Distribution of Eg5 and TPX2 in mitosis: Insight from CRISPR tagged cells.

The mitotic spindle is a dynamic bipolar structure that mediates chromosome segregation in mitosis. In most organisms, spindle formation requires the action of kinesin-5 motor proteins that generate outward force on antiparallel microtubules to establish spindle bipolarity. Previous work has shown that Eg5 and TPX2, a spindle microtubule-associated protein that suppresses Eg5 motor activity, are enriched on parallel microtubules near spindle poles. This distribution is inconsistent with the requirement for Eg5-dependent force production during mitosis. To investigate this, we used CRISPR/Cas9 gene editing to tag Eg5 and TPX2 with EGFP and quantify protein distribution throughout mitosis. The results show that at metaphase both Eg5-EGFP and TPX2-EGFP are enriched toward spindle poles, but only TPX2-EGFP is enriched relative to microtubules. Eg5-EGFP and TPX2-EGFP show distinct localization patterns in anaphase, with Eg5-EGFP relocalizing to the midzone earlier than TPX2-EGFP. Analysis of spindles oriented at 90° to the coverslip confirmed that Eg5-EGFP was present on bridge microtubules in metaphase and anaphase; in contrast, TPX2 was not enriched, or enriched at later times, on these microtubules. Overall, TPX2 was present at 3.6X the level of Eg5 on the spindle and Eg5 was locally enriched at the prophase centrosome (~7×) compared to the whole cell. Our results show that using cells with fluorescent tags at the endogenous locus can provide novel insight into protein distribution during mitosis.

Structure and content of the Entamoeba histolytica genome.

The intestinal parasite Entamoeba histolytica is one of the first protists for which a draft genome sequence has been published. Although the genome is still incomplete, it is unlikely that many genes are missing from the list of those already identified. In this chapter we summarise the features of the genome as they are currently understood and provide previously unpublished analyses of many of the genes.

The structural gene for a phosphorus-repressible phosphate permease in Neurospora crassa can complement a mutation in positive regulatory gene nuc-1.

van+, a gene encoding a phosphorus-repressible phosphate permease, was isolated by its ability to complement nuc-1, a positive regulatory locus that normally regulates van+ expression. This was unexpected because the nuc-1 host already contained a resident van+ gene. Plasmids carrying van+ complemented a nuc-2 mutation as well. Probing of RNA from untransformed wild-type (nuc-1+) and constitutive (nuc-1c) strains by van+ probes indicated that levels of the van+ transcript were subject to control by nuc-1+. Probing of the same RNAs with a cosmid clone, containing approximately 15 kilobases of upstream and downstream DNA, revealed no other detectable phosphorus-regulated transcripts within this 40-kilobase region of the chromosome.

Regulation of adherence and virulence by the Entamoeba histolytica lectin cytoplasmic domain, which contains a beta2 integrin motif.

Killing of human cells by the parasite Entamoeba histolytica requires adherence via an amebic cell surface lectin. Lectin activity in the parasite is regulated by inside-out signaling. The lectin cytoplasmic domain has sequence identity with a region of the beta2 integrin cytoplasmic tail implicated in regulation of integrin-mediated adhesion. Intracellular expression of a fusion protein containing the cytoplasmic domain of the lectin has a dominant negative effect on extracellular lectin-mediated cell adherence. Mutation of the integrin-like sequence abrogates the dominant negative effect. Amebae expressing the dominant negative mutant are less virulent in an animal model of amebiasis. These results suggest that inside-out signaling via the lectin cytoplasmic domain may control the extracellular adhesive activity of the amebic lectin and provide in vivo demonstration of the lectin's role in virulence.

Nucleotide sequence of pho-4+, encoding a phosphate-repressible phosphate permease of Neurospora crassa.

The nucleotide (nt) sequence of the Neurospora crassa pho-4+ gene, which encodes a phosphate-repressible phosphate permease, has been determined. The gene specifies a protein of 590 amino acids (aa) and contains two introns. Two RNA transcripts of 3.3 and 2.4 kb have been identified, and transcription start points (tsp) and termination sites and/or processing sites have been located. The 3.3-kb message is initiated about 890 nt upstream from the tsp for the 2.4-kb transcript. A hydropathy profile of the aa sequence suggests ten to twelve membrane-spanning helices with a large hydrophilic domain between the eighth and ninth helices. This model for the predicted secondary structure of the protein is very similar to models proposed for other sequenced integral membrane proteins from both prokaryotes and eukaryotes. Since very few permease-encoding genes of eukaryotes have been examined in molecular detail, it will be of interest to compare the sequence of pho-4+ with those encoding other anion transport proteins, as they become available.

