The epididymal amyloid matrix: structure and putative functions.

BACKGROUND: We previously demonstrated the normal mouse epididymal lumen contains a non-pathological amyloid matrix that surrounds spermatozoa and plays important roles in sperm maturation and protection. OBJECTIVE: The objective herein was to present a review of this work, including studies showing the amyloid structures of four members of the CRES (cystatin-related epididymal spermatogenic) subgroup are integral and essential components of the amyloid matrix. METHODS: We used conformation-dependent reagents that recognize the cross-ß-sheet structure characteristic of amyloid, including thioflavin S (ThS), thioflavin T (ThT), anti-amyloid antibodies, and X-ray diffraction, as well as negative-stain transmission electron microscopy (TEM) to visualize amyloid structures in the epididymal lumen. Antibodies that specifically detect each CRES subgroup family member were also used in indirect immunofluorescence analysis. RESULTS AND DISCUSSION: The epididymal lumen contains an amyloid matrix that surrounds maturing spermatozoa and represents a functional amyloid. Alterations in the structure of the amyloid matrix by the loss of the CRES subgroup members or the overexpression of cystatin C result in epididymal pathologies, including infertility. Preliminary data suggest the epididymal amyloid matrix is structurally and functionally similar to bacterial biofilms. CONCLUSION: Together, these results suggest the amyloid matrix serves important roles in epididymal function including sperm maturation and protection.

Minocycline reduces ethanol drinking.

Alcoholism is a disease characterized by continued alcohol consumption despite recurring negative consequences. Thus, medications that reduce the drive to consume alcohol can be beneficial in treating alcoholism. The neurobiological systems that regulate alcohol consumption are complex and not fully understood. Currently, medications are available to treat alcoholism that act either by causing accumulation of a toxic metabolite of ethanol, or by targeting specific transmitter receptors. The purpose of our study was to investigate a new potential therapeutic pathway, neuroimmune interactions, for effects on ethanol consumption. We hypothesized that neuroimmune activity of brain glia may have a role in drinking. We utilized minocycline, a second generation tetracycline antibiotic that has immune modulatory actions, to test our hypothesis because it is known to suppress microglia, and to a lesser extent astroglia, activity following many types of insults to the brain. Treatment with 50mg/kg minocycline significantly reduced ethanol intake in male and female C57Bl/6J mice using a free choice voluntary drinking model. Saline injections did not alter ethanol intake. Minocycline had little effect on water intake or body weight change. The underlying mechanism whereby minocycline reduced ethanol intake requires further study. The results suggest that drugs that alter neuroimmune pathways may represent a new approach to developing additional therapies to treat alcoholism.

Oviductin (Muc9) is expressed in rabbit endocervix.

While isolating and characterizing cervical mucin glycoproteins, oviductin (Muc9) was identified in the rabbit endocervix. Following tissue homogenization, endocervical proteins were fractionated by exclusion chromatography (Sepharose CL-4B). High molecular weight components of the void volume were resolved by density gradient centrifugation using cesium bromide and dissociative conditions (4 M guanidinium chloride). High density fractions (rho = 1.40 - 1.56 g/ml) were deglycosylated with anhydrous trifluoromethane sulfonic acid and sent to Harvard Microchemistry where in situ digestion and tryptic peptide separation were performed. Out of an HPLC map, microsequence (KLIMGFPTYGR) from peak 51 was 100% identical to mouse oviductin, and microsequence (KSTGHNFPLP) from peak 70 was 90% identical to hamster oviductin. Temporal expression of oviductin transcripts (2.4-kilobase) was negligible during the first three months of postnatal cervical differentiation. Transcripts were minimally detectable in the cervices of 4-month-old juveniles. Strong expression in the endocervices of adults was eliminated by ovariectomy and restored by estrogen treatment. The presence of oviductin in the rabbit endocervix indicates this glycoprotein may have multiple functions, and it can no longer be considered oviduct-specific.

Cloning and characterization of an atypical Type IV P-type ATPase that binds to the RING motif of RUSH transcription factors.

