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Biomedical subjects

J Sanford

Publications and source records attributed to J Sanford.

At least 19 recordsLinked to original sources
PubMed1992

Dissection of a pollen-specific promoter from maize by transient transformation assays.

We have previously reported the isolation and characterization of a gene (Zm13) from Zea mays which shows a pollen-specific pattern of expression. Stably transformed tobacco plants containing a reporter gene linked to portions of the Zm13 5' flanking region show correct temporal and spatial expression of the gene. Here we present a more detailed analysis of the 5' regions responsible for expression in pollen by utilizing a transient expression system. Constructs containing the beta-glucuronidase (GUS) gene under the control of various sized fragments of the Zm 13 5' flanking region were introduced into Tradescantia and Zea mays pollen via high-velocity microprojectile bombardment, and monitored both visually and with a fluorescence assay. The results suggest that sequences necessary for expression in pollen are present in a region from -100 to -54, while other sequences which amplify that expression reside between -260 and -100. The replacement of the normal terminator with a portion of the Zm13 3' region containing the putative polyadenylation signal and site also increased GUS expression. While the -260 to -100 region contains sequences similar to other protein-binding domains reported for plants, the -100 to -54 region appears to contain no significant homology to other known promoter fragments which direct pollen-specific expression. The microprojectile bombardment of Tradescantia pollen appears to be a good test system for assaying maize and possibly other monocot promoter constructs for pollen expression.

Gene Expression↗
PubMed1991

Gamma-subunits of G proteins, but not their alpha- or beta-subunits, are polyisoprenylated. Studies on post-translational modifications using in vitro translation with rabbit reticulocyte lysates.

Lipid modifications that may be introduced into several subunits of G proteins were explored by in vitro translation of recombinant mRNAs in reticulocyte lysates. In agreement with studies by others, myristic acid was incorporated into alpha i's and alpha o, but not alpha s, beta, or gamma's. In contrast, mevalonate (Mev) was incorporated only into gamma-subunits. Both, the gamma-subunit of transducin (gamma T) and that of other G proteins (gamma G) were modified by the lysates but with different characteristics. Labeled gamma T was unstable and was rapidly proteolyzed. Labeled gamma G was stable. The Mev-derivative in gamma G was sensitive to methyliodide and, after cleavage and chromatographic analysis, comigrated with the C20 polyisoprenol geranylgeraniol. This indicated that gamma G had been geranylgeranylated and that this polyisoprenoid was attached to the protein through a thioether linkage. It is thought that polyisoprenylation is defined by the COOH-terminal sequence Cys-A-A-X, where A is an aliphatic acid and X is any amino acid. Replacement by mutation of the Cys of the COOH-terminal -Cys-Ala-Ile-Leu sequence of gamma G with Ser abolished Mev incorporation, suggesting this Cys as the site of attachment of the geranylgeranyl moiety. Yet, Mev incorporation was less than 10% as much into gamma G with the Cys-A-A-X sequence -Cys-Ala-Ile-Trp. Consistent with geranylgeranylation, the C15 farnesyl moiety of farnesyl pyrophosphate was not incorporated into gamma G unless the incubations were fortified with Mev. In contrast, the farnesyl moiety was incorporated in an Mev-independent manner into gamma T (COOH terminus: -Cys-Val-Ile-Ser) and c-Ha-ras (COOH terminus: -Cys-Val-Leu-Ser) which are both farnesylated rather than geranylgeranylated. Thus, 1) separate enzymes appear to be involved in transferring farnesyl and geranylgeranyl groups to proteins, 2) structural factors other than the CAAX box contribute to the activity of the polyisoprenylating enzymes, and 3) this type of lipidation may be part of a proteolytic signaling system. Polyisoprenylation, which increases hydrophobicity of the derivatized protein, may play a role in anchoring not only ras but also G proteins to membranes.

Animals↗
PubMed1991

Grading written essays: a reliability study.

