HIT Family Research Articles

Profil Ketahanan Ekonomi Rumah Tangga di Masa Pandemi Covid-19 Berdasarkan Tingkat Pendidikan dan Jenis Pekerjaan (Studi Kasus Kecamatan Sukajadi Kota Bandung)

In the microeconomic aspect, one of the impacts of the Covid-19 pandemic has hit family economic resilience. Economic resilience is a condition of the nation's economic life where people must be able to survive in overcoming all threats, disturbances, obstacles and challenges. This qualitative study aims to analyze the economic resilience of families in Sukajadi District, Bandung City. The technique for collecting data is using a convenience sampling technique using a questionnaire instrument, then data analysis using a descriptive method. The number of respondents was 832 heads of household. The variables used are the level of education and type of work. The results showed that the pandemic had an impact on all heads of households with education levels ranging from elementary to university and any type of work. However, in Sukajadi Sub-district, the education level of SMA/equivalent is the most affected by the COVID-19 pandemic, while the type of work most affected is the type of non-permanent work. Following up on the findings of this study, the government can participate in formulating policies that will be made to increase household economic resilience.

Development of mismatch amplification mutation assays for the differentiation of MS1 vaccine strain from wild-type Mycoplasma synoviae and MS-H vaccine strains.

Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain.

Functional and Structural Insights of the Zinc-Finger HIT protein family members Involved in Box C/D snoRNP Biogenesis

Zf–HIT family members share the zf–HIT domain (ZHD), which is characterized by a fold in “treble-clef” through interleaved CCCC and CCHC ZnF motifs that both bind a zinc atom. Six proteins containing ZHD are present in human and three in yeast proteome, all belonging to multimodular RNA/protein complexes involved in gene regulation, chromatin remodeling, and snoRNP assembly. An interesting characteristic of the cellular complexes that ensure these functions is the presence of the RuvBL1/2/Rvb1/2 ATPases closely linked with zf–HIT proteins. Human ZNHIT6/BCD1 and its counterpart in yeast Bcd1p were previously characterized as assembly factors of the box C/D snoRNPs. Our data reveal that the ZHD of Bcd1p is necessary but not sufficient for yeast growth and that the motif has no direct RNA-binding capacity but helps Bcd1p maintain the box C/D snoRNAs level in steady state. However, we demonstrated that Bcd1p interacts nonspecifically with RNAs depending on their length. Interestingly, the ZHD of Bcd1p is functionally interchangeable with that of Hit1p, another box C/D snoRNP assembly factor belonging to the zf–HIT family. This prompted us to use NMR to solve the 3D structures of ZHD from yeast Bcd1p and Hit1p to highlight the structural similarity in the zf–HIT family. We identified structural features associated with the requirement of Hit1p and Bcd1p ZHD for cell growth and box C/D snoRNA stability under heat stress. Altogether, our data suggest an important role of ZHD could be to maintain functional folding to the rest of the protein, especially under heat stress conditions.

Combinatorial phenotypic screen uncovers unrecognized family of extended thiourea inhibitors with copper-dependent anti-staphylococcal activity.

The continuous rise of multi-drug resistant pathogenic bacteria has become a significant challenge for the health care system. In particular, novel drugs to treat infections of methicillin-resistant Staphylococcus aureus strains (MRSA) are needed, but traditional drug discovery campaigns have largely failed to deliver clinically suitable antibiotics. More than simply new drugs, new drug discovery approaches are needed to combat bacterial resistance. The recently described phenomenon of copper-dependent inhibitors has galvanized research exploring the use of metal-coordinating molecules to harness copper's natural antibacterial properties for therapeutic purposes. Here, we describe the results of the first concerted screening effort to identify copper-dependent inhibitors of Staphylococcus aureus. A standard library of 10 000 compounds was assayed for anti-staphylococcal activity, with hits defined as those compounds with a strict copper-dependent inhibitory activity. A total of 53 copper-dependent hit molecules were uncovered, similar to the copper independent hit rate of a traditionally executed campaign conducted in parallel on the same library. Most prominent was a hit family with an extended thiourea core structure, termed the NNSN motif. This motif resulted in copper-dependent and copper-specific S. aureus inhibition, while simultaneously being well tolerated by eukaryotic cells. Importantly, we could demonstrate that copper binding by the NNSN motif is highly unusual and likely responsible for the promising biological qualities of these compounds. A subsequent chemoinformatic meta-analysis of the ChEMBL chemical database confirmed the NNSNs as an unrecognized staphylococcal inhibitor, despite the family's presence in many chemical screening libraries. Thus, our copper-biased screen has proven able to discover inhibitors within previously screened libraries, offering a mechanism to reinvigorate exhausted molecular collections.

