Practical usefulness of atypical endometrial cell categories within the new classification of endometrial cytology when applied to conventional smears.

OBJECTIVE: Endometrial cytology has been widely used as a screening tool in Japan. Traditionally, a three-tier reporting system, consisting of 'negative', 'suspicious' and 'positive' categories, has been used. However, a more descriptive system, the New Terminology in Endometrial Cytology (NTEMC), which is based on the Bethesda System for uterine cervical cytology, was introduced recently. The objective of this study was to validate the NTEMC criteria. METHODS: Endometrial cytology specimens that had been categorised as 'suspicious' were collected in our hospital between 2003 and 2013, and from these, 106 specimens with corresponding histological results, were re-evaluated according to the NTEMC criteria. Diagnostic categories were assigned based on that chosen by the majority of the examining members. RESULTS: Negative, atypical endometrial cells, of undetermined significance (ATEC-US), atypical endometrial cells for which atypical endometrial hyperplasia or worse cannot be excluded (ATEC-A), endometrial hyperplasia, atypical endometrial hyperplasia and malignancy were selected as the diagnostic categories for 9 (8.5%), 34 (32.1%), 17 (16%), 34 (32.1%), 5 (4.7%) and 7 (6.6%) specimens, respectively. Corresponding histological categories of benign, endometrial hyperplasia, atypical endometrial hyperplasia and malignancy were established in 28 (82.4%), 1 (2.9%), 2 (5.9%) and 3 (8.8%) ATEC-US specimens, respectively, and in 6 (35.3%), 3 (17.6%), 2 (11.8%) and 6 (35.3%) ATEC-A specimens, respectively. The histological category distribution differed significantly (P = 0.001), and there was a significant correlation between corresponding cytological and histological categories (P = 0.005). CONCLUSION: The ATEC category of NTEMC system works well in a practical setting and resembles the Bethesda reporting system ASC (atypical squamous cells) category for cervical cytology.

Bovine milk contains microRNA and messenger RNA that are stable under degradative conditions.

We previously reported that microRNA (miRNA) is present in human breast milk. Recently, other groups have reported that bovine milk also contains miRNA; however, these reports are few. We therefore investigated bovine milk miRNA using microarray and quantitative PCR analyses to identify the differences between colostrum and mature milk. The RNA concentration in a colostrum whey fraction was higher than that in a mature milk whey fraction. In total, 102 miRNA were detected in bovine milk by microarray analysis (100 in colostrum and 53 in mature milk; 51 were common to both). Among these miRNA, we selected several immune- and development-related miRNA, including miR-15b, miR-27b, miR-34a, miR-106b, miR-130a, miR-155, and miR-223. These miRNA were detected in bovine milk by quantitative PCR, and each of these miRNA was significantly more highly expressed in colostrum than in mature milk. We also confirmed the presence of some mRNA in bovine milk. Nevertheless, synthesized miRNA spiked in the raw milk whey were degraded, and naturally existing miRNA and mRNA in raw milk were resistant to acidic conditions and RNase treatment. The RNA molecules in milk were stable. We also detected miRNA and mRNA in infant formulas purchased from Japanese markets. It is still unknown whether milk-derived RNA molecules play biological roles in infants; however, if milk-derived RNA do show functions in infants, our data will help guide future studies.

Reactions of the Tridentate and Tetradentate Amine Ligands di-(2-picolyl)(N-ethyl)amine and 2,5-Bis-(2-pyridylmethyl)-2,5 diazohexane with Technetium Nitrosyl Complexes.

The reaction of the Tc(II) nitrosyl complex (Bu(4)N)[Tc(NO)Cl(4)] with Di-(2-picolyl)(NEt)amine in methanol yields the neutral complex [Tc(NO)Cl(py-N(Et)-py)]. The reaction of the Tc(I) nitrosyl complex [Tc(NO)Cl(2)(HOMe)(PPh(3))(2)] with this tridentate ligand yields cationic [Tc(NO)Cl(py-N(Et)-py)(PPh(3))]Cl. These two complexes have been structurally characterized. The reaction of [Tc(NO)Cl(2)(HOMe)(PPh(3))(2)] with the tetradentate ligand 1,4-Bis(2-pyridylmethyl)-1,4-diazobutane yields a mixture of products including cationic [Tc(NO)Cl(py-NH-NH-py)]Cl and cationic [Tc(NO)Cl(PPh(3))(py-NH-NH~py)]Cl, with a pyridyl terminus left dangling.