RUSH proteins are SWI/SNF-related transcription factors with RING finger signatures near their COOH termini. Long suspected of mediating protein-protein interactions, the RING motif was used to clone a binding partner. The RING finger binding protein (RFBP) is a Type IV P-type ATPase, a putative phospholipid pump, with conserved sequences for two loop segments, an ATP-binding site, a phosphorylation domain, and transmembrane passes potentially involved in substrate binding and translocation. However, RFBP differs from all other Type IV P-type ATPases in three ways. It has only three of four highly conserved NH(2)-terminal transmembrane passes, it is located in the inner nuclear membrane, and it binds the RING domain. Topographically the orientation of the adjacent hydrophilic domains and the determinants of transport specificity are altered. As a result, the small, hydrophilic loop extends into the perinuclear space that is contiguous with the lumen of the endoplasmic reticulum. The large, conformationally flexible loop extends into the nucleoplasm to contact euchromatin. Competitive reverse transcriptase-polymerase chain reaction and high performance liquid chromatography analysis revealed that endometrial RFBP mRNA expression is hormonally regulated. The physical association of a hormone-dependent RING finger-binding protein with transcriptionally active chromatin supports the speculation that RFBP plays a role in the subnuclear trafficking of transcription factors with RING motifs.

Expression of RUSH transcription factors in developing and adult rabbit gonads.

The RUSH transcription factors 1alpha and 1beta bind to the Rabbit Uteroglobin promoter and are members of the SWI/SNF complex that facilitates transcription by remodeling chromatin (Helicase). To characterize gonadal expression of RUSH, a cRNA probe that recognizes both isoforms was used for in situ hybridization studies. We found RUSH mRNA to be abundant in Sertoli cells from embryonic, neonatal, prepubertal, and pubertal rabbit testes. In adults, RUSH mRNA was detected in tubules with preleptotene spermatocytes and mature spermatids lining the lumen. However, RUSH was undetectable in tubules that contained leptotene spermatocytes and that lacked mature spermatids. In females, RUSH was expressed in presumptive granulosa cells of embryonic and neonatal ovaries before follicle organization. Abundant RUSH mRNA was detected in granulosa and theca cells surrounding preantral follicles of prepubertal and adult ovaries. Expression of RUSH remained high in granulosa cells of antral follicles in mature ovaries but was negligible in late-stage atretic follicles and in corpora lutea. Western blot analysis confirmed the RUSH-1alpha isoform predominated in both testicular and ovarian tissues. The expression pattern of RUSH indicates transcriptional activity in Sertoli cells and during multiple stages of differentiating granulosa cells, especially those of primordial follicles, which heretofore were considered to be dormant.

Uteroglobin gene transcription: what's the RUSH?

Prolactin enhances progesterone-dependent transcription of the rabbit uteroglobin gene. RUSH transcription factors are implicated in the signal transduction pathway. The RUSH acronym identifies key features of these nuclear phosphoproteins, that is, RING-finger motif, binds the uteroglobin promoter, structurally related to the SWI/SNF family of transcription factors, and helicase-like. Cloned by recognition site screening, RUSH proteins bind to an 85-bp region (-170/-85) of the uteroglobin promoter that was subsequently identified as a novel prolactin-responsive region by promoter deletion analysis. Gel shift and linker-scanning assays further reduced the RUSH target site to -160/-110. A hexameric core of MCWTDK was identified as the RUSH-specific DNA-binding site (-126/-121) by CASTing. This site overlaps authentic HNF3 beta and OCT-1 binding sites. A unique Type IV P-type ATPase that is embedded in the inner nuclear membrane binds the RING domain of RUSH. The conformationally flexible loop portion of this RING-finger binding protein (RFBP) extends into the nucleoplasm to contact euchromatin. The physical association of RFBP with transcriptionally active chromatin supports the speculation that RFBP targets RUSH transcription factors to the active uteroglobin promoter.

Altered sodium pump alpha and gamma subunit gene expression in nephron segments from hypertensive rats.

