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PubMed1997

Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form.

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

Adenomatous Polyposis Coli↗
PubMed2006

Investigation of the electron transfer site of p-benzoquinone in isolated photosystem II particles and thylakoid membranes using alpha- and beta-cyclodextrins.

The electron transfer sites of p-benzoquinone (pBQ) and 2,6-dichloro-p-benzoquinone (DCBQ) were investigated in thylakoid membranes and isolated photosystem II (PSII) particles from barley (Hordeum vulgare) using alpha- and beta-cyclodextrins (CD) at concentrations up to 16 mM. In CD-treated thylakoid membranes incubated with DCBQ the electron transport through PSII, estimated as oxygen evolution (OE), is largely enhanced according to a S-shaped (sigmoidal) dose-response curve displaying a sharp inflection point, or transition. The maxima percent OE enhancement at cyclodextrin concentrations above 14 mM are about 100% (alpha-CD) and 190% (beta-CD). On the contrary, in thylakoid membrane preparations incubated with pBQ as electron acceptor one observes an OE inhibition of about 30% which might result from the depletion of the thylakoid membrane of its plastoquinone content. It was also found that in isolated PSII particles incubated with either pBQ or DCBQ the cyclodextrins induce only a small OE enhancement. Moreover, the observation in CD-treated thylakoid membranes incubated with pBQ of a residual, non-inhibited oxygen-evolving activity of about 70% puts a twofold question. That is, either the plastoquinone depletion was not complete, or, pBQ binds to electron acceptor sites of different nature. From this and data published in the literature, it is concluded that in the thylakoid membrane (i) DCBQ binds to Q(B), as is generally accepted, and (ii) pBQ binds to the plastoquinol molecules in the PQ pool and most likely also to Q(B), thereby in accord with Satoh et al.'s model [K. Satoh, M. Ohhashi, Y. Kashino, H. Koike, Plant Cell Physiol. 36 (1995) 597-605]. An attractive alternative hypothesis is the direct interaction of pBQ with the non-haem Fe(2+) between Q(A) and Q(B).

Benzoquinones↗
PubMed2002

Transimmunization and the evolution of extracorporeal photochemotherapy.

We are now aware that extracorporeal photopheresis (ECP) - in which a patient's leukocytes are isolated, passed through an ultrathin clear plastic plate, and exposed to 8-methoxypsoralen (8-MOP) and ultraviolet A light prior to reinfusion - is a simple and efficient dendritic cell (DC) therapy and the first FDA approved selective immunotherapy for cancer. DCs, as the most effective antigen presenting cells (APCs), are central to many ongoing efforts to stimulate immune responses to cancer cells. Moreover, ECP has not only demonstrated efficacy in the treatment of a T cell malignancy--namely cutaneous T-cell lymphoma (CTCL)--but also in treatment of oligoclonal T-cell-mediated diseases such as graft-versus-host-disease (GVHD) and organ transplant rejection. Recent advances in the understanding of DC/T-cell interactions provide insight into how ECP-induced DCs (EI-DCs) can be utilized to stimulate specific T-cell (i.e. anti-tumor) responses, or down-regulate a pre-existing potent T-cell response. The mechanism of this apparent paradox of EI-DC functionality is likely dependent on several fundamental principles: (1) the status of existing in vivo T-cell reactions, (2) the temporal stage of EI-DC differentiation, and (3) the affinity of the available repertoire of T-cell receptors (TCRs) for the antigen(s) in question. Further investigation into DC/T-cell interactions will help to shape the future of ECP and the ability to optimize this therapy for the desired immune effect. To this end, we are developing and testing Transimmunization to replace conventional ECP.

Dendritic Cells↗
PubMed2002

Human l1 retrotransposition is associated with genetic instability in vivo.

