the uterus internus are characterized by an extensive cytoplasm and an elongated, cone-shaped nucleus. The male genital system of I. lannaianum consists of thick testes and thin convoluted vasa deferentia that open into the wide ductus ejaculatorius. The voluminous globular palpal bulb is filled with seminal fluid consisting of a globular secretion in which only a few spermatozoa are embedded. The spermatozoa are encapsulated by a sheath produced in the genital system. The secretions in females may at least partly consist of male secretions that could be involved in the building of the secretory balls or play a role in sperm activation. The male secretions could also afford nutriments to the spermatozoa.

A degradation-sensitive anionic trypsinogen (PRSS2) variant protects against chronic pancreatitis

Chronic pancreatitis is a common inflammatory disease of the pancreas. Mutations in the genes encoding cationic trypsinogen (PRSS1) and the pancreatic secretory trypsin inhibitor (SPINK1) are associated with chronic pancreatitis. Because increased proteolytic activity owing to mutated PRSS1 enhances the risk for chronic pancreatitis, mutations in the gene encoding anionic trypsinogen (PRSS2) may also predispose to disease. Here we analyzed PRSS2 in individuals with chronic pancreatitis and controls and found, to our surprise, that a variant of codon 191 (G191R) is overrepresented in control subjects: G191R was present in 220/6,459 (3.4%) controls but in only 32/2,466 (1.3%) affected individuals (odds ratio 0.37; P = 1.1 x 10(-8)). Upon activation by enterokinase or trypsin, purified recombinant G191R protein showed a complete loss of trypsin activity owing to the introduction of a new tryptic cleavage site that renders the enzyme hypersensitive to autocatalytic proteolysis. In conclusion, the G191R variant of PRSS2 mitigates intrapancreatic trypsin activity and thereby protects against chronic pancreatitis.

A polymorphism within the equine CRISP3 gene is associated with stallion fertility in Hanoverian warmblood horses

Fertility of stallions is of high economic importance, especially for large breeding organisations and studs. Breeding schemes with respect to fertility traits and selection of stallions at an early stage may be improved by including molecular genetic markers associated with traits. The genes coding for equine cysteine-rich secretory proteins (CRISPs) are promising candidate genes because previous studies have shown that CRISPs play a role in the fertilising ability of male animals. We have previously characterised the three equine CRISP genes and identified a non-synonymous polymorphism in the CRISP1 gene. In this study, we report one non-synonymous polymorphism in the CRISP2 gene and four non-synonymous polymorphisms in the CRISP3 gene. All six CRISP polymorphisms were genotyped in 107 Hanoverian breeding stallions. Insemination records of stallions were used to analyse the association between CRISP polymorphisms and fertility traits. Three statistical models were used to evaluate the influence of single mutations, genotypes and haplotypes of the polymorphisms. The CRISP3 AJ459965:c.+622G>A SNP leading to the amino acid substitution E208K was significantly associated with the fertility of stallions. Stallions heterozygous for the CRISP3 c.+622G>A SNP had lower fertility than homozygous stallions (P = 0.0234). The pregnancy rate per cycle in these stallions was estimated to be approximately 7% lower than in stallions homozygous at this position.

Sphingosylphosphorylcholine acts in an anti-inflammatory manner in renal mesangial cells by reducing interleukin-1beta-induced prostaglandin E2 formation

Sphingosylphosphorylcholine (SPC) is a bioactive lipid that binds to G protein-coupled-receptors and activates various signaling cascades. Here, we show that in renal mesangial cells, SPC not only activates various protein kinase cascades but also activates Smad proteins, which are classical members of the transforming growth factor-beta (TGFbeta) signaling pathway. Consequently, SPC is able to mimic TGFbeta-mediated cell responses, such as an anti-inflammatory and a profibrotic response. Interleukin-1beta-stimulated prostaglandin E(2) formation is dose-dependently suppressed by SPC, which is paralleled by reduced secretory phospholipase A(2) (sPLA(2)) protein expression and activity. This effect is due to a reduction of sPLA(2) mRNA expression caused by inhibited sPLA(2) promoter activity. Furthermore, SPC upregulates the profibrotic connective tissue growth factor (CTGF) protein and mRNA expression. Blocking TGFbeta signaling by a TGFbeta receptor kinase inhibitor causes an inhibition of SPC-stimulated Smad activation and reverses both the negative effect of SPC on sPLA(2) expression and the positive effect on CTGF expression. In summary, our data show that SPC, by mimicking TGFbeta, leads to a suppression of proinflammatory mediator production and stimulates a profibrotic cell response that is often the end point of an anti-inflammatory reaction. Thus, targeting SPC receptors may represent a novel therapeutic strategy to cope with inflammatory diseases.