OBJECTIVE: To determine the qualitative and quantitative expression of alpha and gamma sodium pump subunits in whole kidney and nephron segment RNA from Sprague Dawley rats, spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats. DESIGN: A novel reverse transcription polymerase chain reaction technique was devised which provides accurate and precise measurement of the number of molecules of specific transcript abundance, a measurement of gene expression. This allows the quantitative comparison of multiple samples across multiple subjects and, since the estimates are accurate rather than relative, can also be used to make quantitative comparisons across expressed genes, such as isoforms and subunits of the heterotrimeric renal sodium pump. METHODS: We examined which catalytic isoforms were expressed and then quantified transcript abundance in whole kidney and convoluted and straight segments of the proximal tubule. RESULTS: Alpha 1 and gamma transcripts, but not alpha 2, alpha 3 or alpha 4 isoforms, were consistently observed in nephron segments. Levels of alpha 1 were lower in kidney RNA from 15-16-week-old SHR than in WKY rats of the same age (P = 0.001), but were not different between SHR and WKY in 4-5-week-old animals. No significant difference was observed in gamma subunit abundance in kidney RNA from 4-5-week-old animals; however, at 15-16 weeks, the expression in SHR was one-third that in WKY rats (P = 0.003). In proximal convoluted tubules from 4-5-week-old animals, the level of alpha 1 RNA expression was lower (P = 0.03) in SHR than in WKY rats. In addition, levels of alpha 1 in proximal straight tubule from the 4-5-week-old SHR were also lower than in WKY rats (P = 0.02). This difference was even greater in 15-16-week-old animals: in SHR, alpha 1 expression was less than 20% of the level of expression in WKY rats (P = 0.0003). Expression of the gamma subunit exhibited a similar pattern of downregulation in SHR. In RNA from proximal convoluted tubules and proximal straight tubules from both 4-5- and 15-16-week-old animals, expression of the gamma subunit was demonstrated to be significantly lower in SHR than in WKY rats. CONCLUSION: The results indicate a coordinate reduction in the abundance of sodium pump alpha and gamma subunits in the proximal tubules of SHR, which occurs early during the development of hypertension.

After chromatin is SWItched-on can it be RUSHed?

Repressive chromatin must be remodeled to allow for transcriptional activation of genes in eukaryotic cells. Factors that alter chromatin structure to permit access of transcriptional activators, RNA polymerase II and the polymerase-associated general transcription factors to nucleosomal promoter sequences are as highly conserved as the basic mechanism of transcription. One group of promoter restructuring factors that perturbs chromatin in an ATP-dependent manner includes NURF, CHRAC, ACF, the SWI/SNF complex, and SWI/SNF-related proteins. Each member of this group contains a subunit homologous to the DNA-dependent ATPase; however, their individual mechanisms of action are unique. The small amount of SWI/SNF complex (100-200 copies/cell), its affiliation with a select number of inducible genes, and its interaction with the glucocorticoid and estrogen receptors, suggests the SWI/SNF complex might be preferentially targeted to active promoters. The SWI/SNF-related family of RUSH proteins which includes RUSH-1alpha and beta, hHLTF, HIP116, Zbu1, P113, and the transcription factor RUSH-1alpha isolog has been implicated as a highly conserved DNA binding site-specific ATPase.

Zinc finger proteins RUSH in where others fear to tread.

The uteroglobin (UG) gene family encodes a fascinating group of secreted proteins that includes rat prostatic steroid-binding protein subunit C3, human Clara cell 10-kDa protein, human mammaglobin, and rabbit UG. In the rabbit, UG is a progesterone-dependent preimplantation uterine protein with peak availability on Day 5 of pregnancy. Progesterone stimulates UG synthesis in estrous rabbits and in short-term (3 day to 4 wk) ovariectomized rabbits. However, with increased time after ovariectomy, the uterus becomes increasingly refractory to progesterone challenge, such that after 12 weeks, normal levels of UG synthesis could not be measured. The treatment of these long-term ovariectomized rabbits with either prolactin or progesterone resulted in a dramatic increase in the receptor for the other hormone. Sequential treatment with prolactin plus progesterone increased the endometrial UG mRNA content and stimulated UG production to a concentration equal to that found on the fifth day of pregnancy. Because the increase in UG mRNA could result from an increased rate of transcription, gel shift assays, Southwestern blots, and UV cross-linking were used to show that prolactin augments the binding of four progesterone-dependent proteins to an 85-base pair (bp) 5'-flanking region (-170/-85) of the UG gene. The cDNAs for two of these UG promoter-binding proteins, RUSH-1alpha (113 kDa) and -1beta (95 kDa), were cloned by recognition site screening and identified as new members of the SWI/SNF superfamily of nuclear receptor coactivators. RUSH-1alpha and -1beta result from alternative splicing of a 57-bp exon, and each phosphoprotein has the novel RING-finger motif near its C terminus. Competitive reverse transcription-polymerase chain reaction and HPLC analysis showed that RUSH-1alpha is the progesterone-dependent splice variant. When an endometrial cell line (HRE-H9) was transfected with either the full-length UG construct, pUG3.1-LUC, or the deletion mutant, pUG3.1 deltaRUSH-LUC, progesterone increased the transcriptional activity of pUG3.1. Transcription of pUG3.1-LUC was further increased when cells were treated with prolactin plus progesterone. Prolactin alone had no effect. Progesterone also increased the transcriptional activity of pUG3.1 deltaRUSH-LUC. However, deletion of the proximal promoter region (pUG3.1 deltaRUSH-LUC) eliminated the prolactin effect and implicated RUSH proteins as prolactin signal transducers.

