A Novel Technique-Bone Splitting and Bone Grafting in an Hourglass-shaped Bone Following Distraction Osteogenesis.

AIM: We describe a novel technique of bone splitting and bone grafting in managing hypoplastic or hourglass-shaped regenerated bone in distraction osteogenesis. BACKGROUND: Hourglass-shaped regenerated bone is a potential complication during distraction osteogenesis which is vulnerable to fracture when loaded. Our novel technique overcomes this by increasing the diameter of new bone formation via bone splitting and bone grafting. CASE DESCRIPTION: We report three cases with hypoplastic regenerated bone following distraction osteogenesis. It was treated with bone splitting and bone grafting. Although one case was complicated with an iatrogenic transverse fracture during the surgery, all three cases achieved the goal of increasing bone diameter during the subsequent consolidation phase. CONCLUSION: This relatively simple and novel surgical intervention can overcome the hourglass-shaped appearance, thus preventing potential fracture. CLINICAL SIGNIFICANCE: We emphasise the importance of identifying hypoplastic regenerate bone before the consolidation phase of distraction osteogenesis. The novel technique described is a simple surgical intervention which can prevent potential fracture through the newly formed bone. HOW TO CITE THIS ARTICLE: Lee HS, Tasarib R, Hamzah FC, et al. A Novel Technique-Bone Splitting and Bone Grafting in an Hourglass-shaped Bone Following Distraction Osteogenesis. Strategies Trauma Limb Reconstr 2020;15(3):175-178.

Computational study of bindings of olive leaf extract (OLE) to HIV-1 fusion protein gp41.

Recent experimental study found that OLE (olive leaf extract) has anti-HIV activity by blocking the HIV virus entry to host cells [Lee-Huang, S., Zhang, L., Huang, P.L., Chang, Y. and Huang, P.L. (2003) Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307, 1029; Lee-Huang, S., Huang, P.L., Zhang, D., Lee, J.W., Bao, J., Sun, Y., Chang, Y.-Tae, Zhang, J.Z.H. and Huang, P.L. (2007) Discovery of small-molecule HIV-1 fusion and integrase inhibitors oleuropein and hydroxytyrosol. Biochem. Biophys. Res. Commun. 354, 872-878, 879-884]. As part of a joint experimental and theoretical effort, we report here computational study to help identify and characterize the binding complexes of several main compounds of OLE (olive leaf extract) to HIV-1 envelop protein gp41. A number of possible binding modes are found by docking oleuropein and its metabolites, aglycone, elenolic acid and hydroxytyrosol, onto the hydrophobic pocket on gp41. Detailed OLE-gp41 binding interactions and free energies of binding are obtained through molecular dynamics simulation and MM-PBSA calculation. Specific molecular interactions in our predicted OLE/gp41 complexes are identified and hydroxytyrosol is identified to be the main moiety for binding to gp41. This computational study complements the corresponding experimental investigation and helps establish a good starting point for further refinement of OLE-based gp41 inhibitors.

Crystallization and preliminary X-ray analysis of GAP 31. A protein which inhibits the life cycle of HIV-1.

GAP 31 is an anti-HIV plant protein that we have identified and purified to homogeneity from Gelonium multiflorum. It is the first reported example of an anti-HIV agent capable of acting against multiple stages of the viral life cycle, on viral infection and viral replication. GAP 31 is a unique paragon of multi-functional protein. In addition to anti-HIV activity, it also exhibits anti-tumor action, DNA binding, RNA binding and ribosome inactivation. The present crystals diffract up to 2.0 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 49.30(2) A, b = 44.57(2) A, c = 137.78(7) A and beta = 98.32(3) degrees. There are two molecules of molecular weight 31 kDa in an asymmetric unit with a solvent content of 49%.

Anti-HIV and anti-tumor protein MAP30, a 30 kDa single-strand type-I RIP, shares similar secondary structure and beta-sheet topology with the A chain of ricin, a type-II RIP.

