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T Sherwin

Publications and source records attributed to T Sherwin.

At least 19 recordsLinked to original sources
PubMed2003

Involvement of corneal nerves in the progression of keratoconus.

Keratoconus is a debilitating corneal thinning disease that principally develops in the second and third decades of life. Our group previously developed a novel approach to studying keratoconus, based on the observation that there is a gradient of damage across the keratoconic cone. We identified a number of cellular characteristics of keratoconus such as discrete incursions of fine cellular processes from the anterior keratocytes in association with localised indentation of the basal epithelium, and increased levels of the lysosomal enzymes Cathepsin B and G in aberrant keratocytes, located beneath compromised regions of Bowman's layer, but also deeper in the stroma. Enzyme activity by these cells seemed to be causing localised structural degradation of the anterior stroma, leading to near-complete destruction of both Bowman's layer and the stroma, often necessitating a full-thickness corneal graft for sight restoration. This current study extends our initial findings by investigating the role of corneal nerves passing between the stroma and epithelium at the sites of early degradative change observed previously, and may be facilitating the keratocyte-epithelial interactions in this disease. Cells in sections of normal and keratoconic human corneas were labelled with the fixable fluorescent viability dye 5-chloromethylfluorescein diacetate, antibodies to alpha-tubulin (nerves), alpha3beta1 integrin, Cathepsin B and G, and the nuclear dye DAPI, and then examined with a confocal microscope. Anterior keratocyte nuclei were seen wrapping around the nerves as they passed through the otherwise acellular Bowman's layer, and as the disease progressed and Bowman's layer degraded, these keratocytes were seen to express higher levels of Cathepsin B and G, and become displaced anteriorly into to the epithelium. Localised nerve thickenings also developed within the epithelium in association with Cathepsin B and G expression, and appeared to be very destructive to the cornea. Insight into the molecular mechanisms of keratoconic disease pathogenesis and progression can be gained from the process of extracellular matrix remodelling known from studies of connective tissues other than the cornea, and wound healing studies in the cornea. Further studies are required to determine how well this model fits the actual molecular basis of the pathogenesis of keratoconus.

Cathepsin B↗
PubMed2002

Cellular incursion into Bowman's membrane in the peripheral cone of the keratoconic cornea.

Analysis of corneal tissue from normal and keratoconic donors has revealed differences which may represent early signs in the pathogenesis of keratoconus. Peripheral areas of keratoconic tissue obtained from transplant surgery were targeted to ascertain cellular disposition and morphological changes which may be masked within the extensive damage of the central keratoconic cone. Peripheral keratoconic corneae exhibited discrete incursion of fine cellular processes into Bowman's membrane. These processes originated from keratocytes and were often observed in conjunction with a defined indentation from the basal epithelium. Comparison of the lysosomal enzymes cathepsin B and G with constitutively expressed cytoplasmic esterase determined that both cathepsins were elevated within keratocytes of keratoconic tissue compared with normal tissue. Some clusters of keratoconic keratocytes had elevated levels of cathepsin exceeding all others. Cathepsin-rich keratocytes localized with morphologically compromised regions of Bowman's membrane. The presence of cell nests deeper within the stroma indicated that the catabolic changes, which are visible within the acellular Bowman's membrane, are probably also occurring deeper within the stroma, but are masked and not readily detectable.

Cathepsin B↗
PubMed2001

A trypanosome structure involved in transmitting cytoplasmic information during cell division.

African trypanosomes are protozoan parasites that cause sleeping sickness in humans through a tsetse fly vector. The procyclic form of Trypanosoma brucei has a single, attached flagellum that describes a helical path along the cell from posterior to anterior. During division, a specific flagellum-flagellum connection is elaborated between the new and old flagellum. This connector was present only during cell duplication and was found to be involved in the replication of the helical cell pattern and polarity. This finding implicates the concept of cytotaxis in cell morphogenesis in trypanosomes.

Animals↗
PubMed2001

Differential diagnosis of corneal oedema assisted by in vivo confocal microscopy.

The purpose of this study was to demonstrate microstructural differences between clinically similar, but aetiologically different, cases of corneal oedema in four subjects. In vivo confocal microscopy highlighted oedema of the basal epithelium, prominent nerve-keratocyte interactions, and typical 'epithelialization' of the endothelium in a case of iridocorneal endothelial syndrome; however, a similar microstructural appearance was observed in a case of presumed herpetic disciform keratitis. The latter diagnosis was subsequently revised on this basis. Confocal examination of Fuchs' endothelial dystrophy demonstrated oedema of the basal epithelium, prominent wing cells, anterior stromal alterations, fibrosis of Descemet's membrane and a typical 'strawberry' appearance of the endothelium. In contrast, in vivo microstructural examination of bilateral keratoconus with hydrops confirmed oedema mainly involving the epithelium and anterior stroma. In vivo confocal microscopy allows the clinician to observe the living cornea at a microstructural level and to better diagnose and differentiate borderline or unusual cases of corneal oedema.