Extracellular fibrillar structure of latent TGF beta binding protein-1: role in TGF beta-dependent endothelial-mesenchymal transformation during endocardial cushion tissue formation in mouse embryonic heart.

Transforming growth factor-beta (TGF beta) is a dimeric peptide growth factor which regulates cellular differentiation and proliferation during development. Most cells secrete TGF beta as a large latent TGF beta complex containing mature TGF beta, latency associated peptide, and latent TGF beta-binding protein (LTBP)-1. The biological role of LTBP-1 in development remains unclear. Using a polyclonal antiserum specific for LTBP-1 (Ab39) and three-dimensional collagen gel culture assay of embryonic heart, we examined the tissue distribution of LTBP-1 and its functional role during the formation of endocardial cushion tissue in the mouse embryonic heart. Mature TGF beta protein was required at the onset of the endothelial-mesenchymal transformation to initiate endocardial cushion tissue formation. Double antibody staining showed that LTBP-1 colocalized with TGF beta 1 as an extracellular fibrillar structure surrounding the endocardial cushion mesenchymal cells. Immunogold electronmicroscopy showed that LTBP-1 localized to 40-100 nm extracellular fibrillar structure and 5-10-nm microfibrils. The anti-LTBP-1 antiserum (Ab39) inhibited the endothelial-mesenchymal transformation in atrio-ventricular endocardial cells cocultured with associated myocardium on a three-dimensional collagen gel lattice. This inhibitory effect was reversed by administration of mature TGF beta proteins in culture. These results suggest that LTBP-1 exists as an extracellular fibrillar structure and plays a role in the storage of TGF beta as a large latent TGF beta complex.

Effects of milk casein-derived peptides on absolute oxyhaemoglobin concentrations in the prefrontal area and on work efficiency after mental stress loading in male students.

This study examined the influence of milk casein-derived peptides on cerebral activity after mental stress loading. In a crossover study, 16 male students were given a drink containing peptides (peptide group), or water (control group) before stress loading. The oxyhaemoglobin (HbO(2)) concentration in the prefrontal area of the brain and work efficiency were measured as indicators of cerebral activity and differences in these parameters were examined according to type A or type B personality. Type A behaviour was defined as: aggression-hostility, hard-driving-time-urgency and speed-power, whereas type B behaviour did not have these characteristics. Peptide intake resulted in a significant increase in both HbO(2) concentration and work efficiency, whilst a similar increase was not seen in the control group. When divided into type A or type B personality, the changes in HbO(2) concentration for the control group differed significantly in the right prefrontal area. Moreover, in type A subjects the HbO(2) concentration in the right prefrontal area following intake was significantly different between the peptide and control groups.

MKK1 and MKK2, which encode Saccharomyces cerevisiae mitogen-activated protein kinase-kinase homologs, function in the pathway mediated by protein kinase C.

The PKC1 gene of Saccharomyces cerevisiae encodes a homolog of mammalian protein kinase C that is required for normal growth and division of yeast cells. We report here the isolation of the yeast MKK1 and MKK2 (for mitogen-activated protein [MAP] kinase-kinase) genes which, when overexpressed, suppress the cell lysis defect of a temperature-sensitive pkc1 mutant. The MKK genes encode protein kinases most similar to the STE7 product of S. cerevisiae, the byr1 product of Schizosaccharomyces pombe, and vertebrate MAP kinase-kinases. Deletion of either MKK gene alone did not cause any apparent phenotypic defects, but deletion of both MKK1 and MKK2 resulted in a temperature-sensitive cell lysis defect that was suppressed by osmotic stabilizers. This phenotypic defect is similar to that associated with deletion of the BCK1 gene, which is thought to function in the pathway mediated by PCK1. The BCK1 gene also encodes a predicted protein kinase. Overexpression of MKK1 suppressed the growth defect caused by deletion of BCK1, whereas an activated allele of BCK1 (BCK1-20) did not suppress the defect of the mkk1 mkk2 double disruption. Furthermore, overexpression of MPK1, which encodes a protein kinase closely related to vertebrate MAP kinases, suppressed the defect of the mkk1 mkk2 double mutant. These results suggest that MKK1 and MKK2 function in a signal transduction pathway involving the protein kinases encoded by PKC1, BCK1, and MPK1. Genetic epistasis experiments indicated that the site of action for MKK1 and MKK2 is between BCK1 and MPK1.