MAP30 is a 30 kDa single-stranded, type-I ribosome inactivating protein (RIP) possessing anti-tumor and anti-HIV activities. It binds both ribosomal RNA and the HIV-1 long-terminal repeat DNA. To understand the structural basis for MAP30 activities, we undertook the study of MAP30 by solution NMR spectroscopy. We report nearly complete 1H, 13C, and 15N chemical shift assignments of its 263 amino acids. Based upon an analysis of secondary 13C chemical shifts, 3J(HNHA) coupling constants, hydrogen exchange data, and nuclear Overhauser effect patterns, we find that the secondary structure and beta-sheet topology of MAP30 are very similar to those of the ricin A chain, a subunit of the well-known type-II RIP, even though two proteins display distinct activities. We therefore suggest that MAP30 and ricin A chain share a similar three-dimensional fold, and that the reported functional differences between two proteins arise primarily from differences in local three-dimensional structure and other structural properties such as surface electrostatic potentials.

The human erythropoietin-encoding gene contains a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5' flanking region.

We have reported the cloning and expression of a human erythropoietin (hEp)-encoding cDNA [Lee-Huang, Proc. Natl. Acad. Sci. USA 81 (1984) 2708-2712]. Using this hEp cDNA as a probe, we isolated a 9.3-kb BamHI genomic Ep clone from a human leukocyte library soon thereafter. The size and restriction map of this clone is in agreement with restriction analysis of human genomic DNA probed with the hEp cDNA, demonstrating that this clone is representative of the single hEp gene. This clone is unique in that it extends beyond any reported hEp genomic clone by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. The promoter function of the newly described 5' flanking region has been demonstrated by the expression of biologically active hEp in transfected cells. We find that, despite reports to the contrary, hEp does contain classic canonical TATA boxes and a CAAT box. The 5'-flanking region also contains cytokine-responsive consensus sequences, tissue-specific and metal-responsive elements, CRE and GRE sites, and binding sites for transcription factors, including AP1, NF-kappa beta and Sp1. These regulatory elements have not been found in the hEp genomic clones thus far reported. The identification of these elements and their precise localization in hEp should be useful in studying the regulation of hEp expression, as well as in gene therapy and physiologic modulation of this hormone.

The 3' flanking region of the human erythropoietin-encoding gene contains nitrogen-regulatory/oxygen-sensing consensus sequences and tissue-specific transcriptional regulatory elements.

We have reported the identification of a classical canonical CAAT box, TATA boxes and other transcriptional regulatory elements in the 5' flanking region of the human erythropoietin (hEp)-encoding gene [Lee-Huang et al., Gene 128 (1993) 227-236]. These elements were not found in the hEp genomic clones reported by others. Our genomic clone extends in both directions beyond any reported clones, by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. Many important regulatory elements are found in these extended flanking regions. We report here the genomic structure of the extended 3' flanking region of hEp. This region contains the following regulatory elements: nitrogen-regulatory/oxygen-sensing consensus sequences, 5'-TTTTGCA and 5'-CCCTGCA; tissue-specific regulatory elements, including binding sites for A-activator, 5'-GTGGTGCAA; for DBP, 5'-TGATTTTGT; for HNF, 5'-T(A/G)TTTGT; and for C/EBP, 5'-T(T/G) (T/G)TGCAAT; a lymphokine-responsive element, 5'-GTGAAACCCC (Rev), as well as binding sites for AP and Sp1. In addition, the nucleotide (nt) sequence in this region is rich in inverted repeats (palindromes) that allow the formation of hairpin loops. A total of 14 potential stem loops with a maximum loop size of 20 nt are found. The identification of these regulatory elements in hEp should provide further insight into the tissue-specific and inducible expression of hEp. Such knowledge should be useful in the clinical modulation of erythropoiesis under physiologic and pathologic conditions.

Anti-HIV and anti-tumor activities of recombinant MAP30 from bitter melon.

