Tracheostomy wound myiasis in a patient with hypoxic encephalopathy.

•Tracheostomy wound myiasis is rarely observed in unconscious and immobile patients.•Maggots in the vicinity of the tracheostomy site should be closely monitored.•Controlling myiasis in hospitals requires fly control and patient fluid management.

Molecular cloning of LIM homeodomain transcription factor Lhx2 as a transcription factor of porcine follicle-stimulating hormone beta subunit (FSHß) gene.

We cloned the LIM-homeodomain protein LHX2 as a transcription factor for the porcine follicle-stimulating hormone ß subunit gene (Fshß) by the Yeast One-Hybrid Cloning System using the upstream region of -852/-746 bases (b) from the transcription start site, called Fd2, as a bait sequence. The reporter assay in LßT2 and CHO cells revealed the presence of an LHX2-responsive region other than Fd2. A potential LHX2 binding sequence was confirmed as AATTAAT containing a consensus homeodomain binding core sequence AATT by Systematic Evolution of Ligands by Exponential Enrichment analysis. DNase I footprinting demonstrated three AATTAAT sequences located at regions -835/-829, -818/-812 and -806/-800 b in the Fd2 region and 12 binding sites in the distal and proximal regions mostly containing an AATT-core sequence. RT-PCR analysis of Lhx2 expression during porcine fetal and postnatal pituitary development showed a gradual increase from fetal day (f) 40 to postnatal day (p) 8 followed by a slight decrease to p230, suggesting that LHX2 may play its role largely in the late fetal and postnatal periods. The analyses of Lhx2 expression in pituitary tumor-derived cell lines showed their expressions in cell lines including αT31, LßT2 and others. Since LHX2 was previously identified as a transcription factor for Cga and the in vitro experiments in the present study suggested that LHX2 regulated the expression of Fshß, it is possible that LHX2 controls the synthesis of FSH at the transcription level.

PROP1 coexists with SOX2 and induces PIT1-commitment cells.

Prophet of PIT1 (PROP1) is a pituitary-specific factor and responsive gene for the combined pituitary hormone deficiency in Ames dwarf mice and human patients. Our immunohistochemical studies demonstrated that PROP1 is consistently expressed in SOX2-expressing stem/progenitor cells in the rat pituitary from embryonic (E) to postnatal periods. At E13.5, all the cells in Rathke's pouch, the primordium of the pituitary, express PROP1. Afterward, PROP1-positive cells localize along the marginal cell layer, a putative stem cell niche in the pituitary, and stratify in the parenchyma of the anterior pituitary. In the embryonic period, PROP1 coexists transiently with PIT1, which is the anterior pituitary-specific factor and is a target of PROP1, but not any hormones. Thus, the present results imply a regulatory role of PROP1 not only in pituitary organogenesis but also in conversion of PIT1-lineage cells.

HSV type 1 thymidine kinase protein accumulation in round spermatids induces male infertility by spermatogenesis disruption and apoptotic loss of germ cells.

HSV type 1 thymidine kinase (HSV1-TK)-introduced transgenic rodents and HSV-infected humans were reported to suffer male infertility. The present study aimed to find novel clues to clarify the cause of HSV1-TK-induced male infertility using an HSV1-tk transgenic rat line. Two truncated HSV1-TK proteins, 37 and 39kDa, were produced and accumulated in the round spermatids, and their transcription initiation site was identified for the first time at the 65 base downstream of the translation start point of the full-length 43kDa HSV1-TK. Spermatozoa from those young transgenic rats showed malformed heads, looped tails, and missing cell membrane in heads and tails. Furthermore, age-dependent germ cell loss was observed. TUNEL assay suggested that this germ cell loss is caused by increased apoptotic germ cell death. These results suggest that the expression of HSV1-TK in testes brings about not only abnormal spermiogenesis but also a loss of germ cells due to apoptosis. These findings could provide a novel clue to elucidate the molecular mechanism underlying male infertility in transgenic animals and HSV-infected patients.

Pituitary homeodomain transcription factors HESX1 and PROP1 form a heterodimer on the inverted TAAT motif.