Bovine alpha-lactalbumin stimulates mucus metabolism in gastric mucosa.

Bovine alpha-lactalbumin (alpha-LA), a major milk protein, exerts strong gastroprotective activity against rat experimental gastric ulcers induced by ethanol or stress. To elucidate the mechanisms underlying this activity, the influence of alpha-LA on gastric mucus metabolism was investigated in vitro and in vivo. For the in vitro study, RGM1 cells (a rat gastric epithelial cell line) were selected for observation of the direct activity of alpha-LA on gastric mucosal cells and cultured in the presence of either alpha-LA or ovalbumin (OVA), a reference protein showing no gastroprotective activity. Amounts of synthesized and secreted mucin, a major component of mucus, were determined using [3H]glucosamine as a tracer, and prostaglandin E2 (PGE2) levels in the culture medium were determined by RIA. For the in vivo study, the thickness of the mucus gel layer, a protective barrier for gastric mucosa, was evaluated histochemically in rat gastric mucosa. alpha-Lactalbumin (3 mg/mL) significantly stimulated mucin synthesis and secretion in RGM1 cells and also increased PGE2 levels in the culture medium. In contrast, OVA showed no enhancing effects under identical conditions. Neither indomethacin, a cyclo-oxygenase inhibitor, nor AH23848, a prostaglandin EP4 receptor antagonist, affected alpha-LA-induced enhancement of mucin synthesis and secretion. In vivo, oral administration of alpha-LA (300 mg/kg x 3 times/d x 7 d) increased the thickness of the mucus gel layer in rats. These results indicate that alpha-LA fortifies the mucus gel layer by stimulating mucin production and secretion in gastric mucus-producing cells, and that this enhancing effect is independent of endogenous PGE2. Comparison of the efficacy of alpha-LA with OVA suggests that the activities observed in RGM1 cells are closely related to the gastroprotective effects in rat gastric ulcer models. In conclusion, alpha-LA stimulates mucus metabolism, and this action may be responsible for its gastroprotective activity.

Interplay of signal mediators of decapentaplegic (Dpp): molecular characterization of mothers against dpp, Medea, and daughters against dpp.

Decapentaplegic (Dpp) plays an essential role in Drosophila development, and analyses of the Dpp signaling pathway have contributed greatly to understanding of the actions of the TGF-beta superfamily. Intracellular signaling of the TGF-beta superfamily is mediated by Smad proteins, which are now grouped into three classes. Two Smads have been identified in Drosophila. Mothers against dpp (Mad) is a pathway-specific Smad, whereas Daughters against dpp (Dad) is an inhibitory Smad genetically shown to antagonize Dpp signaling. Here we report the identification of a common mediator Smad in Drosophila, which is closely related to human Smad4. Mad forms a heteromeric complex with Drosophila Smad4 (Medea) upon phosphorylation by Thick veins (Tkv), a type I receptor for Dpp. Dad stably associates with Tkv and thereby inhibits Tkv-induced Mad phosphorylation. Dad also blocks hetero-oligomerization and nuclear translocation of Mad. We also show that Mad exists as a monomer in the absence of Tkv stimulation. Tkv induces homo-oligomerization of Mad, and Dad inhibits this step. Finally, we propose a model for Dpp signaling by Drosophila Smad proteins.

Expression of neurofilaments in a neoplastic human salivary intercalated duct cell line or its derivatives and effect of nerve growth factor on the cellular proliferation and phenotype.