Exon splice enhancer mutation (GH-E32A) causes autosomal dominant growth hormone deficiency

CONTEXT AND OBJECTIVE: Alteration of exon splice enhancers (ESE) may cause autosomal dominant GH deficiency (IGHD II). Disruption analysis of a (GAA) (n) ESE motif within exon 3 by introducing single-base mutations has shown that single nucleotide mutations within ESE1 affect pre-mRNA splicing. DESIGN, SETTING, AND PATIENTS: Confirming the laboratory-derived data, a heterozygous splice enhancer mutation in exon 3 (exon 3 + 2 A-->C) coding for GH-E32A mutation of the GH-1 gene was found in two independent pedigrees, causing familial IGHD II. Because different ESE mutations have a variable impact on splicing of exon 3 of GH and therefore on the expression of the 17.5-kDa GH mutant form, the GH-E32A was studied at the cellular level. INTERVENTIONS AND RESULTS: The splicing of GH-E32A, assessed at the protein level, produced significantly increased amounts of 17.5-kDa GH isoform (55% of total GH protein) when compared with the wt-GH. AtT-20 cells coexpressing both wt-GH and GH-E32A presented a significant reduction in cell proliferation as well as GH production after forskolin stimulation when compared with the cells expressing wt-GH. These results were complemented with confocal microscopy analysis, which revealed a significant reduction of the GH-E32A-derived isoform colocalized with secretory granules, compared with wt-GH. CONCLUSION: GH-E32A mutation found within ESE1 weakens recognition of exon 3 directly, and therefore, an increased production of the exon 3-skipped 17.5-kDa GH isoform in relation to the 22-kDa, wt-GH isoform was found. The GH-E32A mutant altered stimulated GH production as well as cell proliferation, causing IGHD II.

Evaluation of the biological activity of a growth hormone (GH) mutant (R77C) and its impact on GH responsiveness and stature

CONTEXT AND OBJECTIVE: A single missense mutation in the GH-1 gene converting codon 77 from arginine (R) to cysteine (C) yields a mutant GH-R77C peptide, which was described as natural GH antagonist. DESIGN, SETTING, AND PATIENTS: Heterozygosity for GH-R77C/wt-GH was identified in a Syrian family. The index patient, a boy, was referred for assessment of his short stature (-2.5 SD score) and partial GH insensitivity was diagnosed. His mother and grandfather were also carrying the same mutation and showed partial GH insensitivity with modest short stature. INTERVENTIONS AND RESULTS: Functional characterization of the GH-R77C was performed through studies of GH receptor binding and activation of Janus kinase 2/Stat5 pathway. No differences in the binding affinity and bioactivity between wt-GH and GH-R77C were found. Similarly, cell viability and proliferation after expression of both GH peptides in AtT-20 cells were identical. Quantitative confocal microscopy analysis revealed no significant difference in the extent of subcellular colocalization between wt-GH and GH-R77C with endoplasmic reticulum, Golgi, or secretory vesicles. Furthermore studies demonstrated a reduced capability of GH-R77C to induce GHR/GHBP gene transcription rate when compared with wt-GH. CONCLUSION: Reduced GH receptor/GH-binding protein expression might be a possible cause for the partial GH insensitivity with delay in growth and pubertal development found in our patients. In addition, this group of patients deserves further attention because they could represent a distinct clinical entity underlining that an altered GH peptide may also have a direct impact on GHR/GHBP gene expression causing partial GH insensitivity.

