Reducing the number of accepted species in Aspergillus series Nigri.

The Aspergillus series Nigri contains biotechnologically and medically important species. They can produce hazardous mycotoxins, which is relevant due to the frequent occurrence of these species on foodstuffs and in the indoor environment. The taxonomy of the series has undergone numerous rearrangements, and currently, there are 14 species accepted in the series, most of which are considered cryptic. Species-level identifications are, however, problematic or impossible for many isolates even when using DNA sequencing or MALDI-TOF mass spectrometry, indicating a possible problem in the definition of species limits or the presence of undescribed species diversity. To re-examine the species boundaries, we collected DNA sequences from three phylogenetic markers (benA, CaM and RPB2) for 276 strains from series Nigri and generated 18 new whole-genome sequences. With the three-gene dataset, we employed phylogenetic methods based on the multispecies coalescence model, including four single-locus methods (GMYC, bGMYC, PTP and bPTP) and one multilocus method (STACEY). From a total of 15 methods and their various settings, 11 supported the recognition of only three species corresponding to the three main phylogenetic lineages: A. niger, A. tubingensis and A. brasiliensis. Similarly, recognition of these three species was supported by the GCPSR approach (Genealogical Concordance Phylogenetic Species Recognition) and analysis in DELINEATE software. We also showed that the phylogeny based on benA, CaM and RPB2 is suboptimal and displays significant differences from a phylogeny constructed using 5 752 single-copy orthologous proteins; therefore, the results of the delimitation methods may be subject to a higher than usual level of uncertainty. To overcome this, we randomly selected 200 genes from these genomes and performed ten independent STACEY analyses, each with 20 genes. All analyses supported the recognition of only one species in the A. niger and A. brasiliensis lineages, while one to four species were inconsistently delimited in the A. tubingensis lineage. After considering all of these results and their practical implications, we propose that the revised series Nigri includes six species: A. brasiliensis, A. eucalypticola, A. luchuensis (syn. A. piperis), A. niger (syn. A. vinaceus and A. welwitschiae), A. tubingensis (syn. A. chiangmaiensis, A. costaricensis, A. neoniger and A. pseudopiperis) and A. vadensis. We also showed that the intraspecific genetic variability in the redefined A. niger and A. tubingensis does not deviate from that commonly found in other aspergilli. We supplemented the study with a list of accepted species, synonyms and unresolved names, some of which may threaten the stability of the current taxonomy. Citation: Bian C, Kusuya Y, Sklenár F, D'hooge E, Yaguchi T, Ban S, Visagie CM, Houbraken J, Takahashi H, Hubka V (2022). Reducing the number of accepted species in Aspergillus series Nigri. Studies in Mycology 102: 95-132. doi: 10.3114/sim.2022.102.03.

Taxonomy of Aspergillus series Versicolores: species reduction and lessons learned about intraspecific variability.