Adult↗
PubMed2001

Microstructural assessment of rare corneal dystrophies using real-time in vivo confocal microscopy.

PURPOSE: To analyse and describe three cases of rare corneal dystrophy and highlight their in vivo microstructural features. METHODS: Subject 1 was diagnosed with a posterior stromal fleck corneal dystrophy Two of her three children were also affected. Subjects 2 and 3 exhibited an almost identical clinical appearance on biomicroscopic examination, such that both clinically were diagnosed as having pre-Descemet's dystrophies. All subjects underwent in vivo confocal microscopy and approximately 300 sequential digital images were obtained and analysed for each cornea. RESULTS: In vivo confocal microscopy of subject 1 demonstrated an abnormal appearance of numerous large ovoid particles, measuring 50-70 microm in diameter in the mid and posterior stroma as well as smaller hyperreflective dot-like intracellular deposits, of less than 1 microm diameter. Despite the near-identical clinical appearance, subjects 2 and 3 could be clearly differentiated by in vivo confocal microscopy. Subject 2 exhibited small, irregular, optically dense particles, mainly in the anterior stroma, whereas subject 3 possessed classical involvement of the stroma immediately adjacent to Descemet's membrane, with numerous regular small, hyperreflective particles. CONCLUSIONS: The ability of in vivo confocal microscopy to localize and accurately measure various elements in different corneal layers may help to resolve whether abnormalities are intra- or extracellular, and aid clearer differentiation of rare corneal disorders.

Adult↗
PubMed2000

Cell-cycle and developmental regulation of TbRAB31 localisation, a GTP-locked Rab protein from Trypanosoma brucei.

Rab proteins are small GTPases that control the direction and timing of vesicle fusion during intracellular trafficking between membraneous compartments. Genome sequencing and EST analysis of Trypanosoma brucei indicates that the trypanosome Rab (TbRAB) gene family, and hence complexity of intracellular transport pathways, is intermediate between Saccharomyces cerevisiae and mammals. TbRAB31 is a constitutively expressed T. brucei Rab protein (formerly Trab7p) and is the product of one of two closely linked TbRAB genes, the other being TbRAB2 (TbRab2p, in: Field H, Ali BRS, Sherwin T, Gull K, Croft SL, Field MC. TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells. J Cell Sci 1999; 112:147-156), involved in ER to Golgi transport. TbRAB31 has high homology to members of the Sec4/Ypt1 subfamily of Rab proteins from S. cerevisiae and to Rab13 and Rab11 from higher eukaryotes. Recombinant TbRAB31 binds GTP but, unusually for a Rab protein, has undetectable GTPase activity resulting in a constitutively GTP-bound protein. Antibodies against TbRAB31 recognise a discrete structure located between the kinetoplast and nucleus in interphase procyclic cells; by contrast the structure is morphologically more complex in bloodstream form (BSF) parasites, consisting of at least two foci. TbRAB31 behaviour was also studied during the cell cycle; TbRAB31 always localised to a discrete structure that duplicated very early in mitosis and relocated to daughter cells in a coordinate manner with the basal body and kinetoplast, suggesting the involvement of microtubules. Additional evidence suggests that TbRAB31 localises to the trypanosome Golgi complex. Firstly, the interphase position of TbRAB31 is consistent with a Golgi location. Secondly, the TbRAB31 structure is also recognised by cross-reacting antibodies to mammalian beta-coatomer protein (beta-COP), which localises to the Golgi in mammalian cells. Thirdly, the fluorescent ceramide analogue, BODIPY-TR-ceramide, a reliable marker of the mammalian Golgi apparatus, exhibited overlapping distribution with TbRAB31. The location of BODIPY-TR-ceramide was confirmed at the trypanosome Golgi by histochemistry with diaminobenzidine and electron microscopy.

Animals↗
PubMed2000

Architecture of the Trypanosoma brucei nucleus during interphase and mitosis.

