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Biomedical subjects

G J Thomas

Publications and source records attributed to G J Thomas.

At least 19 recordsLinked to original sources
PubMed2006

Alphavbeta6 integrin in wound healing and cancer of the oral cavity.

Integrins are a family of heterodimeric cell surface receptors, which are expressed on most cells where they mediate cell-cell and cell-extracellular matrix (ECM) interactions. The alphavbeta6 integrin is epithelial-specific and binds to the ECM proteins fibronectin, vitronectin and tenascin, and also to the latency associated peptide of TGF-beta. Unlike most epithelial integrins, alphavbeta6 is not expressed constitutively by healthy oral epithelia, but is up-regulated during tissue remodelling, including that accompanying wound healing and carcinogenesis. Although, the data at present have been generated principally from in vitro studies, there is increasing evidence to suggest that alphavbeta6 may promote carcinoma progression: alphavbeta6 has been shown to modulate invasion, inhibit apoptosis, regulate protease expression and activate TGF-beta1. This review examines the current literature, and discusses the possible role of alphavbeta6 in wound healing, and in the development and progression of oral squamous cell carcinoma.

Antigens, Neoplasm↗
PubMed2005

Alpha v beta 6 integrin down-regulates the MMP-13 expression in oral squamous cell carcinoma cells.

The integrin alphavbeta6, a receptor for fibronectin, vitronectin, tenascin and TGF-beta latency-associated peptide (LAP), is not detectable on normal oral epithelium but is neo-expressed in oral squamous cell carcinomas (OSCC) and epithelial dysplasia. Previously it has been shown that alphavbeta6 integrin can up-regulate MMP-3 and -9 expression in OSCC cells. Using beta6-transfected and control OSCC cells we demonstrate that alphavbeta6 integrin down-regulates MMP-13 expression at both mRNA and protein level. Although expressing less MMP-13, beta6-transfected cells were found to have similar collagenolytic activity as control cells and invade at similar levels through type I collagen. Growth of the tumour cells in organotypic culture and confocal microscopy confirmed low levels of MMP-13 in cells with high alphavbeta6 expression. Furthermore, human squamous cell carcinomas of the tongue with high expression of alphavbeta6 showed lower MMP-13 levels than carcinomas with low levels of alphavbeta6. Our results suggest that alphavbeta6 down-regulates MMP-13 expression in OSCC cells and that MMP-13 is not essential for the degradation of type I collagen by OSCC cells.

Antigens, Neoplasm↗
PubMed2005

Development of a quantitative method to analyse tumour cell invasion in organotypic culture.

Tumour invasion is a dynamic process occurring in three dimensions and involving interactions between both tumour and stromal cells. Experimental analysis of squamous carcinoma cell invasion has often used the organotypic gel culture system, which is generated by plating tumour cells on to a synthetic stroma composed of a collagen gel embedded with fibroblasts. Unfortunately, quantitation of invasion in these organotypic gels has relied largely on subjective pathological opinion, which may be influenced by different patterns of tumour cell infiltration. Therefore a computer-assisted digital image analysis system that assesses invasion objectively and provides a numerical 'Invasion Index' was developed. The Invasion Index, by combining depth and pattern of invasion in a single value, establishes a quantitative value that allows assessment of the influences of positive and negative regulation of tumour invasion. These data demonstrate that the organotypic gel system is a robust, accurate, and reproducible method for measuring tumour cell invasion. They also show that the Invasion Index can be used after organotypic gels have been implanted in mice for up to 6 weeks. Illustrative examples of how various factors influence the invasion of squamous carcinoma cells in three dimensions both in vitro and in vivo are provided.

Animals↗
PubMed2005

Assessment of bony resection margins in oral cancer using elastic scattering spectroscopy: a study on archival material.

