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1.
Cultur Divers Ethnic Minor Psychol ; 29(2): 267-278, 2023 Apr.
Article in English | MEDLINE | ID: mdl-34735169

ABSTRACT

OBJECTIVE: Primary caregivers (e.g., parents, grandparents, other family members) from low-income and ethnically minoritized families tend to face a host of barriers when participating in their children's school activities. Research suggests that demographic match and quality communication between caregivers and teachers could support minoritized families' school-based engagement. This study examined the associations among caregiver-teacher demographic match, caregivers' perceived communication quality with the teacher and caregivers' perceived barriers to school-based engagement. METHOD: Caregivers (n = 565) from 49 Head Start classrooms completed the parent-report versions of the surveys: Barriers to Family Engagement, reporting resource, cultural/relational, and program/context barriers to school-based engagement; and Family-Teacher Communication, reporting communication quality with their children's lead and assistant teachers (n = 102). Caregivers and teachers also completed demographic surveys to provide information about their family background, such as race/ethnicity, primary language, and education level. RESULTS: Multilevel modeling results showed that among the three demographic match variables (i.e., race/ethnicity, primary language, formal education), only language match was associated with caregivers' perception of fewer cultural/relational barriers. Latine and Black non-Latine caregivers reported more cultural/relational and program/context carriers than White, non-Latine caregivers. Finally, caregivers who perceived better communication with their children's teachers reported fewer cultural/relational and program/context barriers. CONCLUSIONS: Primary language match and high-quality communication between families and teachers appear essential in creating a welcoming preschool environment that could alleviate some of the barriers to engagement typically faced by ethnically minoritized and low-income families. (PsycInfo Database Record (c) 2023 APA, all rights reserved).


Subject(s)
Communication , Parents , Child , Humans , Child, Preschool , Schools , Language , Caregivers , Parent-Child Relations
2.
Matern Child Health J ; 25(10): 1615-1625, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34328588

ABSTRACT

OBJECTIVE: To determine whether embedding educational information about child development and optimal parenting practices from the Bright Futures Guidelines for Health Supervision into baby books can reduce women's depressive symptoms and parenting stress during the first 18 months of motherhood. METHODS: A sample of 167 low-income, primarily African-American, first-time mothers were randomized into three conditions while pregnant: an educational book group, a non-educational book group, or a no-book group. Depressive symptoms and parenting stress were assessed using two questionnaires during home visits when women were in their third trimester of pregnancy (baseline) and when their child was 2, 4, 6, 9, 12, and 18 months of age. RESULTS: Hierarchical Linear Models showed that women who received books with educational information shed depressive symptoms and parenting stress at a faster rate than women who received similar books without educational information and women who did not receive books. CONCLUSIONS: These findings indicate that providing women with child development and parenting information via baby books can be an effective strategy for promoting new mothers' emotional wellbeing. Since feelings of sadness and stress are risky for both mothers and their infants, this easy to implement intervention holds great promise.


Subject(s)
Depression , Mothers , Books , Depression/prevention & control , Emotions , Female , Humans , Infant , Infant, Newborn , Male , Parenting , Pregnancy
3.
Acad Pediatr ; 21(4): 620-628, 2021.
Article in English | MEDLINE | ID: mdl-32673762

ABSTRACT

OBJECTIVE: This study investigated how caregivers' mobile device use influenced the quality of their interactions with their children. The associations between quality of interactions and the type of activity (eg, typing/swiping, looking at screen), setting, caregiver-child proximity, and child behaviors were also examined. METHOD: Researchers anonymously and systematically observed and coded the behavior of 98 caregiver-child dyads in public settings (eg, parks, food courts) during real-time, naturally occurring interactions using time sampling. RESULTS: Caregivers who used a mobile device for the entire observation engaged in less joint attention and were less responsive than caregivers who used the device some of the time. When looking at patterns within caregivers who used the device intermittently, the probability that they would engage in joint attention, initiate interactions with their child, talk, and display positive emotions was lower when they used a mobile device than when they did not. Child talking and positive affect were unrelated to caregiver device use. Activity type with the device, caregiver-child proximity and setting also related to interaction quality. CONCLUSIONS: Caregiver device use was negatively associated with adult behaviors that are key components of high-quality caregiver-child interactions. Additionally, setting, activity type, and caregiver-child proximity are factors that should be considered because they relate to the quality of caregiver-child interactions in the context of mobile screen technologies.