Quantification of alternatively spliced RUSH mRNA isoforms by QRT-PCR and IP-RP-HPLC analysis: a new approach to measuring regulated splicing efficiency.

Quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR) and the ion-pair reverse-phase (IP-RP)-HPLC product purification and detection system were developed to facilitate the isolation and proportional quantification of alternatively spliced RUSH mRNAs. RUSH isoforms result from alternative splicing of a 57-bp exon and encode SNF/SWI-related proteins that bind to the uteroglobin promoter. QRT-PCR was performed using total RNA, and a pair of primers designed to flank the 57-bp exon. When more than one splice variant was expressed, IP-RP-HPLC identified the specific homoduplex products, as well as the heteroduplexes formed as a consequence of partial sequence complementarity between the products. Data analysis included the correct re-allocation of heteroduplex components to achieve accurate quantitation of changes in the relative levels of RUSH message isoforms. The preferential expression of the RUSH-1alpha isoform by all the tissues except estrous uterine endometrium and lactating mammary gland indicates RUSH pre-mRNAs are alternatively spliced in a tissue-specific manner. A 61-fold difference in the relative rate of RUSH pre-mRNA splicing is indicated by the difference in the ratios of RUSH mRNA isoforms from uterine endometrium and testis. Clearly, QRT-PCR and IP-RP-HPLC are powerful and versatile tools for the detection and quantitation of mRNA splice variants.

Molecular cloning and hormone-dependent expression of rabbit Muc1 in the cervix and uterus.

The microenvironment of the endocervix is regulated by secretory (> 10-15 x 10(6) Da) and cell-surface (< or = 1 x 10(6) Da) mucins. A survey with antisense and sense (control) oligonucleotides for eight epithelial mucin genes revealed that only MUC1, an anti-adhesive protein believed to modulate immune function and the only transmembrane mucin for which molecular probes exist, is expressed in the rabbit cervix. pMUC7, a 500-base pair cDNA probe to the tandem repeat domain of human MUC1, was used to clone the cDNA for the rabbit homologue. Although the GC-rich tandem repeat of rabbit Muc1 had only 40% sequence identity with its human counterpart, its transmembrane domain and cytoplasmic tail maintained 93% sequence identity with the same regions of human MUC1. Detection of restriction length polymorphisms confirmed the Lagomorpha Muc1 gene, like the human, is polymorphic. Temporal expression of Muc1 transcripts (2.4 kilobases) correlated closely with epithelial differentiation in the cervix. However, compared with the adult cervix, uterine endometrium expressed the highest levels of Muc1. In the endometrium, Muc1 was increased (p < 0.0004) by progesterone and returned to estrous levels by treatment with estrogen. Muc1 levels in the cervix were unaffected by ovarian steroids, confirming tissue-specific regulation of Muc1 in the lower reproductive tract.

Novel elements in the uteroglobin promoter are a functional target for prolactin signaling.