Oral squamous cell carcinoma has been shown to infiltrate local bone necessitating either a local or segmental resection for clearance. The current decalcification process takes several weeks before histological examination of the margins is possible. The aim of this study was to determine whether elastic scattering spectroscopy (ESS) could be used to identify bone resection margins positive for tumour. We used an ESS biopsy (optical biopsy) system to assess formalin fixed bone margins resected for squamous cell carcinoma of the oral cavity and compared the results with the histopathological diagnosis. Archival specimens obtained in oral cancer resections over the last 10 years were used, the ESS spectra were obtained from residual resection margins immediately adjacent to the area from which sections were cut and the results correlated with the histopathological diagnosis. Three hundred and forty-one spectra were used in this study taken from the mandibular specimens of 21 patients, of which 231 spectra were taken from histologically positive sites and the rest were of normal tissue. Two different sets of spectra were obtained and using a linear discriminant analysis, a sensitivity of 87% and a specificity of 80% were obtained. These results suggest that ESS may identify tumour involvement of resection margins. This study, on formalin fixed tissue, was shown to be reliable and may significantly reduce pathology workload. If these findings can be applied in vivo, this would be an accurate and instant mechanism for assessment of margins in clinical practice.

Carcinoma, Squamous Cell↗
PubMed2005

Osteomyelitis of the mandible during pregnancy.

A 21-year-old woman who was 9 weeks pregnant presented with osteomyelitis of the mandible that resolved spontaneously after delivery. To our knowledge a link between osteomyelitis of the jaws and pregnancy has not been reported previously.

Adult↗
PubMed2004

Tumour-derived TGF-beta1 modulates myofibroblast differentiation and promotes HGF/SF-dependent invasion of squamous carcinoma cells.

The development of an altered stromal microenvironment is a common feature of many tumours including squamous cell carcinoma (SCC), and there is increasing evidence that these changes in the stroma, which include increased expression of proteases and cytokines, may actually promote tumour progression. A common finding is that stromal fibroblasts become 'activated' myofibroblasts, expressing smooth muscle actin and secreting cytokines, proteases and matrix proteins. We show that myofibroblasts are commonly found in the stroma of oral SCC and are often concentrated at the invasive margin of the tumour. Using oral SCC cells and primary oral fibroblasts, we demonstrate that tumour cells directly induce a myofibroblastic phenotype, and that this transdifferentiation is dependent on SCC-derived TGF-beta1. In turn, myofibroblasts secrete significantly higher levels of hepatocyte growth factor/scatter factor compared with fibroblast controls, and this cytokine promotes SCC invasion through Matrigel, a mixture of basement membrane proteins. This is the first time that this double paracrine mechanism has been demonstrated between squamous carcinoma cells and fibroblasts, and emphasises that cancer invasion can be promoted indirectly by the release of tumour-induced host factors from stroma.

Carcinoma, Squamous Cell↗
PubMed2004

Polymorphous low-grade adenocarcinoma--a rare and aggressive entity in adolescence.

Polymorphous low-grade adenocarcinoma (PLGA) is an uncommon tumour that usually affects the minor salivary glands, particularly in the palate. It is rare in young patients, and here we report a case in a teenage girl. She presented at the age of 16, although the lesion had been noticed 2 years previously. The tumour showed histopathological features of PLGA, but recurred locally, behaved aggressively, and ultimately metastasised to cervical lymph nodes. This was accompanied by an altered histological picture, with a papillary cystic pattern and necrosis becoming progressively more prominent. PLGA is not always a low-grade lesion and some tumours, notably those with a papillary cystic growth pattern, may require more aggressive treatment.

Adenocarcinoma↗
PubMed2004

Detection of cervical intranodal metastasis in oral cancer using elastic scattering spectroscopy.

While histopathology remains the gold standard for tissue diagnosis, several new diagnostic techniques are being developed that rely on physical and biochemical changes that mirror or precede malignant change within tissue. The aim of this study was to compare findings of elastic scattering spectroscopy (ESS) with histopathology on formalin fixed neck dissection specimens to see if this technique could be used as an adjunct or alternative to histopathology in defining nodal involvement. The technique involves the use of Mie scattering and is a simple non-invasive method of tissue interrogation. One hundred and thirty lymph nodes were examined from 13 patients who underwent neck dissection. The nodes were formalin fixed, bivalved and examined by ESS using a pulsed xenon lamp. The intensity of the spectrum at 4 points was considered for comparison; at 360, 450, 630 and 690 nm. The nodes were then routinely processed and haematoxylin and eosin-stained sections examined histopathologically, and the results compared. Using this technique, a sensitivity of 98% and a specificity of 68% were obtained.

