The PHD domain is required to link Drosophila Pygopus to Legless/β-catenin and not to histone H3

In Drosophila Pygopus (Pygo) and Legless (Lgs)/BCL9 are integral components of the nuclear Wnt/Wg signaling machine. Despite intense research, ideas that account for their mode of action remain speculative. One proposition, based on a recently discovered function of PHD fingers, is that Pygo, through its PHD, may decipher the histone code. We found that human, but not Drosophila, Pygo robustly interacts with a histone-H3 peptide methylated at lysine-4. The different binding behavior is due to a single amino acid change that appears unique to Drosophilidae Pygo proteins. Rescue experiments with predicted histone binding mutants showed that in Drosophila the ability to bind histones is not essential. Further experiments with Pygo–Lgs fusions instead demonstrated that the crucial role of the PHD is to provide an interaction motif to bind Lgs. Our results reveal an interesting evolutionary dichotomy in Pygo structure–function, as well as evidence underpinning the chain of adaptors model.     

Spermiogenesis and chromatin condensation in the common tree shrew, <Emphasis Type="Italic">Tupaia glis</Emphasis>

We have investigated the cellular characteristics, especially chromatin condensation and the basic nuclear protein profile, during spermiogenesis in the common tree shrew, Tupaia glis. Spermatids could be classified into Golgi phase, cap phase, acrosome phase, and maturation phase. During the Golgi phase, chromatin was composed of 10-nm and 30-nm fibers with few 50-nm to 60-nm knobby fibers. The latter were then transformed into 70-nm knobby fibers during the cap phase. In the acrosome phase, all fibers were packed into the highest-order knobby fibers, each about 80–100 nm in width. These chromatin fibers became tightly packed in the maturation phase. In a mature spermatozoon, the discoid-shaped head was occupied by the acrosome and completely condensed chromatin. H3, the core histone, was detected by immunostaining in all nuclei of germ cell stages, except in spermatid steps 15–16 and spermatozoa. Protamine, the basic nuclear protein causing the tight packing of sperm chromatin, was detected by immunofluorescence in the nuclei of spermatids at steps 12–16 and spermatozoa. Cross-immunoreactivity of T. glis H3 and protamine to those of primates suggests the evolutionary resemblance of these nuclear basic proteins in primate germ cells. This work was supported by the Thailand Research Fund (Senior Research Fellowship to Prof. Prasert Sobhon).     

Expression and characterization of recombinant interferon gamma (IFN-gamma) from the nine-banded armadillo (Dasypus novemcinctus) and its effect on Mycobacterium leprae-infected macrophages

Armadillos (Dasypus novemcinctus) manifest the full histopathological spectrum of leprosy, and are hosts of choice for in vivo propagation of Mycobacterium leprae. Though potentially useful as a model of leprosy pathogenesis, few armadillo-specific reagents exist. We have identified a region of high homology to the interferon gamma (IFN-γ) of other mammals within the recently published armadillo whole genomic sequence. cDNA was made from ConA-stimulated armadillo peripheral blood mononuclear cells (PBMC), amplified, and cloned into a pET expression vector for transformation and over-expression in Escherichia coli. The recombinant protein (rDnIFN-γ) was characterized by western blot and its biological function confirmed with bioassays including intracellular killing of Toxoplasma gondii and induction of indoleamine 2, 3-dioxygenase activity. In using rIFN-γ to activate macrophages from mice, humans or armadillos, similar to humans, rIFN-γ-activated armadillo MΦ did not produce nitrite and or inhibit the viability of M. leprae in vitro. Conversely, murine rIFN-γ-activated mouse MΦ produced high levels of nitrite and killed intracellular M. leprae in vitro. These data indicate that the response of armadillo MΦ to rDnIFN-γ is similar to that which occurs in humans, and demonstrates a potentially important value of the armadillo as a model in leprosy research.     