Retrotransposons have shaped eukaryotic genomes for millions of years. To analyze the consequences of human L1 retrotransposition, we developed a genetic system to recover many new L1 insertions in somatic cells. Forty-two de novo integrants were recovered that faithfully mimic many aspects of L1s that accumulated since the primate radiation. Their structures experimentally demonstrate an association between L1 retrotransposition and various forms of genetic instability. Numerous L1 element inversions, extra nucleotide insertions, exon deletions, a chromosomal inversion, and flanking sequence comobilization (called 5' transduction) were identified. In a striking number of integrants, short identical sequences were shared between the donor and the target site's 3' end, suggesting a mechanistic model that helps explain the structure of L1 insertions.

5' Flanking Region↗
PubMed2002

Embryonic development in the primitive bilaterian Neochildia fusca: normal morphogenesis and isolation of POU genes Brn-1 and Brn-3.

Neochildia fusca is a member of the taxon Acoela, a group of flatworms that, according to some recent molecular phylogenetic analyses, are distinct from other flatworms and constitute a basal branch with a sister taxon relationship to the rest of the Bilateria. In this paper, we analyze early neural development in this species and report the sequence and expression of two Pit-Oct-Unc (POU) genes, NeocBrn-1 and NeocBrn-3. Homologs of these highly conserved genes play a role in neural fate determination in vertebrates, Drosophila and Caenorhabditis elegans. Acoels, including Neochildia, have a unique invariant pattern of early cleavage called duet spiral cleavage. In subsequent cell divisions descendants of the first three micromere duets form an outer layer of epidermal and neural progenitors surrounding the meso/endoderm progenitors, which are themselves descended from the macromere duet 4A, B and the micromere duet 4a, b. Organ formation begins at mid-embryonic stages with the epidermal primordium adopting a ciliated epithelial shape. Sub-epidermally, a bilaterally symmetric brain primordium can be seen at the anterior pole. Laterally and posteriorly, myoblasts form a thin layer underneath the epidermis. In late embryos and juveniles of Neochildia, the brain is formed by a 3-4 cell-diameter-thick layer of neurons forming a cortex surrounding a neuropile that is relatively free of cell bodies. A highly regular "orthogonal" array of muscle fibers penetrates the brain. We have isolated and partially sequenced homologs of the vertebrate Brn-1 and Brn-3 genes, which we call NeocBrn-1 and NeocBrn-3, respectively. These sequences contain and span portions of the POU-specific domain and a homeodomain, and are sequence similar to their respective homologs in vertebrates and Drosophila. RT-PCR reveals that NeocBrn-1 and NeocBrn-3 are expressed from mid-embryonic to adult stages. Whole-mount in situ hybridization shows expression of both genes in distinct subsets of nerve cells in juvenile and adult worms. NeocBrn-1 also appears in a subset of intra-epidermal gland cells. These observations are an initial step towards reconstructing the neural development of a key group of bilaterians, the Acoela. These flatworms, by virtue of their distinct morphology, development and phylogenetically basal placement, are likely to provide key insights into the interpretation of the evolution of metazoan neural architecture.

Amino Acid Sequence↗
PubMed2003

Effects of steroid hormones on five functional parameters of Tetrahymena: evolutionary conclusions.

The unicellular Tetrahymena pyriformis was studied for chemotaxis, chemotactic selection, phagocytosis, growth and body shape changes in the presence of water soluble (beta-cyclodextrin-coupled) steroid hormones (testosterone, estradiol, progesterone, hydrocortisone and dexamethasone). Testosterone was chemoattractant over a wide range of concentrations, while progesterone and dexamethasone were active only at one concentration (10(-5) and 10(-6) mg ml(-1) respectively) and were either neutral or repellent at other concentrations. Hydrocortisone and estradiol were unambiguously chemorepellent. Chemotactic selection enhanced the effect of testosterone and estradiol, while in the case of hydrocortisone the action was reversed. The other parameters were mildly influenced by the steroid hormones. The results call attention to the fine molecular recognition capacity of Tetrahymena and to the possible rapid effects of steroid hormones at membrane receptors at a very low evolutionary eukaryotic level.