Elasticity↗
PubMed2003

The role of nitric oxide in oral diseases.

Although previously regarded as a toxic pollutant gas, nitric oxide (NO) is a short-lived molecule that plays a key role in many physiological and pathological processes. It is produced in vivo from the amino acid L-arginine by a complex family of enzymes termed nitric oxide synthase (NOS). Since its discovery as a biological messenger in 1987, NO has been implicated in many disease processes, ranging from septic shock to cancer. It is a highly reactive free radical and causes concentration-dependent conformational changes in proteins, enzymes and DNA, predominantly by its reaction with transition metals and thiol residues. Although high concentrations of NO are cytotoxic, the levels produced in many human cancers possibly facilitate tumour growth and dissemination. The interest in this molecule by scientists and clinicians involved with the oral cavity and head and neck regions is fairly recent, and only a tiny minority of 50,000 papers currently cited on NO relate to diseases in this anatomical area. This review gives an overview of NO, outlining its basic chemistry, formation by NOS and its possible roles in the oral diseases studied to date. The implications for possible therapeutic manipulation of NO are also discussed.

Humans↗
PubMed2002

Binding of TGF-beta1 latency-associated peptide (LAP) to alpha(v)beta6 integrin modulates behaviour of squamous carcinoma cells.

The integrin alpha(v)beta6 is not detectable on normal keratinocytes in vivo but expression is increased significantly in oral squamous cell carcinoma where this heterodimer has been shown to play a role in cell migration, invasion and protease expression. Although regarded initially as a fibronectin receptor, alpha(v)beta6 may bind to arginine-glycine-aspartic acid sequences in other matrix molecules including tenascin and vitronectin. Interestingly, alpha(v)beta6 has also been shown to have high affinity for the TGF-beta1 latency associated peptide and to participate in the activation of the TGF-beta1 latent complex. Since TGF-beta1 is present in squamous carcinomas, it is possible that latency associated peptide may modulate malignant keratinocyte behaviour independently from the classical TGF-beta signalling pathways through its interaction with integrins. We show here that when latency associated peptide is immobilised onto a surface, it acts as an alpha(v)beta6-specific ligand for oral squamous carcinoma cells promoting adhesion and haptotactic migration in addition to alpha(v)beta6-dependent increase in pro-MMP-9 expression. In contrast, even very low concentrations of soluble latency associated peptide (0.1 microg ml(-1)) inhibited alpha(v)beta6-dependent adhesion, migration and invasion. Thus alpha(v)beta6-dependent processes of oral squamous cell carcinoma, is likely to be modulated, not only by the local concentration of latency associated peptide in the stroma, but also whether it is immobilised in the matrix or released as a soluble protein.

Antigens, Neoplasm↗
PubMed2001

Structure of bacteriophage P22 portal protein in relation to assembly: investigation by Raman spectroscopy.

Salmonella phage P22, which serves as an assembly paradigm for icosahedral double-stranded DNA viruses, packages its viral genome through a capsid channel (portal) comprising 12 copies of a 725-residue subunit. Secondary and tertiary structures of the portal subunit in monomeric and dodecameric states have been investigated by Raman spectroscopy using a His6-tagged recombinant protein that self-assembles in vitro [Moore, S. D., and Prevelige, P. E., Jr. (2001) J. Biol. Chem. 276, 6779-6788]. The portal protein exhibits Raman secondary structure markers typical of a highly alpha-helical subunit fold that is little perturbed by assembly. On the other hand, Raman markers of subunit side chains change dramatically with assembly, an indication of extensive changes in side chain environments. The cysteinyl Raman signature of the portal consists of a complex pattern of sulfhydryl stretching bands, revealing diverse hydrogen-bonding states for the four S-H groups per subunit (Cys 153, Cys 173, Cys 283, and Cys 516). Upon assembly, the population of strongly hydrogen-bonded S-H groups decreases, while the population of weakly hydrogen-bonded S-H groups increases, implying that specific intrasubunit S-H.X hydrogen bonds must be weakened to effect dodecamer assembly and that the molecular mechanism involves reorganization of subunit domains without appreciable changes in domain conformations. Comparison with other viral protein assemblies suggests an assembly process not requiring metastable intermediates. The recently published X-ray structure of the phi29 portal [Simpson, A. A., et al. (2000) Nature 408, 745-750] shows that residues 125-225 lining the channel surface form alpha-helical modules spaced by short beta-strands and turns; a surprisingly close secondary structure homology is predicted for residues 240-350 of the P22 portal, despite no apparent sequence homology. This motif is proposed as an evolutionarily conserved domain involved in DNA translocation.