Spermatogenesis and distinctive mature sperm in the giant freshwater prawn, Macrobrachium rosenbergii (De Man, 1879)

The structures of differentiating male germ cells in the testis of the giant freshwater prawn, Macrobrachium rosenbergii, were studied by light and electron microscopy. Based on ultrastructural characteristics, the developing male germ cells are classified into 12 stages, including spermatogonia, six phases of primary spermatocytes (leptotene, zygotene, pachytene, diplotene, diakinesis and metaphase), secondary spermatocyte, three stages of spermatids and mature sperm. During spermatogenesis, the differentiating germ cells have characteristics similar to those of other invertebrates, but they exhibit some unique characteristics during spermiogenesis. In particular, an early spermatid has a round nucleus with highly condensed heterochromatin, appearing as thick interconnecting cords throughout the nucleus. In contrast to most invertebrates and vertebrates, the chromatin begins to decondense in one-half of the nucleus at the mid spermatid stage. In the late spermatid, the chromatin becomes almost entirely decondensed with only a small crescent-shaped heterochromatin patch remaining at the anterior pole of the nucleus. Mature sperm possess an everted umbrella-shaped plate with a spike covering the anterior pole of the nucleus, whose chromatin is totally decondensed as only small traces of histones H3 and H2B remain. The acrosome appears at the ruffled border of the spike plate as small sac-like structures. Few mitochondria remain in the cytoplasm at the posterior pole.     

TGF-β type I receptor Alk5 regulates tooth initiation and mandible patterning in a type II receptor-independent manner

TGF-β superfamily members signal through a heteromeric receptor complex to regulate craniofacial development. TGF-β type II receptor appears to bind only TGF-β, whereas TGF-β type I receptor (ALK5) also binds to ligands in addition to TGF-β. Our previous work has shown that conditional inactivation of Tgfbr2 in the neural crest cells of mice leads to severe craniofacial bone defects. In this study, we examine and compare the defects of TGF-β type II receptor (Wnt1-Cre;Tgfbr2^fl/fl) and TGF-β type I receptor/Alk5 (Wnt1-Cre;Alk5^fl/fl) conditional knockout mice. Loss of Alk5 in the neural crest tissue resulted in phenotypes not seen in the Tgfbr2 mutant, including delayed tooth initiation and development, defects in early mandible patterning and altered expression of key patterning genes including Msx1, Bmp4, Bmp2, Pax9, Alx4, Lhx6/7 and Gsc. Alk5 controls the survival of CNC cells by regulating expression of Gsc and other genes in the proximal aboral region of the developing mandible. We conclude that ALK5 regulates tooth initiation and early mandible patterning through a pathway independent of Tgfbr2. There is an intrinsic requirement for Alk5 signal in regulating the fate of CNC cells during tooth and mandible development.     

Pivotal role for TGF-beta in infectious heart disease: The case of Trypanosoma cruzi infection and consequent Chagasic myocardiopathy

This paper summarizes recent data from the literature suggesting that transforming growth factor-β (TGF-β) participates at least in four different processes influencing development of myocardiopathy in Chagas disease, a major parasitic illness caused by Trypanosoma cruzi infection: (a) invasion of cardiac fibroblasts and myocytes; (b) intracellular parasite cycle; (c) regulation of inflammation and immune response; (d) fibrosis and heart remodeling during acute and chronic disease. All these effects point to an important role of TGF-β in Chagas disease myocardiopathy and suggest that monitoring the circulating levels of this cytokine could be of help in clinical prognosis and management of patients. Moreover, TGF-β-interfering therapies appear as interesting adjuvant interventions during acute and chronic phases of T. cruzi infection.     

Kaempferol triosides from Silphium perfoliatum

Two apiose-containing kaempferol triosides, together with nine known flavonoids were isolated from the leaves of Silphium perfoliatum L. Their structures were elucidated by acid hydrolysis and spectroscopic methods including UV, LSI MS, FAB MS, CI MS, ¹H, ¹³C and 2D-NMR, DEPT, HMQC and HMBC experiments. The two new compounds were identified as kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1′→6)-O-β- -galactopyranoside and kaempferol 3-O-β- -apiofuranoside 7-O-α- -rhamnosyl-(1→ 6)-O-β- (2-O-E-caffeoylgalactopyranoside).     