Characterization of hydrogen peroxide-resistant Acinetobacter species isolated during the Mars Phoenix spacecraft assembly.

The microbiological inventory of spacecraft and the associated assembly facility surfaces represent the primary pool of forward contaminants that may impact the integrity of life-detection missions. Herein, we report on the characterization of several strains of hydrogen peroxide-resistant Acinetobacter, which were isolated during the Mars Phoenix lander assembly. All Phoenix-associated Acinetobacter strains possessed very high catalase specific activities, and the specific strain, A. gyllenbergii 2P01AA, displayed a survival against hydrogen peroxide (no loss in 100 mM H2O2 for 1 h) that is perhaps the highest known among Gram-negative and non-spore-forming bacteria. Proteomic characterizations reveal a survival mechanism inclusive of proteins coupled to peroxide degradation (catalase and alkyl hydroperoxide reductase), energy/redox management (dihydrolipoamide dehydrogenase), protein synthesis/folding (EF-G, EF-Ts, peptidyl-tRNA hydrolase, DnaK), membrane functions (OmpA-like protein and ABC transporter-related protein), and nucleotide metabolism (HIT family hydrolase). Together, these survivability and biochemical parameters support the hypothesis that oxidative tolerance and the related biochemical features are the measurable phenotypes or outcomes for microbial survival in the spacecraft assembly facilities, where the low-humidity (desiccation) and clean (low-nutrient) conditions may serve as selective pressures. Hence, the spacecraft-associated Acinetobacter, due to the conferred oxidative tolerances, may ultimately hinder efforts to reduce spacecraft bioburden when using chemical sterilants, thus suggesting that non-spore-forming bacteria may need to be included in the bioburden accounting for future life-detection missions.

Application of encoded library technology (ELT) to a protein–protein interaction target: Discovery of a potent class of integrin lymphocyte function-associated antigen 1 (LFA-1) antagonists

The inhibition of protein–protein interactions remains a challenge for traditional small molecule drug discovery. Here we describe the use of DNA-encoded library technology for the discovery of small molecules that are potent inhibitors of the interaction between lymphocyte function-associated antigen 1 and its ligand intercellular adhesion molecule 1. A DNA-encoded library with a potential complexity of 4.1 billion compounds was exposed to the I-domain of the target protein and the bound ligands were affinity selected, yielding an enriched small-molecule hit family. Compounds representing this family were synthesized without their DNA encoding moiety and found to inhibit the lymphocyte function-associated antigen 1/intercellular adhesion molecule-1 interaction with submicromolar potency in both ELISA and cell adhesion assays. Re-synthesized compounds conjugated to DNA or a fluorophore were demonstrated to bind to cells expressing the target protein.

Identification of Drosophila and Human 7-Methyl GMP-specific Nucleotidases

Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m(7)GMP) or diphosphate (m(7)GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5' nucleotidase from Drosophila that cleaves m(7)GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m(7)GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5' nucleotidase activity with a high affinity for m(7)GMP. CMP is a slightly better substrate but again with a higher K(m). The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m(7)GMP as a substrate. We suggest that the m(7)G-specific nucleotidases protect cells against undesired salvage of m(7)GMP and its incorporation into nucleic acids.

The design and synthesis of a pyrrole-carbaldehyde hit family as HIV-1 integrase inhibitors

Screening of an HIV-1 IN model against database-derived chemical structures identified pyrrole-carbaldehydes as targets for further development. Chemical synthesis using a facile one-pot reaction resulted in the generation of a compound library. Preliminary toxicity and efficacy assays of the water-soluble HCl-salt derivatives of each compound support the in silico predictions.

