Evidence from disruption of the lmcpb gene array of Leishmania mexicana that cysteine proteinases are virulence factors.

The mammalian form of the protozoan parasite Leishmania mexicana contains high activity of a cysteine proteinase (LmCPb) encoded on a tandem array of 19 genes (lmcpb). Homozygous null mutants for lmcpb have been produced by targeted gene disruption. All life-cycle stages of the mutant can be cultured in vitro, demonstrating that the gene is not essential for growth or differentiation of the parasite. However, the mutant exhibits a marked phenotype affecting virulence-- its infectivity to macrophages is reduced by 80%. The mutants are as efficient as wild-type parasites in invading macrophages but they only survive in a small proportion of the cells. However, those parasites that successfully infect these macrophages grow normally. Despite their reduced virulence, the mutants are still able to produce subcutaneous lesions in mice, albeit at a slower rate than wild-type parasites. The product of a single copy of lmcpb re-expressed in the null mutant was enzymatically active and restored infectivity toward macrophages to wild-type levels. Double null mutants created for lmcpb and lmcpa (another cathepsin L-like cysteine proteinase) have a similar phenotype to the lmcpb null mutant, showing that LmCPa does not compensate for the loss of LmCPb.

MyoD forms micelles which can dissociate to form heterodimers with E47: implications of micellization on function.

MyoD is a member of a family of DNA-binding transcription factors that contain a helix-loop-helix (HLH) region involved in protein-protein interactions. In addition to self-association and DNA binding, MyoD associates with a number of other HLH-containing proteins, thereby modulating the strength and specificity of its DNA binding. Here, we examine the interactions of full-length MyoD with itself and with an HLH-containing peptide portion of an E2A gene product, E47-96. Analytical ultracentrifugation reveals that MyoD forms micelles that contain more than 100 monomers and are asymmetric and stable up to 36 degrees C. The critical micelle concentration increases slightly with temperature, but micelle size is unaffected. The micelles are in reversible equilibrium with monomer. Addition of E47-96 results in the stoichiometric formation of stable MyoD-E47-96 heterodimers and the depletion of micelles. Micelle formation effectively holds the concentration of free MyoD constant and equal to the critical micelle concentration. In the presence of micelles, the extent of all interactions involving free MyoD is independent of the total MyoD concentration and independent of one another. For DNA binding, the apparent relative specificity for different sites can be affected. In general, heterodimer-associated activities will depend on the self-association behavior of the partner protein.

Evolutionary conservation of human TATA-binding-polypeptide-associated factors TAFII31 and TAFII80 and interactions of TAFII80 with other TAFs and with general transcription factors.

Human transcription initiation factor TFIID is composed of the TATA-binding polypeptide (TBP) and at least 13 TBP-associated factors (TAFs) that collectively or individually are involved in activator-dependent transcription. To investigate protein-protein interactions involved in TFIID assembly and in TAF-mediated activator functions, we have cloned and expressed cDNAs encoding human TAFII80 and TAFII31. Coimmunoprecipitation assays showed that TAFII80 interacted with TAFII250, TAFII31, TAFII20, and TBP, but not with TAFII55. Similar assays showed that TAFII80 interacted with TFIIE alpha and with TFIIF alpha (RAP74) but not with TFIIB, TFIIE beta, or TFIIF beta (RAP30). Further studies with TAFII80 mutations revealed three distinct interaction domains which fall within regions conserved in human TAFII80, Drosophila TAFII60, and yeast TAFII60. The N terminus of TAFII80 (residues 1-100) interacts with both TAFII31 and TAFII20, while two C-terminal regions are involved, respectively, in interactions with TAFII250 and TFIIF alpha (RAP74) (residues 203-276) and with TBP and TFIIE alpha (residues 377-505). The interactions between TAFII80 and general factors TFIIE alpha and TFIIF alpha (RAP74) could be important for recruitment of GTFs during activator-dependent transcription. Because TAFs 80, 31, and 20 show sequence similarities to histones H4, H3, and H2B, as well as some parallel interactions, this subset of TAFs may form a related core structure within TFIID.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

Differential tubulogenic and branching morphogenetic activities of growth factors: implications for epithelial tissue development.

