The G-protein Coupled Receptor GPR8 Regulates Secondary Metabolism in Trichoderma reesei.

Changing environmental conditions are of utmost importance for regulation of secondary metabolism in fungi. Different environmental cues including the carbon source, light and the presence of a mating partner can lead to altered production of compounds. Thereby, the heterotrimeric G-protein pathway is of major importance for sensing and adjustment of gene regulation. Regulation of secondary metabolism is crucial in the biotechnological workhorse Trichoderma reesei for knowledge-based adjustment in industrial fermentations, but also with respect to the potential use as a host for heterologous compound production. We investigated the function of the class VII G-protein coupled receptor (GPCR) gene gpr8 that is localized in the vicinity of the SOR cluster, which is responsible for biosynthesis of sorbicillinoids. GPR8 positively impacts regulation of the genes in this cluster in darkness. Accordingly, abundance of trichodimerol and dihydrotrichotetronine as well as other secondary metabolites is decreased in the deletion mutant. Transcriptome analysis moreover showed the major role of GPR8 being exerted in darkness with a considerable influence on regulation of secondary metabolism. Genes regulated in Δgpr8 overlap with those regulated directly or indirectly by the transcription factor YPR2, especially concerning genes related to secondary metabolism. The predicted FAD/FMN containing dehydrogenase gene sor7, one of the positive targets of the cascade triggered by GPR8, has a positive effect on secondary metabolite production, but also cellulase gene expression. Hence SOR7 has some overlapping, but also additional functions compared to GPR8. The G-protein coupled receptor GPR8 exerts a light dependent impact on secondary metabolism, which is in part mediated by the transcription factor YPR2 and the function of SOR7. Hence, T. reesei may apply GPR8 to adjust production of secondary metabolites and hence chemical communication to signals from the environment.

The Kinase USK1 Regulates Cellulase Gene Expression and Secondary Metabolite Biosynthesis in Trichoderma reesei.

The complex environment of fungi requires a delicate balance between the efforts to acquire nutrition, to reproduce, and to fend off competitors. In Trichoderma reesei, an interrelationship between regulation of enzyme gene expression and secondary metabolism was shown. In this study, we investigated the physiological relevance of the unique YPK1-type kinase USK1 of T. reesei. Usk1 is located in the vicinity of the SOR cluster and is involved in regulation of several genes from this secondary metabolite cluster as well as dihydrotrichotetronine and other secondary metabolites. Moreover, USK1 is required for biosynthesis of normal levels of secondary metabolites in liquid culture. USK1 positively influences cellulase gene regulation, secreted cellulase activity, and biomass formation upon growth in constant darkness on cellulose. Positive effects of USK1 on transcript abundance of the regulator of secondary metabolism, vel1, and the carbon catabolite repressor gene cre1 are in agreement with these functions. In summary, we found that with USK1, T. reesei comprises a unique kinase that adds an additional layer of regulation to the connection of secondary metabolism and enzyme production in fungi.

Gene regulation associated with sexual development and female fertility in different isolates of Trichoderma reesei.

BACKGROUND: Trichoderma reesei is one of the most frequently used filamentous fungi in industry for production of homologous and heterologous proteins. The ability to use sexual crossing in this fungus was discovered several years ago and opens up new perspectives for industrial strain improvement and investigation of gene regulation. RESULTS: Here we investigated the female sterile strain QM6a in comparison to the fertile isolate CBS999.97 and backcrossed derivatives of QM6a, which have regained fertility (FF1 and FF2 strains) in both mating types under conditions of sexual development. We found considerable differences in gene regulation between strains with the CBS999.97 genetic background and the QM6a background. Regulation patterns of QM6a largely clustered with the backcrossed FF1 and FF2 strains. Differential regulation between QM6a and FF1/FF2 as well as clustering of QM6a patterns with those of CBS999.97 strains was also observed. Consistent mating type dependent regulation was limited to mating type genes and those involved in pheromone response, but included also nta1 encoding a putative N-terminal amidase previously not associated with development. Comparison of female sterile QM6a with female fertile strains showed differential expression in genes encoding several transcription factors, metabolic genes and genes involved in secondary metabolism. CONCLUSIONS: Evaluation of the functions of genes specifically regulated under conditions of sexual development and of genes with highest levels of transcripts under these conditions indicated a relevance of secondary metabolism for sexual development in T. reesei. Among others, the biosynthetic genes of the recently characterized SOR cluster are in this gene group. However, these genes are not essential for sexual development, but rather have a function in protection and defence against competitors during reproduction.