Animals↗
PubMed1998

Shaping animal body plans in development and evolution by modulation of Hox expression patterns.

Most animals exhibit distinctive and diverse morphological features on their anterior-posterior body axis. However, underneath the variation in design and developmental strategies lies a shared ancient structural blueprint that is based on the expression patterns of Hox genes. Both the establishment and maintenance of the spatial and temporal distribution of Hox transcripts play an important role in determining axial pattern. The study of many animal systems, both vertebrate and invertebrate, suggests that the mechanisms used to establish Hox transcription are nearly as diverse as the body plans they specify. The strategies for maintenance of Hox expression pattern seem more conserved among different phyla, and rely on the action of Pc and trx group genes as well as auto- and cross-regulation among Hox genes. In mice, the sharing of regulatory elements coupled with auto- and cross-regulation could explain the conservation of the clustered arrangement of Hox genes. In contrast, fly Hox genes seem to have evolved insulators or boundary elements to avoid sharing regulatory regions. Differences in Hox transcription patterns can be correlated with morphological modifications in different species, and it seems likely that evolutionary variation of Hox cis-regulatory elements has played a major role in the emergence of novel body plans in different taxa of the animal kingdom.

Animals↗
PubMedDate not supplied

Life cycle of Trypanosoma cruzi (Y strain) in mice.

Since 1958, we have studied experimental Chagas' disease (CD) by subcutaneous inoculation of 1,000 blood forms of Trypanosoma cruzi (Y strain) in Balb/C. mice. Evolution of parasitemia remained constant, beginning on the 5th and 6th day of the disease, increasing progressively, achieving a maximum on about the 30th day. After another month, only a few forms were present, and they disappeared from the circulation after the third month, as determined from direct examination of slides and the use of a Neubauer Counting Chamber. These events coincided with the appearance of amastigote nests in the tissues (especially the cardiac ones), starting the first week, and following the Gauss parasitemia curve, but they were not in parallel until the chronic stage. In 1997, we began to note the following changes: Parasites appeared in the circulation during the first week and disappeared starting on the 7th day, and there was a coincident absence of the amastigote nests in the tissues. A careful study verified that young forms in the evolutionary cycle of T. cruzi (epi + amastigotes) began to appear alongside the trypomastigotes in the circulation on the 5th and 7th post-inoculation day. At the same time, rounded, oval, and spindle shapes were seen circulating through the capillaries and sinusoids of the tissues, principally of the hematopoietic organs. Stasis occurs because the diameter of the circulating parasites is greater than the vessels, and this makes them more visible. Examination of the sternal bone marrow revealed young cells with elongated forms and others truncated in the shape of a "C" occupying the internal surface of the blood cells that had empty central portions (erythrocytes?). We hypothesize that there could be a loss of virulence or mutation of the Y strain of Trypanosoma cruzi.

Animals↗
PubMed2001

Morphological and molecular evidence that Culex nigripalpus baculovirus is an unusual member of the family Baculoviridae.

We present evidence that a newly discovered mosquito virus from Culex nigripalpus is an unusual member of the family BACULOVIRIDAE: Development of this virus was restricted to nuclei of midgut epithelial cells in the gastric caeca and posterior stomach. The globular occlusion bodies were not enveloped, measured around 400 nm in diameter, occurred exclusively in nuclei of infected cells and typically contained four, sometimes up to eight, virions. The developmental sequence involved two virion phenotypes: an occluded form (ODV) that initiated infection in the midgut epithelial cells, and a budded form that spread the infection in the midgut. Each ODV contained one rod-shaped enveloped nucleocapsid (40x200 nm). The double-stranded DNA genome was approximately 105-110 kbp with an estimated GC content of 52%. We have sequenced approximately one-third of the genome and detected 96 putative ORFs of 50 amino acids or more including several genes considered to be unique to baculoviruses. Phylogenetic analysis of the amino acid sequences of DNApol and p74 placed this virus in a separate clade from the genera NUCLEOPOLYHEDROVIRUS: and GRANULOVIRUS: We provisionally assign this virus in the genus NUCLEOPOLYHEDROVIRUS:, henceforth abbreviated as CuniNPV (for Culex nigripalpus nucleopolyhedrovirus), and suggest that, awaiting additional data to clarify its taxonomic status, it may be a member of a new genus within the family BACULOVIRIDAE:

Amino Acid Motifs↗
PubMed1999

Helianthus tuberosus lectin reveals a widespread scaffold for mannose-binding lectins.