The faithful citizen: popular Christian media and gendered civic identities

Kristy Maddux. The Faithful Citizen: Popular Christian Media and Gendered Civic Identities. Waco: Baylor University Press, 2010. 282 pp. $22.76. ISBN: 160258253X The Faithful Citizen is an intertextual analysis of ways in which Christian-themed popular media texts model forms of for Christian viewers. Kristy Maddux pays close attention to construction and deployment of gender in each of her major case studies, which include films, a popular Christian fiction series, and a hit family television show. Breaking from social scientific model of analyzing faith-based through measured reception studies, Maddux uses rhetorical criticism as well as feminist theory and close textual analysis to find conflicted images of engagement with five different ideologies of gender, which, taken together, suggest richness of contemporary ideals of participation (6). To Maddux, these models of include narrow definitions of citizenship, shifting impulses between moral reform and social justice, and influence of new media in making visible new ways of participating in life (6-7). Maddux organizes her book around media texts and their corresponding notions of gendered participation. Amazing Grace (chapter two) illustrates genteel masculinity in William Wilberforce's fight for abolition; The Passion of Christ (chapter three) models submission and valorizes suffering, while blurring lines between liberation theology and perceived (white) victimhood; Left Behind (chapter four) idealizes brutish masculinity in novel's apocalyptic setting; 7th Heaven (chapter five) depicts Camden family's charity, suggesting that faith communities can (and should) provide for welfare needs of their neighbours when federal government cannot; and The DaVinci Code (chapter six) depicts civic nonparticipation, or relegation of human sexuality and religious faith to private sphere. Maddux's final chapter explores limitations and implications of each model of gendered for other identity categories, such as race. It is important to note that Maddux does not treat these texts as repositories of ideas but, rather, as texts that construct, disseminate, and popularize these ideologies and identities (24). Maddux seeks to identify what these texts do in their social world, not simply what they mean about their social world (24). By performing close readings and parsing out various conventions work, Maddux not only makes notions of gendered explicit but also legible to readers. One example of this kind of legibility is found in Maddux's chapter about The DaVinci Code where she clearly deciphers how gender, religion, and civic nonparticipation intersect in a popular text, which many leaders of Christian denominations viewed as being radically feminist. While The DaVinci Code's historical narrative and surface-level plot celebrate the sacred feminine, Maddux contends that its focus on women's capacity to engage in heterosexual reproduction and motherhood is made at expense of any other feminine qualities (160). …

Affinity of Dinucleotide Cap Analogues for Human Decapping Scavenger (hDcpS)

Eukaryotic cells utilize scavenger decapping enzymes to degrade cap structure following 3′-5′ mRNA decay. Human DcpS recently has been described as a highly specific hydrolase (a member of the HIT family) that catalyses the cleavage of m 7 GpppG and short capped oligoribonucleotides. We have demonstrated here that cap-1 (m 7 GpppGm) is a preferred substrate among several investigated dinucleotide cap analogues m 7 Gp n N (n = 3–5, N is a purine or pyrimidine base) and m 7 GMP is always one of the reaction product. Cap analogues containing pyrimidine base instead of guanine or diphosphate chain are resistant to hydrolysis catalyzed by human scavenger. Contrary to the other enzymes of HIT family, hDcpS activity is not stimulated by Mg 2+.

Identification of an mRNA-Decapping Regulator Implicated in X-Linked Mental Retardation

Two major decapping enzymes are involved in the decay of eukaryotic mRNA, Dcp2 and DcpS. Despite the detection of robust DcpS decapping activity in cell extract, minimal to no decapping is detected from human Dcp2 (hDcp2) in extract. We now demonstrate that one reason for the lack of detectable hDcp2 activity in extract is due to the presence of inhibitory trans factor(s). Furthermore, we demonstrate that a previously identified testis-specific protein of unknown function implicated in nonspecific X-linked mental retardation, VCX-A, can function as an inhibitor of hDcp2 decapping in vitro and in cells. VCX-A is a noncanonical cap-binding protein that binds to capped RNA but not cap structure lacking an RNA. Its cap association is enhanced by hDcp2 to further augment the ability of VCX-A to inhibit decapping. Our data demonstrate that VCX-A can regulate mRNA stability and that it is an example of a tissue-specific decapping regulator.