Aspergillus series Versicolores members occur in a wide range of environments and substrates such as indoor environments, food, clinical materials, soil, caves, marine or hypersaline ecosystems. The taxonomy of the series has undergone numerous re-arrangements including a drastic reduction in the number of species and subsequent recovery to 17 species in the last decade. The identification to species level is however problematic or impossible in some isolates even using DNA sequencing or MALDI-TOF mass spectrometry indicating a problem in the definition of species boundaries. To revise the species limits, we assembled a large dataset of 518 strains. From these, a total of 213 strains were selected for the final analysis according to their calmodulin (CaM) genotype, substrate and geography. This set was used for phylogenetic analysis based on five loci (benA, CaM, RPB2, Mcm7, Tsr1). Apart from the classical phylogenetic methods, we used multispecies coalescence (MSC) model-based methods, including one multilocus method (STACEY) and five single-locus methods (GMYC, bGMYC, PTP, bPTP, ABGD). Almost all species delimitation methods suggested a broad species concept with only four species consistently supported. We also demonstrated that the currently applied concept of species is not sustainable as there are incongruences between single-gene phylogenies resulting in different species identifications when using different gene regions. Morphological and physiological data showed overall lack of good, taxonomically informative characters, which could be used for identification of such a large number of existing species. The characters expressed either low variability across species or significant intraspecific variability exceeding interspecific variability. Based on the above-mentioned results, we reduce series Versicolores to four species, namely A. versicolor, A. creber, A. sydowii and A. subversicolor, and the remaining species are synonymized with either A. versicolor or A. creber. The revised descriptions of the four accepted species are provided. They can all be identified by any of the five genes used in this study. Despite the large reduction in species number, identification based on phenotypic characters remains challenging, because the variation in phenotypic characters is high and overlapping among species, especially between A. versicolor and A. creber. Similar to the 17 narrowly defined species, the four broadly defined species do not have a specific ecology and are distributed worldwide. We expect that the application of comparable methodology with extensive sampling could lead to a similar reduction in the number of cryptic species in other extensively studied Aspergillus species complexes and other fungal genera. Citation: Sklenár F, Glässnerová K, Jurjevic Z, Houbraken J, Samson RA, Visagie CM, Yilmaz N, Gené J, Cano J, Chen AJ, Nováková A, Yaguchi T, Kolarík M, Hubka V (2022). Taxonomy of Aspergillus series Versicolores: species reduction and lessons learned about intraspecific variability. Studies in Mycology 102 : 53-93. doi: 10.3114/sim.2022.102.02.

A monograph of Aspergillus section Candidi.

Aspergillus section Candidi encompasses white- or yellow-sporulating species mostly isolated from indoor and cave environments, food, feed, clinical material, soil and dung. Their identification is non-trivial due to largely uniform morphology. This study aims to re-evaluate the species boundaries in the section Candidi and present an overview of all existing species along with information on their ecology. For the analyses, we assembled a set of 113 strains with diverse origin. For the molecular analyses, we used DNA sequences of three house-keeping genes (benA, CaM and RPB2) and employed species delimitation methods based on a multispecies coalescent model. Classical phylogenetic methods and genealogical concordance phylogenetic species recognition (GCPSR) approaches were used for comparison. Phenotypic studies involved comparisons of macromorphology on four cultivation media, seven micromorphological characters and growth at temperatures ranging from 10 to 45 °C. Based on the integrative approach comprising four criteria (phylogenetic and phenotypic), all currently accepted species gained support, while two new species are proposed (A. magnus and A. tenebricus). In addition, we proposed the new name A. neotritici to replace an invalidly described A. tritici. The revised section Candidi now encompasses nine species, some of which manifest a high level of intraspecific genetic and/or phenotypic variability (e.g., A. subalbidus and A. campestris) while others are more uniform (e.g., A. candidus or A. pragensis). The growth rates on different media and at different temperatures, colony colours, production of soluble pigments, stipe dimensions and vesicle diameters contributed the most to the phenotypic species differentiation. Taxonomic novelties: New species: Aspergillus magnus Glässnerová & Hubka; Aspergillus neotritici Glässnerová & Hubka; Aspergillus tenebricus Houbraken, Glässnerová & Hubka. Citation: Glässnerová K, Sklenár F, Jurjevic Z, Houbraken J, Yaguchi T, Visagie CM, Gené J, Siqueira JPZ, Kubátová A, Kolarík M, Hubka V (2022). A monograph of Aspergillus section Candidi. Studies in Mycology 102: 1-51. doi: 10.3114/sim.2022.102.01.

Host-driven subspeciation in the hedgehog fungus, Trichophyton erinacei, an emerging cause of human dermatophytosis.

