Biomedical subjects

A A Amin

Publications and source records attributed to A A Amin.

At least 19 recordsLinked to original sources

PubMed2005

The use of autologous chondrocyte implantation following and combined with anterior cruciate ligament reconstruction.

We report our experience of using autologous chondrocyte implantation (ACI) to treat osteochondral defects of the knee in combination with anterior cruciate ligament (ACL) reconstruction. The outcome of symptomatic osteochondral lesions treated with ACI following previous successful ACL reconstruction is also reviewed. Patients were followed for a mean of 23 months. Nine patients underwent ACL reconstruction in combination with ACI. Mean modified Cincinnati knee scores improved from 42 to 69 following surgery. Seven patients described their knee as better and two as the same. A second group of nine patients underwent ACI for symptomatic articular cartilage defects following previous ACL reconstruction. In this group, the mean modified Cincinnati knee score improved from 53 to 62 after surgery. Six patients described their knee as better and three as worse. Combined treatment using ACI with ACL reconstruction is technically feasible and resulted in sustained improvement in pain and function. The results following previous ACL reconstruction also resulted in clinical improvement, although results were not as good as following the combined procedure.

Adult↗

PubMed2005

The quality of sulphadoxine-pyrimethamine and amodiaquine products in the Kenyan retail sector.

BACKGROUND AND OBJECTIVE: Malaria is a disease of major public health importance in Kenya killing 26,000 children under 5 years of age annually. This paper seeks to assess the quality of sulphadoxine-pyrimethamine (SP) and amodiaquine (AQ) products available over-the-counter to communities in Kenya as most malaria fevers are self-medicated using drugs from the informal retail sector. METHODS: A retail audit of 880 retail outlets was carried in 2002 in four districts in Kenya, in which antimalarial drug stocks and their primary wholesale sources were noted. In addition, the expiry dates on audited products and the basic storage conditions were recorded on a proforma. The most commonly stocked SP and AQ products were then sampled from the top 10 wholesalers in each district and samples subjected to standard United States Pharmacopoeia (USP) tests of content and dissolution. RESULTS AND DISCUSSION: SP and AQ were the most frequently stocked antimalarial drugs, accounting for approximately 75% of all the antimalarial drugs stocked in the four districts. Of 116 SP and AQ samples analysed, 47 (40.5%) did not meet the USP specifications for content and/or dissolution. Overall, approximately 45.3% of SP and 33.0% of AQ samples were found to be sub-standard. Of the sub-standard SP products, 55.2% were suspensions while 61.1% of the substandard AQ products were tablets. Most SP failures were because of the pyrimethamine component. CONCLUSION: There is a need to strengthen post-marketing surveillance systems to protect patients from being treated with sub-standard and counterfeit antimalarial drugs in Kenya.

Amodiaquine↗

PubMed2002

Inhibition of methylnitrosourea (MNU) induced oxidative stress and carcinogenesis by orally administered bee honey and Nigella grains in Sprague Dawely rats.

We studied the protective effect of bee honey and Nigella grains as nutraceuticals on the oxidative stress and carcinogenesis induced by methylnitrosourea (MNU) in Sprague Dawely rats. Four groups of animals were used and fed ad-libitum. The first group was a control (n=8), the second (n=8), the third (n=15) and the fourth groups (n=12) were injected MNU (single i.v. dose 50 mg/kg body weight). After one week the third and fourth groups were given orally 0.2 g ground Nigella grains and 0.2 g Nigella with 5 g honey/rat/day, respectively. After six months all animals were sacrificed except two from the second group (MNU-injected rats) that died one-week before the end of the experiment. We observed that MNU injected in the second group produced a variety of oxidative stresses ranging from severe inflammatory reaction in lung and skin to colon adenocarcinoma in four out of six animals. There was an associated elevation of malondialdehyde (MDA) and nitric oxide (NO) in sera obtained from animals of this group compared to the control one. Nigella sativa grains given orally protected against MNU-induced oxidative stress and carcinogenesis by 80% (12/15) and combated this effect by lowering MDA and NO. Whereas honey from bees and Nigella sativa together protected 100% (12/12) against MNU-induced oxidative stress, carcinogenesis and abolished the NO and MDA elevations shown in sera of animals who did not receive these nutrients. These results showed that supplementation of diet with honey and Nigella sativa has a protective effect against MNU-induced oxidative stress, inflammatory response and carcinogenesis.

Adenocarcinoma↗

PubMed2000

Identification of domains of the HPV11 E1 protein required for DNA replication in vitro.