A neoplastic salivary cell line with an ultrastructure similar to that of an intercalated duct cell of the salivary gland, established from a human submandibular salivary gland, has been used in our laboratory as a model for studying mechanisms regulating cytodifferentiation in salivary glands. The expression of neurofilaments (Mr 200,000, 160,000, and 68,000) in the neoplastic human salivary intercalated duct cell line and its derivatives was found by the immunofluorescence staining technique, immunoblotting, or immunoelectron microscopy. In addition, these cells stained with Bodian impregnation and expressed specific antigens such as tubulin alpha and beta chain, HNK-1 antigen, and laminin. When these cells were cultured in the presence of nerve growth factor, only the cells with a myoepithelial cell phenotype formed the long cytoplasmic processes which were densely packed with ample microfibrils in addition to microtubule bundles, and they exhibited marked suppression of anchorage-independent and anchorage-dependent growths. These findings indicate that the characteristics of neoplastic human salivary intercalated duct cell line and its derivatives are similar to those of neuronal cells.

FTZ-F1alpha is expressed in the developing gonad of frogs.

Fushi tarazu transcription factor-1 (FTZ-F1), a member of the nuclear hormone receptor superfamily, is a regulator for fushi tarazu gene expression in Drosophila. Its expression pattern during organogenesis in vertebrates, however, is not known yet. In this study, we cloned a frog FTZ-F1 homologue (rrFTZ-F1alpha) and analyzed its expression and localization during gonadal development of the frog Rana rugosa. Cloned rrFTZ-F1alpha cDNA encoded a protein of 501 amino acids including the regions I-III and FTZ-F1 box that are evolutionally conserved in the FTZ-F1 superfamily. rrFTZ-F1alpha shared high similarity at the amino acid level with mouse LRH-1 (76%), human FTF (92%), chicken OR2.0 (92%), Xenopus laevis FF1rA (94%) and zebrafish FF1A (82%). Northern blot analysis showed that the rrFTZ-F1alpha mRNA at a size of 7.4 kb was the most prominent in the testis among various tissues of adult frogs examined. The RT-PCR analysis revealed that the expression of rrFTZ-F1alpha was weak in the gonad of tadpoles before stage XVI, but it became stronger in the testis of froglets at stage XXV and much higher in the testis of frogs 2 months after metamorphosis. In addition, in situ hybridization analysis revealed that the rrFTZ-F1alpha gene was transcribed in germ cells except for sperm in the testis, and in oocytes at stage A in the ovary of frogs 2 months after metamorphosis. Together, these results suggest that FTZ-F1alpha probably plays an important role in differentiation of germ cells in the gonad of frogs in both sexes.

Lactoferrin inhibits cholesterol accumulation in macrophages mediated by acetylated or oxidized low-density lipoproteins.

When macrophages are incubated with acetylated or oxidized low-density lipoproteins (Ac- or OxLDL), cellular cholesteryl esters (CE) increase significantly. In the present study, we investigated the effect of whey protein on Ac- or OxLDL mediated accumulation of CE in macrophages and found that lactoferrin (Lf), a minor protein component of whey, inhibits the accumulation of CE dose-dependently. In the presence of bovine Lf (1 mg/ml), CE accumulation in macrophages incubated with AcLDL (100 micrograms of protein/ml) decreased by more than 80%. Human Lf was less potent than bovine Lf, and bovine transferrin had no effect. Binding of 125I-AcLDL to macrophages was also inhibited by Lf. Agarose gel electrophoresis revealed that Lf binds to Ac- or OxLDLs and neutralizes their negative charges. These results indicate that Lf inhibits the binding of modified LDLs to macrophages by direct interaction with modified LDLs, resulting in their loss of function as ligands of the scavenger receptor. Modification of the arginine residues of Lf with 1,2-cyclohexanedione abolished its ability to bind to AcLDL, suggesting that a region rich in basic amino acid residues near the N-terminus of Lf, which resembles the ligand-binding site of the scavenger receptor, may be responsible for this binding ability. As a result, the inhibitory effect of Lf on CE accumulation in macrophages was significantly weakened by this modification. Our results suggest the possibility that Lf in the blood stream may act as an anti-atherogenic agent in vivo.

Identification of the bactericidal domain of lactoferrin.