Trichophyton erinacei is a main cause of dermatophytosis in hedgehogs and is increasingly reported from human infections worldwide. This pathogen was originally described in the European hedgehog (Erinaceus europaeus) but is also frequently found in the African four-toed hedgehog (Atelerix albiventris), a popular pet animal worldwide. Little is known about the taxonomy and population genetics of this pathogen despite its increasing importance in clinical practice. Notably, whether there are different populations or even cryptic species associated with different hosts or geographic regions is not known. To answer these questions, we collected 161 isolates, performed phylogenetic and population-genetic analyses, determined mating-type, and characterised morphology and physiology. Multigene phylogeny and microsatellite analysis supported T. erinacei as a monophyletic species, in contrast to highly incongruent single-gene phylogenies. Two main subpopulations, one specific mainly to Atelerix and second to Erinaceus hosts, were identified inside T. erinacei, and slight differences in the size of microconidia and antifungal susceptibilities were observed among them. Although the process of speciation into two lineages is ongoing in T. erinacei, there is still gene flow between these populations. Thus, we present T. erinacei as a single species, with notable intraspecies variability in genotype and phenotype. The data from wild hedgehogs indicated that sexual reproduction in T. erinacei and de novo infection of hedgehogs from soil are probably rare events and that clonal horizontal spread strongly dominates. The molecular typing approach used in this study represents a suitable tool for further epidemiological surveillance of this emerging pathogen in both animals and humans. The results of this study also highlighted the need to use a multigene phylogeny ideally in combination with other independent molecular markers to understand the species boundaries of dermatophytes. Citation: Cmoková A, Kolarík M, Guillot J, et al. 2022. Host-driven subspeciation in the hedgehog fungus, Trichophyton erinacei, an emerging cause of human dermatophytosis. Persoonia 48: 203-218. https://doi.org/10.3767/persoonia.2022.48.06.

Re-examination of species limits in Aspergillus section Flavipedes using advanced species delimitation methods and description of four new species.

Since the last revision in 2015, the taxonomy of section Flavipedes evolved rapidly along with the availability of new species delimitation techniques. This study aims to re-evaluate the species boundaries of section Flavipedes members using modern delimitation methods applied to an extended set of strains (n = 90) collected from various environments. The analysis used DNA sequences of three house-keeping genes (benA, CaM, RPB2) and consisted of two steps: application of several single-locus (GMYC, bGMYC, PTP, bPTP) and multi-locus (STACEY) species delimitation methods to sort the isolates into putative species, which were subsequently validated using DELINEATE software that was applied for the first time in fungal taxonomy. As a result, four new species are introduced, i.e. A. alboluteus, A. alboviridis, A. inusitatus and A. lanuginosus, and A. capensis is synonymized with A. iizukae. Phenotypic analyses were performed for the new species and their relatives, and the results showed that the growth parameters at different temperatures and colonies characteristics were useful for differentiation of these taxa. The revised section harbors 18 species, most of them are known from soil. However, the most common species from the section are ecologically diverse, occurring in the indoor environment (six species), clinical samples (five species), food and feed (four species), droppings (four species) and other less common substrates/environments. Due to the occurrence of section Flavipedes species in the clinical material/hospital environment, we also evaluated the susceptibility of 67 strains to six antifungals (amphotericin B, itraconazole, posaconazole, voriconazole, isavuconazole, terbinafine) using the reference EUCAST method. These results showed some potentially clinically relevant differences in susceptibility between species. For example, MICs higher than those observed for A. fumigatus wild-type were found for both triazoles and amphotericin B for A. ardalensis, A. iizukae, and A. spelaeus whereas A. lanuginosus, A. luppiae, A. movilensis, A. neoflavipes, A. olivimuriae and A. suttoniae were comparable to or more susceptible as A. fumigatus. Finally, terbinafine was in vitro active against all species except A. alboviridis.

Unravelling species boundaries in the Aspergillus viridinutans complex (section Fumigati): opportunistic human and animal pathogens capable of interspecific hybridization.