The HPV E1 and E2 proteins along with cellular factors, are required for replication of the viral genome. In this study we show that in vitro synthesized HPV11 E1 can support DNA replication in a cell-free system and is able to cooperate with E2 to recruit the host polymerase alpha primase to the HPV origin in vitro. Deletion analysis revealed that the N-terminal 166 amino acids of E1, which encompass a nuclear localization signal and a cyclin E-binding motif, are dispensable for E1-dependent DNA replication and for recruitment of pol alpha primase to the origin in vitro. A shorter E1 protein lacking the N-terminal 190 amino acids supported cell-free DNA replication at less than 25% the efficiency of wild-type E1 and was active in the pol alpha primase recruitment assay. An even shorter E1 protein lacking a functional DNA-binding domain due to a truncation of the N-terminal 352 amino acids was inactive in both assays despite the fact that it retains the ability to associate with E2 or pol alpha primase in the absence of ori DNA. We provide additional functional evidence that E1 interacts with pol alpha primase through the p70 subunit of the complex by showing that p70 can be recruited to the HPV origin by E1 and E2 in vitro, that the domain of E1 (amino acids 353-649) that binds to pol alpha primase in vitro is the same as that needed for interaction with p70 in the yeast two-hybrid system, and that exogenously added p70 competes with the interaction between E1 and pol alpha primase and inhibits E1-dependent cell-free DNA replication. On the basis of these results and the observation that pol alpha primase competes with the interaction between E1 and E2 in solution, we propose that these three proteins assemble at the origin in a stepwise process during which E1, following its interaction with E2, must bind to DNA prior to interacting with pol alpha primase.

Animals↗

PubMed1998

Venous flap ischemia: microcirculatory changes in experimental flaps in a rabbit model.

This study was designed to investigate the simultaneous changes in blood flow and microcirculation in an island flap during venous occlusion (venous ischemia), in an ischemia/reperfusion injury model in the rabbit. An island groin flap based on the inferior epigastric vessels was harvested in 15 rabbits. The flap was rendered ischemic for 3 hr (n = 5) or 4 hr (n = 10, 5 heparinized and 5 not), by applying a microvascular clamp to the inferior epigastric vein. Transonic Doppler and laser Doppler were used to monitor blood flow in the epigastric artery and microcirculation of the flap for 1 hr after flap elevation, 1 hr after occlusion, and for 3 hr at the end of the ischemic period. Venous occlusion was followed by a rapid decrease of blood flow and microcirculation readings. After ischemia, both blood flow and microcirculation readings in the flap were significantly decreased, compared to pre-ischemic values in all groups. In the 3-hr ischemia group, blood flow readings returned to pre-stress values, while microcirculation remained significantly lower. In the 4-hr ischemia group treated with heparin, blood flow in the artery settled at levels significantly lower than pre-stress readings; however, microcirculation of the flap was ultimately fully restored to pre-ischemic values. In the 4-hr ischemia group, both blood flow and microcirculation in the flap settled at levels significantly lower than pre-stress values. The authors concluded that tolerance for venous ischemia is time-dependent in this model and that venous ischemia is more deleterious than global ischemia. Administration of heparin may alter the time frame of ischemia/reperfusion injury and may prevent the harmful effects of injury at the microcirculatory level.

Animals↗

PubMed1998

Reconstruction of complex radiation-induced injuries using free-tissue transfer.

The purpose of this study was to evaluate the use of free-tissue transfers for the reconstruction of radiation-induced complex injuries. The case files for patients who underwent reconstruction for radiation-induced injuries between May 1988 and November 1995 at The University of Texas M.D. Anderson Cancer Center were retrospectively reviewed. Thirty patients in whom 33 free flaps were done were included. Radiation-induced defects were located in the head and neck (n=23), extremities (n=4), chest wall (n=2), and inguinal area (n=1) The mean period between irradiation and injury was 78 months (range: 4 months to 38 years). Free-tissue transfer was successful in 97 percent (32/33) of patients. The overall complication rate was 40 percent (12/30). Flap donor sites included the fibula (n=12), latissimus dorsi (n=6), rectus abdominis (n=6), iliac crest (n=4), scapula (n=3), and radial forearm (n=2). Large-caliber vessels in the cervical, axillary, or inguinal regions were most commonly used to revascularize flaps. Vein grafts were used in five cases for the artery (2/5) or vein (3/5). Pedicle thrombosis occurred in three cases in recipient vessels located within the irradiated field. Two flaps were salvaged; one was lost, and the patient required a second free-flap reconstruction The mean follow-up was 40 months (range: 2.5 to 83 months). The disease-free survival rate was 67 percent (20/30), local failures occurred in 10 percent (3/30) of patients, and 23.3 percent (7/30) of patients either died or were lost to follow-up. Healing of radiation-induced wounds may be achieved using free-tissue transfers, but complications are frequent. Large-caliber irradiated vessels may be used to revascularize flaps, but there may be an increased risk of pedicle thrombosis.