Targeted cell replacement with bone marrow cells for airway epithelial regeneration

It has been suggested that some adult bone marrow cells (BMC) can localize to the lung and develop tissue-specific characteristics including those of pulmonary epithelial cells. Here, we show that the combination of mild airway injury (naphthalene-induced) as a conditioning regimen to direct the site of BMC localization and transtracheal delivery of short-term cultured BMC enhances airway localization and adoption of an epithelial-like phenotype. Confocal analysis of airway and alveolar-localized BMC (fluorescently labeled) with epithelial markers shows expression of the pulmonary epithelial proteins, Clara cell secretory protein, and surfactant protein C. To confirm epithelial gene expression by BMC, we generated transgenic mice expressing green fluorescent protein (GFP) driven by the epithelial-specific cytokeratin-18 promoter and injected BMC from these mice transtracheally into wild-type recipients after naphthalene-induced airway injury. BMC retention in the lung was observed for at least 120 days following cell delivery with increasing GFP transgene expression over time. Some BMC cultured in vitro over time also expressed GFP transgene, suggesting epithelial transdifferentiation of the BMC. The results indicate that targeted delivery of BMC can promote airway regeneration.

Role of protein translocation pathways across the endoplasmic reticulum in Trypanosoma brucei

The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.

Impairment of gastric acid secretion and increase of embryonic lethality in Foxq1-deficient mice

The mouse Foxq1 gene, also known as Hfh1, encodes a winged helix/forkhead transcription factor. In adult mice, Foxq1 is highly expressed in kidney and stomach. Here, we report that Foxq1 is expressed during prenatal and postnatal stomach development and the transcripts are restricted to acid secreting parietal cells. Mice homozygous for a deletion of the Foxq1 locus on a 129/Sv x C57BL/6J hybrid genetic background display variable phenotypes consistent with requirement of the gene during embryogenesis. Approximately 50% of Foxq1-/- embryos die in utero. Surviving homozygous mutants are normal and fertile, and have a silky shiny coat. Although the parietal cell development is not affected in the absence of Foxq1, there is a lack of gastric acid secretion in response to various secretagogue stimuli. Ultrastructural analysis suggests that the gastric acid secretion defect in Foxq1-deficient mice might be due to impairment in the fusion of cytoplasmic tubulovesicles to the apical membrane of secretory canaliculi.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Sensitization to wasp venom does not induce autoantibodies leading to infertility

Antigenic cross-reactivity has been described between the venom allergen (antigen 5) and mammalian testis proteins. Based on an allergen database we have previously shown that allergens can be represented by allergen motifs. A motif group was found containing venom antigen 5 sequences from different vespids. Using an optimized amino acid profile based on antigen 5 sequences for searching cross-reactive proteins, three human semen proteins belonging to the family of cysteine-rich secretory proteins (hCRISP) were found in the Swiss Protein database. To analyze antigenic cross-reactivity between antigen 5 and hCRISPs, antigen 5 from yellow jacket venom (Ves v 5) and two hCRISPs (CRISP-2 and -3) were chosen and produced as recombinant proteins in E. coli. A correlation was found between antibodies reacting with rVes v 5 and rhCRISP-2, -3 in a small human sera population indicating the presence of cross-reactive antibodies in human serum. Using intravenous immunoglobulin (IVIg), a therapeutic multidonor IgG preparation, cross-reactive antibodies were isolated that recognize rVes v 5, hCRISP-2 and -3 suggesting the presence of common epitopes between Ves v 5 and hCRISPs. However this cross-reactivity seems not to be linked to allergy to wasp venom as we could show no correlation between increasing CAP-class IgE level to wasp venom and IgG to sperm extract and hCRISPs. These data suggest that higher sensitization to wasp venom does not induce more antibodies against autoantigens and might not represent a higher risk to develop autoantibodies leading to infertility.

The immune geography of IgA induction and function

The production of immunoglobulin A (IgA) in mammals exceeds all other isotypes, and it is mostly exported across mucous membranes. The discovery of IgA and the realization that it dominates humoral mucosal immunity, in contrast to the IgG dominance of the systemic immune system, was early evidence for the distinct nature of mucosal immunology. It is now clear that IgA can function in high-affinity modes for neutralization of toxins and pathogenic microbes, and as a low-affinity system to contain the dense commensal microbiota within the intestinal lumen. The basic map of induction of IgA B cells in the Peyer's patches, which then circulate through the lymph and bloodstream to seed the mucosa with precursors of plasma cells that produce dimeric IgA for export through the intestinal epithelium, has been known for more than 30 years. In this review, we discuss the mechanisms underlying selective IgA induction of mucosal B cells for IgA production and the immune geography of their homing characteristics. We also review the functionality of secretory IgA directed against both commensal organisms and pathogens.