We report the existence of a previously unknown antimicrobial domain near the N-terminus of lactoferrin in a region distinct from its iron-binding sites. A single active peptide representing this domain was isolated following gastric pepsin cleavage of human lactoferrin, and bovine lactoferrin, and sequenced by automated Edman degradation. The antimicrobial sequence was found to consist mainly of a loop of 18 amino acid residues formed by a disulfide bond between cysteine residues 20 and 37 of human lactoferrin, or 19 and 36 of bovine lactoferrin. Synthetic analogs of this region similarly exhibited potent antibacterial properties. The active peptide of bovine lactoferrin was more potent than that of human lactoferrin having effectiveness against various Gram-negative and Gram-positive bacteria at concentrations between 0.3 microM and 3.0 microM, depending on the target strain. The effect of the isolated domain was lethal causing a rapid loss of colony-forming capability. Our studies suggest this domain is the structural region responsible for the bacterial properties of lactoferrin.

Pregnenolone, pregnenolone sulfate, and cytochrome P450 side-chain cleavage enzyme in the amphibian brain and their seasonal changes.

To clarify whether the amphibian brain synthesizes de novo neurosteroids, we examined pregnenolone, pregnenolone sulfate ester, and cytochrome P450 side-chain cleavage enzyme (cytochrome P450scc), an enzyme converting cholesterol to pregnenolone, using amphibians. Pregnenolone and its sulfate ester in the brain, gonad, and plasma of Xenopus laevis were measured by a specific pregnenolone RIA. The concentrations of these two steroids in the female brain were significantly larger than those in the ovary and plasma. A similar tendency was evident in the male. In both sexes, pregnenolone and its sulfate ester were concentrated more highly in the cerebellum than in the telencephalon, diencephalon, or midbrain. An immunoreactive protein band of electrophoretic mobility in the proximity of bovine adrenal P450scc was detected in the Xenopus brain as well as the testis by Western blot analysis. Immunohistochemical analysis indicated that Purkinje cells in the Xenopus cerebellum were specifically immunostained with the P450scc antibody. P450scc-like immunoreactive cells were further found in several telencephalic and diencephalic regions, such as the pallium mediale and nucleus preopticus, in the Xenopus brain. A similar localization of P450scc-like immunoreactive cells was evident in Rana nigromaculata, a seasonally breeding amphibian. In the present study, seasonal changes in pregnenolone and its sulfate ester were further examined as a possible physiological change using R. nigromaculata. In both sexes, pregnenolone concentrations in the brain were almost constant during the seasonally breeding cycle. In contrast, the pregnenolone sulfate concentration in the brain was significantly lower in the hibernating quiescent phase and higher in the breeding and postbreeding active phases, independent of the plasma steroid level. These results taken together suggest that the amphibian brain possesses steroidogenic enzyme P450scc and produces pregnenolone and its sulfate ester. Pregnenolone sulfate may function well during the breeding and postbreeding active phases of the year in the seasonal breeder.

Expression of Dax-1 during gonadal development of the frog.

Dax-1, a member of the nuclear hormone receptor superfamily of transcription factors, is known to be involved in gonadal development in mammals. To date, Dax-1 has only been isolated in reptiles, birds and mammals. The expression of Dax-1 is down-regulated in the developing testis, but persists in the ovary of mice (Swain et al., Nat. Genet. 12 (1996) 404) and chicken (Smith et al., J. Mol. Endocrinol. 24 (2000) 23). Curiously, there is no sex difference in the expression patterns of Dax-1 in the American alligator (Western et al., Gene 241 (2000) 223). To understand its role(s) in gonadal development in vertebrates, molecular cloning of Dax-1 in amphibians is required. In this study, we cloned an amphibian Dax-1 homologue of the frog Rana rugosa and examined its expression profile during gonadal development. Cloned Dax-1 cDNA encoded a protein of 287 amino acids. Unlike mammalians that possess the three and one half repeat elements representing the putative DNA binding domain in the predicted sequence of Dax-1 protein, the frog had a single poorly conserved copy of the repeat unit. By RT-PCR analysis, the Dax-1 mRNA was detected in the liver and pancreas, but not in the testis and ovary of adult frogs. However, Dax-1 expression was seen first in the embryo at stage 12 and became stronger in tadpoles until stage X. The Dax-1 was transcribed in the testis stronger than in the ovary of frogs at stage XXV (just after completion of metamorphosis). In the gonad of frogs 2 months after metamorphosis (at this stage postmeiotic cells can be seen in the seminiferous tubules), the Dax-1 was expressed only in males. In addition, the Dax-1 transcription declined gradually as ovarian development proceeded, but its expression was down-regulated and then up-regulated rapidly when female-to-male sex reversal was caused by administration of testosterone into female tadpoles. Taken together, the results suggest that the Dax-1 may be closely involved in testicular development of amphibians.