MAP30 is an anti-HIV plant protein that we have identified and purified to homogeneity from bitter melon (Momordica charantia). It is capable of acting against multiple stages of the viral life cycle, on acute infection as well as replication in chronically infected cells. In addition to antiviral action, MAP30 also possesses anti-tumor activity, topological inactivation of viral DNA, inhibition of viral integrase and cell-free ribosome-inactivation activities. We have cloned and expressed the MAP30 gene. The objective of this study is to characterize recombinant MAP30 (re-MAP30) and to determine its anti-HIV, anti-tumor and other activities. We report here that re-MAP30 inhibits HIV-1 and certain human tumors to the same extent as its native counterpart, natural MAP30 (nMAP30). The anti-HIV activity was measured by quantitative focal syncytium formation on CEM-ss cell monolayers, viral core protein p24 expression and viral-associated reverse transcriptase activity in HIV-1-infected H9 cells. The anti-tumor activity was measured by metabolic labeling of protein synthesis in tumor cells. In the dose range of the assay, re-MAP30 exhibits little toxicity to the uninfected viral target cells and other normal human cells. Identical to nMAP30, re-MAP30 is also active in topological inactivation of viral DNA, inhibition of viral DNA integration and cell-free ribosome inactivation. The cloning and expression of the gene encoding biologically active re-MAP30 provides an abundant source of homogeneous material for clinical investigations, as well as structure-function studies of this novel antiviral and anti-tumor agent.

The non-steroidal anti-inflammatory drug, indomethacin, as an inhibitor of HIV replication.

Indomethacin, a common non-steroidal anti-inflammatory drug (NSAID), has been used to treat rheumatoid arthritis. Although indomethacin has also been used as an immunopotentiator and symptomatic NSAID in AIDS, its effect on HIV replication is unknown. MT-4 lymphocytes were inoculated with HIV in the presence of indomethacin and tested for p24 expression by ELISA. The 50% inhibition (IC50) was 10 microM, corresponding to plasma levels after administration of 50 mg oral indomethacin. The antiviral effect appears to be specific since no toxicity has been observed at the IC50 dose, and unrelated NSAIDs have not shown the activity at clinical doses. Indomethacin may, thus, represent a new class of anti-HIV drug.

A new class of anti-HIV agents: GAP31, DAPs 30 and 32.

Three inhibitors of human immunodeficiency virus (HIV) have been isolated and purified to homogeneity from Euphorbiaceae himalaya seeds (Gelonium multiflorum) and carnation leaves (Dianthus caryophyllus). These proteins, GAP 31 (Gelonium Anti-HIV Protein 31 kDa) and DAPs 30 and 32 (dianthus anti-HIV proteins, 30 and 32 kDa), inhibit HIV-1 infection and replication in a dose-dependent manner with little toxicity to target cells. The therapeutic indices of these compounds are in the order 10(4), suggesting that they may be clinically important agents in the treatment of AIDS. The N-terminal amino acid sequences of these proteins show little homology to those of previously described anti-HIV proteins. The structure-function features of these HIV inhibitors, based on the 40-60 amino acid residues of N-terminal sequences, are examined.

MAP 30: a new inhibitor of HIV-1 infection and replication.

A new inhibitor of human immunodeficiency virus (HIV) has been isolated and purified to homogeneity from the seeds and fruits of the Momordica charantia. This compound, MAP 30 (Momordica Anti-HIV Protein), is a basic protein of about 30 kDa. It exhibits dose-dependent inhibition of cell-free HIV-1 infection and replication as measured by: (i) quantitative focal syncytium formation on CEM-ss monolayers; (ii) viral core protein p24 expression; and (iii) viral-associated reverse transcriptase (RT) activity in HIV-1 infected H9 cells. The doses required for 50% inhibition (ID50) in these assays were 0.83, 0.22 and 0.33 nM, respectively. No cytotoxic or cytostatic effects were found under the assay conditions. These data suggest that MAP 30 may be a useful therapeutic agent in the treatment of HIV-1 infections. The sequence of the N-terminal 44 amino acids of MAP 30 has been determined.

Protective effect of interferon-alpha against cell-mediated human immunodeficiency virus transmission resulting from coculture of infected lymphocytes with fetal trophoblasts.