At least two kidney epithelial cell lines, the Madin-Darby canine kidney (MDCK) and the murine inner medullary collecting duct line mIMCD-3, can be induced to form branching tubular structures when cultured with hepatocyte growth factor (HGF) plus serum in collagen I gels. In our studies, whereas MDCK cells remained unable to form tubules in the presence of serum alone, mIMCD-3 cells formed impressive branching tubular structures with apparent lumens, suggesting the existence of specific factors in serum that are tubulogenic for mIMCD-3 cells but not for MDCK cells. Since normal serum does not contain enough HGF to induce tubulogenesis, these factors appeared to be substances other than HGF. This was also suggested by another observation: when MDCK cells or mIMCD-3 cells were cocultured under serum-free conditions with the embryonic kidney, both cell types formed branching tubular structures similar to those induced by HGF; however, only in the case of MDCK cells could this be inhibited by neutralizing antibodies against HGF. Thus, the embryonic kidney produces growth factors other than HGF capable of inducing tubule formation in the mIMCD-3 cells. Of a number of growth factors examined, transforming growth factor alpha (TGF-alpha) and epidermal growth factor (EGF) were found to be tubulogenic for mIMCD-3 cells. Whereas only HGF was a potent tubulogenic factor for MDCK cells, HGF, TGF-alpha, and EGF were potent tubulogenic factors for mIMCD-3 cells. Nevertheless, there were marked differences in the capacity of these tubulogenic factors to induce tubulation as well as branching events in those tubules that did form (HGF >> TGF-alpha > EGF). Thus, at least three different growth factors can induce tubulogenesis and branching in a specific epithelial cell in vitro (though to different degrees), and different epithelial cells that are capable of forming branching tubular structures demonstrate vastly different responses to tubulogenic growth factors. The results are discussed in the context of branching morphogenesis during epithelial tissue development.

Seasonal development and yield of native plants on the upper Snake River Plains and their relation to certain climatic factors /

Issued also in microfilm form, as thesis, University of Minnesota, under title: Seasonal development and yield of native plants on the upper Snake River Plains of Idaho and their relation to climatic factors, especially precipitation and temperature.

Frequency, Form, and Distribution of Illocutionary Speech Acts in Swedish Parent-Child Interaction

In this study, young children’s development of speech acts was examined. Interaction between six Swedish-speaking parents and their children was observed. The frequency, form and distribution of speech acts in the output from the parents were compared with the frequency, form and distribution of the children’s speech acts. The frequency was measured by occurrences per analysed session. The aim of the analysis was to examine if the parent’s behaviour could be treated as a baseline for the child’s development. Both the parents’ and the children’s illocutionary speech acts were classified. Each parent-child dyad was observed at four different occasions, when the children were 1;0, 1;6, 2;0, and 2;6 years of age. Similar studies have previously shown that parents keep a consistent frequency of speech acts within a given time span of interaction, though the distribution of different types of speech acts may shift, depending on contextual factors. The form, in terms of Mean Length of Speech Act in Words (MLSAw), were correlated with the longitudinal result of the children’s MLSAw. The distribution of the parents’ speech acts showed extensive individual differences. The result showed that the children’s MLSAw move significantly closer the MLSAw of their parents. Since the parent’s MLSAw showed a wide distribution, these results indicate that the parent’s speech acts can be treated as a baseline for certain aspects of the children’s development, though further studies are needed.

