Search PubMed⌕ Search

Biomedical subjects

John Mills

Publications and source records attributed to John Mills.

17 recordsLinked to original sources
PubMed2006

Identifying the bacterial community on the surface of Intralox belting in a meat boning room by culture-dependent and culture-independent 16S rDNA sequence analysis.

We examined the bacterial community present on an Intralox conveyor belt system in an operating lamb boning room by sequencing the 16S ribosomal DNA (rDNA) of bacteria extracted in the presence or absence of cultivation. RFLP patterns for 16S rDNA clone library and cultures were generated using HaeIII and MspI restriction endonucleases. 16S rDNA amplicons produced 8 distinct RFLP pattern groups. RFLP groups I-IV were represented in the clone library and RFLP groups I and V-VIII were represented amongst the cultured isolates. Partial DNA sequences from each RFLP group revealed that all group I, II and VIII representatives were Pseudomonas spp., group III were Sphingomonas spp., group IV clones were most similar to an uncultured alpha proteobacterium, group V was similar to a Serratia spp., group VI with an Alcaligenes spp., and group VII with Microbacterium spp. Sphingomonads were numerically dominant in the culture-independent clone library and along with the group IV alpha proteobacterium were not represented amongst the cultured isolates. Serratia, Alcaligenes and Microbacterium spp. were only represented with cultured isolates. Pseudomonads were detected by both culture-dependent (84% of isolates) and culture-independent (12.5% of clones) methods and their presence at high frequency does pose the risk of product spoilage if transferred onto meat stored under aerobic conditions. The detection of sphingomonads in large numbers by the culture-independent method demands further analysis because sphingomonads may represent a new source of meat spoilage that has not been previously recognised in the meat processing environment. The 16S rDNA collections generated by both methods were important at representing the diversity of the bacterial population associated with an Intralox conveyor belt system.

Animals↗
PubMed2006

Fourier transform near-infrared vibrational circular dichroism used for on-line monitoring the epimerization of 2,2-dimethyl-1,3-dioxolane-4-methanol: A pseudo racemization reaction.

Near-infrared (near-IR) Fourier transform vibrational circular dichroism (FT-VCD) spectroscopy has been used to monitor the epimerization of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (S-DDM). The near-IR-VCD spectra display clear isolated VCD bands at the range of 4700-5050 cm(-1) resulting from the OH stretch-bend combination bands of S-DDM, which were found to decrease in intensity with increasing reaction time. The near-IR-VCD spectra of 10 reference samples obtained were subjected to partial least-squares (PLS) regression analysis, and the results were used to build predictive models for enantiomeric excess (EE) determination. Multivariate regression was carried out on three different sets of spectra, corresponding to the epimerization of S-DDM in three different solvents: methylcyclohexane, carbon tetrachloride and tetrahydrofuran. The effects of solvent in DDM epimerization are discussed in terms of the relative stabilization of the reaction intermediate of the DDM epimerization reaction. The results of these near-IR-VCD studies for the determination of EE highlights the potential of VCD for in situ real-time process monitoring of the reaction kinetics of chiral molecules in solution.

Calibration↗
PubMed2006

Quantitation of chromosome 1p and 19q deletions in glial tumours by interphase FISH on formalin-fixed paraffin-embedded tissue.

Deletions on chromosomes 1p and 19q identify oligodendroglial tumours that are likely to have a complete response to some chemotherapy regimens and are associated with prolonged patient survival. Detection of these chromosomal alterations is becoming increasingly important in the evaluation of glial tumours. Interphase fluorescence in situ hybridisation (FISH) is one of a number of techniques for detecting deletions, and is an efficient method for screening large numbers of tumours. We used FISH to detect 1p and 19q deletions in formalin-fixed paraffin sections of 11 oligodendrogliomas, 11 oligoastrocytomas, two astrocytomas and four glioblastomas multiforme. 1p and 19q deletion and non-deletion ratios were tabulated in 200 nuclei in each tumour. We found considerable variation between tumours in the fraction of cells with deletions. This variation has not been reported previously and its clinical significance will be clarified with patient follow-up. There was little variation between regions within the same tumour.

Brain Neoplasms↗
PubMed2006

Adverse effects of antiretroviral drugs on HIV-1-infected and -uninfected human monocyte-derived macrophages.