Although Aspergillus fumigatus is the major agent of invasive aspergillosis, an increasing number of infections are caused by its cryptic species, especially A. lentulus and the A. viridinutans species complex (AVSC). Their identification is clinically relevant because of antifungal drug resistance and refractory infections. Species boundaries in the AVSC are unresolved since most species have uniform morphology and produce interspecific hybrids in vitro. Clinical and environmental strains from six continents (n = 110) were characterized by DNA sequencing of four to six loci. Biological compatibilities were tested within and between major phylogenetic clades, and ascospore morphology was characterised. Species delimitation methods based on the multispecies coalescent model (MSC) supported recognition of ten species including one new species. Four species are confirmed opportunistic pathogens; A. udagawae followed by A. felis and A. pseudoviridinutans are known from opportunistic human infections, while A. felis followed by A. udagawae and A. wyomingensis are agents of feline sino-orbital aspergillosis. Recently described human-pathogenic species A. parafelis and A. pseudofelis are synonymized with A. felis and an epitype is designated for A. udagawae. Intraspecific mating assay showed that only a few of the heterothallic species can readily generate sexual morphs in vitro. Interspecific mating assays revealed that five different species combinations were biologically compatible. Hybrid ascospores had atypical surface ornamentation and significantly different dimensions compared to parental species. This suggests that species limits in the AVSC are maintained by both pre- and post-zygotic barriers and these species display a great potential for rapid adaptation and modulation of virulence. This study highlights that a sufficient number of strains representing genetic diversity within a species is essential for meaningful species boundaries delimitation in cryptic species complexes. MSC-based delimitation methods are robust and suitable tools for evaluation of boundaries between these species.

Prognostic value of tumor-infiltrating lymphocytes differs depending on histological type and smoking habit in completely resected non-small-cell lung cancer.

BACKGROUND: T-cell infiltration in tumors has been used as a prognostic tool in non-small-cell lung cancer (NSCLC). However, the influence of smoking habit and histological type on tumor-infiltrating lymphocytes (TILs) in NSCLC remains unclear. PATIENTS AND METHODS: We evaluated the prognostic significance of TILs (CD4+, CD8+, CD20+, and FOXP3+) according to histological type and smoking habit using automatic immunohistochemical staining and cell counting in 218 patients with NSCLC. RESULTS: In multivariate survival analyses of clinical, pathological, and immunological factors, a high ratio of FOXP3+ to CD4+ T cells (FOXP3/CD4) [hazard ratio (HR): 4.46, P < 0.01 for overall survival (OS); HR: 1.96, P < 0.05 for recurrence-free survival (RFS)] and a low accumulation of CD20+ B cells (HR: 2.45, P = 0.09 for OS; HR: 2.86, P < 0.01 for RFS) were identified as worse prognostic factors in patients with adenocarcinoma (AD). In non-AD, a low number of CD8+ T cells were correlated with an unfavorable outcome (HR: 7.69, P < 0.01 for OS; HR: 3.57, P < 0.02 for RFS). Regarding smoking habit in AD, a high FOXP3/CD4 ratio was poorly prognostic with a smoking history (HR: 5.21, P < 0.01 for OS; HR: 2.38, P < 0.03 for RFS), whereas a low accumulation of CD20+ B cells (HR: 4.54, P = 0.03 for OS; HR: 2.94, P < 0.01 for RFS) was confirmed as an unfavorable factor in non-smokers with AD. CONCLUSIONS: A low number of CD8+ T cells in non-AD, a high FOXP3/CD4 ratio in smokers with AD, and a low number of CD20+ B cells in non-smokers with AD were identified as independent unfavorable prognostic factors in resected NSCLC. Evaluating the influence of histological type and smoking habit on the immunological environment may lead to the establishment of immunological diagnosis and appropriate individualized immunotherapy for NSCLC.

Immunosuppression through constitutively activated NF-κB signalling in human ovarian cancer and its reversal by an NF-κB inhibitor.