Socio-Historical Factors Mediating Collaborative Teaching and Learning: A Design-Based Investigation and Intervention

Thesis (Ph.D.)--University of Washington, 2016-06

Factors affecting the formation of a skin reservoir for topically applied solutes

The reservoir function of the skin is an important determinant of the duration of action of a topical solute. The reservoir can exist in the stratum corneum, in the viable avascular tissue (viable epidermis and supracapillary dermis) and in the dermis. A steroid reservoir in the stratum corneum has been demonstrated by the reactivation of a vasoconstrictor effect by occlusion or application of a placebo cream to the skin some time after the original topical application of steroid. Other solutes have also been reported to show a reservoir effect in the skin after topical application. A simple compartmental model is used to understand why reactivation of vasoconstriction some time after a topical steroid application shows dependency on time, topical solute concentration and the product used to cause reactivation. The model is also used to show which solutes are likely to show a reservoir effect and could be potentially affected by desquamation, especially when the turnover of the skin is abnormally rapid. A similar form of the model can be used to understand the promotion of reservoir function in the viable tissue and in the dermis in terms of effective removal by blood perfusing the tissues. Copyright (C) 2004 S. Karger AG, Basel.

Analysis of articulatory dynamics in dysarthria following brain injury in childhood using electromagnetic articulography and electropalatography

Two physiological assessments, electromagnetic articulography (EMA) and electropalatography (EPG), were used simultaneously to investigate the articulatory dynamics in an 18-year-old male with dysarthria 9 years following traumatic brain injury (TBI). Eight words consisting of /t, s, integral, k/ in word initial and word final positions were produced up to 10 times. A nonneurologically impaired male served as a control subject. Six parameters were analyzed using EMA: velocity, acceleration, deceleration, distance, duration, and motion path of tongue movements. Using EPG, the pattern and amount of tongue-to-palate contact and the duration of the closure/constriction phase of each consonant produced were assessed. Timing disturbances in the TBI speaker's speech were highlighted in perceptual assessments in the form of prolonged phonemes and a reduced speech rate. EMA analysis revealed that the approach and release phase durations of the consonant productions were within normal limits. Kinematic strategies such as decreased velocity and decreased distances traveled by the tongue, however, may have counterbalanced each other to produce these appropriate results. EPG examination revealed significantly longer closure/constriction phase periods, which may have contributed to the prolonged phonemes and reduced speech rate observed. The implications of these findings for the development of treatment programs for dysarthria subsequent to TBI will be highlighted.

Factors affecting the design and construction of Lamella suspen-dome systems

The suspen-dome system is a new structural form that has become popular in the construction of long-span roof structures. These domes are very slender and lightweight, their configuration is complicated, and hence sequential consideration in the structural design is needed. This paper focuses on these considerations, which include the method for designing cable prestress force, a simplified analysis method, and the estimation of buckling capacity. Buckling is one of the most important problems for dome structures. This paper presents the findings of an intensive buckling study of the Lamella suspen-dome system that takes geometric imperfection, asymmetric loading, rise-to-span ratio, and connection rigidity into consideration. Finally, suggested design and construction guidelines are given in the conclusion of this paper. (c) 2005 Elsevier Ltd. All rights reserved.

Factor structure and internal consistency of the Young Schema Questionnaire (Short Form) in Korean and Australian samples

We investigated cross-cultural differences in the factor structure and psychometric properties of the 75-item Young Schema Questionnaire-Short Form (YSQ-SF). Participants were 833 South Korean and 271 Australian undergraduate students. The South Korean sample was randomly divided into two sub-samples. Sample A was used for Exploratory Factor Analysis (EFA) and sample B was used for Confirmatory Factor Analysis (CFA). EFA for the South Korean sample revealed a 13-factor solution to be the best fit for the data, and CFA on the data from sample B confirmed this result. CFA on the data from the Australian sample also revealed a 13-factor solution. The overall scale of the YSQ-SF demonstrated a high level of internal consistency in the South Korean and Australian groups. Furthermore, adequate internal consistencies for all subscales in the South Korean and Australian samples were demonstrated. In conclusion, the results showed that YSQ-SF with 13 factors has good psychometric properties and reliability for South Korean and Australian University students. Korean samples had significantly higher YSD scores on most of the 13 subscales than the Australian sample. However, limitations of the current study preclude the generalisability of the findings to beyond undergraduate student populations. (c) 2006 Elsevier B.V. All rights reserved.