Adult↗

PubMed1998

Second free flaps in head and neck reconstruction.

Over the past decade, free-tissue transfer has greatly improved the quality of oncology-related head and neck reconstruction. As this technique has developed, second free flaps have been performed for aesthetic improvement of the reconstructed site. This study evaluated the indications for and the success of second free flaps. Medical files for patients who underwent second free flaps for head and neck reconstruction at the University of Texas M.D. Anderson Cancer Center, from May 1, 1988 to November 30, 1996, were reviewed. The flaps were classified as being either immediate (done within 72 hr) or delayed (done within 2 years) reconstructions. Indications, risk factors, recipient vessels, outcome, and complications were analyzed. Of the 28 patients included in this study, 12 had immediate (nine as salvage after primary free flap failure, and three for reconstruction of a soft-tissue defect), and 16 had delayed second free flaps (two for reconstruction of a defect resulting from excision of recurrent tumors, and 14 for aesthetic improvement). Reconstruction sites included the oral cavity in 18 patients; the midface in six; the skull base in two; and the scalp in two. The success rate for the second free flaps was 96 percent. Five patients had significant wound complications. In a substantial number of cases, identical recipient vessels were used for both the first and second free flaps. The authors conclude that second free flaps can play an important role in salvaging or improving head and neck reconstruction in selected patients. In many cases, the same recipient vessels can be used for both the first and second flaps.

Adolescent↗

PubMed1997

Isolation of human and fission yeast homologues of the budding yeast origin recognition complex subunit ORC5: human homologue (ORC5L) maps to 7q22.

Orc5p is a subunit of the origin recognition complex in the budding yeast Saccharomyces cerevisiae, which has been shown to play a critical role in both chromosomal DNA replication and transcriptional silencing. We have cloned cDNAs from both human and fission yeast Schizosaccharomyces pombe that encode proteins homologous to the budding yeast and Drosophila Orc5p. Human Orc5p showed 35.1, 22.3, and 19.4% identity to the Drosophila, S. pombe, and S. cerevisiae Orc5p, respectively. We have localized the human ORC5 gene (ORC5L) to chromosome 7 using Southern and PCR analysis of DNA isolated from a panel of human/rodent somatic cell hybrids and mapped the gene locus to 7q22 using fluorescence in situ hybridization. We have identified a YAC clone that contains human ORC5L and maps to chromosome band 7q22.1. We have identified the S. pombe ORC5 gene and located it in a cosmid mapped on chromosome II.

Amino Acid Sequence↗

PubMed1996

Transcription-positive cofactor 4 forms complexes with HSSB (RPA) on single-stranded DNA and influences HSSB-dependent enzymatic synthesis of simian virus 40 DNA.

The replication of simian virus 40 (SV40) DNA in vitro requires a trimeric single-stranded DNA (ssDNA)-binding protein called HSSB or RPA. HSSB supports the unwinding of DNA containing the SV40 origin in the presence of the viral-encoded T antigen and is required for the initiation of RNA primer synthesis as well as processive elongation of DNA catalyzed by the DNA polymerase delta holoenzyme. In this report we show that the transcription positive cofactor 4 (PC4), a ssDNA-binding protein, forms complexes with HSSB on ssDNA and markedly affects the replication functions of HSSB. PC4 supports T antigen-catalyzed unwinding of SV40 origins in lieu of HSSB but inhibits both RNA primer synthesis and polymerase delta-catalyzed DNA chain elongation reactions. These inhibitory effects can be reversed by the addition of excess HSSB. Depending on the concentration of HSSB, PC4 is capable of either inhibiting or activating SV40 DNA replication measured in both mono- and dipolymerase systems. The possible role of PC4 in the initiation of DNA replication is discussed.

Antigens, Viral, Tumor↗

PubMed1996

Purification, gene cloning, and reconstitution of the heterotrimeric single-stranded DNA-binding protein from Schizosaccharomyces pombe.