Krüppel Like Factors Family Expression in Cervical Cancer Cells.

BACKGROUND AND AIMS: Krüppel Like Factors (KLF) refers to a family of seventeen members of transcription factors. Involved in several cellular processes. As other cancer types, Cervical Cancer (CC) presents molecular deregulations in transcription factors, but especially Human Papilloma Virus (HPV) sequences. Here in this work we analyzed the mRNA expression of all KLF family members in CC-derived cell lines and CC tissues. METHODS: The cell lines used were HeLa, INBL, RoVa, C4-I, Ms751, ViPa, CaLo, SiHa, CaSki, C33a and ViBo and the non-tumorigenic HaCaT. mRNA expression was analyzed by means of expression microarray and RT-PCR, and KLF5 protein by immunofluorescence. RESULTS: The cell lines were grouped according to HPV genotype as HPV16, HPV18 positive or HPV negative cells. Heterogeneous expression was observed among the cell lines. Despite the heterogeneous expression profile, KLF3, -5, -12, -15 and -16 transcripts were present in all cell lines, KLF4 and -10 which were not expressed in CaSki; KLF11 and 13 were not expressed by Vipa and C4-I, and KLF7 was not expressed by C4-I and Rova. The CC tissue analysis shows expression of most of the KLF members, such as KLF5. KLF5 immunosignal was positive in the three cell lines analyzed. CONCLUSIONS: We suggest that KLF expression could not be related to HPV presence/genotype, at least at transcriptional level, and the expression of KLF family members may be necessary in the biology of the CC cells.

A CRE1- regulated cluster is responsible for light dependent production of dihydrotrichotetronin in Trichoderma reesei.

Changing light conditions, caused by the rotation of earth resulting in day and night or growth on the surface or within a substrate, result in considerably altered physiological processes in fungi. For the biotechnological workhorse Trichoderma reesei, regulation of glycoside hydrolase gene expression, especially cellulase expression was shown to be a target of light dependent gene regulation. Analysis of regulatory targets of the carbon catabolite repressor CRE1 under cellulase inducing conditions revealed a secondary metabolite cluster to be differentially regulated in light and darkness and by photoreceptors. We found that this cluster is involved in production of trichodimerol and that the two polyketide synthases of the cluster are essential for biosynthesis of dihydrotrichotetronine (syn. bislongiquinolide or bisorbibutenolide). Additionally, an indirect influence on production of the peptaibol antibiotic paracelsin was observed. The two polyketide synthetase genes as well as the monooxygenase gene of the cluster were found to be connected at the level of transcription in a positive feedback cycle in darkness, but negative feedback in light, indicating a cellular sensing and response mechanism for the products of these enzymes. The transcription factor TR_102497/YPR2 residing within the cluster regulates the cluster genes in a light dependent manner. Additionally, an interrelationship of this cluster with regulation of cellulase gene expression was detected. Hence the regulatory connection between primary and secondary metabolism appears more widespread than previously assumed, indicating a sophisticated distribution of resources either to degradation of substrate (feed) or to antagonism of competitors (fight), which is influenced by light.