Subject(s)
Caregivers , Child Behavior , Adult , Attention , Child , Computers, Handheld , Humans , Parent-Child Relations
4.
Front Psychol ; 11: 570712, 2020.
Article in English | MEDLINE | ID: mdl-33162913

ABSTRACT

Young children's use of mobile screens is increasing despite the American Academy of Pediatrics' recommendations to limit screen use. Research on TV has found that maternal beliefs about the effects of screens on children's learning and parental socioeconomic status influence children's media consumption. However, few studies have explored parents' beliefs about mobile screens and whether there are differences in beliefs by socioeconomic status, particularly within the largest ethnic minoritized group - Latines. Because Latines are a socioeconomically and linguistically heterogenous group, but are often represented by low-income mothers in research, it is important to understand whether there are socioeconomic and linguistic differences on how and why Latine mothers AND fathers permit their children to use mobile screens. This study used in-depth, semi-structured interviews to understand how and why Latine mothers (low-income = 10, middle-to-high income = 10) and fathers (low-income = 10, middle-to-high income = 10) permitted their children (0-4 years) to use mobile screens. Specifically, we discussed their beliefs about how mobile screens support and hinder their children's learning and how their children used them. Results from qualitative content analysis showed that mothers and fathers, across income, education levels, and language use, believed that they, as parents, were the key decision-makers in determining the extent to which mobile screens supported and hindered their young children's learning. They described mediation strategies of selecting appropriate content, setting time limits, and monitoring use, to ensure that their children primarily benefited from device use. However, two distinctions were noted. Parents with a high school diploma or beyond stressed the importance of co-using devices with their children. This was not mentioned by less formally educated parents. Additionally, low-income parents with diverse educational levels, mentioned the importance of continuously monitoring device use to avoid their children encountering inappropriate content. Findings can inform work seeking to promote optimal media habits among Latine families.

5.
J Child Lang ; : 1-25, 2019 Jul 09.
Article in English | MEDLINE | ID: mdl-31284888

ABSTRACT

This study examines language mixing in 26 Spanish-English dual language learners over the course of their first year of preschool. The children's patterns of language choice while interacting in monolingual language contexts were analyzed at age 3;6 and 4;5 to examine: (1) whether the frequency of language mixing changed during the year; (2) whether mixing was related to proficiency as measured by utterance length and lexical diversity; and (3) whether there were different subgroups of children, among the participants, with similar proficiency and language use patterns. The results indicate that language mixing, which was low at both ages, was related to limited lexical resources only at 3;6. However, by age 4;5, language choice was more constrained by sociolinguistic variables - children's awareness of the language prescribed by the majority culture - than by proficiency. An exploratory cluster analysis further reveals different profiles of learners sharing similar proficiency and language mixing characteristics.

6.
Acad Pediatr ; 19(8): 969-977, 2019.
Article in English | MEDLINE | ID: mdl-30904582

ABSTRACT

OBJECTIVE: To understand the experiences of diverse families when taking their young children to the dentist and to document their prevalence. METHODS: An exploratory sequential design was used. First, 4 focus groups (N = 33) comprised of low-income female caregivers of children under 6 years of age were conducted in English and Spanish. Discussions centered around facilitators and barriers to taking children to the dentist. Themes derived from the groups were then used to create a survey that was given to 1184 caregivers in English, Spanish, or Vietnamese. RESULTS: Thematic coding of focus groups found little support for typically reported barriers to pediatric oral health care utilization (eg, transportation, cost, knowledge); instead, caregivers reported negative experiences (eg, restraint, separation) as barriers. In the surveys, 66% of caregivers reported being separated from their children, 25% reported that their children were restrained (53.7% for cleanings), 26% of children were given sedating medication for cleanings, and 22% of the caregivers reported experiences that made them not want to return to the dentist. The prevalence of these experiences differed significantly among Latino, Asian, and Caucasian families and for annual incomes under or above $50,000. CONCLUSIONS: Families with lower incomes and/or from ethnic and linguistic minority groups were more likely to report negative experiences at the dentist than higher income and Caucasian families. These data document the high prevalence of negative experiences and suggest ethnic, financial, and linguistic disparities in the quality of experiences. More research is needed on the role of dentists in facilitating or hindering oral health care utilization among diverse families.