BACKGROUND: Heltuba, a tuber lectin from the Jerusalem artichoke Helianthus tuberosus, belongs to the mannose-binding subgroup of the family of jacalin-related plant lectins. Heltuba is highly specific for the disaccharides Man alpha 1-3Man or Man alpha 1-2Man, two carbohydrates that are particularly abundant in the glycoconjugates exposed on the surface of viruses, bacteria and fungi, and on the epithelial cells along the gastrointestinal tract of lower animals. Heltuba is therefore a good candidate as a defense protein against plant pathogens or predators. RESULTS: The 2.0 A resolution structure of Heltuba exhibits a threefold symmetric beta-prism fold made up of three four-stranded beta sheets. The crystal structures of Heltuba in complex with Man alpha 1-3Man and Man alpha 1-2Man, solved at 2.35 A and 2.45 A resolution respectively, reveal the carbohydrate-binding site and the residues required for the specificity towards alpha 1-3 or alpha 1-2 mannose linkages. In addition, the crystal packing reveals a remarkable, donut-shaped, octahedral assembly of subunits with the mannose moieties at the periphery, suggesting possible cross-linking interactions with branched oligomannosides. CONCLUSIONS: The structure of Heltuba, which is the prototype for an extended family of mannose-binding agglutinins, shares the carbohydrate-binding site and beta-prism topology of its galactose-binding counterparts jacalin and Maclura pomifera lectin. However, the beta-prism elements recruited to form the octameric interface of Heltuba, and the strategy used to forge the mannose-binding site, are unique and markedly dissimilar to those described for jacalin. The present structure highlights a hitherto unrecognized adaptability of the beta-prism building block in the evolution of plant proteins.

Agglutinins↗
PubMed1998

Biochemical and phylogenetic characterization of the dUTPase from the archaeal virus SIRV.

The derived amino acid sequence from a 474-base pair open reading frame in the genome of the Sulfolobus islandicus rod-shaped virus SIRV shows striking similarity to bacterial dCTP deaminases and to dUTPases from eukaryotes, bacteria, Poxviridae, and Retroviridae. The putative gene was expressed in Escherichia coli, and dUTPase activity of the recombinant enzyme was demonstrated by hydrolysis of dUTP to dUMP. Deamination of dCTP by the enzyme was not detected. Phylogenetic analysis based on amino acid sequences of the characterized enzyme and its homologues showed that the dUTPase-encoding dut genes and the dCTP deaminase-encoding dcd genes constitute a paralogous gene family. This report is the first identification and functional characterization of an archaeal dUTPase and the first phylogeny derived for the dcd-dut gene family.

Amino Acid Sequence↗
PubMed2003

Histologic findings in left ventricle papillary muscle arteries from human hearts.

We describe original histologic findings of left ventricle papillary muscle (LVPM) arteries in people under 30 years of age. We examined 666 samples taken from the tip, mid-portion and base of papillary muscles in 56 males and 55 females, as well as several samples from the rest of the left ventricle. The number of smooth muscle cells (SMC) in the tunica media of the LVPM arteries led us to divide the samples examined into three groups: (i) group 1, 355 samples (53%) with a normal number of SMC and a normal lumen (the number of group 1 samples increased from the tip (21%) to the base (47%)); (ii) group 2, 252 samples (38%) with a mild to moderately increased number of SMC (the number of these samples decreased from the tip (44%) to the base (22%)); and (iii) group 3, 59 samples (9%) with abundant SMC that were more than twofold greater in size and number of normal arteries, in contrast with the other two groups. The shape of the SMC in group 3 samples was round and the extremely narrow, centrally located lumen of these SMCE had a round or oval shape. These changes were restricted only to papillary muscle arteries and the number of group 3 samples decreased from the tip (63%) to the mid-portion (37%). No inflammatory reaction or chronic ischemic changes were found in the LVPM arteries and surrounding area. The SMC changes in groups 2 and 3 were found in subjects aged more than 2 months. These findings will provide anatomists, cardiologists, pathologists and physiologists with valuable knowledge and will trigger further investigation into the etiology of the structural changes observed and their evolution with age.

