Biomedical subjects

T M Smith

Publications and source records attributed to T M Smith.

At least 19 recordsLinked to original sources

PubMed2006

FLASH is an essential component of Cajal bodies.

Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.

Animals↗

PubMed2006

Molar development in common chimpanzees (Pan troglodytes).

Numerous studies have reported on enamel and dentine development in hominoid molars, although little is known about intraspecific incremental feature variation. Furthermore, a recent histological study suggested that there is little or no time between age at chimpanzee crown completion and age at molar eruption, which is unlikely given that root growth is necessary for tooth eruption. The study presented here redefines growth standards for chimpanzee molar teeth and examines variation in incremental features. The periodicity of Retzius lines in a relatively large sample was found to be 6 or 7 days. The number of Retzius lines and cuspal enamel thickness both vary within a cusp type, among cusps, and among molars, resulting in marked variation in formation time. Daily secretion rate is consistent within analogous cuspal zones (inner, middle, and outer enamel) within and among cusp types and among molar types. Significantly increasing trends are found from inner to outer cuspal enamel (3 to 5 microns/day). Cuspal initiation and completion sequences also vary, although sequences for mandibular molar cusps are more consistent. Cusp-specific formation time ranges from approximately 2 to 3 years, increasing from M1 to M2, and often decreasing from M2 to M3. These times are intermediate between radiographic studies and a previous histological study, although both formation time within cusps and overlap between molars vary considerably. Cusp-specific (coronal) extension rates range from approximately 4 to 9 microns/day, and root extension rates in the first 5 mm of roots range from 3 to 9 microns/day. These rates are greater in M1 than in M2 or M3, and they are greater in mandibular molars than in respective maxillary molars. This significant enlargement of comparative data on nonhuman primate incremental development demonstrates that developmental variation among cusp and molar types should be considered during interpretations and comparisons of small samples of fossil hominins and hominoids.

Age Determination by Teeth↗

PubMed2006

Modern human molar enamel thickness and enamel-dentine junction shape.

This study examines cross-sections of molar crowns in a diverse modern human sample to quantify variation in enamel thickness and enamel-dentine junction (EDJ) shape. Histological sections were generated from molars sectioned buccolingually across mesial cusps. Enamel cap area, dentine area, EDJ length, and bi-cervical diameter were measured on micrographs using a digitizing tablet. Nine landmarks along the EDJ were defined, and X and Y coordinates were digitized in order to quantify EDJ shape. Upper molars show greater values for the components of enamel thickness, leading to significantly greater average enamel thickness than in lower molars. Average enamel thickness increased significantly from M1 to M3 in both molar rows, due to significantly increasing enamel cap area in upper molars, and decreasing dentine area in lower molars. Differences in EDJ shape were found among maxillary molars in combined and individual populations. Sex differences were also found; males showed significantly greater dentine area, EDJ length, and bi-cervical diameters in certain tooth types, which resulted in females having significantly thicker average enamel. Differences in enamel thickness and EDJ shape within molars were also found among populations, although few consistent trends were evident. This study demonstrates that enamel thickness and EDJ shape vary among molars, between sexes, and among populations; these factors must be considered in the categorization and comparison of ape and human molars, particularly when isolated teeth or fossil taxa are included. Human relative enamel thickness encompasses most values reported for fossil apes and humans, suggesting limited taxonomic value when considered alone.

Animals↗

PubMed2006

Experimental determination of the periodicity of incremental features in enamel.

Vital labelling of hard tissues was used to examine the periodicity of features of dental enamel microstructure. Fluorescent labels were administered pre- and postnatally to developing macaques (Macaca nemestrina), which were identified histologically in dentine and related to accentuated lines in enamel, allowing for counts of features within known-period intervals. This study demonstrates that cross-striations represent a daily rhythm in enamel secretion, and suggests that intradian lines are the result of a similar 12-h rhythm. Retzius lines were found to have a regular periodicity within individual dentitions, and laminations appear to represent a daily rhythm that also shows 12-h subdivisions. The inclusion of intradian lines and laminations represents the first empirical evidence for their periodicities in primates; these features frequently complicate precise measurements of secretion rate and Retzius line periodicity, which are necessary for determination of crown formation time. The biological basis of incremental feature formation is not completely understood; long-period features may result from interactions between short-period rhythms, although this does not explain the known range of Retzius line periodicities within humans or among primates. Studies of the genetic, neurological and hormonal basis of incremental feature formation are needed to provide more insight into their physiological and structural basis.

Animals↗

PubMed2006

The accuracy of histological assessments of dental development and age at death.