Two isoforms of FTZ-F1 messenger RNA: molecular cloning and their expression in the frog testis.

FTZ-F1, a member of the orphan nuclear receptors, is a transcriptional factor regulating the expression of the fushi tarazu gene in Drosophila (Lavorgna et al., 1991. Science 252, 848-851). Previously, we cloned a frog homologue of FTZ-F1 (rrFTZ-F1alpha; GenBank Accession No. AB035498). In this study, we isolated the rrFTZ-F1beta cDNA encoding a protein of 469 amino acids. Then, expressions of two types (alpha and beta) of rrFTZ-F1 mRNAs were examined during development of embryos and gonads in the frog Rana rugosa. They were expressed in the embryo at stage 12. Expressions of both the alpha and beta mRNAs became stronger in the testis of frogs at stage XXV and were most prominent in that of frogs 2months after metamorphosis. In the former testis, spermatogonia were the only germ cells in the seminiferous tubules, whilst postmeiotic cells were observed in the latter testis. Expression of the typealpha mRNA was more prominent. In addition, we cloned the regions with either exon I or II of the rrFTZ-F1 gene. Genomic structure analysis revealed that rrFTZ-F1beta is a partial exon I-truncated variant of rrFTZ-F1alpha. The results suggest that rrFTZ-F1alpha and -beta are expressed from the same gene by alternative splicing and that they may play an important role(s) in differentiation of premeiotic germ cells in the testis of the frog R. rugosa.

Cloning and sequencing of the cDNA encoding tyrosinase of the Japanese pond frog, Rana nigromaculata.

We cloned and sequenced the cDNA encoding tyrosinase (TYN) of the Japanese pond frog, Rana nigromaculata. The 3511-bp cDNA contained a 54-bp 5'-noncoding region, a 1596-bp open reading frame encoding TYN of 532 amino acids (aa), and a 1861-bp 3'-noncoding region. The aa sequence of frog TYN predicted from the cDNA sequence was homologous to that of mouse and human TYNs. The aa sequence including the copper-binding domain, which is likely the active center of TYN, was highly conserved among these three species and Neurospora crassa, Streptomyces antibioticus, and S. glaucescens. The frog TYN also contains possible glycosylation sites and conserved Cys at sites similar to those in the mouse and human TYNs. There are two hydrophobic regions at the N-terminus and near the C-terminus, which are likely the signal (leader) peptide and a transmembrane domain, respectively.

Molecular cloning and expression of the SF-1/Ad4BP gene in the frog, Rana rugosa.

SF-1/Ad4BP is a transcriptional factor that was originally found to be a mammalian homologue of the Drosophila Ftz-F1 (fushi tarazu factor 1) (), and transcribed from a gene designated the Ftz-F1 gene (). Ftz-F1 gene-deficient mice lack adrenal glands and gonads. Besides mammals, however, the SF-1/Ad4BP cDNA has only been isolated to date in fish and birds. To understand its role(s) for adrenal and gonadal development in vertebrates, cloning of this gene in animals other than mammals is required. In this study, we succeeded to isolate frog (Rana rugosa) SF-1/Ad4BP cDNA from a testis lambdagt10 cDNA library. It encoded a protein of 468 amino acids, and its open reading frame (ORF) shared 70% similarity with that of chicken OR2.1 (a SF-1/Ad4BP homologue) and 62% with bovine SF-1/Ad4BP. SF-1/Ad4BP mRNA was expressed in the testes, brains, adrenals/kidneys and spleens, but not ovaries, of adult frogs. In addition, we also cloned the 5'-untranslated region (4.6kb) of the SF-1/Ad4BP gene with exons I and II. Genomic structure analysis revealed that frog SF-1/Ad4BP was also transcribed from the same gene as that of mammals. However, many Ftz-F1-related proteins have been reported so far. The Ftz-F1 gene does not encode all of those Ftz-F1-related proteins. Thus, the name of Ftz-F1 is not adequate for the gene coding SF-1/Ad4BP. Here, we propose the use of SF-1/Ad4BP instead of Ftz-F1 for the gene that encodes SF-1/Ad4BP in vertebrates.