We have purified a single-stranded DNA-binding protein (SSB) from Schizosaccharomyces pombe (Sp) and have shown that it is composed of three subunits of 68, 30, and 12 kDa. The SpSSB supports T antigen-dependent unwinding of SV40 ori containing DNA, but is not functional in the SV40 in vitro replication reaction. All three genes that encode the SpSSB subunit have been isolated. The cloned cDNA of the ssb1(+), encoding the p68 subunit, contains 609 amino acids (68.3 kDa), while that of the ssb2(+), encoding the p30 subunit, contains a 279 amino acids (30.3 kDa). The genomic DNA clone of the p12 subunit gene (ssb3(+)) has 2 introns and an open reading frame of 104 amino acids (11.8 kDa). Significant homology is observed among the largest and middle subunits of eukaryotic SSBs, but there is poor homology among the smallest subunits. In addition, we have reconstituted the SpSSB complex by coexpression of all three subunits in Escherichia coli. The reconstituted complex is active in single-stranded DNA binding and the T antigen-dependent unwinding of SV40 ori DNA. Finally, we observed a cell cycle-dependent phosphorylation pattern of the p30 subunit of SpSSB, which is similar to that observed for the human and Saccharomyces cerevisiae SSB.

Amino Acid Sequence↗

PubMed1995

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

DNA Repair↗

PubMed1994

Phosphorylation of the p34 subunit of human single-stranded-DNA-binding protein in cyclin A-activated G1 extracts is catalyzed by cdk-cyclin A complex and DNA-dependent protein kinase.

The human single-stranded-DNA-binding protein (HSSB, also called RP-A) is a trimeric complex (p70, p34, and p14) required for multiple functions in DNA transactions. We report here that the p34 subunit of HSSB was hyperphosphorylated by kinase activities present in G1 extract (obtained from HeLa cells in G1 phase) preincubated with human cyclin A. This hyperphosphorylated HSSB product included at least four species of p34 that migrated more slowly through denaturing polyacrylamide gels than the hypophosphorylated form. Fractionation of cyclin A-activated G1 extract identified two kinases involved in the hyperphosphorylation of HSSB p34: cdk-cyclin A complex and DNA-dependent p350 protein kinase (DNA-PK). Kinetic analysis revealed that in cyclin A-activated G1 extract, p34 was first phosphorylated by cdk-cyclin A prior to the action of DNA-PK. Addition of p21cip1, a specific inhibitor of cdk-cyclin A but not DNA-PK, nearly abolished the hyperphosphorylation of HSSB p34 in G1 extract preincubated with cyclin A. This suggests a requirement of the cdk-cyclin A activity for the phosphorylation of p34 by DNA-PK in G1 extract.

CDC2-CDC28 Kinases↗

PubMed1994

Initiation of DNA replication by simian virus 40 T antigen is inhibited by the p107 protein.

The p107 protein is related to the Rb protein by a 60-kDa region of homology called the pocket domain that binds cellular proteins such as E2F and cyclins A and E as well as the viral oncoproteins E1A, E7, and SV40 T antigen. The p107 and Rb proteins have both been implicated as negative regulators of cell growth. We have examined the effect of the unphosphorylated pocket domain of p107 on specific stages of the T antigen-mediated replication of SV40 DNA in vitro. The pocket domain inhibited replication by preventing the assembly of T antigen at the SV40 core origin DNA containing binding site II. However, both proteins formed a ternary complex with DNA containing T antigen-binding site I. The pocket domain of p107 did not inhibit the oligomerization of T antigen in the absence of DNA, and the p107 derivative is bound to the intermediates of this reaction. In the unwinding assay, once T antigen was preassembled as hexamers at the core origin, the pocket domain bound to and stabilized the complex, resulting in an increase in the yield of unwound product. Preformed T antigen hexamers complexed with the pocket domain bind to a synthetic replication fork, and this complex supported unwinding. Consistent with this, the p107 pocket domain had no effect on the helicase activity of T antigen in an assay using a partial duplex substrate. However, a complex containing p107 and T antigen assembled at the core origin did not support SV40 DNA replication in HeLa cell crude extracts or in the monopolymerase reaction. This inhibition is due to the inability of the complex to bind to DNA polymerase alpha, which is required for the initiation of DNA synthesis. The data suggest pathways by which the pocket domain of p107 can negatively regulate T antigen-mediated replication in vitro. The binding of T antigen to p107 is discussed with respect to its role in mitigating negative cell growth control, resulting in viral mediated transformation.

Animals↗

PubMed1992

Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein-terB complex of Escherichia coli.

The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined. In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems. With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient. These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner. We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence. The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen. With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E. coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation. However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked. Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.