BACKGROUND: Although T-cell immunity is thought to be involved in the prognosis of epithelial ovarian cancer (EOC) patients, immunosuppressive conditions hamper antitumour immune responses. Thus, their mechanisms and overcoming strategies need to be investigated. METHODS: The role of NF-κB in human EOC cells and macrophages was evaluated by in vitro production of immunosuppressive IL-6 and IL-8 by EOC cells and in vivo analysis of immune responses in nude mice implanted with human EOC cells using an NF-κB inhibitor DHMEQ. RESULTS: In EOC patients, increased plasma IL-6, IL-8, and arginase were observed. The NF-κB inhibitor DHMEQ inhibited the production of IL-6 and IL-8 by EOC cell lines. Immunosuppression of human DCs and macrophages by culture supernatant of EOC cells was reversed with the pretreatment of DHMEQ. Administration of DHMEQ to nude mice implanted with human EOC resulted in the restoration of T-cell stimulatory activity of murine DCs along with the reduction of tumour accumulation and arginase expression of MDSCs. Nuclear factor-κB inhibition in tumour-bearing mice also enhanced antitumour effects of transferred murine naive T cells. CONCLUSIONS: NF-κB is involved in the immunosuppression induced by human EOC, and its inhibitor may restore antitumour immune responses, indicating that NF-κB is an attractive target for EOC treatment.

Identification and nomenclature of the genus Penicillium.

Penicillium is a diverse genus occurring worldwide and its species play important roles as decomposers of organic materials and cause destructive rots in the food industry where they produce a wide range of mycotoxins. Other species are considered enzyme factories or are common indoor air allergens. Although DNA sequences are essential for robust identification of Penicillium species, there is currently no comprehensive, verified reference database for the genus. To coincide with the move to one fungus one name in the International Code of Nomenclature for algae, fungi and plants, the generic concept of Penicillium was re-defined to accommodate species from other genera, such as Chromocleista, Eladia, Eupenicillium, Torulomyces and Thysanophora, which together comprise a large monophyletic clade. As a result of this, and the many new species described in recent years, it was necessary to update the list of accepted species in Penicillium. The genus currently contains 354 accepted species, including new combinations for Aspergillus crystallinus, A. malodoratus and A. paradoxus, which belong to Penicillium section Paradoxa. To add to the taxonomic value of the list, we also provide information on each accepted species MycoBank number, living ex-type strains and provide GenBank accession numbers to ITS, ß-tubulin, calmodulin and RPB2 sequences, thereby supplying a verified set of sequences for each species of the genus. In addition to the nomenclatural list, we recommend a standard working method for species descriptions and identifications to be adopted by laboratories working on this genus.

Phylogeny, identification and nomenclature of the genus Aspergillus.

Aspergillus comprises a diverse group of species based on morphological, physiological and phylogenetic characters, which significantly impact biotechnology, food production, indoor environments and human health. Aspergillus was traditionally associated with nine teleomorph genera, but phylogenetic data suggest that together with genera such as Polypaecilum, Phialosimplex, Dichotomomyces and Cristaspora, Aspergillus forms a monophyletic clade closely related to Penicillium. Changes in the International Code of Nomenclature for algae, fungi and plants resulted in the move to one name per species, meaning that a decision had to be made whether to keep Aspergillus as one big genus or to split it into several smaller genera. The International Commission of Penicillium and Aspergillus decided to keep Aspergillus instead of using smaller genera. In this paper, we present the arguments for this decision. We introduce new combinations for accepted species presently lacking an Aspergillus name and provide an updated accepted species list for the genus, now containing 339 species. To add to the scientific value of the list, we include information about living ex-type culture collection numbers and GenBank accession numbers for available representative ITS, calmodulin, ß-tubulin and RPB2 sequences. In addition, we recommend a standard working technique for Aspergillus and propose calmodulin as a secondary identification marker.

The linoleic acid derivative FR236924 facilitates hippocampal synaptic transmission by enhancing activity of presynaptic alpha7 acetylcholine receptors on the glutamatergic terminals.

