The Influence of Heights of Power Arm for Controlled Anterior Teeth Movement in Sliding Mechanics: A 3D FEM Study.

INTRODUCTION: The effectiveness of orthodontic treatment depends on precise control of the anterior teeth during retraction in sliding mechanics. That is why researchers were interested in finding the appropriate loading conditions for sliding mechanics to govern anterior teeth's movement, such as the height of retraction force application and its location on the arch wire. METHODS:  A FEM study was conducted to evaluate the type of movement of anterior teeth during en-masse retraction by applying 200 gms of force to each side of the maxillary arch with power arms set at various levels (4 mm, 6 mm, 8 mm) mesial to canine from mini-screw placed at a height of 8 mm between maxillary first molar and second premolar. A 3D model (FEM) of the maxillary arch was constructed to study the amount of labio-lingual tipping and bodily displacement of maxillary central incisor achieved. RESULTS:  When a 200-gm power was applied to the power arm, controlled lingual crown tipping was seen at the levels of 4 mm and 6 mm, in contrast to the level of 8 mm, where bodily displacement occurred, according to this evaluation.  Conclusion: In sliding mechanics, the height of the power arm plays an important role in obtaining controlled lingual crown tipping or bodily displacement during retraction of anterior teeth.

Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev.

The HIV-1 Rev protein facilitates the nuclear export of mRNA containing the Rev response element (RRE) through binding to the export receptor CRM-1. Here we show that a cellular nuclear protein, Sam68 (Src-associated protein in mitosis), specifically interacts with RRE and can partially substitute for as well as synergize with Rev in RRE-mediated gene expression and virus replication. Differential sensitivity to leptomycin B, an inhibitor of CRM-1, indicates that the export pathways mediated by Rev and Sam68 are distinct. C-terminally deleted mutants of Sam68 inhibited the transactivation of RRE-mediated expression by both wild-type Sam68 and Rev. They were retained in the cytoplasm and impeded the nuclear localization of Rev in co-expressed cells. These mutants also inhibited wild-type HIV-1 replication to the same extent as the RevM10 mutant, and may be useful as anti-viral agents in the treatment of AIDS.

Identification and validation of the mode of action of the chalcone anti-mycobacterial compounds.

OBJECTIVES: The search for new TB drugs has become one of the great challenges for modern medicinal chemistry. An improvement in the outcomes of TB chemotherapy can be achieved by the development of new, shorter, cheap, safe and effective anti-TB regimens. METHODS: Chalcones (1a-1o) were synthesized and evaluated for their antimycobacterial activity against Mycobacterium bovis BCG using growth inhibition assays. Compound 1a was selected as a 'hit' compound. The mode of action of compound 1a, was identified by mycolic acid methyl esters (MAMEs) and fatty acid methyl esters (FAMEs) analysis using thin layer chromatography. Dose dependent experiments were conducted by over-expressing components of FAS-II in M. bovis BCG to confirm the target. Ligand binding using intrinsic tryptophan assay and molecular docking were used to further validate the target. RESULTS: MAMEs and FAMEs analysis showed dose-dependent reduction of MAMEs with the overall abundance of FAMEs suggesting that compound 1a targets mycolic acid biosynthesis. Direct binding of 1a to InhA was observed using an intrinsic tryptophan fluorescence binding assay, and a 2-fold IC50 shift was observed with an InhA overexpressing strain confirming InhA as the cellular target. CONCLUSION: The chalcone 1a exhibits potent antimycobacterial activity, displays a good safety profile and is a direct inhibitor of InhA, a key component in mycolic acid synthesis, validating this series for further anti-TB drug development.

A single point mutation in the nuclear localization domain of Sam68 blocks the Rev/RRE-mediated transactivation.