A tetracycline-inducible gene expression system in Entamoeba histolytica.

We have developed an episomal inducible gene expression system in Entamoeba histolytica based on the TetR repressor. The tetR gene was placed under control of 5' and 3' ferredoxin (fdx) regulatory sequences on a plasmid encoding the hygromycin resistance gene directed by 5' and 3' hgl sequences. The reporter luciferase constructs were introduced on a second episome bearing the neomycin resistance gene controlled by 5' and 3' actin sequences. The reporter constructs were driven by the hgl5 promoter in which the tetO sequence was introduced. We found that the optimal tetO location for induction by tetracycline was +4 from the start of transcription. The efficiency of repression and the induction ratio could be improved by increasing hygromycin levels, presumably by increasing tetR plasmid levels. Under these conditions, maximal induction of reporter luciferase could be effected with 5 micrograms/ml tetracycline in 18 h. This system permits regulated expression of the reporter gene over two orders of magnitude and should be useful in the analysis of gene function.

Analysis of the gene family encoding the Entamoeba histolytica galactose-specific adhesin 170-kDa subunit.

The 170-kDa or heavy subunit of the galactose binding adhesin of Entamoeba histolytica is seminal in target cell binding and lysis. To determine the existence and complexity of the 170-kDa subunit gene family, hgl, an amebic genomic library in lambda phage was hybridized with DNA fragments from the 5' or 3' ends of hgl1. Termini from three distinct heavy subunit genes were identified including hgl1, hgl2, and a third, unreported gene designated hgl3. The open reading frame of hgl3 was sequenced in its entirety. Non-stringent hybridization of a genomic Southern blot with heavy subunit specific DNA labeled only those bands predicted by hgl1-3. The amino acid sequence of hgl3 was 95.2% identical to hgl1 and 89.4% identical to hgl2. All 97 cysteine residues present in the heavy subunit were conserved in hgl1-3. Analysis of amebic RNA showed that all three heavy subunit genes were expressed in the amebae and that hgl message became less abundant as the amebae entered a stationary growth phase.

Upstream regulatory elements controlling expression of the Entamoeba histolytica lectin.

Entamoeba histolytica genomic organization and putative promoter elements appear to be distinct from both metazoan and better characterized protozoan organisms. The recent development of DNA-mediated transfection for E. histolytica enabled characterization of cis-acting promoter elements required for gene expression. A deletion and replacement analysis was conducted on the promoter of an E. histolytica gene encoding the heavy subunit of the N-acetyl-beta-D-galactosamine-specific adhesin (hgl5). Deletion of the DNA from -1000 bases to -272 bases upstream from the start of transcription of hgl5 did not decrease reporter gene expression. Subsequent nested deletions and 10-bp replacement mutagenesis identified four positive upstream regulatory elements between bases -219 to -200, -189 to -160, -69 to -60, and -49 to -40. A negative upstream regulatory element between bases -89 to -80 was conserved upstream of three other E. histolytica genes. Mutation of the previously unidentified 'GAAC' element conserved within the putative core promoter decreased reporter gene expression by 75%. Site directed mutagenesis of the putative TATA element decreased reporter gene expression by greater than 50%, while mutation of the putative initiator element resulted in a more modest decrease. This analysis suggests that E. histolytica promoters are unlike other protozoan promoters, with AT-rich upstream regulatory elements, a non-consensus TATA element, the "GAAC' element, and an unusual initiator element.

Seroepidemiology of Entamoeba histolytica in a slum in northeastern Brazil.

Infection with the human pathogenic parasite Entamoeba histolytica has not been well-characterized in northeastern Brazil. In this study, the prevalence of E. histolytica infection in a slum in northeastern Brazil was assayed using an enzyme-linked immunosorbent assay (ELISA) for antibodies against the galactose/N-acetyl-D-galactosamine (Gal/GalNAc)-inhibitable adherence lectin of E. histolytica. Sera from a total of 335 individuals were examined for anti-Gal/GalNAc lectin antibodies. The overall seropositivity was 24.7%; 29.4% of females and 19.4% of males were positive. Among different age groups there was a peak of 40% positivity in the 6-14-year-old age group. There was also familial clustering of seropositivity. To examine colonization, stool samples from 155 people were examined microscopically for the presence of the parasite. Fourteen of 155 stools (9.0%) were identified as containing E. histolytica or nonpathogenic E. dispar. These 14 positive stools were analyzed with an ELISA that detects Gal/GalNAc lectin antigen and can distinguish between E. histolytica and E. dispar. Four stools (29%) were positive for E. histolytica and the remaining 10 were identified as E. dispar-positive. Although the overall colonization rate by microscopy was only 9%, with a third identified as E. histolytica, up to 40% of older children develop serologic evidence of having experienced pathogenic E. histolytica infection. The results of this study demonstrate that this community in northeastern Brazil is highly endemic for E. histolytica with infection rates similar to other developing nations.