The hypothesis that the low transmission rate of HIV in utero may be due, in part, to the protective effect of IFN-producing placental trophoblasts was explored in vitro. The model consisted of H9 lymphocytes, as surrogates of maternal HIV-infected T cells, incubated for 3 h with JEG-3 trophoblasts in the presence of 10-fold dilutions of leukocyte-derived IFN-alpha (from 1000 to 0.1 IU/ml). The dose effect was monitored either directly, by measuring the levels of proviral DNA by PCR after a single round of infection, or indirectly, by coculturing infected JEG-3 with cord blood-derived MT-4 lymphocytes and determining the levels of p24 production by ELISA. Both assays revealed a dose-dependent blocking effect of IFN-alpha on cell-mediated HIV transmission. The complete inhibition of HIV infection was observed in the presence of 100 IU IFN-alpha. The efficacy of such a low dose could not be attributed to insufficient viral load because up to 10(8) infectious particles could be transmitted during cell-cell contact. An adhesion assay ruled out the possibility that IFN-alpha acts through prevention of lymphocyte-trophoblast contact. The results suggest that physiologic levels of IFN-alpha, present in the placental environment, may contribute to the protection of the fetus against HIV infection.

Anti-HIV effect of beta subunit of human chorionic gonadotropin (beta hCG) in vitro.

Human chorionic gonadotropin (hCG)--a pregnancy-associated immunomodulating hormone--has been recently shown in vitro to suppress reverse transcriptase activity in chronically HIV-infected lymphocytes and monocytes and to block viral transmission resulting from cell-cell contact between virus-carrying lymphocytes and placental trophoblasts. In further pursuit of the query into the mechanism of action, purified alpha and beta subunits of hCG were tested for the inhibition of p24 gag protein synthesis in virus-producing ACH-2 lymphocytes and U1 monocytes. Unlike the alpha subunit, beta-hCG displayed a distinct U-shaped dose response, characteristic of the effect of dimer hCG. Maximum inhibition of viral expression has been achieved at 10-100 ng/ml, the concentration corresponding to blood levels of beta-hCG in pregnant women. The doses that were several logs higher of normal levels seemed to increase viral production in monocytes. The data presented supports our original observations regarding the effect of intact hCG on HIV replication. While the mechanism of action remains to be established, the results suggest that the virus-interfering activity of hCG is determined by hormone-specific beta chain but not by the alpha subunit--shared with the family of glycoprotein hormones from the pituitary--follicle-stimulating hormone, luteinizing hormone and thyrotropin.

The antiviral agents, MAP30 and GAP31, are not toxic to human spermatozoa and may be useful in preventing the sexual transmission of human immunodeficiency virus type 1.

OBJECTIVE: To investigate the effects of two virucidal compounds, MAP30 (Momordica anti-human immunodeficiency virus [HIV] protein; molecular weight, 30 kd) and GAP31 (Gelonium anti-HIV protein; molecular weight, 31 kd), obtained from Momordica charantia and Gelonium multiflorum, respectively, on the motility and vitality of human spermatocytes. DESIGN: Prospective, controlled study. SETTING: New York University School of Medicine. PATIENT(S): Ten healthy men undergoing evaluation for infertility provided 10 semen specimens. INTERVENTION(S): Human sperm were treated with the anti-HIV agents, MAP30 and GAP3 1. Nonoxynol-9, a commonly used spermicide, and phosphate-buffered saline were used as the positive and negative controls, respectively. MAIN OUTCOME MEASURE(S): The motility and vitality of human spermatocytes treated with MAP30 and GAP31 at doses that inhibit HIV-1 and herpes simplex virus. RESULT(S): MAP30 and GAP31 did not inhibit the motility or vitality of human sperm cells over a dose range of 100-0.1 microg/mL, whereas nonoxynol-9 demonstrated spermicidal action on all 10 samples over the same dose range. CONCLUSION(S): The antiviral agents, MAP30 and GAP31, were not toxic to human sperm cells at the doses at which they inhibit HIV-1 and herpes simplex virus. They had no effect on the motility of spermatozoa, even at a dose of 1,000 times the maximum effective concentration. These results indicate that MAP30 and GAP31 may be useful as nonspermicidal protection against sexually transmitted diseases.