Two Sox9 messenger RNA isoforms: isolation of cDNAs and their expression during gonadal development in the frog Rana rugosa.

Sox is a family of SRY-related testis-determining genes. We have isolated two different mRNA isoforms of the frog Sox9 gene from adult frog testis cDNAs. One form (Sox9 alpha) encodes a 482 amino acid protein containing the HMG box, whereas the other form (Sox9 beta), which completely lacks the HMG box, is a truncated 265 amino acid protein of Sox9 alpha. Sox9 alpha is 82% similar to mouse, 86% to chicken, and 77% to trout Sox9 at the amino acid level. Sox9 expression was upregulated in embryos after stage 16, and was seen in both developing testes and ovaries. The size of Sox9 transcripts was determined to be 7.8 knt by Northern blot analysis. In addition, Sox9 alpha expression was found prominently in the testis and brain among various tissues of adult frogs examined, and was considerably higher than Sox9 beta. The fact that Sox9 is expressed in both sexes suggests that this gene is involved in gonadal development of male and female frogs. This is dissimilar to the pattern in birds and mammals, in which Sox9 expression is male-specific.

Lactoferricin derived from milk protein lactoferrin.

Lactoferricin (LFcin) was initially identified as an antimicrobial peptide derived by pepsin digestion of lactoferrin (LF), a multifunctional innate-defense protein in milk. Various synthetic analogs of LFcin have also been reported. LFcin inhibits a diverse range of microorganisms such as gram-negative bacteria, gram-positive bacteria, yeast, filamentous fungi, and parasitic protozoa, including some antibiotic-resistant pathogens. LFcin kills target organisms by membrane perturbation and acts synergistically with some antimicrobial agents. LFcin exhibits numerous biological activities in common with those of LF. Whereas LFcin suppresses the activation of innate immunity by microbial components such as lipopolysaccharide (LPS) and CpG DNA, the peptide itself activates immunity. Administration of LFcin analogs has been shown to protect the host via direct antimicrobial activity and immunostimulatory effects in several infection models of mice. Here we present a comprehensive review of investigations of LFcin and related peptides.

Expression of Dmrt1 protein in developing and in sex-reversed gonads of amphibians.

Many genes are known to be involved in gonadal differentiation in vertebrates. Dmrt1, a gene that encodes a transcription factor with a DM-domain, is considered to be one of the essential genes controlling testicular differentiation in mammals, birds, reptiles, amphibians and fish. However, it still remains unknown which testicular cells of animals other than mice and chicks express Dmrt1 protein. For an explanation of its role(s) in testicular differentiation in vertebrates, the expression of the Dmrt1 protein needs to be studied. For this purpose, we conducted an immunohistochemical study of this protein in an amphibian by using an antibody specific for Dmrt1. No positive signal was found in the indifferent gonad of tadpoles of Rana rugosa at early stages. However, in the testis of tadpoles at later stages (XV-XXV) and in frogs one month after metamorphosis, this protein was expressed in interstitial cells and Sertoli cells. In the testis of adult frogs, germ cells also expressed Dmrt1 protein. RT-PCR analysis revealed that the gene for this protein was not transcribed at any time during ovarian development, but was expressed in the female to male sex-reversed gonad. This was true when immunohistological studies were performed. In addition, Southern blot analysis showed DMRT1 to be an autosomal gene. Taken together, our findings indicate that Dmrt1 protein is expressed by interstitial cells, Seroli cells and germ cells in the testis of R. rugosa. Dmrt1 may thus be very involved in the testicular differentiation of amphibians.