Effect of retinoic acid signaling on Wnt/β-catenin and FGF signaling during body axis extension

Cell–cell signaling regulated by retinoic acid (RA), Wnt/β-catenin, and fibroblast growth factor (FGF) is important during body axis extension, and interactions between these pathways have been suggested. At early somite stages, Wnt/β-catenin and FGF signaling domains exist both anterior and posterior to the developing trunk, whereas RA signaling occurs in between in the trunk under the control of the RA-synthesizing enzyme retinaldehyde dehydrogenase-2 (Raldh2). Previous studies demonstrated that vitamin A deficient quail embryos and Raldh2^−/− mouse embryos lacking RA synthesis exhibit ectopic expression of Fgf8 and Wnt8a in the developing trunk. Here, we demonstrate that Raldh2^−/− mouse embryos display an expansion of FGF signaling into the trunk monitored by Sprouty2 and Pea3 expression, and an expansion of Wnt/β-catenin signaling detected by expression of Axin2, Tbx6, Cdx2, and Cdx4. Following loss of RA signaling, the caudal expression domains of Fgf8, Wnt8a, and Wnt3a expand anteriorly into the trunk, but no change is observed in caudal expression of Fgf4 or Fgf17 plus caudal expression of Fgf18 and Cdx1 is reduced. These findings suggest that RA repression of Fgf8, Wnt8a, and Wnt3a in the developing trunk functions to down-regulate FGF signaling and Wnt/β-catenin signaling as the body axis extends.     

A new ecdysteroid and other constituents from two Dioscorea species

Phytochemical investigation of the rhizome of Dioscorea dumetorum has led to the isolation by several chromatographic steps on normal and reversed phase silica gel of a new ecdysteroid, (20R)-5β,11α,20-trihydroxyecdysone (1), and two known ecdysteroids, ajugasterone C (2) and herkesterone (3). Their structures were determined by spectroscopic methods including 1D- and 2D-NMR (COSY, TOCSY, HSQC and HMBC). This is the first report on the occurrence of phytoecdysteroids in the Dioscoreaceae family. These compounds were devoid of antifungal activity against three Candida species (Candida albicans, Candida glabrata and Candida tropicalis, MIC > 200 μg/ml).     

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as η subunit of chaperonin containing T-complex protein 1

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.     

Patterns of importin-α expression during Drosophila spermatogenesis

Importin-α proteins do not only mediate the nuclear import of karyophilic proteins but also regulate spindle assembly during mitosis and the assembly of ring canals during Drosophila oogenesis. Three importin-α genes are present in the genome of Drosophila. To gain further insights into their function we analysed their expression during spermatogenesis by using antibodies raised against each of the three Importin-α proteins identified in Drosophila, namely, Imp-α1, -α2, and -α3. We found that each Imp-α is expressed during a specific and limited period of spermatogenesis. Strong expression of Imp-α2 takes place in spermatogonial cells, persists in spermatocytes, and lasts up to the completion of meiosis. In growing spermatocytes, the intracellular localisation of Imp-α2 appears to be dependent upon the rate of cell growth. In pupal testes Imp-α2 is essentially present in the spermatocyte nucleus but is localised in the cytoplasm of spermatocytes from adult testes. Both Imp-α1 and -α3 expression initiates at the beginning of meiosis and ends during spermatid differentiation. Imp-α1 expression extends up to the onset of the elongation phase, whereas that of Imp-α3 persists up to the completion of nuclear condensation when the spermatids become individualised. During meiosis Imp-α1 and -α3 are dispersed in the karyoplasm where they are partially associated with the nuclear spindle, albeit not with the asters. At telophase they aggregate around the chromatin. During sperm head differentiation, both Imp-α1 and -α3 are nuclear. These data indicate that each Imp-α protein carries during Drosophila spermatogenesis distinct, albeit overlapping, functions that may involve nuclear import of proteins, microtubule organisation, and other yet unknown processes.     