The purpose of this study was to examine the interrater reliability of grades obtained by physical therapy and occupational therapy tutors in rating their students' term papers. This study was carried out in two phases. In phase 1, four student essay papers (two physical therapy students' papers and two occupational therapy students' papers) with grades that had been assigned from the previous year's course were randomly selected from a bank of papers. These papers were independently rated by three course planners (who were responsible for planning, coordinating, and tutoring in the course), and agreement as to the assignment of grades for each paper was established. In phase 2, the same students' essays were rated independently by eight course tutors. To test for differences among students' written essay papers and for differences among the raters in the subcategories of discipline (physical therapy versus occupational therapy) and level of expertise (novice versus experienced) in grading essays, a three-way analysis of variance was performed. An intraclass correlation coefficient (ICC) was calculated for interrater reliability. Although there were no statistically significant differences among the tutors with regard to their discipline and expertise, the reliability analysis produced an ICC of .79. Strategies to enhance the reliability of grading essays are discussed.

Educational Measurement↗
PubMed1990

At least three alternatively spliced mRNAs encoding two alpha subunits of the Go GTP-binding protein can be expressed in a single tissue.

Hybridization blot (Northern) analysis of mRNA coding for alpha subunits of the Go signal-transducing protein detects three bands at 5.7, 4.2, and 3.2 kilobases (kb). We showed previously that the largest is a splice variant coding for the type 2 form of the polypeptide (alpha o2) and the two smaller RNAs react with a probe specific for the seventh of the eight exons that code for the type 1 form (alpha o1). In the present work we demonstrate that the 3.2- and 4.2-kb mRNAs also result from alternative splicing, the splice site being located 31 nucleotides downstream from the termination codon of the open reading frame, and that therefore the alpha o mRNA is made up of at least nine exons. All three alpha o mRNAs are expressed in both heart and brain, more in the latter than the former, as well as in the hamster insulin-secreting tumor (HIT) cell from which the cDNAs encoding the splice variants had been cloned. In contrast, in lung and testis we found only the 5.7-kb alpha o2 mRNA. The same analysis was unable to detect alpha o-specific sequences in either kidney, pancreas (whole), spleen, or liver, while at the same time detecting strong bands for alpha s mRNA. A comparison of the nucleotide sequences of the 5'- and 3'-untranslated regions of the hamster cDNAs cloned here indicated that previously cloned alpha o cDNAs all belong to the same alpha o1A slice subclass derived from 3.2-kb mRNA. The comparison also revealed that the sequences of the untranslated regions are highly conserved among three species (rat, hamster, and brain). Their 3' tails are 99.1% (HIT versus bovine, 200 known bases) and 99.7% (HIT versus rat, 229 bases) identical, and their 5' leader sequences are 92.7% (HIT versus bovine, 165 known bases) and 90.7% (HIT versus rat, 670 bases) identical. This indicates that untranslated regions of mRNAs need not exhibit high degrees of species variation.

Animals↗
PubMed1990

Molecular cloning of a novel splice variant of the alpha subunit of the mammalian Go protein.

We screened a HIT (hamster insulin-secreting tumor) cell cDNA library constructed in lambda gt11 with a Go-specific oligonucleotide probe and isolated six recombinant phages. The inserts of these phages encoded two forms of alpha o, called here alpha o1 and alpha o2. The deduced amino acid sequence of alpha o1 is identical in all of its 354 amino acids to that reported previously for rat and bovine alpha o; that of alpha o2, also of 354 amino acids, is identical to alpha o1 up to and including amino acid 248 and differs thereafter in 26 amino acids. At the nucleotide level, alpha o1 and alpha o2 are identical up to and including the second base of the codon that specifies amino acid 243 and differs thereafter in 88 nucleotides of the remaining open reading frame and has no similarity to alpha o1 in its 3'-untranslated region. We propose that alpha o1 and alpha o2 result as a consequence of alternative splicing of a single alpha o transcript. Northern analysis with specifically designed oligonucleotides indicates that both forms of alpha o are expressed in normal tissues, e.g. brain. After in vitro transcription and translation, the peptides encoded in the alpha o1 and alpha o2 cDNAs could be ADP-ribosylated by pertussis toxin in the presence of added beta gamma dimers. The count of distinct G proteins keeps increasing.