Adolescent↗
PubMed2002

The cell morphogenesis gene ANGUSTIFOLIA encodes a CtBP/BARS-like protein and is involved in the control of the microtubule cytoskeleton.

The ANGUSTIFOLIA (AN) gene is required for leaf hair (trichome) branching and is also involved in polarized expansion underlying organ shape. Here we show that the AN gene encodes a C-terminal binding proteins/brefeldin A ADP-ribosylated substrates (CtBP/BARS) related protein. AN is expressed at low levels in all organs and the AN protein is localized in the cytoplasm. In an mutant trichomes, the organization of the actin cytoskeleton is normal but the distribution of microtubules is aberrant. A role of AN in the control of the microtubule cytoskeleton is further supported by the finding that AN genetically and physically interacts with ZWICHEL, a kinesin motor molecule involved in trichome branching. Our data suggest that CtBP/BARS-like protein function in plants is directly associated with the microtubule cytoskeleton.

Alcohol Oxidoreductases↗
PubMed2001

Does nonneutral evolution shape observed patterns of DNA variation in animal mitochondrial genomes?

Early studies of animal mitochondrial DNA (mtDNA) assumed that nucleotide sequence variation was neutral. Recent analyses of sequences from a variety of taxa have brought the validity of this assumption into question. Here we review analytical methods used to test for neutrality and evidence for nonneutral evolution of animal mtDNA. Evaluations of mitochondrial haplotypes in different nuclear backgrounds identified differences in performance, typically favoring coevolved mitochondrial and nuclear genomes. Experimental manipulations also indicated that certain haplotypes have an advantage over others; however, biotic and historical effects and cyto-nuclear interactions make it difficult to assess the relative importance of nonneutral factors. Statistical analyses of sequences have been used to argue for nonneutrality of mtDNA; however, rejection of neutral patterns in the published literature is common but not predominant. Patterns of replacement and synonymous substitutions within and between species identified a trend toward an excess of replacement mutations within species. This pattern has been viewed as support for the existence of mildly deleterious mutations within species; however, other alternative explanations that can produce similar patterns cannot be eliminated.

Animals↗
PubMed2003

The evolution of transcriptional regulation in eukaryotes.

Gene expression is central to the genotype-phenotype relationship in all organisms, and it is an important component of the genetic basis for evolutionary change in diverse aspects of phenotype. However, the evolution of transcriptional regulation remains understudied and poorly understood. Here we review the evolutionary dynamics of promoter, or cis-regulatory, sequences and the evolutionary mechanisms that shape them. Existing evidence indicates that populations harbor extensive genetic variation in promoter sequences, that a substantial fraction of this variation has consequences for both biochemical and organismal phenotype, and that some of this functional variation is sorted by selection. As with protein-coding sequences, rates and patterns of promoter sequence evolution differ considerably among loci and among clades for reasons that are not well understood. Studying the evolution of transcriptional regulation poses empirical and conceptual challenges beyond those typically encountered in analyses of coding sequence evolution: promoter organization is much less regular than that of coding sequences, and sequences required for the transcription of each locus reside at multiple other loci in the genome. Because of the strong context-dependence of transcriptional regulation, sequence inspection alone provides limited information about promoter function. Understanding the functional consequences of sequence differences among promoters generally requires biochemical and in vivo functional assays. Despite these challenges, important insights have already been gained into the evolution of transcriptional regulation, and the pace of discovery is accelerating.