Inhibition of MDA-MB-231 human breast tumor xenografts and HER2 expression by anti-tumor agents GAP31 and MAP30.

GAP31 (Gelonium protein of 31 kDa) and MAP30 (Momordica protein of 30 kDa) are agents isolated from the medicinal plants Gelonium multiflorum and Momordica charantia, respectively. The current study was conducted to investigate the efficacy of GAP31 and MAP30 on estrogen-independent and highly metastatic human breast tumor MDA-MB-231 both in vitro and in vivo. The effect of these agents on the expression of breast tumor antigen HER2 (also known as neu or as c-erbB 2) was also examined. Treatment of MDA-MB-231 breast cancer cells with GAP31 and MAP30 resulted in inhibition of cancer cell proliferation as well as inhibition of the expression of HER2 gene in vitro. When MDA-MB-231 human breast cancer cells were transferred into SCID mice, the mice developed extensive metastases and all mice succumbed to tumor by day 46. Treatment of the human breast cancer bearing SCID mice with GAP31 or MAP30 at 10 micrograms/injection EOD for 10 injections resulted in significant increases in survival, with 20-25% of the mice remaining tumor free for 96 days. Thus, anti-tumor agents GAP31 and MAP30 are effective against human breast cancer MDA-MB-231 in vitro and in vivo. These agents may therefore be a potential therapeutic use against breast carcinomas.

Anti-HIV effect of immunomodulating agent, levamisole, in vitro.

An anthelminthic agent, levamisole, also known as a potent immunomodulator, has been successfully used for adjuvant therapy of malignancies and chronic infections underlined by immunodeficiency. We have tested the effect of this drug on de novo viral infection by exposing MT-4 T lymphocytes to HIV in the presence of serial ten-fold dilutions of levamisole (range 10(-3)-10(-9) M). The results indicate that 50% reduction in viral infectivity (IC50) of levamisole starts from as low as 10(-7) M, whereas even the highest millimolar dose of the drug has not shown any appreciable cytotoxicity. Although the mechanism of levamisole action remains unknown, our observation in vitro suggests that levamisole, a clinically established immunomodulator, can be potentially effective for treatment of HIV infection.

Inhibitory effect of the oral immune response modifier, bestatin, on cell-mediated and cell-free HIV infection in vitro.

The antiviral effect of the immunomodulating anti-cancer agent, bestatin, was examined in vitro by exposing MT-4 lymphocytes to HIV in the presence of 10-fold dilutions of drug (range 100 micrograms-100 pg/ml). The reduction in infectivity was measured by p24 ELISA and compared to the effect of established anti-HIV drugs-azidothymidine (AZT) and dextran sulfate. The results indicate that low doses of bestatin (1 microgram/ml) can completely inhibit viral infection resulting either from inoculation with free virus or coculture with infected lymphocytes. Unlike AZT or dextran sulfate, bestatin prevents HIV infection without interfering with the rate of cell growth. No appreciable decrease in HIV production was observed when chronically infected virus-producing T cell lines ie, H9, MOLT-4, HPB-ALL, 8E5 and MT-2 were treated with bestatin. Bestatin appears to act in the early stages of viral penetration, possibly through inhibition of lymphocyte-associated aminopeptidases.

Comparative in vitro study of contraceptive agents with anti-HIV activity: gramicidin, nonoxynol-9, and gossypol.

Gramicidin, a polypeptide antibiotic derived from Bacillus brevis, was compared in vitro with the established contraceptive virucidal agents nonoxynol-9 and gossypol for activity against human immunodeficiency virus (HIV) infection. The effective antiviral 10 ng/ml concentration of gramicidin required for complete HIV inactivation was a thousand-fold lower than the dose observed for nonoxynol-9 or gossypol. Gramicidin, routinely used as a contraceptive agent in the former Soviet Union, should be considered for in vivo trials as a spermicide with potent antiviral activity.