Nucleosome transactions on the<Emphasis Type="Italic"> Hypocrea jecorina</Emphasis> (<Emphasis Type="Italic"> Trichoderma reesei</Emphasis>) cellulase promoter<Emphasis Type="Italic"> cbh2</Emphasis> associated with cellulase induction

The 5 regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes –1 and –2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome –1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5 regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome –1.Communicated by C. A. M. J. J. van den Hondel     

Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.     

Drosophila CK2 phosphorylates Hairy and regulates its activity in vivo

Hairy is a repressor that regulates bristle patterning, and its loss elicits ectopic bristles (neural hyperplasia). However, it has remained unknown whether Hairy is regulated by phosphorylation. We describe here the interaction of protein kinase CK2 and Hairy. Hairy is robustly phosphorylated by the CK2-holoenzyme (CK2-HoloE) purified from Drosophila embryos, but weakly by the catalytic CK2α-subunit alone, suggesting that this interaction requires the regulatory CK2β-subunit. Consistent with this, Hairy preferentially forms a direct complex with CK2-HoloE. Importantly, we demonstrate genetic interactions between CK2 and hairy (h). Thus, flies trans-heterozygous for alleles of CK2α and h display neural hyperplasia akin to homozygous hypomorphic h alleles. In addition, we show that similar phenotypes are elicited in wild-type flies upon expression of RNAi constructs against CK2α/β, and that these defects are sensitive to h gene dosage. Together, these studies suggest that CK2 contributes to repression by Hairy.     

-Amino acid contents of mitochondria and some purple bacteria

The endosymbiont most likely to have given rise to mitochondria is an aerobic bacterium belonging to the α subdivision of the so-called purple bacteria such as Rickettsia, Bradythizobium and Agrobacterium [1 and 2]. Contents of the -enantiomers of serine, alanine, proline, glutamate and aspartate in rat liver whole mitochondria, mitochondrial outer membranes, inner membranes and matrix, soluble proteins and free amino acids were detected. These values for -amino acid content were compared with those in soluble proteins and free amino acids from the purple bacteria Paracoccus denitrificans, Pseudomonas aeruginosa and Escherichia coli, members, respectively of the α, β, and γ subdivisions, to find any similarity between mitochondria and these purple bacteria. A similarity was observed in protein -amino acid contents which were low (<1.5%, D-type/D-type+L-type) both in the membrane and soluble protein fractions from mitochondria and in soluble protein from bacteria. Oddly, substantial amounts of free -serine and free -aspartate (around 2%) were found for the first time in mitochondria. The contents of -serine and -aspartate were higher than those of -alanine, -proline and -glutamate. In purple bacteria, the concentration of -serine (<2%) was the lowest of the five amino acids examined, and those of -alanine (27–32%) and -glutamate (7–26%) were high. Therefore, no similarity was shown in the free -amino acid content between mitochondria and any of the three purple bacteria.     

Cell death induction in Giardia lamblia: Effect of beta-lapachone and starvation

Giardia lamblia is a protozoan that parasitizes the small intestine of vertebrates. It is a cause of intestinal infection and diarrhea and infects millions of people worldwide. This protozoan presents many characteristics common to eukaryotic cells but it lacks organelles found in most eukaryotes (e.g., peroxisomes, typical Golgi complex and mitochondria). Also it presents mitosomes, a relic organelle that appears to be a mitochondrial remnant. Cell death in Giardia was induced by the drug β-Lapachone and by starvation. Giardia behavior was followed by scanning, transmission and fluorescence microscopy, quantification of cell metabolism using MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide), changes in lipid rafts, using DiIC₁₆ and cholera toxin. Cell shrinkage, chromatin condensation, membrane blebbing and vacuolization provided ultrastructural evidence of apoptosis, whereas the myelinic figures in large vacuoles and LC-3 staining suggested an autophagic process. Lipids rafts were altered by drug treatment and co-localized with regions containing membrane blebbing. The treatment with β-Lap induced encystation. A search for sequence similarities in databases and protein alignments was carried out. Although Giardia is an amitochondrial organism, it presented some autophagic-like cell death characteristics and several, but not all, apoptotic characteristics, induced by β-Lapachone and starvation.