The marine algae Sargassum spp. (Sargassaceae) as feed for sheep in tropical and subtropical regions

The objective of this study was to evaluate Sargassum meal as feed for sheep through the measures of in vivo digestibility, dry matter degradability, pH, ammonia and volatile fatty acids in rumen. The Sargassum algae used in this experiment were collected at the end of spring, when they are more abundant, bigger, and have completed their reproductive cycle. Four tons (wet weigth) were collected manually from the intertidal zone of La Paz bay, Baja California Sur, Mexico. These algae were sun-dried and ground in a hammer mill to obtain the Sargassum meal. Four fistulated Pelibuey sheep, were fed daily with diets containing the marine algae (MA) at different levels (0, 10, 20 and 30 %), using a 4 x 4 Latin-square design experiment. Feed intake was not affected (p>0.05). Water consumption and urine excretion increased with MA (p<0.05; r²=0.54 and r²=0.74, respectively). In all treatments dry matter digestibility was of 74%-79%, and crude protein digestibility was of 85%-88%. Acid detergent fiber (59%-65%) and neutral detergent fiber (55%-66%) digestibility were greater in all treatments with MA. Ruminal pH was greater in all groups fed with MA (p<0.05). Ammonium concentration was not influenced (p>0.05) by MA. Ruminal volatile fatty acids decreased in all MA groups (p<0.05). The marine algae Sargassum spp. can be used as a feed supplement for sheep, especially in tropical and subtropical regions where these marine algae are available. Rev. Biol. Trop. 57 (4): 1271-1281. Epub 2009 December 01.

El objetivo de este estudio fue evaluar la harina del alga marina Sargassum como alimento para ovejas, midiendo la digestibilidad in vivo, la degradabilidad de la materia seca, así como el pH y los ácidos grasos volátiles en rumen. El alga Sargassum utilizada en este experimento, fue recolectada a finales de la primavera, cuando esta alga es más abundante, alcanza su mayor talla y ha completado su ciclo reproductivo. Se recolectaron manualmente, cuatro toneladas (peso húmedo) de la zona intermareal en la Bahía de La Paz, Baja California Sur, México. Estas algas fueron secadas directamente al sol y molidas en un molino de martillos, para obtener la harina. Se utilizaron cuatro borregos Pelibuey fistulados, distribuidos en un arreglo factorial de 4 x 4. Los animales fueron alimentados diariamente con dietas que contenían el alga marina (AM) Sargassum a diferentes niveles (0, 10, 20 y 30%). El consumo de alimento no se vio afectado con la inclusión del alga (p> 0.05). El consumo de agua y la excreción de orina se incrementaron conforme aumentó la concentración de AM en las dietas (p<0.05; r²=0.54 and r²=0.74, respectivamente). En todos los tratamientos la digestibilidad de la materia seca fue de 74% a 79%, la digestibilidad de la proteína cruda fue de 85% a 88%. La digestibilidad de la fibra ácido detergente (59%-65%) y de la neutro detergente (55%-66%) fue mayor en todos los tratamientos con AM, lo mismo ocurrió con el pH en rumen (p<0.05). La concentración de amonio en rumen no se vio afectada por AM (p>0.05). La concentración de ácidos grasos volátiles se redujo en todos los tratamientos con AM (p<0.05). El alga marina Sargassum spp. puede ser usada como complemento alimenticio para ovejas, especialmente en las regiones tropicales y subtropicales donde está disponible.

The marine algae Sargassum spp. (Sargassaceae) as feed for sheep in tropical and subtropical regions.

The objective of this study was to evaluate Sargassum meal as feed for sheep through the measures of in vivo digestibility, dry matter degradability, pH, ammonia and volatile fatty acids in rumen. The Sargassum algae used in this experiment were collected at the end of spring, when they are more abundant, bigger, and have completed their reproductive cycle. Four tons (wet weigth) were collected manually from the intertidal zone of La Paz bay, Baja California Sur, Mexico. These algae were sun-dried and ground in a hammer mill to obtain the Sargassum meal. Four fistulated Pelibuey sheep, were fed daily with diets containing the marine algae (MA) at different levels (0, 10, 20 and 30 %), using a 4 x 4 Latin-square design experiment. Feed intake was not affected (p>0.05). Water consumption and urine excretion increased with MA (p<0.05; r2=0.54 and r2=0.74, respectively). In all treatments dry matter digestibility was of 74%-79%, and crude protein digestibility was of 85%-88%. Acid detergent fiber (59%-65%) and neutral detergent fiber (55%-66%) digestibility were greater in all treatments with MA. Ruminal pH was greater in all groups fed with MA (p<0.05). Ammonium concentration was not influenced (p>0.05) by MA. Ruminal volatile fatty acids decreased in all MA groups (p<0.05). The marine algae Sargassum spp. can be used as a feed supplement for sheep, especially in tropical and subtropical regions where these marine algae are available.