Histological analyses of dental development have been conducted for several decades despite few studies assessing the accuracy of such methods. Using known-period incremental features, the crown formation time and age at death of five pig-tailed macaques (Macaca nemestrina) were estimated with standard histological techniques and compared with known ages. Estimates of age at death ranged from 8.6% underestimations to 15.0% overestimations, with an average 3.5% overestimate and a 7.2% average absolute difference. Several sources of error were identified relating to preparation quality and section obliquity. These results demonstrate that histological analyses of dental development involving counts and measurements of short- and long-period incremental features may yield accurate estimates, particularly in well-prepared material. Values from oblique sections (or most naturally fractured teeth) should be regarded with caution, as obliquity leads to inflated cuspal enamel formation time and underestimated imbricational formation time. Additionally, Shellis's formula for extension rate and crown formation time estimation was tested, which significantly overestimated crown formation time due to underestimated extension rate. It is suggested that Shellis' method should not be applied to teeth with short, rapid periods of development, and further study is necessary to validate this application in other material.

Age Determination by Teeth↗

PubMed2003

Validation of fermentation processes.

The ability to prepare consistent biopharmaceutical products depends extensively on possession of banked and characterized cell substrates and on development of production processes which can be validated. While the attributes that define cell characterization have been extensively detailed by ICH and the regulatory agencies in the past decade, little has been specified regarding process validation for biological processes. The extent to which validation concepts can be applied to biological processes varies depending on the nature of the process, the nature of the product, and the level of knowledge regarding the relationship between process parameters and product quality. Expectations concerning the rigour of the validation programme should be adjusted accordingly. There is no single approach that is appropriate for all processes and products. At a minimum, there should be an attempt to define which process parameters are critical, and to focus the attention of validation efforts on these parameters.

Biotechnology↗

PubMed2001

The importance of histidine residues in human ecto-nucleoside triphosphate diphosphohydrolase-3 as determined by site-directed mutagenesis.

Most ecto-nucleoside triphosphate diphosphohydrolases (eNTPDases) are inhibited by the histidine reagent diethyl pyrocarbonate (DEPC), while being resistant to inhibition by many other chemical modification agents. We used site-directed mutagenesis to investigate the sites of modification responsible for DEPC inhibition. First, we constructed the mutations H135A and R67H in eNTPDase-3 to address the possibility that, in eNTPDase-3, histidine 135 compensates for the lack of a histidine in apyrase conserved region (ACR) 1, present in all other membranous eNTPDases (but replaced by R67 in ACR1 of eNTPDase-3). We found histidine 135 is a major, but not the sole, target for DEPC-induced inhibition in eNTPDase-3. In addition, analysis of the R67H mutant led us to conclude that this site is important for DEPC inactivation of other eNTPDases. We also mutated singly and collectively three of the most conserved histidine residues present in eNTPDase-3 (129, 257 and 447) to alanine. None of the single, conserved histidine mutations nor the triple histidine mutation inactivated the enzyme or decreased susceptibility to DEPC inhibition. However, changes in the tendency of monomers to self-associate were noted, and the triple histidine mutant exhibited a higher nucleotidase specific activity than the wild-type.

Animals↗

PubMed2001

Site-directed mutagenesis of human nucleoside triphosphate diphosphohydrolase 3: the importance of residues in the apyrase conserved regions.

Ecto-nucleoside triphosphate diphosphohydrolase 3 (eNTPDase-3, also known as HB6 and CD39L3) is a membrane-associated ecto-apyrase. Only a few functionally significant residues have been elucidated for this enzyme, as well as for the whole family of eNTPDase enzymes. Four highly conserved regions (apyrase conserved regions, ACRs) have been identified in all the members of eNTPDase family, suggesting their importance for biological activity. In an effort to identify those amino acids important for the catalytic activity of the eNTPDase family, as well as those residues mediating substrate specificity, 11 point mutations of 7 amino acid residues in ACR1-4 of eNTPDase-3 were constructed by site-directed mutagenesis. Mutagenesis of asparagine 191 to alanine (N191A), glutamine 226 to alanine (Q226A), and arginine 67 to glycine (R67G) resulted in an increase in the rates of hydrolysis of nucleoside diphosphates relative to triphosphates. Mutagenesis of arginine 146 to proline (R146P) essentially converted the eNTPDase-3 ecto-apyrase to an ecto-ATPase (eNTPDase-2), mainly by decreasing the hydrolysis rates for nucleoside diphosphates. The Q226A mutant exhibited a change in the divalent cation requirement for nucleotidase activity relative to the wild-type and the other mutants. Mutation of glutamate 182 to aspartate (E182D) or glutamine (E182Q), and mutation of serine 224 to alanine (S224A) completely abolished enzymatic activity. We conclude that the residues corresponding to eNTPDase-3 glutamate 182 in ACR3 and serine 224 in ACR4 are essential for the enzymatic activity of eNTPDases in general, and that arginine 67, arginine 146, asparagine 191, and glutamine 226 are important for determining substrate specificity for human ecto-nucleoside triphosphate diphosphohydrolase 3.