Antiretroviral drugs approved for treatment of HIV-1 infection include nucleoside reverse transcriptase inhibitors (NRTIs) and protease inhibitors (PIs). Use of these drugs in combinations (highly active antiretroviral therapy) has delayed disease progression. However, long-term therapy is associated with potentially serious adverse effects. NRTIs are thought to contribute to these adverse effects via depletion of mtDNA. Inasmuch as macrophages (major targets for HIV-1) are highly metabolically active with large numbers of mitochondria, we investigated the effects of NRTIs (didanosine, stavudine, lamivudine, and zidovudine) on the viability and function of HIV-1-infected and -uninfected human monocyte-derived macrophages (MDMs). We demonstrate that the combinations didanosine/stavudine and lamivudine/zidovudine decrease mtDNA content in MDMs, with HIV-1-infected MDMs displaying a greater reduction than uninfected cells. This decrease correlated with decreased complement-mediated phagocytosis (C'MP) by MDMs, a process dependent on mitochondrial function. Inasmuch as PIs have previously been reported to interact with cellular proteases and given that cellular proteases are involved in the phagocytic process, we investigated the effects of the PI indinavir on C'MP. We demonstrate that indinavir augments C'MP by uninfected MDMs, but not HIV-1-infected MDMs. This study provides additional understanding on the effects of commonly used antiretroviral drugs on cellular immune function.

Cell Survival↗
PubMed2005

Interaction between the respiratory syncytial virus G glycoprotein cytoplasmic domain and the matrix protein.

Paramyxovirus assembly at the cell membrane requires the movement of viral components to budding sites and envelopment of nucleocapsids by cellular membranes containing viral glycoproteins, facilitated by interactions with the matrix protein. The specific protein interactions during assembly of respiratory syncytial virus (RSV) are unknown. Here, the postulated interaction between the RSV matrix protein (M) and G glycoprotein (G) was investigated. Partial co-localization of M with G was demonstrated, but not with a truncated variant lacking the cytoplasmic domain and one-third of the transmembrane domain, in cells infected with recombinant RSV or transfected to express G and M. A series of G mutants was constructed with progressively truncated or modified cytoplasmic domains. Data from co-expression in cells and a cell-free binding assay showed that the N-terminal aa 2-6 of G play a key role in G-M interaction, with serine at position 2 and aspartate at position 6 playing key roles.

Amino Acid Sequence↗
PubMed2004

Longitudinal analysis of nef/long terminal repeat-deleted HIV-1 in blood and cerebrospinal fluid of a long-term survivor who developed HIV-associated dementia.

We studied the evolution and compartmentalization of nef/long terminal repeat (nef/LTR)-deleted human immunodeficiency virus type 1 (HIV-1) from a long-term survivor who developed HIV-associated dementia (HIVD). Analysis of sequential blood-derived HIV-1 isolated before and during HIVD revealed a persistent R5X4 phenotype and a progressive loss of nef/LTR sequence; in contrast, HIV-1 present in cerebrospinal fluid during HIVD had an R5 phenotype, distinct nef/LTR sequence of unique deletions and additional nuclear factor- kappa B sites and specificity factor-1 sites, and enhanced transcriptional activity, compared with the blood-derived isolates. Thus, nef/LTR-deleted HIV-1 strains may undergo compartmentalized evolution in long-term survivors and cause neurologic disease.

AIDS Dementia Complex↗
PubMed2002

Coreceptor phenotype of natural human immunodeficiency virus with nef deleted evolves in vivo, leading to increased virulence.

The Sydney Blood Bank Cohort is a group of patients with slowly progressive infection by a human immunodeficiency virus strain containing spontaneous deletions within the nef long terminal repeat region. In 1999, 18 years after the initial infection, one of the members (D36) developed AIDS. In this work, we used an ex vivo human lymphoid cell culture system to analyze two viral isolates obtained from this patient, one prior to the onset of AIDS in 1995 and one after disease progression in 1999. Both D36 isolates were less potent in depleting CD4(+) T cells than a reference dualtropic, nef-bearing viral isolate. However, the 1999 isolate was measurably more cytotoxic to CD4(+) T cells than the 1995 isolate. Interestingly, although both isolates were nearly equally potent in depleting CCR5(+) CD4(+) T cells, the cytotoxic effect of the 1999 isolate toward CCR5(-) CD4(+) T cells was significantly higher. Furthermore, GHOST cell infection assays and blocking experiments with the CXCR4 inhibitor AMD3100 showed that the later D36 1999 isolate could infect both CCR5(+) and CCR5(-) CXCR4(+) cells efficiently, while infection by the 1995 isolate was nearly completely restricted to CCR5(+) cells. Sequence analysis of the V1/V2 and V3 regions of the viral envelope protein gp120 revealed that the more efficient CXCR4 usage of the later isolate might be caused by an additional potential N-glycosylation site in the V1/V2 loop. In conclusion, these data show that an in vivo evolution of the tropism of this nef-deleted strain toward an X4 phenotype was associated with a higher cytopathic potential and progression to AIDS.