We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, we have shown that the C-terminal deletion mutants of Sam68 act with a transdominant negative phenotype in HIV replication. Previously, an Arginine429 mutation within the C-terminal domain of Sam68 has been reported to be critical for the localization of Sam68 in the nucleus. However, these studies were done in the context of truncated protein in which C-terminal amino acids 420 - 443 of Sam68 were fused to GFP. In contrast, we now report that the full length Sam68 protein having the same mutation (Arginine429-->Alanine) is completely localized in the nucleus while another Sam68 (Proline439-->Arginine) mutant is found in the cytoplasm. The localization of these Sam68 mutant proteins also correlates with their function in RRE-mediated reporter gene expression, i.e. Sam68 mutant protein that is localized in the cytoplasm failed to enhance RRE-mediated transactivation. Furthermore, we demonstrate that Sam68 P439-->R inhibited the transactivation of RRE-mediated gene expression by both wild type Sam68 and Rev. These results indicate that the proline residue at position 439 unlike arginine at position 429, may play a critical role in targeting Sam68 protein to nucleus. We propose that these negative dominant mutants of Sam68 may have potential as anti-viral agents to combat AIDS.

Functional interaction of the HTLV-1 transactivator Tax with activating transcription factor-4 (ATF4).

The Tax protein of the Human T-cell Leukemia Virus (HTLV) activates the expression of viral mRNA through a three 21 bp repeat enhancer located within the HTLV-1 LTR. Since Tax does not bind to the 21 bp DNA repeats directly, it has been speculated that Tax interacts with cellular protein(s) which mediate binding to the enhancer. We employed the yeast two hybrid system to identify host proteins that are potentially relevant to Tax transactivation. We identified a Tax binding protein encoded from a cDNA expression library derived from peripheral blood lymphocytes. The corresponding cDNA has sequence identity with a known transcription factor, activating factor-4 (ATF-4). ATF-4 also binds to GST-Tax fusion protein in vitro. Tax mutants that did not transactivate the HTLV-1 LTR also failed to bind ATF-4. The critical domain for Tax binding resides in a 85 amino acid stretch in the C-terminus of ATF-4, which contains the basic domain and leucine zipper. We further demonstrated that both full length and N-terminal truncated ATF-4 were able to enhance Tax transactivation. Thus, ATF-4 may act as an adapter between Tax and the TRE (Tax responsive element), and play an important role in Tax-mediated transactivation.

General effect of Sam68 on Rev/Rex regulated expression of complex retroviruses.

We have previously demonstrated that overexpression of Sam68 functionally substitutes for, as well as synergizes with, HIV-1 Rev in RRE-mediated gene expression and virus replication. In addition, C-terminal deletion mutants of Sam68 exhibit a transdominant negative phenotype in HIV replication. We now report that Sam68 also enhances the activities of Rev-like proteins of other complex retroviruses (e.g. HTLV-1 and EIAV) on their respective RNA targets. Furthermore, we demonstrate that Sam68 can function alone as well as synergize with Rev-MS2 and/or Rex-MS2 chimeric proteins on expression mediated by the corresponding RRE-MS2 fusion RNA element. Additionally, dominant negative mutants of Sam68 also repressed the synergistic activation of Sam68 with Rex, E-Rev, and/or Rev-MS2/Rex-MS2 on their corresponding RNA targets. Thus, Sam68 may play an important role in the post-transcriptional regulation of all complex retroviruses. Oncogene (2000) 19, 4071 - 4074

Sam68, RNA helicase A and Tap cooperate in the post-transcriptional regulation of human immunodeficiency virus and type D retroviral mRNA.