PIP: At the New York University Medical Center, researchers used an antiviral assay, p24 ELISA, and cytoxicity assays to compare the antiviral activity of the newly-discovered anti-HIV compound gramicidin with established spermicides that have demonstrated antiviral activity. A 10 ng/ml of gramicidin completely inhibited productive HIV infection in MT-4 lymphocytes, while a 1000-fold higher dose of nonoxynol-9 and gossypol (10 mcg/ml) was needed to achieve the same effect. A possible mechanism is impaired permeability for cations, resulting in inhibition of virus-induced fusion. The study did not try to determine the biochemical mode of gramicidin action, however. Gramicidin is used in the former USSR as an active component of spermicidal gels and foams and as a topical, non-irritating antibiotic often used to treat ocular infections. The results of this in vitro study call for more in vitro studies to determine the biochemical mechanism of gramicidin action and its clinical potential as a vaginal spermicide with antiviral activity.

Acrosin inhibitor, 4'-acetamidophenyl 4-guanidinobenzoate, an experimental vaginal contraceptive with anti-HIV activity.

Serine proteases are involved in a wide variety of seemingly unrelated physiological functions including capacitation of the spermatozoa and potentiation of human immunodeficiency virus (HIV) infection. The experimental vaginal contraceptives derived from 4-guanidinobenzoic acid act through inhibition of acrosin--a serine protease from the sperm. The serial ten-fold dilutions of 4'-acetamidophenyl 4-guanidinobenzoate (AGB) were tested in vitro for the effect against HIV infection by assaying the suppression of de novo p24 synthesis in virus-inoculated MT-4 T lymphocytes. The results reveal that complete inhibition of HIV occurred at 100 micrograms/ml--a dose corresponding to previously reported concentrations responsible for preventing fertilization in rabbits. These findings suggest that serine protease inhibitors and in particular the guanidinobenzoates, reported to be up to 100-fold more potent and less irritating than nonoxynol-9, can be potentially operative against sexual transmission of HIV.

PIP: Biochemists at the New York University Medical Center assayed suppression of de novo p24 synthesis in HIV-inoculated MT-4 T lymphocytes to compare the effects of 4'-acetamidophenyl 4-guanidinobenzoate hydrochloride (AGB), Nalpha-p-tosyl-L-lysine chloro methyl ketone (TLCK), and nonoxynol-9 against HIV infection. Specifically, they wanted to determine the possibility that AGB, an antagonist of acrosin (a serine protease critical to sperm capacitation and to fortification of HIV infection), could launch a new class of vaginal spermicidal agents with anti-HIV activity. 100 mcg/ml AGB (a nontoxic dose) induced complete inhibition of HIV but did not affect the viability of MT-4 T lymphocytes. AGB had a 100 times more potent effect against HIV and had a lower cytotoxic effect (i.e., less irritating) than nonoxynol 9. These findings suggest that serine protease inhibitors, especially the guanidinobenzoates, have the potential to be effective vaginal contraceptives with anti-HIV activity.

Anti-HIV plant proteins catalyze topological changes of DNA into inactive forms.

GAP 31, DAP 32 and DAP 30 comprise a new class of plant proteins with potent anti-HIV activity and insignificant cytotoxicity. We report here the identification and characterization of a new DNA enzyme activity in these three proteins. They irreversibly relax and decatenate supercoiled DNA, as well as catalyze double-stranded breakage to form linear DNA. The relaxed molecules are topologically inactive and no longer serve as substrates for DNA gyrase to form supercoils, phenomena similar to those of cellular topoisomerases in the presence of topoisomerase poisons. The ability of these anti-HIV agents to interrupt essential topological interconversions of DNA may provide a novel mechanism for their antiviral and antitumor actions. The presence of this new DNA topological enzyme activity in these plant proteins also suggests that their anti-HIV activity may not be merely a consequence of ribosome inactivation previously recognized.