Subject(s)
Dental Care for Children , Ethnicity , Health Knowledge, Attitudes, Practice , Healthcare Disparities/ethnology , Hypnotics and Sedatives/therapeutic use , Mothers , Restraint, Physical , Adult , Aged , Asian , California , Child, Preschool , Dentist-Patient Relations , Female , Focus Groups , Grandparents , Health Expenditures , Health Services Accessibility , Healthcare Disparities/statistics & numerical data , Hispanic or Latino , Humans , Income/statistics & numerical data , Infant , Informed Consent , Male , Middle Aged , Parents , Poverty , Surveys and Questionnaires , White People , Young Adult
7.
J Cell Sci ; 125(Pt 18): 4333-42, 2012 Sep 15.
Article in English | MEDLINE | ID: mdl-22641695

ABSTRACT

Toxoplasma gondii, a human pathogen and a model apicomplexan parasite, actively and rapidly invades host cells. To initiate invasion, the parasite induces the formation of a parasite-cell junction, and progressively propels itself through the junction, inside a newly formed vacuole that encloses the entering parasite. Little is known about how a parasite that is a few microns in diameter overcomes the host cell cortical actin barrier to achieve the remarkably rapid process of internalization (less than a few seconds). Using correlative light and electron microscopy in conjunction with electron tomography and three-dimensional image analysis we identified that toxofilin, an actin-binding protein, secreted by invading parasites correlates with localized sites of disassembly of the host cell actin meshwork. Moreover, quantitative fluorescence speckle microscopy of cells expressing toxofilin showed that toxofilin regulates actin filament disassembly and turnover. Furthermore, Toxoplasma tachyzoites lacking toxofilin, were found to be impaired in cortical actin disassembly and exhibited delayed invasion kinetics. We propose that toxofilin locally upregulates actin turnover thus increasing depolymerization events at the site of entry that in turn loosens the local host cell actin meshwork, facilitating parasite internalization and vacuole folding.


Subject(s)
Actin Capping Proteins/metabolism , Actin Cytoskeleton/metabolism , Actin Cytoskeleton/parasitology , Host-Parasite Interactions , Protozoan Proteins/metabolism , Toxoplasma/physiology , Up-Regulation , Actin Cytoskeleton/ultrastructure , Actin Depolymerizing Factors/metabolism , Actins/metabolism , Animals , Biomechanical Phenomena , Cell Line , Cell Survival , Gene Knockout Techniques , Humans , Kinetics , Life Cycle Stages , Phosphorylation , Phosphoserine/metabolism , Protein Transport , Rats , Secretory Vesicles/metabolism , Secretory Vesicles/parasitology , Toxoplasma/growth & development , Toxoplasma/ultrastructure
8.
J Struct Biol ; 172(3): 363-71, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20599510

ABSTRACT

Filamentous fungus Fusarium poae is a worldwide cause of the economically important disease Fusarium head blight of cereal grains. The fungus is itself commonly infected with a bisegmented dsRNA virus from the family Partitiviridae. For this study, we determined the structure of partitivirus Fusarium poae virus 1 (FpV1) to a resolution of 5.6Å or better by electron cryomicroscopy and three-dimensional image reconstruction. The main structural features of FpV1 are consistent with those of two other fungal partitiviruses for which high-resolution structures have been recently reported. These shared features include a 120-subunit T=1 capsid comprising 60 quasisymmetrical capsid protein dimers with both shell and protruding domains. Distinguishing features are evident throughout the FpV1 capsid, however, consistent with its more massive subunits and its greater phylogenetic divergence relative to the other two structurally characterized partitiviruses. These results broaden our understanding of conserved and variable elements of fungal partitivirus structure, as well as that of vertebrate picobirnavirus, and support the suggestion that a phylogenetic subcluster of partitiviruses closely related to FpV1 should constitute a separate taxonomic genus.