Interleukin-1 beta (IL-1beta) induces tumor necrosis factor alpha (TNF-alpha) expression on mouse myeloid multipotent cell line 32D cl3 and inhibits their proliferation.

Interleukin-1 alpha (IL-1alpha) and beta (IL-1beta) are well known factors that stimulate hematopoiesis, nevertheless there are reports that show that they can also inhibit this activity. While both IL-1alpha and IL-1beta induce the expression of hematopoietic cytokines, such as growth factors and their receptors on myeloid cells, helping thus to regulate hematopoiesis, it is not known if their inhibitory activity is also mediated through the induction of other specific cytokines. In this work we show that recombinant human IL-1beta (rhIL-1beta) inhibits the proliferation of a mouse IL-3-dependent myeloid multipotent cell line (32D cl3), without inducing its differentiation. We show that rhIL-1beta induces in 32D cl3 cells the expression of the tumor necrosis factor alpha (TNF-alpha) gene, a well known growth inhibitor, and that the rhIL-1beta growth inhibition property on 32D cl3 cells is partially due to this secreted TNF-alpha, hinting thus that the inhibition of hematopoiesis by IL-1 is mediated through other induced cytokines.

Neocarzinostatin induces an effective p53-dependent response in human papillomavirus-positive cervical cancer cells.

Human papillomavirus (HPV) E6 viral oncoprotein plays an important role during cervical carcinogenesis. This oncoprotein binds the tumor suppressor protein p53, leading to its degradation via the ubiquitin-proteasome pathway. Therefore, it is generally assumed that in HPV-positive cancer cells p53 function is completely abolished. Nevertheless, recent findings suggest that p53 activity can be recovered in cells expressing endogenous E6 protein. To investigate whether p53-dependent functions controlling genome integrity, cell proliferation, and apoptosis can be reactivated in cervical cancer cells, we examined the capacity of HeLa, INBL, CaSki, C33A, and ViBo cell lines to respond to neocarzinostatin (NCS), a natural product which induces single- and double-strand breaks in DNA. We found that NCS treatment inhibits cellular proliferation through G2 cell cycle arrest and apoptosis induction. This effect was preceded by nuclear accumulation of p53 protein and by an increase of p21 transcripts. Although apoptosis was blocked in ViBo cells (HPV-negative), nuclear accumulation of transcriptionally active p53 and inhibition of cell proliferation are observed after NCS treatment. These results suggest that HPV-positive cervical cancer cells are capable of responding efficiently to DNA damage provoked by NCS treatment through a p53-dependent pathway in spite of the presence of E6 protein.

Chromosomal imbalances in four new uterine cervix carcinoma derived cell lines.

BACKGROUND: Uterine cervix carcinoma is the second most common female malignancy worldwide and a major health problem in Mexico, representing the primary cause of death among the Mexican female population. High risk human papillomavirus (HPV) infection is considered to be the most important risk factor for the development of this tumor and cervical carcinoma derived cell lines are very useful models for the study of viral carcinogenesis. Comparative Genomic Hybridization (CGH) experiments have detected a specific pattern of chromosomal imbalances during cervical cancer progression, indicating chromosomal regions that might contain genes that are important for cervical transformation. METHODS: We performed HPV detection and CGH analysis in order to initiate the genomic characterization of four recently established cervical carcinoma derived cell lines from Mexican patients. RESULTS: All the cell lines were HPV18 positive. The most prevalent imbalances in the cell lines were gains in chromosomes 1q23-q32, 3q11.2-q13.1, 3q22-q26.1, 5p15.1-p11.2, this alteration present as a high copy number amplification in three of the cell lines, 7p15-p13, 7q21, 7q31, 11q21, and 12q12, and losses in 2q35-qter, 4p16, 6q26-qter, 9q34 and 19q13.2-qter. CONCLUSIONS: Analysis of our present findings and previously reported data suggest that gains at 1q31-q32 and 7p13-p14, as well as losses at 6q26-q27 are alterations that might be unique for HPV18 positive cases. These chromosomal regions, as well as regions with high copy number amplifications, coincide with known fragile sites and known HPV integration sites. The general pattern of chromosomal imbalances detected in the cells resembled that found in invasive cervical tumors, suggesting that the cells represent good models for the study of cervical carcinoma.