Bacteriophage P22↗
PubMed2001

AlphaVbeta6 integrin promotes invasion of squamous carcinoma cells through up-regulation of matrix metalloproteinase-9.

The integrin alphaVbeta6 is a fibronectin receptor, which is not detectable on normal epithelium but is neo-expressed in oral epithelial dysplasia and oral squamous-cell carcinoma (SCC), suggesting a role in promoting malignant behaviour and tumour progression. We used transfection and retroviral infection to create a panel of SCC cell lines expressing various levels of alphaVbeta6 to examine this possibility. We found that increased expression of alphaVbeta6 in malignant keratinocytes up-regulates MMP-9 and MMP-2 expression and promotes invasion in an MMP-9-dependent manner. Our results suggest a possible mechanism for the involvement of alphaVbeta6 in squamous carcinoma in vivo.

Animals↗
PubMed2001

Distinct cysteine sulfhydryl environments detected by analysis of Raman S-hh markers of Cys-->Ser mutant proteins.

Very little is known about the character or functional relevance of hydrogen-bonded cysteine sulfhydryl (S-H) groups in proteins. The Raman S-H band is a unique and sensitive probe of the local S-H environment. Here, we report the use of Raman spectroscopy combined with site-specific mutagenesis to document the existence of five distinguishable hydrogen-bonded states of buried cysteine sulfhydryl groups in a native protein. The 666 residue subunit of the Salmonella typhimurium bacteriophage P22 tailspike contains eight cysteine residues distributed through the elongated structure. The tailspike cysteine residues display an unusual Raman S-H band complex (2500-2600 cm(-1) interval) indicative of diverse S-H hydrogen-bonding interactions in the native trimeric structure. To resolve specific Cys contributions to the complex Raman band we characterized a set of tailspike proteins with each cysteine replaced by a serine. The mutant proteins, once folded, were structurally and functionally indistinguishable from wild-type tailspikes, except for their Raman S-H signatures. Comparison of the Raman spectra of the mutant and wild-type proteins reveals the following hydrogen-bond classes for cysteine sulfhydryl groups. (i) Cys613 forms the strongest S-H...X bond of the tailspike, stronger than any heretofore observed for a protein. (ii) Cys267, Cys287 and Cys458 form robust S-H...X bonds. (iii) Moderate S-H...X bonding occurs for Cys169 and Cys635. (iv) Cys290 and Cys496 form weak hydrogen bonds. (v) It is remarkable that Cys287 contributes two Raman S-H markers, indicating the population of two distinct hydrogen-bonding states. The sum of the S-H Raman signatures of all eight mutants accurately reproduces the composite Raman band of the wild-type tailspike. The diverse cysteine states may be an outcome of the folding and assembly pathway of the tailspike, which though lacking disulfide bonds in the native state, utilizes transient disulfide bonds in the maturation pathway. This Raman study represents the first detailed assessment of local S-H hydrogen bonding in a native protein and provides information not obtainable directly by other structural probes. The method employed here should be applicable to a wide range of cysteine-containing proteins.