Subject(s)
Picobirnavirus/ultrastructure , Capsid/ultrastructure , Cryoelectron Microscopy , Imaging, Three-Dimensional , Picobirnavirus/classification , Virion/ultrastructure
9.
Biophys J ; 99(2): 685-94, 2010 Jul 21.
Article in English | MEDLINE | ID: mdl-20643089

ABSTRACT

Most dsRNA viruses have a genome-enclosing capsid that comprises 120 copies of a single coat protein (CP). These 120 CP subunits are arranged as asymmetrical dimers that surround the icosahedral fivefold axes, forming pentamers of dimers that are thought to be assembly intermediates. This scheme is violated, however, in recent structures of two dsRNA viruses, a fungal virus from family Partitiviridae and a rabbit virus from family Picobirnaviridae, both of which have 120 CP subunits organized as dimers of quasisymmetrical dimers. In this study, we report the CP backbone trace of a second fungal partitivirus, determined in this case by electron cryomicroscopy and homology modeling. This virus also exhibits quasisymmetrical CP dimers that are connected by prominent surface arches and stabilized by domain swapping between the two CP subunits. The CP fold is dominated by alpha-helices, although beta-strands mediate several important contacts. A dimer-of-dimers assembly intermediate is again implicated. The disordered N-terminal tail of each CP subunit protrudes into the particle interior and likely interacts with the genome during packaging and/or transcription. These results broaden our understanding of conserved and variable aspects of partitivirus structure and reflect the growing use of electron cryomicroscopy for atomic modeling of protein folds.


Subject(s)
Capsid Proteins/chemistry , Capsid Proteins/ultrastructure , Cryoelectron Microscopy , RNA Viruses/ultrastructure , Structural Homology, Protein , Amino Acid Sequence , Animals , Crystallography, X-Ray , Genome, Viral/genetics , Models, Molecular , Molecular Sequence Data , Particle Size , Picobirnavirus/genetics , Protein Multimerization , Protein Structure, Secondary , RNA Viruses/genetics , Rabbits , Transcription, Genetic
10.
Virology ; 388(1): 185-90, 2009 May 25.
Article in English | MEDLINE | ID: mdl-19344926

ABSTRACT

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NDelta52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T=1, T=3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.


Subject(s)
Antigens/metabolism , Capsid/metabolism , Epitopes/metabolism , Tombusvirus/physiology , Vaccines , Amino Acid Sequence , Animals , Cell Line , Insecta , Mice , Mice, Inbred BALB C , Models, Molecular , Protein Conformation , Recombinant Fusion Proteins/metabolism , Ricin/immunology , Ricin/metabolism
11.
Proc Natl Acad Sci U S A ; 106(11): 4225-30, 2009 Mar 17.
Article in English | MEDLINE | ID: mdl-19246376

ABSTRACT

For most dsRNA viruses, the genome-enclosing capsid comprises 120 copies of a single capsid protein (CP) organized into 60 icosahedrally equivalent dimers, generally identified as 2 nonsymmetricallyinteracting CP molecules with extensive lateral contacts. The crystal structure of a partitivirus, Penicillium stoloniferum virus F (PsV-F), reveals a different organization, in which the CP dimer is related by almost-perfect local 2-fold symmetry, forms prominent surface arches, and includes extensive structure swapping between the 2 subunits. An electron cryomicroscopy map of PsV-F shows that the disordered N terminus of each CP molecule interacts with the dsRNA genome and probably participates in its packaging or transcription. Intact PsV-F particles mediate semiconservative transcription, and transcripts are likely to exit through negatively charged channels at the icosahedral 5-fold axes. Other findings suggest that the PsV-F capsid is assembled from dimers of CP dimers, with an arrangement similar to flavivirus E glycoproteins.


Subject(s)
Capsid/chemistry , RNA Viruses/chemistry , Capsid Proteins/chemistry , Cryoelectron Microscopy , Crystallization , Models, Biological , RNA Viruses/genetics , RNA, Double-Stranded , RNA-Binding Proteins , Transcription, Genetic
12.
Proc Natl Acad Sci U S A ; 105(45): 17526-31, 2008 Nov 11.
Article in English | MEDLINE | ID: mdl-18981418

ABSTRACT

Infectious myonecrosis virus (IMNV) is an emerging pathogen of penaeid shrimp in global aquaculture. Tentatively assigned to family Totiviridae, it has a nonsegmented dsRNA genome of 7,560 bp and an isometric capsid of the 901-aa major capsid protein. We used electron cryomicroscopy and 3D image reconstruction to examine the IMNV virion at 8.0-A resolution. Results reveal a totivirus-like, 120-subunit T = 1 capsid, 450 A in diameter, but with fiber complexes protruding a further 80 A at the fivefold axes. These protrusions likely mediate roles in the extracellular transmission and pathogenesis of IMNV, capabilities not shared by most other totiviruses. The IMNV structure is also notable in that the genome is centrally organized in five or six concentric shells. Within each of these shells, the densities alternate between a dodecahedral frame that connects the threefold axes vs. concentration around the fivefold axes, implying certain regularities in the RNA packing scheme.