The structural basis of mitosis, spindle organisation and chromosome segregation, in the unicellular parasite Trypanosoma brucei is poorly understood. Here, using immunocytochemistry, fluorescent in situ hybridisation and electron microscopy, we provide a detailed analysis of mitosis in this parasite. We describe the organisation of the mitotic spindle during different stages of mitosis, the complex ultrastructure of kinetochores and the identification of a potential spindle-organising centre in the mitotic nucleus. We investigate the dynamics of chromosome segregation using telomeric and chromosome-specific probes. We also discuss the problems involved in chromosome segregation in the light of the fact that the T. brucei karyotype has 22 chromosomes in the apparent presence of only eight ultrastructurally defined kinetochores.

Animals↗
PubMed1999

An alternate technique to close neurosurgical incisions using octylcyanoacrylate tissue adhesive.

The present authors have applied the use of tissue adhesive octylcyanoacrylate, recently approved by the FDA, in the closure of routine neurosurgical cases. The authors find this to be an excellent substitute for nylon, staples, vicryl or steristrip final layer closure of the surgical incision. This is especially useful in the pediatric neurosurgical practice where young children can be emotionally traumatized by the experience of suture or staple removal. We recommend octylcyanoacrylate closure as a safe, simple, quick, cost-effective method of skin closure that has superior applications in pediatric neurosurgical wound closure when used with proper technique.

Child↗
PubMed1999

Genetic dissection of the Leishmania paraflagellar rod, a unique flagellar cytoskeleton structure.

The paraflagellar rod (PFR) is a unique network of cytoskeletal filaments that lies alongside the axoneme in the flagella of most trypanosomatids. While little is known about how two major Leishmania mexicana PFR protein components, PFR1 and PFR2, assemble into this complex structure, previous analysis of PFR2 null mutants demonstrated that the PFR is essential for proper cell motility. The structural roles of PFR1 and PFR2 are now examined through comparison of PFR2 null mutants with new PFR1 null mutant and PFR1/PFR2 double null mutant parasites. Both PFR1 and PFR2 were essential for PFR formation and cell motility. When elimination of one PFR gene prevented assembly of a native PFR structure, the other PFR protein accumulated at the distal flagellar tip. Comparison of PFR substructures remaining in each mutant revealed that: (1) fibers that attach the PFR to the axoneme did not contain PFR1 or PFR2, and assemble in the absence of a PFR. (2) PFR1 was synthesized and transported to the flagella in the absence of PFR2, where it formed a stable association with the axoneme attachment fibers. (3) PFR2 was synthesized and transported to the flagella in the absence of PFR1, though it was not found associated with the axoneme attachment fibers. (4) PFR1 and PFR2 were located throughout the subdomains of the PFR. These data suggest that while PFR filaments contain both PFR1 and PFR2, the PFR is attached to the axoneme by interaction of PFR1 with the axoneme attachment fibers.

Animals↗
PubMed1999

TbRab2p, a marker for the endoplasmic reticulum of Trypanosoma brucei, localises to the ERGIC in mammalian cells.

The Rab family of small GTPases is a subset of the Ras superfamily. Rabs regulate the flux through individual steps of the intracellular membrane trafficking pathway, such as ER-to-Golgi transport, probably by controlling SNARE complex assembly. In Trypanosoma brucei a number of Rab proteins have been isolated by EST analysis; here we characterise one of these, TbRab2p (originally designated Trab1p), which is a member of the Ypt1p subfamily of Rab proteins. Recombinant TbRab2p is capable of hydrolysing GTP and is post-translationally modified in vitro by addition of a geranylgeranyl prenyl group, properties of an authentic Rab GTPase. Antibodies against recombinant TbRab2p show that in trypanosomes TbRab2p is localised primarily to the endoplasmic reticulum (ER) and colocalises with BiP in wild-type trypanosomes. Over expression of TbRab2p in procyclic form T. brucei results in a cell population having a 40-fold increase in TbRab2p expression. In these cells biosynthesis of procyclin, a secretory pathway glycoprotein, is decreased, accompanied by an increase in general protein biosynthesis, suggesting that excess TbRab2p affects ER function. Heterologous expression of TbRab2p in COS cells resulted in targeting to the pre-Golgi transport intermediate (ERGIC), indicating that the targeting information is conserved between mammals and trypanosomes. Clustal and phylogenetic analyses support assignment of TbRab2p as a Rab2 homologue. In addition, over expression of TbRab2p in trypanosomes results in membrane reorganisation and formation of opaque vesicular structures visible by phase contrast microscopy, consistent with accumulation of ER-derived vesicular structures in cells highly overexpressing TbRab2p. Ultrastructural examination by electron microscopy confirmed the presence of a tubulo-vesicular membrane bound compartment in close proximity to the cis-Golgi, probably equivalent to the ERGIC. TbRab2p is therefore a new ER/ERGIC marker for T. brucei.