Amino Acid Substitution↗
PubMed2001

Tyrosine Raman signatures of the filamentous virus Ff are diagnostic of non-hydrogen-bonded phenoxyls: demonstration by Raman and infrared spectroscopy of p-cresol vapor.

p-Cresol is a simple molecular model for the para phenolic side chain of tyrosine. Previously, Siamwiza and co-workers [(1975) Biochemistry 14, 4870-4876] investigated p-cresol solutions to identify Raman spectroscopic signatures for different hydrogen-bonding states of the tyrosine phenoxyl group in proteins. They found that the phenolic moiety exhibits an intense Raman doublet in the spectral interval 820-860 cm(-1) and that the doublet intensity ratio (I2/I1, where I2 and I1 are Raman peak intensities of the higher- and lower-wavenumber members of the doublet) is diagnostic of specific donor and acceptor roles of the phenoxyl OH group. The range of the doublet intensity ratio in proteins (0.30 < I2/I1 < 2.5) was shown to be governed by Fermi coupling between the phenolic ring-stretching fundamental nu1 and the first overtone of the phenolic ring-deformation mode nu(16a), such that when the tyrosine phenoxyl proton is a strong hydrogen-bond donor, I2/I1 = 0.30, and when the tyrosine phenoxyl oxygen is a strong hydrogen-bond acceptor, I2/I1 = 2.5. Here, we interpret the Raman and infrared spectra of p-cresol vapor and extend the previous correlation to the non-hydrogen-bonded state of the tyrosine phenoxyl group. In the absence of hydrogen bonding, the Raman intensity of the higher-wavenumber component of the canonical Fermi doublet is greatly enhanced such that I2/I1 = 6.7. Thus, for the non-hydrogen-bonded phenoxyl, the lower-wavenumber member of the Fermi doublet loses most of its Raman intensity. This finding provides a basis for understanding the anomalous Raman singlet signature (approximately 854 cm(-1)) observed for tyrosine in coat protein subunits of filamentous viruses Ff and Pf1 [Overman, S. A., et al. (1994) Biochemistry 33, 1037-1042; Wen, Z. Q., et al. (1999) Biochemistry 38, 3148-3156]. The implications of the present results for Raman analysis of tyrosine hydrogen-bonding states in other proteins are considered.

Capsid↗
PubMed2001

Orientations of Tyr 21 and Tyr 24 in the capsid of filamentous virus Ff determined by polarized Raman spectroscopy.

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.

Amino Acid Sequence↗
PubMed2001

Characterization of subunit structural changes accompanying assembly of the bacteriophage P22 procapsid.

P22 serves as a model for the assembly and maturation of icosahedral double-stranded DNA viruses. The viral capsid precursor, or procapsid, is assembled from 420 copies of a 47 kDa coat protein subunit (gp5) that is rich in beta-strand secondary structure. Maturation to the capsid, which occurs in vivo upon DNA packaging, is accompanied by shell expansion and a large increase in the level of protection against deuterium exchange of amide NH groups. Accordingly, shell maturation resembles the final step in protein folding, wherein domain packing and an exchange-protected core become more fully developed [Tuma, R., Prevelige, P. E., Jr., and Thomas, G. J., Jr. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9885-9890]. Here, we exploit recent advances in Raman spectroscopy to investigate the P22 coat protein subunit under conditions which stabilize the monomeric state, viz., in solution at very low concentrations. Under these conditions, the monomer exhibits an elongated shape, as demonstrated by small-angle X-ray scattering. Raman spectra allow the identification of conformation-sensitive marker bands of the monomer, as well as the characterization of NH exchange dynamics for comparison with procapsid and capsid shell assemblies. We show that procapsid assembly involves significant ordering of the predominantly beta-strand backbone. We propose that such ordering may mediate formation of the distinct subunit conformations required for assembly of a T = 7 icosahedral lattice. However, the monomer, like the subunit within the procapsid lattice, exhibits a moderate level of protection against low-temperature NH exchange, indicative of a nascent folding core. The environments and exchange characteristics of key side chains are also similar for the monomeric and procapsid subunits, and distinct from corresponding characteristics of the capsid subunit. The monomer thus represents a compact but metastable folding intermediate along the pathway to assembly of the procapsid and capsid.

Amides↗