Antigens, Polyomavirus Transforming↗

PubMed1990

Synaptic intermediates promoted by the FLP recombinase.

We have devised a novel assay to trap nucleoprotein synaptic intermediates of the FLP recombination reaction. DNase I footprinting analysis of these intermediates indicates that synapsis is mediated by protein-protein interactions between FLP molecules bound to each FLP recombination target (FRT) site. Under certain conditions we have observed a synaptic structure in which the FRT sites have come together in an aberrant arrangement. Although our analysis shows that homology between the core sequences of the sites is not a prerequisite for synapsis, the data suggest that homology between cores dictates the directionality of the reaction. Many of the intermediates contain a Holliday junction indicating that the FLP protein has catalysed strand exchanges between the FRT sites. The general scheme of the assay should prove useful to analyse nucleoprotein intermediates in other site-specific recombination systems, and to investigate protein-protein and protein-DNA interactions in intermediates important for DNA replication and transcription.

Base Sequence↗

PubMed1989

Synthesis of an enzymatically active FLP recombinase in vitro: search for a DNA-binding domain.

We have used an in vitro transcription and translation system to synthesize an enzymatically active FLP protein. The FLP mRNA synthesized in vitro by SP6 polymerase is translated efficiently in a rabbit reticulocyte lysate to produce enzymatically active FLP. Using this system, we assessed the effect of deletions and tetrapeptide insertions on the ability of the respective variant proteins synthesized in vitro to bind to the FLP recognition target site and to carry out excisive recombination. Deletions of as few as six amino acids from either the carboxy- or amino-terminal region of FLP resulted in loss of binding activity. Likewise, insertions at amino acid positions 79, 203, and 286 abolished DNA-binding activity. On the other hand, a protein with an insertion at amino acid 364 retained significant DNA-binding activity but had no detectable recombination activity. Also, an insertion at amino acid 115 had no measurable effect on DNA binding, but recombination was reduced by 95%. In addition, an insertion at amino acid 411 had no effect on DNA binding and recombination. On the basis of these results, we conclude that this approach fails to define a discrete DNA-binding domain. The possible reasons for this result are discussed.

Amino Acid Sequence↗

PubMed1987

Yeast centromere sequences do not confer mitotic stability on circular plasmids containing ARS elements of Tetrahymena thermophila rDNA.

We have previously demonstrated that a 657 bp TaqI-XbaI and a 427 bp XbaI-XbaI fragment from the 5' non-transcribed spacer of the extrachromosomal ribosomal DNA of Tetrahymena thermophila function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae. These fragments are adjacent to each other in a region that encompasses the in vivo origin of bidirectional replication of rDNA. The presence of a yeast centromere (CEN) fragment does not confer mitotic stability on these plasmids. A sensitive yeast colony colour assay (Hieter et al. 1985a) has been used to evaluate the cis-acting effect of each ARS segment on the pattern of inheritance of a plasmid containing CEN5:URA3:SUP4. Colonies of transformed cells obtained both in the presence and absence of selection were red with no detectable white or pink sectors. The lack of sectoring indicates that both plasmids are lost at an extremely high rate, likely due to 1:0 segregation events. We conclude that while these ARS elements confer a high frequency transformation phenotype, they lack a function which is required in cis for the maintenance of mitotic stability in the presence of a centromere. This missing cis-acting function may result in the inability of the plasmids to be brought under the control of cell-cycle regulated replication.

Animals↗

PubMed1986

In vitro deletion analysis of ARS elements spanning the replication origin in the 5' non-transcribed spacer of Tetrahymena thermophila ribosomal DNA.

Two adjacent but non-overlapping restriction fragments that encompass the replication origin of the macronuclear copy of rDNA from Tetrahymena thermophila allow autonomous replication of plasmids in the yeast Saccharomyces cerevisiae; i.e. they function as autonomously replicating segments (ARS). Deletions generated in vitro into these fragments yield an 82 bp segment from each as the smallest sequence specifying ARS function. These 82 bp segments are at the 5' end of a 220 bp region of homology between the two original ARS restriction fragments. A 39 bp region of almost complete sequence identity between the two 82 bp fragments is suggested to be a core sequence element necessary for ARS function. This 39 bp sequence contains a region identical or nearly identical to the 11 bp yeast ARS consensus sequence (T/ATTTATPuTTTA/T) which is suggested to be essential for ARS function. Detailed comparisons of the 82 bp segments and of the 39 bp core with other ARS sequences reveal no extensive homologies aside from the consensus.

Animals↗