PH‐Dependence in the Hydrolytic Action of the Human Fragile Histidine Triad

AbstractThe human fragile histidine triad protein (Fhit) is a member of the HIT family of enzymes, which catalyze hydrolysis or nucleotidyltransfer reactions of dinucleoside polyphoshates. Fhit catalyzes the magnesium ion‐dependent hydrolysis of P1–5′‐O‐adenosine‐P3–5′‐O‐adenosine triphosphate (Ap3A) to adenosine‐5′‐O‐phosphate (AMP) and the magnesium complex of adenosine‐5′‐O‐diphosphate (ADP) by a double displacement mechanism, with the formation of an adenylyl enzyme as an intermediate. Fhit also catalyzes the hydrolysis of adenosine‐5′‐phosphoimidazolide (AMP‐Im) and adenosine‐5′‐phospho‐N‐methylimidazolide (AMP‐N‐MeIm). The pH‐dependence of these reactions is reported herein, in which the principal conclusions are as follows: The action of wild‐type Fhit on MgAp3A is diffusion‐limited and on AMP‐Im and AMP‐N‐MeIm largely diffusion‐limited and largely pH‐independent. The actions of specifically mutated H96G‐Fhit on AM‐Im, and on AMP‐N‐MeIm, are not diffusion‐limited and are pH‐dependent. The actions of mutated forms of Fhit, H94G‐Fhit, H98G‐Fhit, and H94/98G‐Fhit, are also not diffusion‐limited and are pH‐dependent. Log plots of kinetic parameters against pH show breaks that indicate a group on the enzyme must be protonated for maximal activity. Extensive analysis shows that the imidazole ring of His94 is not essential for the hydrolysis of MgAp3A or AMP‐imidizolides, and the imidazole ring of His98 engages in binding the substrates. In the hydrolysis of AMP‐Im, Fhit and its His94, His96, and His98 variants bind the monoanionic form of AMP‐Im, and the proton required for formation of imidazole in the hydrolytic process originates with an acid/base group of the enzyme. Fhit and several variants also catalyze the hydrolysis of p‐nitrophenyl‐AMP. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)

HIT family genes: FHIT but not PKCI-1/HINT produces altered transcripts in colorectal cancer.

Forty-five colorectal adenocarcinomas were examined for alterations in the HIT family genes FHIT and PKCI-1/HINT by a combination of reverse transcriptase polymerase chain reaction and DNA sequencing. In all cases a single transcript corresponding to the reported sequence was detected using primers specific for the PKCI-1/HINT gene. In contrast multiple transcripts were detected using primers specific for the FHIT gene transcript. 6% (3/45) of tumours evinced no detectable expression of any FHIT transcript and a further 12% (6/45) produced only the normal full length transcripts. Ninety-six aberrant transcripts were characterized from the remaining tumours. Deviations from the normal full length sequence characterized included deletions, insertions of novel sequences, a point mutation as well as the usage of a putative alternate splice site in exon 10. Message variants were detected with approximately equal frequency in all tumour stages with the exception that templates with insertions were found solely in Dukes’ stage B tumours (P < 0.001). With the exception of the putative alternate splice site, aberrant transcripts were not detected in matched normal mucosa. These results suggest that members of the HIT family of genes are only selectively involved in tumorigenesis and that perturbation of FHIT gene expression is an early event in colorectal tumorigenesis. © 1999 Cancer Research Campaign

Three-dimensional structure of human protein kinase C interacting protein 1, a member of the HIT family of proteins.

The three-dimensional structure of protein kinase C interacting protein 1 (PKCI-1) has been solved to high resolution by x-ray crystallography using single isomorphous replacement with anomalous scattering. The gene encoding human PKCI-1 was cloned from a cDNA library by using a partial sequence obtained from interactions identified in the yeast two-hybrid system between PKCI-1 and the regulatory domain of protein kinase C-beta. The PKCI-1 protein was expressed in Pichia pastoris as a dimer of two 13.7-kDa polypeptides. PKCI-1 is a member of the HIT family of proteins, shown by sequence identity to be conserved in a broad range of organisms including mycoplasma, plants, and humans. Despite the ubiquity of this protein sequence in nature, no distinct function has been shown for the protein product in vitro or in vivo. The PKCI-1 protomer has an alpha+beta meander fold containing a five-stranded antiparallel sheet and two helices. Two protomers come together to form a 10-stranded antiparallel sheet with extensive contacts between a helix and carboxy terminal amino acids of a protomer with the corresponding amino acids in the other protomer. PKCI-1 has been shown to interact specifically with zinc. The three-dimensional structure has been solved in the presence and absence of zinc and in two crystal forms. The structure of human PKCI-1 provides a model of this family of proteins which suggests a stable fold conserved throughout nature.