Acid Anhydride Hydrolases↗

PubMed2000

A phase II study of "decrescendo" interleukin-2 plus interferon-alpha-2a in patients with progressive metastatic melanoma after chemotherapy.

BACKGROUND: The authors tested a biotherapy regimen involving recombinant interferon-alpha-2a (rIFN-alpha-2a) and recombinant human interleukin-2 (rhIL-2), given in a "decrescendo" schedule over 5 days, for its activity and toxicity in 21 patients who previously had received chemotherapy for advanced melanoma. METHODS: Patients (15 men and 6 women) were given intravenous rhIL-2 at a dose of 18 MIU/m(2) over 6 hours, followed by 18 MIU/m(2) over 12 hours, then 18 MIU/m(2) over 24 hours, and finally 4.5 MIU/m(2)/day for 3 consecutive days. rIFN-alpha-2a (10 MIU/m(2)) was given subcutaneously on Days 1-5. Courses were repeated every 4 weeks. Tumor sites were measured every 8 weeks. Toxicity was recorded using National Cancer Institute Common Toxicity Criteria. RESULTS: No major objective responses were noted. The median number of courses given was two. The median time to progression was 2 months and the median survival was 6 months (range, 2-25 months). However, 2 patients with melanoma involving >/= 2 visceral organs (1 with a high baseline serum lactate dehydrogenase level) and a third with soft tissue metastases achieved durable control of disease and were alive a median of 30+ months later. A fourth patient had a palliative response with reversal of melanosis and a survival of 7 months. This regimen was well tolerated and resulted in no serious long term adverse effects. CONCLUSIONS: The response rate for this regimen was no greater than 10% with Type I and II errors each not exceeding 10%. Nevertheless, occasional durable control of disease and the nonoverlapping toxicity profile with prior chemotherapy support consideration of this regimen in these patients who have limited second-line treatment options.

Adult↗

PubMed2000

Measurement of protein interaction bioenergetics: application to structural variants of anti-sCD4 antibody.

This chapter has described a bioenergetic analysis of the interaction of sCD4 with an IgG1 and two IgG4 derivatives of an anti-sCD4 MAb. The MAbs have identical VH and VL domains but differ markedly in their CH and CL domains, raising the question of whether their antigen-binding chemistries are altered. We find the sCD4-binding kinetics and thermodynamics of the MAbs are indistinguishable, which indicates rigorously that the molecular details of the binding interactions are the same. We also showed the importance of using multiple biophysical methods to define the binding model before the bioenergetics can be appropriately interpreted. Analysis of the binding thermodynamics and kinetics suggests conformational changes that might be coupled to sCD4 binding by these MAbs are small or absent.

Antibodies, Monoclonal↗

PubMed1999

Mutagenesis of two conserved tryptophan residues of the E-type ATPases: inactivation and conversion of an ecto-apyrase to an ecto-NTPase.

A human brain E-type ATPase (HB6 ecto-apyrase) was subjected to site-directed mutagenesis to assess the functional significance of two highly conserved tryptophan residues (Trp 187 and Trp 459), the only two tryptophans conserved in nearly all E-type ATPases. Mutation of tryptophan 187 to alanine yielded a poorly expressed ecto-apyrase completely devoid of nucleotidase activity. Immunolocalization of the W187A mutant in mammalian COS cells showed a cellular distribution clearly different from that of the wild-type enzyme, with the majority of the immunoreactivity concentrated in the interior of the cell. Unlike the wild-type enzyme, this mutant did not bind the nucleotide analogue Cibacron Blue and was sensitive to proteolytic digestion by chymotrypsin. These results suggest alteration of the tertiary structure, causing the enzyme to be improperly folded and retained within the cell. In contrast, mutation of tryptophan 459 to alanine resulted in an ecto-apyrase with enhanced NTPase activity, but diminished NDPase activity. Immunolocalization of this active mutant ecto-apyrase revealed a cellular pattern similar to that of the wild-type enzyme, distributed along the cell periphery and in cell processes. Coupling this active W459A mutation to a previously described mutation (D219E) resulted in an enzyme which preferentially hydrolyzes nucleoside triphosphates over diphosphates. The D219E/W459A double mutant had an ATPase:ADPase ratio of 11:1 and a UTPase:UDPase ratio of 148:1. In addition, the double mutant is substantially less sensitive to inhibition by azide, a more potent inhibitor of ecto-apyrases than ecto-ATPases. Thus, mutation of only two amino acids of an E-type ATPase essentially converts an ecto-apyrase to an ecto-NTPase.