Adenosine Diphosphate Ribose↗
PubMed1990

Effect of station examination item sampling on generalizability of student performance.

This article may be of interest to physical therapy educators who are responsible for structuring station or practical examinations used to evaluate physical therapy students. The global intent of the article is to provide information that may be useful in selecting test items. Specifically, the purposes of this study were 1) to examine how two item-sampling strategies (one based on different diagnostic concepts, or diagnostic probes, and the other based on different anatomical sites) influenced the generalizability of a station examination, 2) to determine the interrater reliability during the station examination, and 3) to determine whether the status of the rater (that of observer or simulated patient) influenced the rating. Using a nested study design, 24 physical therapy students were assessed by eight raters. The raters were randomly and equally assigned to four teams. Each team assessed six students. One rater acted as the simulated patient for the first three students in each group, and the other rater acted as observer. This order was reversed for the last three students. Each student performed nine mini-diagnostic patient cases consisting of three diagnostic probes reproduced at three different anatomical sites. The results demonstrate that 1) similar diagnostic concepts can be generalized across anatomical sites, although different concepts or skills cannot be generalized at a given anatomical site or across sites; 2) interrater reliability was excellent; and 3) the status of the raters (ie, simulated patient or observer) did not bias the ratings.(ABSTRACT TRUNCATED AT 250 WORDS)

Clinical Competence↗
PubMed1990

Roles of G proteins in coupling of receptors to ionic channels and other effector systems.

Guanine nucleotide binding (G) proteins are heterotrimers that couple a wide range of receptors to ionic channels. The coupling may be indirect, via cytoplasmic agents, or direct, as has been shown for two K+ channels and two Ca2+ channels. One example of direct G protein gating is the atrial muscarinic K+ channel K+[ACh], an inwardly rectifying K+ channel with a slope conductance of 40 pS in symmetrical isotonic K+ solutions and a mean open lifetime of 1.4 ms at potentials between -40 and -100 mV. Another is the clonal GH3 muscarinic or somatostatin K+ channel, also inwardly rectifying but with a slope conductance of 55 pS. A G protein, Gk, purified from human red blood cells (hRBC) activates K+ [ACh] channels at subpicomolar concentrations; its alpha subunit is equipotent. Except for being irreversible, their effects on gating precisely mimic physiological gating produced by muscarinic agonists. The alpha k effects are general and are similar in atria from adult guinea pig, neonatal rat, and chick embryo. The hydrophilic beta gamma from transducin has no effect while hydrophobic beta gamma from brain, hRBCs, or retina has effects at nanomolar concentrations which in our hands cannot be dissociated from detergent effects. An anti-alpha k monoclonal antibody blocks muscarinic activation, supporting the concept that the physiological mediator is the alpha subunit not the beta gamma dimer. The techniques of molecular biology are now being used to specify G protein gating. A "bacterial" alpha i-3 expressed in Escherichia coli using a pT7 expression system mimics the gating produced by hRBC alpha k.

Animals↗
PubMed1989

Expression of the human chondrocyte phenotype in vitro.

We report a culture scheme in which human epiphyseal chondrocytes lose their differentiated phenotype in monolayer and subsequently reexpress the phenotype in an agarose gel. The scheme is based on a method using rabbit chondrocytes. Culture in monolayer allowed small quantities of cells to be amplified and provided a starting point to study expression of the differentiated human chondrocyte phenotype. The cells cultured in monolayer produced type I procollagen, fibronectin, and small noncartilaginous proteoglycans. Subsequent culture in agarose was associated with the acquisition of typical chondrocyte ultrastructural features and the synthesis of type II collagen and cartilage-specific proteoglycans. The switch from the nonchondrocyte to the differentiated chondrocyte phenotype occurred under these conditions between 1 and 2 wk of agarose culture and was not necessarily homogeneous throughout a culture. This culture technique will facilitate direct investigation of human disorders of cartilage that have been addressed in the past by alternative approaches.