Subject(s)
Capsid Proteins/genetics , Genome, Viral/genetics , Models, Molecular , Penaeidae/virology , Totiviridae/genetics , Virion/ultrastructure , Animals , Aquaculture , Cryoelectron Microscopy
13.
Structure ; 16(5): 776-86, 2008 May.
Article in English | MEDLINE | ID: mdl-18462682

ABSTRACT

Two distinct partitiviruses, Penicillium stoloniferum viruses S and F, can be isolated from the fungus Penicillium stoloniferum. The bisegmented dsRNA genomes of these viruses are separately packaged in icosahedral capsids containing 120 coat-protein subunits. We used transmission electron cryomicroscopy and three-dimensional image reconstruction to determine the structure of Penicillium stoloniferum virus S at 7.3 A resolution. The capsid, approximately 350 A in outer diameter, contains 12 pentons, each of which is topped by five arched protrusions. Each of these protrusions is, in turn, formed by a quasisymmetric dimer of coat protein, for a total of 60 such dimers per particle. The density map shows numerous tubular features, characteristic of alpha helices and consistent with secondary structure predictions for the coat protein. This three-dimensional structure of a virus from the family Partitiviridae exhibits both similarities to and differences from the so-called "T = 2" capsids of other dsRNA viruses.


Subject(s)
Capsid Proteins/chemistry , Capsid/chemistry , Penicillium/virology , RNA Viruses/chemistry , Amino Acid Sequence , Capsid/metabolism , Capsid/ultrastructure , Cryoelectron Microscopy , Dimerization , Imaging, Three-Dimensional , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Structure, Secondary , RNA, Double-Stranded/chemistry , Virion/isolation & purification
14.
J Virol ; 81(3): 1119-28, 2007 Feb.
Article in English | MEDLINE | ID: mdl-17079293

ABSTRACT

Noroviruses (NoVs) are the causative agents of nonbacterial acute gastroenteritis in humans. NoVs that belong to genogroup II (GII) are quite prevalent and prone to undergo recombination, and their three-dimensional structure is not yet known. Protein homology modeling of Sinsiro virus (SV), a member of the GII.3 NoVs, revealed the presence of a surface-exposed 20-amino-acid (aa) insertion in the P2 domain of the capsid protein (CP) relative to the Norwalk virus (NV) CP, which is a well known hot spot for mutations to counter the host immunological response. To further characterize the role of the long insertion in SV, the capsid protein gene was expressed using the recombinant baculovirus system. Trypsinization of the resultant virus-like particles yielded two predominant bands (31.7 and 26.1 kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. N-terminal sequencing and analysis of the mass spectroscopic data indicated that these fragments correspond to residues 1 to 292 (26.1 kDa) and 307 to 544 (31.7 kDa). In addition, the above data taken together with the comparative modeling studies indicated that the trypsin cleavage sites of the Sinsiro virus CP, Arg292 and Arg307, are located at the beginning of and within the 20-aa insertion in the P2 domain, respectively. This study demonstrates that the presence of the surface-exposed loop in the GII.3 NoVs facilitates the trypsinization of the capsid protein in the assembled form. The SV particles remain intact even after trypsin digestion and retain the suggested receptor binding linear epitope of residues 325 to 334. The above results are distinct from those obtained from the trypsinization studies performed earlier on the NV (GI) and VA387 (GII) viruses, both of which lack the large surface insertion and associated basic residues. These new observations may have implications for host receptor binding, cell entry, and norovirus infection in general.


Subject(s)
Capsid Proteins/metabolism , Norovirus/chemistry , Trypsin/metabolism , Virion/chemistry , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Norovirus/genetics , Protein Structure, Secondary
15.
Chem Biol ; 13(7): 771-8, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16873025

ABSTRACT

Chemical and genetic modifications on the surface of viral protein cages confer unique properties to the virus particles with potential nano and biotechnological applications. The enclosed space in the interior of the virus particles further increases its versatility as a nanomaterial. In this paper, we report a simple method to generate a high yield of stable cowpea mosaic virus (CPMV) empty capsids from their native nucleoprotein counterparts by removing the encapsidated viral genome without compromising the integrity of the protein coat. Biochemical and structural comparison of artificially generated empty particles did not reveal any distinguishable differences from CPMV particles containing viral RNA. Preliminary results on the use of artificially produced empty CPMV capsids as a carrier capsule are described.