Acid Anhydride Hydrolases↗

PubMed1999

Glycosylation is essential for functional expression of a human brain ecto-apyrase.

The importance of N-linked glycosylation for the function and oligomerization of an E-type ATPase was examined by using tunicamycin and peptide N-glycosidase F, two agents used to prevent and remove glycosylations, respectively. The cDNA encoding a human ecto-apyrase (HB6), predicted to have seven N-linked glycosylation sites, was transiently expressed in mammalian COS cells and the resulting membrane preparations were treated with peptide N-glycosidase F (PNGase-F). PNGase-F caused a decrease in the apparent molecular weight of the protein (consistent with glycan removal) and a decrease in enzymatic activity over time. The ecto-apyrase was also expressed in the presence of tunicamycin, which completely prevented N-linked glycosylation, resulting in a nonglycosylated core protein devoid of ATP and ADP hydrolyzing activity. However, control and tunicamycin-treated cells expressed the enzyme to similar levels and localization. Interestingly, the quaternary structure of this E-type ATPase appears to be dependent upon the presence of glycan chains. The glycosylated ecto-apyrase exists as a homodimer in situ as assessed by both size-exclusion chromatography of detergent-solubilized ecto-apyrase and cross-linking of membrane-bound ecto-apyrase, in contrast to the enzymatically deglycosylated ecto-apyrase and the tunicamycin-treated ecto-apyrase. These results suggest that glycosylation is necessary for homooligomerization and nucleotide hydrolyzing activity, but not for expression and plasma membrane localization of the E-type ATPase. Similar results were obtained with another human ecto-apyrase, CD39, suggesting that the importance of glycosylation may be generalized to all membrane-bound E-type ATPases.

Adenosine Triphosphatases↗

PubMed1999

Site-directed mutagenesis of a human brain ecto-apyrase: evidence that the E-type ATPases are related to the actin/heat shock 70/sugar kinase superfamily.

On the basis of sequence homologies observed between members of the E-type ATPases and the phosphate binding motifs of the actin/heat shock protein 70/sugar kinase superfamily, a human ecto-apyrase was analyzed by site-directed mutagenesis of conserved amino acids in apyrase conserved regions (ACR) I and IV. The expressed proteins were analyzed to assess the significance of these amino acids. A conserved aspartic acid residue in ACR IV was mutated to alanine, asparagine, and glutamic acid, and the relative activity and Km for ATP and ADP were determined. Mutation of this Asp 219 to Ala or Asn yielded an enzyme severely reduced in ATP hydrolyzing activity (>90%) and completely devoid of ADPase activity, along with a similar extent of inhibition of hydrolysis of other nucleoside di- and triphosphates. Interestingly, mutation of Asp 219 to Glu completely restored the ability of the enzyme to hydrolyze nucleoside triphosphates at levels above that of the wild-type enzyme, while the ability to hydrolyze nucleoside diphosphates was slightly reduced. Mutation of a second conserved aspartic acid in ACR I (Asp 62) and two invariant glycine residues in both ACR I (Gly 64) and ACR IV (Gly 221) also severely disrupted nucleotidase activity. These results demonstrate that the E-type ATPases contain the nucleoside phosphate binding domains present in the actin/heat shock protein/sugar kinase superfamily. Together with analysis of computer-predicted secondary structures, the results suggest that the ecto-ATPases and ecto-apyrases are part of, or closely related to, the actin superfamily of proteins.

Actins↗

PubMed1999

Islet cell tumor of the pancreas associated with tumor thrombus in the portal vein.

We report the MR findings of a 70-year-old man with an islet cell tumor that diffusely involved the body of the pancreas associated with enhancing portal vein tumor thrombus and cavernous transformation. The diffusely infiltrative tumor mass was best shown on early post gadolinium spoiled gradient echo. The tumor thrombus enhanced intensely on early post gadolinium images and was also well shown on true FISP (Fast Imaging with Steady State Precession) images. The extent of liver metastases was best shown on fat suppressed T2-weighted images. The most unusual finding was tumor thrombus involving the SMV and portal vein.