Household epidemiology of Entamoeba histolytica infection in an urban community in northeastern Brazil.

The natural history of infection with Entamoeba histolytica was studied in 2 slum communities in northeastern Brazil. Twenty-eight index patients colonized with E. histolytica were identified. Three stool specimens from the index patients and their household contacts were gathered over a 45-day period and tested for E. histolytica by means of a specific enzyme-linked immunosorbent assay-based detection kit. The detection kit is an antigen capture assay that has been shown to be highly specific for E. histolytica and does not detect nonpathogenic Entamoeba dispar or other enteric organisms. Blood samples were also collected at the start of the study, at 45 days, and at 6 months and analyzed for E. histolytica-specific antibody. High rates of colonization were seen in the family units. Colonization was self-limited, with 85% of colonized patients clearing their infections within 45 days. Reinfection appeared to be low during this time; however, previous seropositivity did not prevent colonization.

Children's concepts of the family.

The past decade has witnessed increased research emphasis on children's developing conceptions of their social environment. In the present study, 64 children in grades 1 through 6 were interviewed to examine age-related changes in their understanding of the concept of family. The study also explored whether children's developing conceptions of the family were mediated by their own family structures. Results indicated five levels of family concept understanding that reflected a growing (i.e., age-related) facility in children's application of kinship criteria across traditional and nontraditional family groupings. However, the sophistication of children's understanding of the family was not related to the structure of their own families. These findings suggest that children's level of cognitive development is more important for their comprehension of the family than the specific experiences derived from living within their own unique family. Implications of these findings for mental health professionals who work with families in transition are discussed.

An investigation of systemic conceptualizations of parent-child coalitions and symptom change.

Two important theoretical assumptions of family therapy were examined: (a) Child behavior problems are associated with cross-generational coalitions, and (b) treatment of these coalitions using family therapy leads to decreases in individual symptomatology. Ss were 45 delinquent adolescents assigned to multisystemic therapy (MST) or individual therapy and 16 well-adjusted adolescents. Pretreatment and posttreatment assessments included measures of observed family relations and self-reported symptoms. Cross-generational coalitions were more evident in families of delinquents vs. families of well-adjusted adolescents. In addition, changes in adolescent and paternal symptoms in the MST group were linked with changes in marital relations. Implications for systemic conceptualizations of symptom maintenance and change are highlighted.

Multisystemic treatment of serious juvenile offenders: long-term prevention of criminality and violence.

This article examined the long-term effects of multisystemic therapy (MST) vs. individual therapy (IT) on the prevention of criminal behavior and violent offending among 176 juvenile offenders at high risk for committing additional serious crimes. Results from multiagent, multimethod assessment batteries conducted before and after treatment showed that MST was more effective than IT in improving key family correlates of antisocial behavior and in ameliorating adjustment problems in individual family members. Moreover, results from a 4-year follow-up of rearrest data showed that MST was more effective than IT in preventing future criminal behavior, including violent offending. The implications of such findings for the design of violence prevention programs are discussed.

Involvement of the actin cytoskeleton and p21rho-family GTPases in the pathogenesis of the human protozoan parasite Entamoeba histolytica.

It has been estimated that infection with the enteric protozoan parasite Entamoeba histolytica kills more than 50,000 people a year. Central to the pathogenesis of this organism is its ability to directly lyse host cells and cause tissue destruction. Amebic lesions show evidence of cell lysis, tissue necrosis, and damage to the extracellular matrix. The specific molecular mechanisms by which these events are initiated, transmitted, and effected are just beginning to be uncovered. In this article we review what is known about host cell adherence and contact-dependent cytolysis. We cover the involvement of the actin cytoskeleton and small GTP-binding proteins of the p21rho-family in the process of cell killing and phagocytosis, and also look at how amebic interactions with molecules of the extracellular matrix contribute to its cytopathic effects.