Unlike cellular mRNA, retroviral mRNA bypasses the tight coupling of the splicing and nuclear export steps to allow the export of intron-containing viral RNA transcripts to the cytoplasm. Two distinct nuclear export pathways for retroviral mRNA have been described: a CRM-1 dependent pathway mediated by the HIV-1 Rev protein and the Rev Response Element (RRE), and a CRM-1 independent pathway mediated by the Constitutive Transport Element (CTE) of type D retroviruses. Two CTE-binding proteins, RNA helicase A (RHA) and Tap, have been implicated in the nuclear export of CTE-containing RNA. Recently, we reported that expression of RRE-containing RNA could also be mediated by a cellular protein, Sam68, independently of Rev. Here we report evidence that Sam68, RHA and Tap cooperate in the nuclear export of both CTE- and RRE-containing RNA. RHA binds to Sam68 and to Tap both in vivo and in vitro. Over-expression of Sam68 activates both RRE- and CTE-regulated reporter gene expression in human cells and in quail cells in the presence of human Tap. This activation was competitively inhibited by the nuclear transport domain (NTD) of RHA and a transdominant negative mutant of Tap. Conversely, the activation of CTE by Tap in quail cells was inhibited by a transdominant mutant of Sam68 and NTD. We propose that both HIV and type D retroviruses may access the same constitutive RNA nuclear export pathway involving RHA, Tap and Sam68, even though HIV also utilizes the Rev protein for more efficient nuclear export. it is likely that this constitutive export pathway is also used by cellular mRNA, but at a different interface with the splicing process.

One-step, highly efficient site-directed mutagenesis by toxic protein selection.

A fast and efficient site-directed mutagenesis method has been developed, using the newly constructed plasmid pTPS19, which expresses the toxic CcdB protein originally encoded by the E. coli F plasmid. Once the target gene is cloned into pTPS19, desired mutations can be introduced with two primers. The first contains the desired mutation, and the second is designed to create a +1 frame shift in the ccdB gene to inactivate the CcdB protein. The mutants can be directly selected on LB plates containing IPTG, through which the toxic CcdB protein is induced, thereby eliminating cells carrying wild-type parental plasmids. Based on stringent selection through the toxic CcdB protein, mutagenesis efficiency of 90%-100% was reached even after one round of transformation.

Molecular cloning and characterization of a TAR-binding nuclear factor from T cells.

The TAt protein of the human immunodeficiency virus type 1 (HIV-1) activates the expression of viral mRNA through a cis-acting element in the LTR termed TAR. TAR RNA forms a stable stem-loop structure. Mutagenesis studies indicate that the stem structure, the primary sequence of the loop, and three unpaired bases in the stem (bulge) are important for Tat activation. Using the in vitro-transcribed TAR RNA as a probe, we have cloned a gene (TARBP-b) that encodes a TAR-binding protein from a cDNA expression library derived from Hut-78 cells. Expression of the 1.4-kb TARBP-b mRNA was observed in all mammalian cell lines tested. TARBP-b binds specifically to the bulge region of TAR RNA and trans-activates the HIV-1 long terminal repeat in the presence of ptat and prev expression plasmids. These results suggest that TARBP-b contributes to tat-mediated trans-activation.

A Novel hnRNP Specifically Interacts with HIV-1 RRE RNA.

We have identified and obtained the full-length clone of RREBP49, a human nuclear factor which specifically interacts with the Rev-responsive element (RRE) sequence of human immunodeficiency virus type 1. Sequence analysis revealed that RREBP49 is highly homologous to hnRNP F protein and contains three repeated RNA-binding domains. Binding assays demonstrated that Rev and RREBP49 bind to different subregions on the RRE sequence and that binding is mutually nonexclusive. Blocking of endogenous RREBP49 expression by an antisense construct increases Rev activity in CV-1 cells, indicating that RREBP49 and Rev may play antagonistic roles in HIV-1 replication. RREBP49 may function as a splicing factor or a nuclear retention factor for unspliced mRNAs. However, only a slight decrease of Rev activity was observed when exogenous RREBP49 was introduced into CV-1 cells by pSVL-RREBP49 expression vector. This may be explained by a high endogenous level of RREBP49 which is above optimal. Alternatively, additional cellular factors may be required for RREBP49-mediated inhibition of Rev. Copyright 1996 S. Karger AG, Basel

Effect of thermal stress on early and late passaged mouse lens epithelial cells.

