Expression and distribution of GnRH, LH, and FSH and their receptors in gastrointestinal tract of man and rat.

Gonadotropin-releasing hormone (GnRH), luteinizing hormone (LH), and follicle-stimulating hormone (FSH) regulate the reproductive axis. Their analogs have been found to influence gastrointestinal activity and enteric neuronal survival. The aims of the study were to investigate expression and cellular distribution of GnRH, LH, and FSH and their receptors in human and rat gastrointestinal tract

A second KRT71 allele in curly coated dogs.

Major characteristics of coat variation in dogs can be explained by variants in only a few genes. Until now, only one missense variant in the KRT71 gene, p.Arg151Trp, has been reported to cause curly hair in dogs. However, this variant does not explain the curly coat in all breeds as the mutant Trp allele, for example, is absent in Curly Coated Retrievers. We sequenced the genome of a Curly Coated Retriever at 22× coverage and searched for variants in the KRT71 gene. Only one protein-changing variant was present in a homozygous state in the Curly Coated Retriever and absent or present in a heterozygous state in 221 control dogs from different dog breeds. This variant, NM_001197029.1:c.1266_1273delinsACA, was an indel variant in exon 7 that caused a frameshift and an altered and probably extended C-terminus of the KRT71 protein NP_001183958.1:p.(Ser422ArgfsTer?). Using Sanger sequencing, we found that the variant was fixed in a cohort of 125 Curly Coated Retrievers and segregating in five of 14 additionally tested breeds with a curly or wavy coat. KRT71 variants cause curly hair in humans, mice, rats, cats and dogs. Specific KRT71 variants were further shown to cause alopecia. Based on this knowledge from other species and the predicted molecular consequence of the newly identified canine KRT71 variant, it is a compelling candidate causing a second curly hair allele in dogs. It might cause a slightly different coat phenotype than the previously published p.Arg151Trp variant and could potentially be associated with follicular dysplasia in dogs

A Mutation in the SUV39H2 Gene in Labrador Retrievers with Hereditary Nasal Parakeratosis (HNPK) Provides Insights into the Epigenetics of Keratinocyte Differentiation.

Peer reviewe

Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16

Severe gastrointestinal dysmotility developed after treatment with gonadotropin-releasing hormone analogs

Background. Sporadic cases of abdominal pain and dysmotility has been described after treatment with gonadotropin-releasing hormone (GnRH) analogs. The aim of the present study was to scrutinize for patients with severe gastrointestinal complaints after treatment with GnRH analogs, to describe the expression of antibodies against progonadoliberin-2, GnRH1, GnRH receptor (GnRHR), luteinizing hormone (LH), and LH receptor in serum in these patients, and to search for possible triggers and genetic factors behind the development of this dysmotility. Methods. Patients suffering from prolonged gastrointestinal complaints after treatment with GnRH analogs at the Department of Gastroenterology, Skane University Hospital, were included. GnRHR and LH receptor (LHCGR) genes were exome-sequenced. Serum was analyzed by enzyme-linked immune sorbent assays for the presence of antibodies. Healthy blood donors and women treated with GnRH analogs because of in vitro fertilization (IVF) were used as controls. Results. Seven patients with severe gastrointestinal complaints after GnRH treatment were identified, of whom six suffered from endometriosis. Several variants were found within the 11 exons of LHCGR. The minor allele G, at the single nucleotide polymorphism rs6755901, was detected in homozygosity in two patients (28.5%) who had developed chronic intestinal pseudo-obstruction and in 5.5% of the IVF controls. Three patients expressed IgM antibodies against progonadoliberin-2 and three against GnRH1 (42.9%) when cut off was set to a titer >97.5th percentile in blood donors. Conclusion. A high prevalence of endometriosis, polymorphism in the LHCGR and GnRH1 and progonadoliberin-2 antibodies in serum was found among the patients with severe dysmotility after treatment with GnRH analogs

Nucleotide substitutions in ASF/SF2 binding site III in the splicing enhancer at SA3358 induced late gene expression from the full-length,episomalform of the HPV-16 genome.