The development and differentiation of the pituitary gland progress through spatial and temporal expressions of many transcription factors. Transcription factor HESX1, which begins to be expressed in the Rathke's pouch at the early stage of pituitary development, acts as a transcription repressor. Another transcription factor, PROP1, which is a pituitary-specific factor and important for the determination of the differentiation of pituitary hormone-producing cells, appears later than HESX1 and is assumed to block the action of HESX1. Both factors are members of the homeodomain family, and the amino acid residue at the 50th position of the homeodomain is glutamine (Gln-50). We recently observed that both factors share the same target sequence through different binding profiles. Hence, using random oligonucleotides and an electrophoretic mobility-shift assay, we have examined the DNA-binding preference of HESX1 by a determination of its binding sequence. HESX1 binds as a monomer to a TAATT motif but not to a TAAT motif. In the presence of PROP1, HESX1 develops to bind to an inverted TAAT motif by forming a heterodimer. Thus, the formation of a heterodimer between HESX1 and PROP1 provides a condition in which, in the early pituitary primordium, HESX1 alters its repressive role to an active one by forming a heterodimer with newly appearing PROP1 so that PROP1 finally replaces HESX1 to advance to the middle stage of pituitary development.

Regulation of porcine pituitary glycoprotein hormone alpha subunit gene with LIM-homeobox transcription factor Lhx3.

The aim of this study was to characterize the promoter activity of the porcine pituitary glycoprotein hormone common alpha gene (Cga) promoter (-1059/+12) and the role of LIM homeodomain transcription factor Lhx3. A transfection assay using Chinese hamster ovary (CHO) cells showed that the -1059/-101 region of the Cga promoter definitely responds to Lhx3 and that the -1059/-240 region exhibits a high basal transcriptional level in a pituitary-derived cell line, LbetaT2. A DNA binding and DNase I footprinting assay demonstrated that Lhx3 has seven binding sites in the -1059/+12 region of Cga, including a pituitary glycoprotein hormone basal element (PGBE) known as a LIM homeodomain factor-binding site. A transfection assay of the sequence of Lhx3-binding sites fused with minimal promoter vector confirmed their Lhx3-dependent stimulations in LbetaT2 cells. RT-PCR analysis of porcine pituitary ontogeny demonstrated that porcine Lhx3 showed striking changes of expression in both sexes during the fetal period but a stable high level of expression after birth. Thus, the porcine Cga promoter is regulated by Lhx3 through seven sites in the distal and proximal regions.

Dimeric PROP1 binding to diverse palindromic TAAT sequences promotes its transcriptional activity.

Mutations in the Prop1 gene are responsible for murine Ames dwarfism and human combined pituitary hormone deficiency with hypogonadism. Recently, we reported that PROP1 is a possible transcription factor for gonadotropin subunit genes through plural cis-acting sites composed of AT-rich sequences containing a TAAT motif which differs from its consensus binding sequence known as PRDQ9 (TAATTGAATTA). This study aimed to verify the binding specificity and sequence of PROP1 by applying the method of SELEX (Systematic Evolution of Ligands by EXponential enrichment), EMSA (electrophoretic mobility shift assay) and transient transfection assay. SELEX, after 5, 7 and 9 generations of selection using a random sequence library, showed that nucleotides containing one or two TAAT motifs were accumulated and accounted for 98.5% at the 9th generation. Aligned sequences and EMSA demonstrated that PROP1 binds preferentially to 11 nucleotides composed of an inverted TAAT motif separated by 3 nucleotides with variation in the half site of palindromic TAAT motifs and with preferential requirement of T at the nucleotide number 5 immediately 3' to a TAAT motif. Transient transfection assay demonstrated first that dimeric binding of PROP1 to an inverted TAAT motif and its cognates resulted in transcriptional activation, whereas monomeric binding of PROP1 to a single TAAT motif and an inverted ATTA motif did not mediate activation. Thus, this study demonstrated that dimeric binding of PROP1 is able to recognize diverse palindromic TAAT sequences separated by 3 nucleotides and to exhibit its transcriptional activity.

Molecular cloning of paired related homeobox 2 (prx2) as a novel pituitary transcription factor.