Adenoma, Islet Cell↗

PubMed1999

Expression and characterization of chicken muscle ecto-ATPase in mammalian COS cells.

Chicken muscle ecto-ATPase has unusual enzyme kinetics and properties not found in many other E-type ATPases. To determine whether the unique properties of the chicken ecto-ATPase are inherent in the protein sequence and not mediated by some unique property of the chicken system, we have spliced together two partial cDNAs encoding the ecto-ATPase. The enzymatic properties of the COS (green monkey kidney) cell-expressed protein are indistinguishable from the purified chicken gizzard ecto-ATPase, including a 2- to 3-fold stimulation of membrane-bound activity by crosslinking and lectins, properties not shared by most other E-type ATPases. The expressed enzyme is specific for nucleotide triphosphates (ATPase:ADPase hydrolysis ratio of 26:1) and is inhibited by Cibacron Blue (IC50 = 10 microM). The active, expressed enzyme can be affinity-purified with Cibacron Blue, is relatively resistant to deglycosylation, and is less stable than other E-type ATPases. Expression in the presence of tunicamycin resulted in an inactive, unfolded enzyme.

Adenosine Triphosphatases↗

PubMed1999

Childhood obesity.

Childhood obesity has become one of the more alarming nutritional problems plaguing the American population, with estimates as high as 25% of all children being obese. Aside from obesity's associated risks, there are psychosocial and emotional burdens carried by obese children as well. Clinicians are encountering many of these children in their clinics everyday for other reasons and yet are failing to address the issue of obesity. The problem is not so much that physicians are not recognizing it, but rather that they are ignoring it, especially if the parent or child is unaware that there is a problem. Unfortunately, much controversy exists regarding the treatment of childhood obesity. This article attempts to sort through the myriad issues surrounding childhood obesity and to dispel some of the rumors and myths surrounding this subject.

Adolescent↗

PubMed1998

Cloning, sequencing, and expression of a human brain ecto-apyrase related to both the ecto-ATPases and CD39 ecto-apyrases1.

An extracellular ATPase (E-type ATPase) clone was isolated from a human brain cDNA library and sequenced. The transcript shows similarity to the previously published chicken smooth muscle and rat brain ecto-ATPase cDNAs, human CD39L1 cDNA (putative human ecto-ATPase), and mammalian CD39 (lymphoid cell activation antigen, ecto-apyrase, ATPDase, ATP-diphosphohydrolase) cDNAs. The full-length human brain cDNA encodes a 529 amino acid glycoprotein with a putative membrane spanning region near each terminus, with the majority of the protein found extracellularly. Expression of this clone in mammalian COS-1 cells yielded NaN3-sensitive ATPase and ADPase activity detectable both on intact cells and cell membrane preparations. The nucleotide hydrolysis ratio of the expressed protein is approx. 2.75:1 (ATPase:ADPase activity), classifying it as an ecto-apyrase. However, this hydrolysis ratio is intermediate between that observed for the ecto-ATPases and the CD39 ecto-apyrases (L. Plesner, Int. Rev. Cytol. 158 (1995) 141-214). Quantitative analyses of amino acid identities and similarities between this ecto-apyrase and other vertebrate E-type ATPases suggest that this human brain enzyme is nearly equally related to the ecto-ATPases and the CD39s, and phylogenetic analysis suggests that it could be an ancestral enzyme from which both ecto-ATPases and CD39 ecto-apyrases are derived.

Adenosine Triphosphatases↗

PubMed1998

Immunological detection of ecto-ATPase in chicken and rat tissues: characterization, distribution, and a cautionary note.

We have generated a polyclonal antibody (CKG2) against native chicken gizzard ecto-ATPase for immunolocalization and immunoprecipitation. Active ecto-ATPase is immunoprecipitated from solubilized chicken and rat membranes and shown to be localized to the plasma membrane of the chicken smooth muscle cells. This antibody is specific for the ecto-ATPases, since the more abundant chicken stomach ecto-apyrase is not recognized in immunoprecipitation, western blot or immunolocalization analyses. The CKG2 antibody cross-reacts with mammalian (rat) ecto-ATPase in western blots, with testis being the most abundant source. Interestingly, when the same rat membranes are analyzed by western blot under non-reducing conditions, the 66 kDa ecto-ATPase is not recognized, instead a 200 kDa protein is detected, previously postulated to be an oligomer of ecto-ATPase. However, this 200 kDa cross-reacting protein is not related to the ecto-ATPases, but is instead an immunoglobulin binding protein, comprised of 50 kDa subunits.

Adenosine Triphosphatases↗