<p>(<b>A</b>). Schematic representation of the HPV-16 genomic plasmids pHPV16ANSL and pHPV16MANSL. The early and late viral promoters p97 and p670 are indicated. Numbers indicate nucleotide positions of 5′- (filled arrow heads) and 3′-splice sites (open arrow heads) or the early and late poly (A) sites pAE and pAL, respectively. L1M represents a previously described mutant HPV-16 L1 sequence in which a number of nucleotide substitutions inactivate splicing silencer elements downstream of late 3′-splice site SA5639. IRES, the poliovirus internal ribosome entry site sequence; sLuc, secreted luciferase gene; LCR, long control region. Sequences below plasmid maps represent wild type HPV-16 ASF/SF2 site III (capitals, black) and nucleotide substitutions (lower case, red) in the various plasmids indicated to the left. (<b>B, C</b>). Cell culture medium ofhuman primary keratinocytes collected at day 5 posttransfection with the various indicated plasmids was subjected to secreted-luciferase assay as described in Materials and Methods. Transfections were performed in the presence of pCAGGS-nlscre. Mean values and standard deviations of sLuc activity in the cell culture med of triplicate transfections are shown.</p

One of the predicted ASF/SF2 binding sites downstream of HPV-16 SA3358 accounts for the majority of the enhancer activity.

<p>(<b>A</b>). CAT protein levels produced in HeLa cells transfected with the indicated mutant pBELMCAT-derived plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Orru1" target="_blank">[38]</a>. CAT was monitored as described in Materials and Methods. Mean values and standard deviations are shown. Note the logarithmic scale. (<b>B</b>) RT-PCR with primers 757s and L1A on cDNA of cytoplasmic RNA extracted from HeLa cells transfected with the indicated plasmids. L1 and L1i mRNAs are indicated. M, molecular weight marker; GAPDH, cDNA amplified as internal control. (<b>C, D</b>) CAT protein levels produced in HeLa cells transfected with the indicated mutant pBELMCAT-derived plasmids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Orru1" target="_blank">[38]</a>. CAT was monitored as described in Materials and Methods. Mean values and standard deviations are shown.</p

Location of a splicing enhancer in the HPV E2/E4 coding region is conserved among HPVs.

<p>(<b>A</b>). Schematic representation of the HPV-16 genome, the subgenomic HPV-16 expression plasmids and the control plasmid pCMVCAT16 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Zhao1" target="_blank">[39]</a>. The early and late viral promoters p97 and p670 are indicated. Numbers indicate nucleotide positions of 5′- (filled circles) and 3′-splice sites (open circles) or the early and late poly (A) sites pAE and pAL, respectively, and the borders of deletions. L1M represents a previously described mutant HPV-16 L1 sequence in which a number of nucleotide substitutions that inactivate splicing silencers have been inserted downstream of SA5639. Boxes indicate position of various HPV sequences inserted between nucleotide positions 3407 and 3627 in HPV-16 plasmid pBELMCAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Orru1" target="_blank">[38]</a>. IRES, the poliovirus internal ribosome entry site sequence; CAT, CAT reporter gene; CMV, human cytomegalovirus immediate early promoter; LCR, long control region. mRNAs produced by the plasmids are indicated. The positions of the RT-PCR primers (arrows) are indicated. (<b>B</b>) CAT protein levels produced in HeLa cells transfected with the indicated plasmids. CAT was monitored as described in Materials and Methods. Mean values and standard deviations are shown. Note the logarithmic scale. (<b>C</b>) RT-PCR with primers 757s and L1A on cDNA of cytoplasmic RNA extracted from HeLa cells transfected with the indicated plasmids. L1 and L1i mRNAs are indicated. M, molecular weight marker; GAPDH, cDNA amplified as internal control. (<b>D, E, F</b>) CAT protein levels produced in HeLa cells transfected with the indicated HPV plasmids in the presence or absence of ASFDRS expression plasmid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone.0072776-Somberg2" target="_blank">[18]</a>. CAT was monitored as described in Materials and Methods. Mean values and standard deviations are shown. Note that results in D and E are from transfections of cells in 24-well plates, whereas results in B and C are from 60 mm plates. (<b>G</b>) Phylogenetic tree of exonic sequences located between the splice sites that correspond to HPV-16 SA3358 and SD3632 of various HPV types. MUT16 represents the mutated HPV-16 sequence shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072776#pone-0072776-g001" target="_blank">Figure 1B</a>.</p

Predicted ASF/SF2 sites that are mutated in the various HPV-16 reporter plasmids.

<p>Predicted ASF/SF2 sites that are mutated in the various HPV-16 reporter plasmids.</p