Planar electromagnetic bandgap structures

This article presents various novel and conventional planar electromagnetic bandgap (EBG)-assisted transmission lines. Both microstrip lines and coplanar waveguides (CPWs) are designed with circular, rectangular, annular, plus-sign and fractal-patterned EBGs and dumbbell-shaped defected ground structure (DGS). The dispersion characteristics and the slow-wave factors of the design are investigated. (c) 2006 Wiley Periodicals, Inc.

Maternal health during pregnancy and perinatal mortality in Bangladesh: evidence form a large-scale community-based clinical trial

Perinatal mortality is very high in Bangladesh. In this setting, few community-level studies have assessed the influence of underlying maternal health factors on perinatal outcomes. We used the data from a community-based clinical controlled trial conducted between 1994 and 1997 in the catchment areas of a large MCH/FP hospital located in Mirpur, a suburban area of Dhaka in Bangladesh, to investigate the levels of perinatal mortality and its associated maternal health factors during pregnancy. A total of 2007 women were followed after recruitment up to delivery, maternal death, or until they dropped out of the study. Of these, 1584 who gave birth formed our study subjects. The stillbirth rate was 39.1 per 1000 births [95% confidence interval (CI) 39.0, 39.3] and the perinatal mortality rate (up to 3 days) was 54.3 per 1000 births [95% CI 54.0, 54.6] among the study population. In the fully adjusted logistic regression model, the risk of perinatal mortality was as high as 2.7 times [95% CI 1.5, 4.9] more likely for women with hypertensive disorders, 5.0 times [95% CI 2.3, 10.8] as high for women who had antepartum haemorrhage and 2.6 times [95% CI 1.2, 5.8] as high for women who had higher haemoglobin levels in pregnancy when compared with their counterparts. The inclusion of potential confounding variables such as poor obstetric history, sociodemographic characteristics and preterm delivery influenced only marginally the net effect of important maternal health factors associated with perinatal mortality. Perinatal mortality in the study setting was significantly associated with poor maternal health conditions during pregnancy. The results of this study point towards the urgent need for monitoring complications in high-risk pregnancies, calling for the specific components of the safe motherhood programme interventions that are designed to manage these complications of pregnancy.

Molecular investigations into vaginal immunization with HIV gp41 antigenic construct H4A in a quick release solid dosage form

A robust vaginal immune response is considered essential for an effective prophylactic vaccine that prevents transmission of HIV and other sexually acquired diseases. Considerable attention has recently focused on the potential of vaginally administered vaccines as a means to induce such local immunity. However, the potential for vaccination at this site remains in doubt as the vaginal mucosa is generally considered to have low immune inductive potential. In the current study, we explored for the first time the use of a quick release, freeze-dried, solid dosage system for practical vaginal administration of a protein antigen. These solid dosage forms overcome the common problem associated with leakage and poor retention of vaginally administered antigen solutions. Mice were immunized vaginally with H4A, an HIV gp41 envelope based recombinant protein, using quick release, freeze-dried solid rods, and the immune responses compared to a control group immunized via subcutaneous H4A injection. Vaginally immunized mice failed to elicit robust immune responses. Our detailed investigations, involving cytokine analysis, the stability of H4A in mouse cervicovaginal lavage, and elucidation of the state of H4A protein in the immediate-release dosage form, revealed that antigen instability in vaginal fluid, the state of the antigen in the dosage form, and the cytokine profile induced are all likely to have contributed to the observed lack of immunogenicity. These are important factors affecting vaginal immunization and provide a rational basis for explaining the typically poor and variable elicitation of immunity at this site, despite the presence of immune responsive cells within the vaginal mucosae. In future mucosal vaccine studies, a more explicit focus on antigen stability in the dosage form and the immune potential of available antigen-responsive cells is recommended.