Amino Acid Sequence↗
PubMed1999

Protein transport and flagellum assembly dynamics revealed by analysis of the paralysed trypanosome mutant snl-1.

The paraflagellar rod (PFR) of Trypanosoma brucei is a large, complex, intraflagellar structure that represents an excellent system in which to study flagellum assembly. Molecular ablation of one of its major constituents, the PFRA protein, in the snl-1 mutant causes considerable alteration of the PFR structure, leading to cell paralysis. Mutant trypanosomes sedimented to the bottom of the flask rather than staying in suspension but divided at a rate close to that of wild-type cells. This phenotype was complemented by transformation of snl-1 with a plasmid overexpressing an epitope-tagged copy of the PFRA gene. In the snl-1 mutant, other PFR proteins such as the second major constituent, PFRC, accumulated at the distal tip of the growing flagellum, forming a large dilation or 'blob'. This was not assembled as filaments and was removed by detergent-extraction. Axonemal growth and structure was unmodified in the snl-1 mutant and the blob was present only at the tip of the new flagellum. Strikingly, the blob of unassembled material was shifted towards the base of the flagellum after cell division and was not detectable when the daughter cell started to produce a new flagellum in the next cell cycle. The dynamics of blob formation and regression are likely indicators of anterograde and retrograde transport systems operating in the flagellum. In this respect, the accumulation of unassembled PFR precursors in the flagellum shows interesting similarities with axonemal mutants in other systems, illustrating transport of components of a flagellar structure during both flagellum assembly and maintenance. Observation of PFR components indicate that these are likely to be regulated and modulated throughout the cell cycle.

Animals↗
PubMed1998

Intravenous ketamine for pediatric sedation in the emergency department: safety profile with 156 cases.

OBJECTIVES: To determine the safety of i.v. ketamine when administered by emergency physicians (EPs) for pediatric procedures, and to contrast the sedation characteristics of the i.v. and i.m. routes. METHODS: The study was a retrospective consecutive case series of children aged < or =15 years given i.v. ketamine in the EDs of a university medical center and an affiliated county hospital over a 9-year period. A protocol for ketamine was used by treating physicians. Records were reviewed for adverse effects, indication, dosing, adjunctive drugs, inadequate sedation, and time to release. Results were contrasted with previously reported data for the i.m. route. RESULTS: During the study period i.v. ketamine was administered 156 times, primarily for laceration repair and fracture reduction. Transient apnea and respiratory depression occurred in one patient each; both were quickly identified and were without sequelae. Laryngospasm or aspiration was not noted in any children. There were 6 children with emesis and 2 with mild agitation during recovery. The median time from initial dose to ED release was 103 minutes (25th to 75th percentiles 76 to 146 minutes). The i.v. and i.m. routes were comparable in terms of adverse effects, inadequate sedation, and time to release. CONCLUSION: I.v. ketamine can be administered safely by EPs to facilitate pediatric procedures when used in a defined protocol. The sedation characteristics of the i.v. and i.m. routes appear comparable.

Adolescent↗
PubMed1997

A motility function for the paraflagellar rod of Leishmania parasites revealed by PFR-2 gene knockouts.

We demonstrate a functional role for the paraflagellar rod (PFR) in motility of Leishmania mexicana. The PFR is a complex cytoskeletal structure running parallel to the axoneme in the flagella of kinetoplastid protozoa. The PFR is composed of a latticework of protein filaments whose major constituents are two related proteins (PFR-1 and PFR-2 in Leishmania). The molecular details of their assembly into PFR filaments are unknown as is the biological function of the PFR. As an approach to understanding the structure and function of the PFR in Leishmania, we made L. mexicana null mutants of PFR-2. PFR-2 minus parasites grow and divide normally in culture and still express the PFR-1 protein. They lack most of the PFR structure demonstrating that the PFR-2 protein is an essential constituent of the PFR. Detailed ultrastructural analysis of the PFR-2 null mutant reveals the presence of a residual inner substructure of the PFR which contains PFR-1 protein, indicating that PFR-1 can polymerize in the absence of PFR-2. The PFR-2 null mutant displays pronounced changes in flagellar beat waveform and forward swimming velocity, compared to wild type parasites consistent with decreased internal elastic bending resistance in PFR-lacking flagella, and indicating a functional role for the PFR in the motility of Leishmania.

Animals↗
PubMed1997

gamma-tubulin in trypanosomes: molecular characterisation and localisation to multiple and diverse microtubule organising centres.