The Differentiation of the Vitelline Envelope of Xenopus Oocytes*: (xenopus/oocyte/vitelline envelope/differentiation/fucosyl glycoproteins).

We have studied the differentiation of the vitelline envelope (V.E.) of the oocyte of the anuran Xenopus laevis. The V.E. precursor material is synthesized by the oocyte concomitantly with the onset of vitellogenesis, and its extrusion reaches a maximum at late vitellogenesis. Oocytes at different stages of growth were incubated in L-[3 H]fucose and the progress of incorporation was followed by kinetic and histoautoradiographic analysis. We found that the highest overall rate of incorporation was exhibited by the vitellogenic oocytes. These oocytes showed clusters of grains in the perinuclear and in the cortical areas. The highest accumulation of grains in the V.E. was associated with the late vitellogenic stage, when the differentiation of the V.E. was almost complete. L-[3 H]Fucose labelled glycoproteins have been identified by electrophoretic analysis of V.E. prepared from late vitellogenic stages.

Effect on Inhibition of Micromere Segregation on the Mitotic Pattern in the Sea Urchin Embryo: (micromeres/mitotic pattern/sea urchin).

Detergent treatment of sea urchin eggs at the mid 4-cell stage results in prevention of micromere segregation at the fourth cleavage. In these embryos not only the formation of the primary mesenchyme is suppressed, but synchrony of cell division, which is the rule during the first four cleavage cycles, continues for several cycles after the 16-cell stage while the typical mitotic phase wave that sets in after micromere segregation is abolished. These results support the hypothesis that micromeres act as coordinators of the mitotic activity of the embryo.

Differentiation of the vitelline coat in the ascidianCiona intestinalis: an ultrastructural study.

We have studied the differentiation of the vitelline coat (VC) of the ascidianCiona intestinalis. In the young previtellogenic oocyte the vitelline coat precursor material (VCPM) makes its first appearance as patches of fibrous material in close apposition to the outer surface of the oocyte. The presence of subcortical vescicles containing a fuzzy electron-dense material and their opening into the oocyte surface parallels the formation of VCPM. Numerous microvillar-like structures emerge from the oocyte surface. When the VCPM completely surrounds the oocyte the microvilli are withdrawn. An overall increase of VCPM parallels the growth of the oocyte. The next step in the differentiation of the vitelline coat consists in the packing of the constituent fibrils in a dense layer at its outer surface, i.e. the one in contact with the follicle cells. At this time the VC is penetrated by microvilli protruding both from the oocyte and follicle cells. The VC reaches its final structure and thickness at the time the test cells are extruded into the perivitelline space.The participation of the follicle cells in VC organization is also discussed.

ACROSOME DIFFERENTIATION IN THE SPERMATOGENESIS OF CIONA INTESTINALIS.

The process of acrosome formation in the course of spermatogenesis of Ciona intestinalis has been investigated. At the flute-beak-shaped tip of the head of the mature spermatozoon a small acrosomal vesicle(s) is described. The vesicles migrate to a region where the outer and inner nuclear membranes fuse thus giving rise to a "dense plate". At the same time the chromatin begins to organize into longitudinally oriented strands which become attached to the inner side of the dense plate. The possible relationships between the dense plate, the formation of the acrosome and the orientation of the chromatin is discussed.