Cell Differentiation↗
PubMed1989

Expression of dipeptidyl peptidase IV in rat tissues is mainly regulated at the mRNA levels.

Dipeptidyl peptidase IV (DPPIV) is a serine peptidase that cleaves N-terminal dipeptides from polypeptides when the second residue is a proline or an alanine. We have recently cloned cDNAs for rat gp110, a membrane glycoprotein with Mr of 110,000 isolated initially from rat liver. Studies reported here establish that the gp110 for which we have cloned cDNAs is DPPIV. Using the antibodies against and cDNA for DPPIV, we have assessed the tissue distribution of DPPIV by molecular approaches. Immunoblot analysis demonstrated that DPPIV is present in the kidney, lung, and small intestine at high levels, in the liver and spleen at moderate levels, and in the heart at low levels. The highest levels of mRNA for DPPIV were detected in the kidney and small intestine as compared to moderate levels found in the lung, liver, and spleen. The lowest levels of DPPIV mRNA were found in the stomach, testis, and heart. No detectable DPPIV protein and mRNA were found in brain or muscle. LDPPIV protein and mRNA are present at much lower levels in fetal livers as compared to the adult liver. Indirect immunofluorescence microscopy demonstrated that DPPIV is localized in the bile canaliculus of hematocytes and in the apical membrane domains of kidney tubule and small intestine. Further studies by Southern blot analysis indicate that DPPIV is encoded by a single gene.

Animals↗
PubMed1987

Disposition and pharmacokinetics of meptazinol in the CSF. Studies after intrathecal administration in the non-human primate Erythrocebus patas.

A preliminary study is reported on the kinetics of meptazinol following intrathecal and i.m. administration in the Patas monkey. Following intrathecal administration (single dose of 0.5 mg) at T12/L1, meptazinol rapidly disappeared from the CSF with a T1/2 of 35 min. At 240 min after intrathecal injection, most of the meptazinol had been distributed within the spinal tissue near the region of the injection, with minimal amounts reaching the brain (less than 5% of the concentrations present in the lumbar and thoracic tissue). Following i.m. administration (16 mg kg-1) peak concentrations were present in the CSF and plasma within 60 min. Appreciable concentrations persisted in the CSF up to 180 min after i.m. administration. The results would suggest that meptazinol should give rapid but short acting pain relief following intrathecal injection with minimal CNS-related side effects.

Animals↗
PubMed1979

A computerized word-processing and data system for histology in a private medical laboratory.

This report describes the use of a word-processing micro-computer, the Lanier Text Editor, for preparation of surgical tissue reports. Gross and microscopic description formats are stored on floppy disks, which may easily be completed and edited on a cathode-ray tube (CRT) for rapid computerized printing. Also described is the use of a PDP 8/E mini-computer for entry, storage, and manipulation of historic, demographic and diagnostic data, printing of numeric and alphabetic listings, cross-indexing of cases by diagnosis, and the accumulation of data for direct patient or physician billing.

Computers↗
PubMed1979

A computer system for cytology in a private laboratory using a PDP 8/E minicomputer.

A computer software package written in Dibol for a PDP 8/E computer is described that is designed for the needs of the cytology department of a moderate-sized private medical laboratory. Computerized accessioning of cases, report preparation and printing, historical filing, activity summary reports, and direct patient and physician billing are provided by the relatively inexpensive system. The software has been in use for over two years in our cytology department and has resulted in increased efficiency, cost-effectiveness, and quality of reports.

Cell Biology↗