Cataract is an age related disease of protein aggregation. It has been suggested that aging affects the cells ability to protect protein integrity. The protein integrity, which is essential for cellular homeostasis, is maintained by a complex system of refolding or degradation of damaged proteins. The heat shock proteins (hsps) are the major contributors in the maintenance of protein integrity. The heat shock transcription factor (HSF-1) is the master regulator of all hsp synthesis in response to stress. This investigation examined the role of HSF-1 in the regulation of hsp synthesis in early and late passaged alphaTN-4 cells. Data collected in this study revealed that the nucleotide sequence of HSF-1 mRNA obtained from early and late passaged alphaTN-4 cells were identical. When early and late passaged cell were exposed to thermal stress, their hsp expression were also similar. HSP-40 expression was detected after 2 h of heat stress, whereas HSP-70 and low molecular weight heat shock protein alphabeta crystallin showed significantly increased synthesis 18 h post heat stress. The late passaged alphaTN-4 cells ability to upregulate hsps in response to heat stress could be due to its high replicative activities. The data presented here suggests a relationship between the presence of functional HSF-1 and sustained proliferative activities of the late passaged alphaTN-4 cell.

Archaebacterial histone-like proteins. Purification and characterization of helix stabilizing DNA binding proteins from the acidothermophile Sulfolobus acidocaldarius.

Four DNA binding histone-like proteins have been purified from the nucleoid of the acidothermophilic archaebacterium Sulfolobus acidocaldarius to homogeneity employing DNA-cellulose chromatography and carboxymethylcellulose chromatography. The molecular weights of these proteins are in the range 8,000-12,500. Immunoblotting results suggest that a few antigenic determinants are common among these proteins which could not be detected by immunodiffusion. Spectroscopic properties of the proteins have been studied. The amino acid compositions of these proteins show both similarities and differences with histones and prokaryotic histone-like proteins. All of the four proteins bind native and denatured DNAs and single stranded RNA with differing affinities. Three of the proteins, denoted by HSNP (helix stabilizing nucleoid protein)-A, HSNP-C, and HSNP-C', show physiologically significant, strong, and synergistic effects in stabilizing duplex DNA against thermal denaturation with Tm increases in the range of 15-30 +/- degrees C.

Life-threatening acute respiratory distress in late pregnancy.

Acute severe ventilatory failure from any cause is always a critical emergency. In advanced pregnancy, such an episode is particularly hazardous. The management of a pregnant patient who presented with an acute episode of respiratory distress is described. Although this was though initially to be an asthmatic attack, there was evidence of upper airway obstruction caused by an enlarged thyroid. The major complicating factors and aetiology are discussed.

A novel shuttle protein binds to RNA helicase A and activates the retroviral constitutive transport element.

The constitutive transport element (CTE) of type D retroviruses mediates the nuclear export of unspliced viral transcripts. We previously showed that RNA helicase A functionally interacts with CTE and contains a bidirectional nuclear transport domain at the carboxyl terminus. Here we report the identification of a novel human protein, helicase A-binding protein 95 (HAP95), which specifically binds to the carboxyl terminus of RNA helicase A. HAP95 is partially homologous to AKAP95, a member of the A kinase-anchoring protein family, but lacks the protein kinase A binding domain characteristic of this family. HAP95 is a nuclear protein at steady state but shuttles between the nucleus and cytoplasm. Overexpression of HAP95 significantly increases CTE-dependent gene expression but has no effect on general gene expression or that mediated by the Rev/Rev response element of human immunodeficiency virus type 1.

Identification and mapping of inhibitory sequences in the human immunodeficiency virus type 2 vif gene.

Human immunodeficiency virus (HIV) regulates the expression of its genes temporally at the mRNA processing step. A subset of the mRNA species which encode the structural and some accessory genes contains inhibitory sequences (INS or CRS elements) which prevent nuclear export of the RNA or its utilization in the cytoplasm. Such inhibition is overridden by the interaction of a viral protein, Rev, with its RNA target sequence, RRE. The vif gene product, which is essential for virus replication in vivo, is encoded by a singly spliced mRNA, and its expression is dependent on rev in infected cells. However, INS elements have not been found in the HIV-1 vif gene itself, although such elements have been observed in Gag, Pol, and Env coding sequences. We have now identified an INS within the 5' half of HIV-2 vif which does not show any homology with cellular mRNAs or other previously identified INS and CRS elements of HIV. These results suggest that retroviral mRNAs have novel labile sequences different from those of cellular mRNAs.