A genomic clone from Trypanosoma brucei, which contains a full length gamma-tubulin gene, was isolated using degenerate oligonucleotide primers. The sequence of this clone predicts a protein of 447 amino acids having a high degree of homology with gamma-tubulins from human and Xenopus laevis (67.2% amino acid identity) and only 57.7% identity with the Plasmodium falciparum gamma-tubulin. Northern blot analysis of poly(A)+ selected RNA from a procyclic culture detects a major transcript of approximately 2.2 kb plus a minor transcript of approximately 3.6 kb. A fusion protein comprising almost the full length gamma-tubulin gene product (amino acids 8-447) plus an amino-terminal histidine tag has been expressed and purified from Escherichia coli and used to raise a polyclonal antibody. Immunofluorescence, using this antibody, shows classical centrosomal localisation in mammalian cells. In T. brucei gamma-tubulin is present in the basal bodies which subtend the flagellum and also at the anterior tip of the cell body where many minus ends of microtubules are located. Furthermore the antibody reveals a small subset of the sub-pellicular microtubules and a discrete dot within the nucleus which alters form with progression through the mitotic cycle. Evidence is also presented for discrete punctate staining within the microtubules of the cell body which may represent the presence of gamma-tubulin on the ends of individual microtubules. Our results indicate that gamma-tubulin is associated with diverse microtubule organising centres and structures in trypanosomes.

Amino Acid Sequence↗
PubMed1995

Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle.

Trypanosoma brucei has a precisely ordered microtubule cytoskeleton whose morphogenesis is central to cell cycle events such as organelle positioning, segregation, mitosis, and cytokinesis. We have defined microtubule polarity and show the + ends of the cortical microtubules to be at the posterior end of the cell. Measurements of organelle positions through the cell cycle reveal a high degree of coordinate movement and a relationship with overall cell extension. Quantitative analysis of the segregation of the replicated mitochondrial genome (the kinetoplast) by the flagellar basal bodies identifies a new G2 cell cycle event marker. The subsequent mitosis then positions one "daughter" nucleus into the gap between the segregated basal bodies/kinetoplasts. The anterior daughter nucleus maintains its position relative to the anterior of the cell, suggesting an effective yet cryptic nuclear positioning mechanism. Inhibition of microtubule dynamics by rhizoxin results in a phenomenon whereby cells, which have segregated their kinetoplasts yet are compromised in mitosis, cleave into a nucleated portion and a flagellated, anucleate, cytoplast. We term these cytoplasts "zoids" and show that they contain the posterior (new) flagellum and associated basal-body/kinetoplast complex. Examination of zoids suggests a role for the flagellum attachment zone (FAZ) in defining the position for the axis of cleavage in trypanosomes. Progression through cytokinesis, (zoid formation) while mitosis is compromised, suggests that the dependency relationships leading to the classical cell cycle check points may be altered in trypanosomes, to take account of the need to segregate two unit genomes (nuclear and mitochondrial) in this cell.

Animals↗
PubMed1995

Emergency department presentation and management of pediatric heart transplant recipients.

The object of this study was to review and delineate the presenting complaints, signs, symptoms, and Emergency Department (ED) management of pediatric heart transplant recipients who presented to Loma Linda University Medical Center's (LLUMC) Emergency Department. A retrospective chart review was made of all of the pediatric heart transplant patients who presented to the ED at LLUMC from January 1986 through February 1993. The department is part of a 600-bed university hospital with an associated 250-bed children's hospital that includes a pediatric heart transplant center and an ED that sees over 38,000 patients per year. The retrospective review collected information relating to chief complaint, physical findings, laboratory analysis, and diagnoses. Forty-seven patients (23%) presented to the ED for a total of 76 visits. The patients presented a median of 278 days (range 19 days to 6.5 years) after transplantation. The most common chief complaints were related to the respiratory tract, and the most common diagnoses (55%) were related to infectious processes. Fever was present in 21% of the visits. Three of 13 blood cultures obtained were positive. Cardiac symptoms were present in 14% of the visits with two rejection episodes. Hospital admission was required for 22 (29%) of the ED visits. Results showed that pediatric heart transplant recipients are most likely to present to the ED with infections. Although infections from opportunistic organisms and bacteremia must be considered, most infections are similar to those in the nontransplanted child. Life-threatening conditions such as graft rejection are less likely. Nevertheless, the emergency physician should maintain caution in the evaluation of these patients. Close cooperation and consultation with the transplant team will assure the optimal outcome for these patients.

Adolescent↗