Subject(s)
Iridoviridae/chemistry , Virion/chemistry , Cryoelectron Microscopy , Crystallography, X-Ray , Electrophoresis, Polyacrylamide Gel , Fluorescent Dyes , Hydrolysis , RNA, Viral/analysis
16.
Virology ; 349(1): 222-9, 2006 May 25.
Article in English | MEDLINE | ID: mdl-16603216

ABSTRACT

Expression of full-length and N-terminal deletion mutants of the coat protein (CP) of tomato bushy stunt virus (TBSV) using the recombinant baculovirus system resulted in spontaneously assembled virus-like particles (VLPs). Deletion of the majority of the R-domain sequence of the CP, residues 1-52 (CP-NDelta52) and 1-62 (CP-NDelta62), produced capsids similar to wild-type VLPs. Interestingly, the CP-NDelta62 mutant that retains the last 3 residues of R-domain is capable of forming both the T = 1 and T = 3 particles. However, between the two types of VLPs, formation of the T = 1 capsids appears to be preferred. Another mutant, CP-NDelta72, in which R-domain (residues 1-65) was completely removed but contains most of the beta-annulus and extended arm (betaA) regions exclusively formed T = 1 particles. These results suggest that as few as 3 residues (63-65) of the R-domain, which includes 2 basic amino acids together with the arm (betaA) and beta-annulus regions, may be sufficient for the formation of T = 3 particles. However, anywhere between 4 to 13 residues of the R-domain may be required for proper positioning of betaA and beta-annulus structural elements of the C-type subunits to facilitate an error free assembly of T = 3 capsids.


Subject(s)
Capsid Proteins/genetics , Tombusvirus/genetics , Virosomes/metabolism , Virosomes/ultrastructure , Amino Acid Sequence , Animals , Capsid/metabolism , Capsid/ultrastructure , Capsid Proteins/isolation & purification , Capsid Proteins/metabolism , Cell Line , Microscopy, Electron, Transmission , Models, Molecular , Molecular Sequence Data , Polymorphism, Genetic , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , RNA, Viral/analysis , Sequence Alignment , Sequence Deletion , Spodoptera , Tombusvirus/ultrastructure
17.
Nano Lett ; 5(4): 597-602, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15826093

ABSTRACT

Five different "HIS tag" mutants of cowpea mosaic virus were made by genetically introducing six contiguous histidine residues at various locations on the virus capsid. The mutant particles showed differential affinity for binding nickel, and their electrostatic properties could be controlled as a function of the protonation state of the exposed histidine sequence. The specific addressability of the HIS tag was corroborated by the selective modification of the histidine sequence with nanogold cross-linked to the Ni-NTA moiety.


Subject(s)
Capsid/chemistry , Comovirus/chemistry , Histidine/chemistry , Nanostructures/chemistry , Amino Acid Sequence , Comovirus/genetics , Cryoelectron Microscopy , Gold/chemistry , Histidine/genetics , Molecular Sequence Data , Mutation/genetics , Nickel/chemistry , Static Electricity
18.
J Mol Biol ; 341(4): 1049-62, 2004 Aug 20.
Article in English | MEDLINE | ID: mdl-15289102