Mapping the functional domains of HAP95, a protein that binds RNA helicase A and activates the constitutive transport element of type D retroviruses.

The complex retroviruses such as human immunodeficiency virus, type 1, employ a virally encoded protein, Rev, to mediate the nuclear export of unspliced and partially spliced mRNA. In contrast, the simian type D retroviruses act through a cis-acting constitutive transport element (CTE) that presumably interacts directly with cellular export proteins. We first reported that RNA helicase A (RHA) is a shuttle protein that binds to functional CTE in vitro and in vivo. Recently, we isolated a novel protein, HAP95, that specifically binds to the nuclear transport domain of RHA and up-regulates CTE-mediated gene expression. Here, using truncation and deletion mutations, we mapped the domains of HAP95 that are important for RHA binding, transactivation of CTE, and nuclear cytoplasmic shuttling. We report evidence for a novel nuclear export signal in HAP95 and showed that the domains involved in RHA binding and nuclear localization are required for CTE activation. Finally, we showed that HAP95 synergizes significantly with RHA on CTE-mediated reporter gene expression and promotes nuclear export of unspliced mRNA in transfected cells. Taken together, these data support the proposal that HAP95 specifically facilitates CTE-mediated gene expression by directly binding to RHA.

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.

A role for RNA helicase A in post-transcriptional regulation of HIV type 1.

Retroviruses must bypass the tight coupling of splicing and nuclear export of mRNA in their replication cycle because unspliced genomic RNA and incompletely spliced mRNA must be exported to the cytoplasm for packaging or translation. This process is mediated by a cis-acting constitutive transport element (CTE) for simple retroviruses and by the trans-acting viral protein Rev in concert with its response element (RRE) for complex retroviruses (e.g., HIV). Recently, we identified RNA helicase A (RHA) as a potential cellular cofactor for CTE. Here, we report that RHA also plays a role in Rev/RRE-mediated gene expression and HIV replication. RHA binds weakly to HIV-1 RRE independently of Rev. Overexpression of RHA, but not of an RHA mutant lacking helicase activity, increased both Rev/RRE- and CTE-dependent gene expression and the levels of unspliced HIV mRNA. Microinjection of antibodies to RHA into nuclei dramatically inhibited both CTE- and Rev-dependent gene expression in human cells. Exogenous RHA cDNA, but not the mutant RHA, rescued this inhibition. We propose that RHA is required to release both CTE- and RRE-containing mRNA from spliceosomes before completion of splicing, thus freeing them for nuclear export.

Interactions of HIV-1 nef with the mu subunits of adaptor protein complexes 1, 2, and 3: role of the dileucine-based sorting motif.

HIV-1 Nef interacts with cellular adaptor protein (AP) complexes and their medium (mu) subunits. However, the role of the dileucine-based sorting motif within Nef in these interactions has been incompletely characterized. Here, yeast two-hybrid assays indicated that HIV-1 Nef interacted not only with the mu subunits of AP-1 and AP-2, but also with that of AP-3. The interactions with mu1 and mu3 were markedly stronger than the interaction with mu2. Leucine residues of the sorting motif were required for the interactions with mu3 and mu2 and contributed to the interaction with mu1. Confocal immunofluorescence microscopy indicated that Nef, AP-1, and AP-3 (but not AP-2) were concentrated in a juxtanuclear region near the cell center, potentially facilitating interaction between Nef and the mu1 and mu3 subunits. However, leucine residues of the sorting motif were not required for this subcellular localization of Nef. These data suggest that the dileucine motif, required for optimal viral replication, functions through interactions with a variety of AP complexes, including AP-3, potentially by recruiting adaptor complexes to subcellular locations specified by additional determinants in the Nef protein.