Child dissociation and the family context.

Forty boys and their parents completed measures of dissociation, state anxiety, and perceptions of parental behaviors. Mothers completed a behavior problems checklist about their sons. The results indicated a correspondence between fathers' and sons' dissociation scores. In addition, links were found between parental dissociation, parental inconsistency and rejection, and child dissociation. The findings are discussed in the context of several recently proposed models of the etiology of child dissociative disorders.

Pediatricians' perceptions of their behavioral and developmental training.

Eighty graduates of a pediatric residency that required training in behavior and development completed a survey of their perceptions regarding the quality of this training, the utility of behavior and development for modern pediatric practice, their competence in applying behavior and development knowledge, and their desire to pursue additional training in behavior and development. Surveys were completed 1 to 5 years (mean = 3.4 years) after graduation from the residency program. Graduates consistently rated child development and anticipatory guidance/counseling as the areas that were best taught, most useful, and in which they felt the most competent. The highest ratings of behavioral/developmental pediatrics (BDP) competence and usefulness were from graduates who received additional BDP training after residency. However, a large percentage of graduates did not endorse the usefulness of several dimensions of behavior and development that have been considered integral components of training. Implications and limitations of the present study are discussed.

Entamoeba histolytica and Entamoeba dispar infections as detected by monoclonal antibody in an urban slum in Fortaleza, Northeastern Brazil.

In this study the authors used the Elisa-based antigen detection tests that distinguish E. histolytica from E. dispar to examine the prevalence of E. histolytica infection in individuals from an urban slum in Fortaleza, Northeastern, Brazil. This test has a sensitivity and specificity that is comparable to PCR and isoenzyme analysis, which is the gold standard. Single stools samples were obtained from 735 individuals. The prevalence of E. histolytica infection was 14.9% (110/735) and 25.4%(187/735) for E. dispar-E. histolytica complex. The most affected age group for E. histolytica /E. histolytica-E. dispar infection was the 1-5 year olds but there was no remarkable decrease with age. There was no significant difference in colonization rates between males and females. The results from this survey demonstrate that E. histolytica is highly prevalent in the Community studied. Furthermore, it offers promise for the antigen detection test as a sensitive and technically simple tool for detecting E. histolytica infection in the field.

The effects of light, temperature, trance length, and time of day on hypnotic depth.

We evaluated predictions derived from the ultradian theory of hypnosis regarding the effects of temperature, light, trance length, and time of day on reported trance depth in 95 college undergraduates. Temperature and light showed no relation to trance depth. However, as predicted by ultradian theory, subjects who were kept in trance for 15 minutes reported greater trance depth than those who experienced a 5-minute trance. Time of day interacted with subjects' self-reported time of peak alertness in the following way: Subjects who reported greatest alertness in the morning achieved greater trance depth in the morning than in the evening, whereas those subjects who reported greater evening alertness reported deeper trance in the evening than in the morning. This latter finding was inconsistent with ultradian theory and prior research. Alternative explanations for this finding are discussed. Overall, the results from the present study do not provide strong support for Rossi's ultradian theory of hypnosis.

A critical review of psychotherapy outcome studies with adolescents: 1978-1988.

Although interest in identifying effective psychotherapies for adolescent mental health problems has increased dramatically in the last decade, there have been few attempts to summarize and integrate the recent empirical literature on psychotherapy outcomes with adolescents. The present paper reviews the methods and findings of adolescent psychotherapy outcome studies published from 1978 to 1988. Evaluations of individual psychotherapy with adolescents generally reveal positive short-term outcomes, but long-term outcomes are still to be determined. Evaluations of systemic therapies (especially family therapy) with adolescents often reveal positive outcomes that are maintained over extended follow-up periods. Recommendations are presented for improving the methodology of future psychotherapy outcome studies with adolescents.

The relationship between parental controlling behavior and perceptions of control of preadolescent children and adolescents.

Previous research has suggested that a noncontrolling, independence-encouraging parenting style is correlated with children's having an internal locus of control. In the present study, children's and adolescents' reports of parent behaviors were used. Parental acceptance and child-centeredness were found to be related to more internal control beliefs in both preadolescent children and adolescents. Parental controlling behavior, however, was related to more internal control beliefs in preadolescent children and more external control beliefs in adolescents. The relationships among structure, parent controlling behavior, and the age and developmental level of children are discussed.