ABSTRACT

The crystal structure of Saccharomyces cerevisiae ScAdh6p has been solved using the anomalous signal from the two zinc atoms found per subunit, and it constitutes the first structure determined from a member of the cinnamyl alcohol dehydrogenase family. ScAdh6p subunits exhibit the general fold of the medium-chain dehydrogenases/reductases (MDR) but with distinct specific characteristics. In the three crystal structures solved (two trigonal and one monoclinic), ScAdh6p molecules appear to be structural heterodimers composed of one subunit in the apo and the second subunit in the holo conformation. Between the two conformations, the relative disposition of domains remains unchanged, while two loops, Cys250-Asn260 and Ile277-Lys292, experience large movements. The apo-apo structure is disfavoured because of steric impairment involving the loop Ile277-Lys292, while in the holo-holo conformation some of the hydrogen bonds between subunits would break apart. These suggest that the first NADPH molecule would bind to the enzyme much more tightly than the second. In addition, fluorimetric analysis of NADPH binding demonstrates that only one cofactor molecule binds per dimer. Therefore, ScAdh6p appears to function according to a half-of-the-sites reactivity mechanism, resulting from a pre-existing (prior to cofactor binding) tendency for the structural asymmetry in the dimer. The specificity of ScAdh6p towards NADPH is mainly due to the tripod-like interactions of the terminal phosphate group with Ser210, Arg211 and Lys215. The size and the shape of the substrate-binding pocket correlate well with the substrate specificity of ScAdh6p towards cinnamaldehyde and other aromatic compounds. The structural relationships of ScAdh6p with other MDR structures are analysed.


Subject(s)
Alcohol Oxidoreductases/chemistry , NADP/metabolism , Alcohol Oxidoreductases/metabolism , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Molecular Sequence Data , Phylogeny , Protein Conformation , Sequence Homology, Amino Acid , Substrate Specificity
19.
Bioconjug Chem ; 15(4): 807-13, 2004.
Article in English | MEDLINE | ID: mdl-15264868

ABSTRACT

Genetic economy leads to symmetric distributions of chemically identical subunits in icosaherdal and helical viruses. Modification of the subunit genes of a variety of viruses has permitted the display of polypeptides on both the infectious virions and virus particles made in expression systems. Icosahedral chimeric particles of this type often display novel properties resulting in high local concentrations of the insert. Here we report an extension of this concept in which entire proteins were chemically cross-linked to lysine and cysteine residues genetically engineered on the coat protein of icosahedral Cowpea mosaic virus particles. Three exogenous proteins, the LRR domain of internalin B, the T4 lysozyme, and the Intron 8 gene product of the of the HER2 tyrosine kinase receptor were derivatized with appropriate bifunctional cross-linkers and conjugated to the virus capsid. Characterization of these particles demonstrated that (1) virtually 100% occupancy of the 60 sites was achieved; (2) biological activity (either enzyme or binding specificity) of the attached protein was preserved; (3) in one case (LRR-internalin B) the attached protein conformed with the icosahedral symmetry to the extent that a reconstruction of the derivatized particles displayed added density with a shape consistent with the X-ray structure of the attached protein. Strategies demonstrated here allow virus particle targeting to specific cell types and the use of an icosahedral virus as a platform for structure determination of small proteins at moderate resolution.


Subject(s)
Comovirus/chemistry , Proteins/analysis , Proteins/chemistry , Bacterial Proteins , Bacteriophage T4/enzymology , Capsid Proteins/chemistry , Capsid Proteins/genetics , Chromatography, Liquid , Comovirus/genetics , Comovirus/isolation & purification , Cross-Linking Reagents/chemistry , Drug Delivery Systems , Membrane Proteins/chemistry , Models, Molecular , Molecular Structure , Muramidase/chemistry , Protein Engineering , Protein Structure, Tertiary , Receptor, ErbB-2/chemistry
20.
Chem Biol ; 11(6): 855-63, 2004 Jun.
Article in English | MEDLINE | ID: mdl-15217618

ABSTRACT

Cowpea mosaic virus (CPMV) is a robust, icosahedrally symmetric platform successfully used for attaching a variety of molecular substrates including proteins, fluorescent labels, and metals. The symmetric distribution and high local concentration of the attached molecules generates novel properties for the 30 nm particles. We report new CPMV reagent particles generated by systematic replacement of surface lysines with arginine residues. The relative reactivity of each lysine on the native particle was determined, and the two most reactive lysine residues were then created as single attachment sites by replacing all other lysines with arginine residues. Structural analysis of gold derivatization not only corroborated the specific reactivity of these unique lysine residues but also demonstrated their dramatically different presentation environment. Combined with site-directed cystine mutations, it is now possible to uniquely double label CPMV, expanding its use as an addressable nanoblock.


Subject(s)
Comovirus/chemistry , Lysine/chemistry , Arginine/analysis , Arginine/chemistry , Comovirus/genetics , Comovirus/physiology , Gold/chemistry , Lysine/analysis , Models, Biological , Molecular Structure , Mutagenesis, Site-Directed , Mutation , Protein Structure, Tertiary
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