Animals↗
PubMed1999

Genetic analyses of visual pigments of the pigeon (Columba livia).

We isolated five classes of retinal opsin genes rh1(Cl), rh2(Cl), sws1(Cl), sws2(Cl), and lws(Cl) from the pigeon; these encode RH1(Cl), RH2(Cl), SWS1(Cl), SWS2(Cl), and LWS(Cl) opsins, respectively. Upon binding to 11-cis-retinal, these opsins regenerate the corresponding photosensitive molecules, visual pigments. The absorbance spectra of visual pigments have a broad bell shape with the peak, being called lambdamax. Previously, the SWS1(Cl) opsin cDNA was isolated from the pigeon retinal RNA, expressed in cultured COS1 cells, reconstituted with 11-cis-retinal, and the lambdamax of the resulting SWS1(Cl) pigment was shown to be 393 nm. In this article, using the same methods, the lambdamax values of RH1(Cl), RH2(Cl), SWS2(Cl), and LWS(Cl) pigments were determined to be 502, 503, 448, and 559 nm, respectively. The pigeon is also known for its UV vision, detecting light at 320-380 nm. Being the only pigments that absorb light below 400 nm, the SWS1(Cl) pigments must mediate its UV vision. We also determined that a nonretinal P(Cl) pigment in the pineal gland of the pigeon has a lambdamax value at 481 nm.

Amino Acid Sequence↗
PubMed2003

Hearing in hooktip moths (Drepanidae: Lepidoptera).

This study presents anatomical and physiological evidence for a sense of hearing in hooktip moths (Drepanoidea). Two example species, Drepana arcuata and Watsonalla uncinula, were examined. The abdominal ears of drepanids are structurally unique compared to those of other Lepidoptera and other insects, by having an internal tympanal membrane, and auditory sensilla embedded within the membrane. The tympanum is formed by two thin tracheal walls that stretch across a teardrop-shaped opening between dorsal and ventral air chambers in the first abdominal segment. There are four sensory organs (scolopidia) embedded separately between the tympanal membrane layers: two larger lateral scolopidia within the tympanal area, and two smaller scolopidia at the medial margin of the tympanal frame. Sound is thought to reach the tympanal membrane through two external membranes that connect indirectly to the dorsal chamber. The ear is tuned to ultrasonic frequencies between 30 and 65 kHz, with a best threshold of around 52 dB SPL at 40 kHz, and no apparent difference between genders. Thus, drepanid hearing resembles that of other moths, indicating that the main function is bat detection. Two sensory cells are excited by sound stimuli. Those two cells differ in threshold by approximately 19 dB. The morphology of the ear suggests that the two larger scolopidia function as auditory sensilla; the two smaller scolopidia, located near the tympanal frame, were not excited by sound. We present a biophysical model to explain the possible functional organization of this unique tympanal ear.

Animals↗
PubMed2000

Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS.

Almost all aphid species (Homoptera, Insecta) have 60-80 huge cells called bacteriocytes, within which are round-shaped bacteria that are designated Buchnera. These bacteria are maternally transmitted to eggs and embryos through host generations, and the mutualism between the host and the bacteria is so obligate that neither can reproduce independently. Buchnera is a close relative of Escherichia coli, but it contains more than 100 genomic copies per cell, and its genome size is only a seventh of that of E. coli. Here we report the complete genome sequence of Buchnera sp. strain APS, which is composed of one 640,681-base-pair chromosome and two small plasmids. There are genes for the biosyntheses of amino acids essential for the hosts in the genome, but those for non-essential amino acids are missing, indicating complementarity and syntrophy between the host and the symbiont. In addition, Buchnera lacks genes for the biosynthesis of cell-surface components, including lipopolysaccharides and phospholipids, regulator genes and genes involved in defence of the cell. These results indicate that Buchnera is completely symbiotic and viable only in its limited niche, the bacteriocyte.

Aerobiosis↗