The present study aimed at understanding the effect of FR236924, a newly synthesized linoleic acid derivative with cyclopropane rings instead of cis-double bonds, on hippocampal synaptic transmission in both the in vitro and in vivo systems. FR236924 increased the rate of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-mediated miniature excitatory postsynaptic currents, without affecting the amplitude, triggered by nicotine in CA1 pyramidal neurons of rat hippocampal slices, that is inhibited by GF109203X, a selective protein kinase C (PKC) inhibitor or alpha-bungarotoxin, an inhibitor of alpha7 acetylcholine (ACh) receptors. FR236924 stimulated glutamate release from rat hippocampal slices and in the hippocampus of freely behaving rats, and the effect was also inhibited by GF109203X or alpha-bungarotoxin. FR236924 induced a transient huge potentiation followed by a long-lasting potentiation in the slope of field excitatory postsynaptic potentials recorded from the CA1 region of rat hippocampal slices, and the latter effect was blocked by GF109203X or alpha-bungarotoxin. Likewise, the compound persistently facilitated hippocampal synaptic transmission in the CA1 region of the intact rat hippocampus. It is concluded from these results that FR236924 stimulates glutamate release by functionally targeting presynaptic alpha7 ACh receptors on the glutamatergic terminals under the influence of PKC, responsible for the facilitatory action on hippocampal synaptic transmission. This may provide evidence for a link between cis-unsaturated free fatty acids and presynaptic alpha7 ACh receptors in hippocampal synaptic plasticity.

[Intrapulmonary aberrant needle; report of a case].

Intrapulmonary aberrant needles are rarely encountered in clinical practice. An intrathoracic aberrant needle should be always surgically removed as soon as possible. We report a case of an intrapulmonary aberrant needle removed with video-assisted thorascopic surgery (VATS) and briefly review the literature. A 47-year-old man referred to us for chest discomfort was found to have an intrapulmonary aberrant needle at the right middle lobe by chest X-ray and computed tomography (CT). We tried simple extraction under thoracoscopy, but a residual fragment was recognized by intraoperative X-ray after the removal of the needle from the surface of the lung. We searched for the residual fragment by real-time fluoroscopic examination. Partial resection was performed to remove the residual needle fragment at the right lower lobe. The postoperative course was uneventful and the man was discharged on the postoperative day 10. Intraoperative chest X-ray is always necessary before closing the chest to avoid leaving the residual fragments in the lung. The real-time fluoroscopy is useful to search for the residual fragment.

Cross-sectional sample preparation by focused ion beam: a review of ion-sample interaction.

A focused ion beam (FIB) was applied for cross-sectional sample preparation with both transmission electron microscopes (TEM) and scanning electron microscopes (SEM). The FIB sample preparation has the advantage of high positioning accuracy for cross sections. On the other hand, a broad ion beam (BIB) has been conventionally used for thinning TEM samples. Although both FIB and BIB use energetic ion beams, they are essentially different from each other in many aspects such as beam size, beam current density, incident angle of the beam with respect to cross sections, and beam scanning (i.e., dynamic or static beam). In this study, FIB cross-sectioning is compared with BIB thinning. We review inherent characteristics such as positioning accuracy and uniformity of cross section, radiation damage, and beam heating. Discussion is held from a view-point of ion beam and sample interaction.

Purification of form L2 RubisCO from a marine obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus strain MH-110.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) was purified from an obligately autotrophic hydrogen-oxidizing bacterium, Hydrogenovibrio marinus MH-110. The protein has a M(r) value of approximately 110,000, and is composed of two identical subunits of 55,000. To our knowledge, the existence of L2-form RubisCO in a chemolithoautotrophic bacterium is first reported in this paper. The N-terminal amino acid sequence determination of the purified enzyme showed high homology with those of the L2-form RubisCO of Rhodospirillum rubrum and the Lx-form RubisCO from Rhodobacter sphaeroides.

Involvement of endogenous PACAP expression in the activity-dependent survival of mouse cerebellar granule cells.