Amino Acid Sequence↗
PubMed2002

Human immunodeficiency virus type 1 Nef binds to tumor suppressor p53 and protects cells against p53-mediated apoptosis.

The nef gene product of human immunodeficiency virus type 1 (HIV-1) is important for the induction of AIDS, and key to its function is its ability to manipulate T-cell function by targeting cellular signal transduction proteins. We reported that Nef coprecipitates a multiprotein complex from cells which contains tumor suppressor protein p53. We now show that Nef interacts directly with p53. Binding assays showed that an N-terminal, 57-residue fragment of Nef (Nef 1-57) contains the p53-binding domain. Nef also interacted with p53 during HIV-1 infection in vitro. As p53 plays a critical role in the regulation of apoptosis, we hypothesized that Nef may alter this process. Nef inhibited UV light-induced, p53-dependent apoptosis in MOLT-4 cells, with Nef 1-57 being as effective as its full-length counterpart. The inhibition by Nef of p53 apoptotic function is most likely due its observed ability to decrease p53 protein half-life and, consequently, p53 DNA binding activity and transcriptional activation. These data show that HIV-1 Nef may augment HIV replication by prolonging the viability of infected cells by blocking p53-mediated apoptosis.

Apoptosis↗
PubMed2002

Mechanical stimulation improves tissue-engineered human skeletal muscle.

Human bioartificial muscles (HBAMs) are tissue engineered by suspending muscle cells in collagen/MATRIGEL, casting in a silicone mold containing end attachment sites, and allowing the cells to differentiate for 8 to 16 days. The resulting HBAMs are representative of skeletal muscle in that they contain parallel arrays of postmitotic myofibers; however, they differ in many other morphological characteristics. To engineer improved HBAMs, i.e., more in vivo-like, we developed Mechanical Cell Stimulator (MCS) hardware to apply in vivo-like forces directly to the engineered tissue. A sensitive force transducer attached to the HBAM measured real-time, internally generated, as well as externally applied, forces. The muscle cells generated increasing internal forces during formation which were inhibitable with a cytoskeleton depolymerizer. Repetitive stretch/relaxation for 8 days increased the HBAM elasticity two- to threefold, mean myofiber diameter 12%, and myofiber area percent 40%. This system allows engineering of improved skeletal muscle analogs as well as a nondestructive method to determine passive force and viscoelastic properties of the resulting tissue.

Biocompatible Materials↗
PubMed1997

Detection and subgrouping of respiratory syncytial virus directly from nasopharyngeal aspirates.

OBJECTIVE: To develop a reverse transcription-polymerase chain reaction (RT-PCR)-based assay to identify the subgroup of the infecting respiratory syncytial virus (RSV) strain directly from nasopharyngeal aspirates (NPAs). METHODS: A total of 154 NPAs which were positive for RSV antigen by direct immunofluorescence were subjected to RT-PCR. The primers used were designed to give products for subgroup A and B which were of different sizes and easily visualized on agarose electrophoresis. The PCR products were further analyzed by restriction analysis using enzymes which were unique or rare cutters within the PCR amplimer. RESULTS: It was possible to confirm RSV infection in 70% of the NPA samples studied. Of these, 92.6% belonged to the A group, and only 7.4% to the B group. Within the A group, six subgroups were identified using restriction analysis, while all B-group samples were identical to the prototype B strain, 18537. CONCLUSION: RT-PCR performed on RNA isolated directly from NPAs provides a quick, easy-to-use, reasonably sensitive method to identify and group the infecting RSV strain.

Journal Article↗