This study aimed to identify protein(s) that bind(s) to the highly AT-rich sequence of porcine Fshb promoter region -852/-746 (named Fd2) by the Yeast One-Hybrid Cloning System and finally a paired related homeodomain transcription factor, Prx2, known as a key factor for skeletogenesis was cloned. RT-PCR analysis of fetal and postnatal porcine pituitaries demonstrated that Prx2 starts to be expressed at around fetal days 40-50 just before the beginning of Lhb-expression and that the level of Prx2 increases after birth. Immunohistochemical analysis of the prepubertal porcine pituitary revealed that some Prx2-positive cells overlap some Lh beta-positive cells. Transient transfection assay using non-pituitary CHO cells and pituitary tumor-derived LbetaT2 cells revealed that Prx2 plays a cell-type dependent role in modulation of the Fshb promoter, showing stimulation in CHO cells and repression in LbetaT2 cells via the regions of Fd2 and -596/-239. The binding ability of Prx2 to the regions of Fd2 and -596/-239 was confirmed by electrophoretic mobility shift assay. DNase I footprinting revealed that broad regions of Fd2 were bound by Prx2 and that -596/-239 contained seven Prx2-binding sites. The SELEX method using a random N15-mer oligonucleotide pool demonstrated that Prx2 monomer binds to a TAATT motif, which is present in Fd2 and -596/-239. However, the binding of Prx2 to TAATT with a single molecule and its inverted repeat with two molecules could not induce transcriptional activation, indicating that the Prx2-dependent transcriptional modulation demonstrated in cultured cells is not introduced by Prx2 alone. Thus, this study demonstrated for the first time that Prx2 is expressed in the pituitary gland and at least in a part of gonadotropes in which Prx2 may play a role in repression of the Fshb gene.

Homeodomain transcription factor Hesx1/Rpx occupies Prop-1 activation sites in porcine follicle stimulating hormone (FSH) beta subunit promoter.

Homeodomain repressor factor Hesx1/Rpx plays a crucial role in the formation of Rathke's pouch at the start of pituitary organogenesis and represses the Prop-1-dependent expression of Pit-1 gene, which promotes the differentiation of Pit-1-dependent hormone producing cells. Recently, we discovered a novel function of Prop-1 by which it activates the porcine follicle stimulating hormone beta subunit (FSHbeta) gene through Fd2 region (-852/-746). The present study aimed to determine whether Hesx1 exerts its role in the Prop-1-dependent activation of FSHbeta gene. Transient transfection assay for the porcine FSHbeta promoter -985/+10, electrophoretic mobility shift assay (EMSA) and DNase I footprinting analysis for Fd2 region were carried out. Transfection assay in GH3 cells demonstrated that expression of Hesx1 alone does not change the promoter activity but the coexpression with Prop-1 represses the Prop-1-dependent activation of FSHbeta promoter. Similar results were obtained for the mutant reporter vector deleting the region -745/-104 indicating that Fd2 region is a target site of Hesx1 as well as Prop-1. EMSA and DNase I footprinting analysis using recombinant Hesx1 and Prop-1 protein demonstrated that Hesx1 and Prop-1 certainly bind to the AT-rich regions in a different manner. These results suggest that Hesx1 blocks the advanced expression of FSHbeta gene in the early stage of pituitary development, and Prop-1 thereafter appears and activates this gene.

Expression of porcine FSHbeta subunit promoter-driven herpes simplex virus thymidine kinase gene in transgenic rats.

A transgenic rat was established using the construct of porcine FSHbeta subunit promoter, the -852/+10 bp region, fused to a Herpes simplex virus thymidine kinase (HSV-TK) gene. Integration of the transgene was confirmed by PCR of tail DNA. RT-PCR of total RNAs of the pituitary, gonad, cerebellum, liver, kidney, adrenal gland, prostate, and uterus revealed that FSHbeta was only expressed in the pituitary. Analysis of the expression of reporter gene, HSV-TK, using two specific primer sets revealed that different transcripts were present in the pituitary and testis. The transcript initiated at the transcription initiation site of the porcine FSHbeta gene was detected in the pituitary, and another within the TK gene was found in the testis, indicating ectopic testis-specific expression. Immunohistochemistry of the pituitary glands of the transgenic rats for FSH and HSV-TK demonstrated that the FSH-producing cells also produced HSV-TK. The results indicated that the -852/+10 bp region of the FSHbeta promoter contains an element(s) that determines the tissue-specific expression. We succeeded in producing FSHbeta promoter-driven HSV-TK transgenic rats and were the first time to do so using an animal other than the mouse. The transgenic rats show male infertility that involves abnormal spermatogenesis. We also observed a decrease in the weight of the testis and epididymis, and both motile and living spermatozoa were absent in the epididymis. Consequently, the FSHbeta-HSV-TK transgenic rat will provide a useful model for studies on FSH function and male infertility.