Membrane depolarization causes Ca2+ influx through L-type voltage-dependent calcium channels (L-VDCC), which promotes the activity-dependent survival of mouse cerebellar granule cells (CGCs). Although exogenously added pituitary adenylate cyclase activating polypeptide (PACAP) is effective in promoting the survival of CGCs, it is unknown whether PACAP is synthesized in CGCs and involved in the activity-dependent survival of CGCs. In this study, we found that the PACAP gene was activated in depolarized CGCs cultured at 25 mM KCl (high K+), independently of de novo protein synthesis. In addition, the PACAP immunoreactivity increased through the activation of L-VDCC in depolarized CGCs, indicating that PACAP is concomitantly produced with PACAP mRNA in an activity-dependent manner. Exogenously added PACAP attenuated the apoptosis of CGCs through a specific interaction with PACAP receptors. Furthermore, a PACAP receptor antagonist, PACAP(6-38), reduced the survival of CGCs at high K+. These findings indicate that endogenous PACAP production induced by Ca2+ signals exerts a survival effect on CGCs via PACAP receptors, which, at least in part, accounts for the activity-dependent survival of CGCs.

In vivo introduction of the interleukin 6 gene into human keratinocytes: induction of epidermal proliferation by the fully spliced form of interleukin 6, but not by the alternatively spliced form.

The achievement of keratinocyte gene therapy in clinical practice requires fundamental experiments using human keratinocytes or skin. We have recently demonstrated that the in vivo introduction of the interleukin 6 (IL-6) gene into rat keratinocytes induces epidermal proliferation and lymphocyte infiltration into the skin. In this study, we first amplified the human IL-6 cDNA from oligo-dT-primed keratinocyte cDNA and then detected the fully spliced (FS) form and the alternatively spliced (AS) form of IL-6 cDNA. Sequence analysis showed that the AS form, which was composed of the IL-6 coding region with all of exon II deleted except for the first guanine, was identical to that reported to be present in lymphocytes. We constructed the expression vectors phIL6 of the FS form and phIL6S of the AS form. We transplanted human skin onto nude rats and introduced phIL6 and phIL6S into the human keratinocytes using the naked DNA method. Keratinocytes prepared 24 h after introduction from the areas treated with them were examined by reverse transcriptase (RT)-PCR and enzyme linked immunosorbent assay (ELISA). RT-PCR showed that the amounts of FS IL-6 mRNA and AS IL-6 mRNA were similar, whereas the ELISA showed that the amount of FS IL-6 peptide was four times that of the AS IL-6 peptide. Histological examination 48 h after introduction showed that the FS form had induced epidermal proliferation, whereas the AS form had not. The epidermal thickening without lymphocyte infiltration induce by the FS form indicates that keratinocyte proliferation is caused by a direct effect of overexpressed IL-6, and not by a secondary effect of infiltrating lymphocytes. This is the first report of the introduction of a human gene into human keratinocytes to produce a biologically active transgenic gene product in human skin using the naked DNA method.

Mitorubrin derivatives on ascomata of some talaromyces species of ascomycetous fungi

Two groups, producers of (+)- and (-)-mitorubrin derivatives, coexist in the series Lutei of the genus Talaromyces. The optical rotations of mitorubrins from T. emodensis, T. hachijoensis, and T. wortmannii var. sublevisporus, which produced mitorubrinol acetate (5), were all positive, whereas those from T. austrocalifornicus and T. convolutus, which produced mitorubrinal (3) and mitorubrinic acid (4), were all negative.

PF1018, a novel insecticidal compound produced by Humicola sp.

A new insecticidal compound PF1018 was isolated from the culture broth of Humicola sp. It exhibited insecticidal activity against a wide range of critical pest species. The structure of PF1018 was determined to be (7aS)-2-((2E)-1-hydroxy-3-((1S,3aR,4R,5R,7aR)-3a,4,5,7 a-tetrahydro-1,3,5,7- tetramethyl-5,1-((3S)-(Z)-2,3-dimethylpropeno)-1H-inden-4-yl )-2- propenylidene)pyrrolizidine-1,3-dione, by NMR spectral analyses coupled with X-ray crystallographic analysis and chemical degradation study.