To quantify uteroglobin (UG) gene expression, varying lengths of 5' flanking sequence were subcloned into a luciferase reporter plasmid and introduced into HRE-H9 uterine epithelial cells. In response to estrogen, prolactin (PRL) and progesterone, transcriptional activity was maximal for the full-length construct, pUG3.1-LUC. Transcriptional activity was reduced (P < 0.05) by the removal of the progesterone receptor binding site, tested with pUG2.3-LUC, and eliminated (P < 0.05) by the removal of the remaining 5' flanking sequences, tested with pUG0.1-LUC. When the effects of PRL +/- progesterone were evaluated, progesterone alone increased (P < 0.05) the transcriptional activity of pUG3.1-LUC, and the deletion mutant, pUG3.1deltaRUSH-LUC. Transcription of pUG3.1-LUC was further increased (P < 0.05) by PRL + progesterone. However, deletion of the proximal promoter region -170/-85, tested with pUG3.1deltaRUSH-LUC, eliminated (P > 0.05) the PRL effect. These data support the speculation that RUSH proteins which bind to this region of the promoter play an important regulatory role in PRL signal transduction.

Cloning, characterization, and steroid-dependent posttranscriptional processing of RUSH-1 alpha and beta, two uteroglobin promoter-binding proteins.

We previously used gel shift assays, Southwestern blots, and UV cross-linking to identify four proteins that bind to the 203-bp 5'-flanking region (-194/ +9) of the rabbit uteroglobin gene. Here we report cloning, by recognition site screening, the cDNAs for two of the uteroglobin promoter-binding proteins (95 kDa and 113 kDa). Their presumptive nucleotide-binding motifs share 61% identity with the SWI2/SNF2 helicase superfamily, and each protein has the novel C3HC4 (RING) zinc-finger signature near its C terminus. RUSH-1 alpha, the 113-kDa protein, is the rabbit homolog of human HIP116, a protein that binds to the human immunodeficiency virus-1 promoter. RUSH-1 beta is a 95-kDa truncated version of RUSH-1 alpha that results from alternative splicing of a 57-bp exon as confirmed by genomic cloning. Northern analysis showed mRNA expression (5.2 kb) was induced by progesterone +/- PRL and antagonized by estrogen. However, because the two proteins result from alternative splicing of a 57-bp exon, the small difference in their mRNA sizes could not be detected by Northern analysis. Therefore, competitive RT-PCR and HPLC were used to quantify differences in the ratios of their mRNAs. Progesterone +/- PRL treatment increased (P < 0.005) the ratio of message for RUSH-1 alpha compared with RUSH-1 beta. Western analysis showed the RUSH-1 alpha protein is increased in response to progesterone +/- PRL and decreased in response to estrogen. The antiserum used for immunoblotting specifically supershifts uteroglobin promoter-protein complexes in gel shift experiments. Because RUSH-1 alpha and beta messages were detected in lung, liver, and HRE-H9 cells, these proteins may regulate genes in numerous cell types.

Prolactin augments progesterone-dependent uteroglobin gene expression by modulating promoter-binding proteins.

The sequence-specific binding of endometrial nuclear proteins to uteroglobin 200 (UG200) (-194/+9), the 203-base pair 5'-flanking region of the rabbit uteroglobin gene, and UG99 (-170/-85), a subfragment of UG200, was compared with gel shift assays. PRL + progesterone treatment of estrous rabbits produced a 6- to 7-fold increase in the primary shift compared to progesterone alone. PRL + progesterone treatment of long-term ovariectomized rabbits increased the primary shift 60% over progesterone alone, which increased the primary shift 30-fold over similarly treated estrous rabbits. The primary shift was eliminated when rabbits were treated with progesterone + estradiol benzoate (E2Bz). Changes in the steady state levels of UG mRNA paralleled changes in the intensity of the primary gel shift. Southwestern blotting revealed four proteins from progesterone-dominated endometrial nuclei that bind UG200. PRL pretreatment produced a 3- to 12-fold increase in each of the proteins. Protein binding was eliminated by E2Bz. A 100-kilodalton (kDa) protein from progesterone-dominated endometrial nuclei was UV cross-linked to UG200 and UG99. An additional protein was detected by each probe with long autoradiographic exposure. PRL pretreatment increased the 100-kDa protein, whereas covalent attachment of the 100-kDa protein to UG promoter DNA was eliminated by E2Bz. UV cross-linking in situ was used to identify the 100-kDa protein as responsible for the primary shift. Collectively, these experiments provide compelling evidence that PRL augments the progesterone-dependent transcription of the UG gene in the uterus by regulating at least one and as many as four proteins that bind to the UG promoter.