Impact of Changing Distance-Learning Formats in Physical Therapy Professional Education.

AIMS: In early 2020, the COVID-19 pandemic caused a majority of higher education to shift content delivery formats to allow for social distancing to decrease spread of the virus. The purpose of this investigation was to discover physical therapy students' perceived impact from changing from a synchronous videoconferencing format to a more hybrid program format. METHODS: A qualitative case study design bounded by the time of switching formats due to the pandemic was chosen (March 2020 through September 2020). Physical therapy students participated in an agreement survey (n=38) and semi-structured interviews (n=12). Survey and interview data were coded and analyzed to form categories and themes of discovered perspectives. RESULTS: Students' perceived proficiency of hands-on skill was negatively affected. Cross-campus student and faculty interactions improved. Students felt changing formats would not result in detrimental effects on their overall learning nor on their career potential as physical therapists. DISCUSSION: Educators in entry-level professional physical therapy programs utilizing distance-education models should consider and adjust timing of hands-on skill instruction to match didactic content to encourage better connection and clinical application. Distance-learning educators should foster more interaction with students who may feel isolated. Interaction between distance-separated cohorts can reduce feelings of competition and inequality between campus locations and create improved learning communities.

Postural sway of subjects wearing the U.S. Army chemical protective ensemble after functional activity.

BACKGROUND: This study assessed the effects of wearing the chemical protective clothing ensemble (CPE) vs. the battle dress uniform (BDU) on postural sway after 18 min of simulated field activity. Postural sway is a measure of static balance where a person maintains his/her center of gravity over his/her base of support by swaying fore to aft usually around the ankle joint axis. HYPOTHESES: Subjects' postural sway would increase more post-exercise while wearing the CPE vs. the BDU. The increase in postural sway while wearing the CPE would be due to decreased visual and somatosensory inputs. METHODS: Static balance was measured on 25 subjects pre- and post-exercise on the NeuroCom SMART Balance Master using the Sensory Organization Test protocol. Following a test-retest, repeated measures design, each subject completed the protocol twice, once while wearing only the BDU and once while wearing the CPE. RESULTS: A 2 x 2 repeated measures, multivariate analysis of variance revealed no significant difference between the static balance of subjects wearing the CPE vs. wearing the BDU pre- or post-exercise. CONCLUSIONS: The authors suggest that the wearing of the CPE does not affect static balance, even after completing 18 min of functional tasks. Future research should objectively quantify the amount of fatigue postexercise and employ a protocol that has been previously shown to increase postural sway.

The pathologic measurement of polyp size is preferable to the endoscopic estimate.

BACKGROUND: There is no standardized technique to measure polyp size. Estimation of polyp size at endoscopy is difficult. Polyp size measurement by pathologists would seem to be an accurate alternative, but tissue fixation may alter polyp size. To evaluate methods of determining polyp size, we compared endoscopists' estimates and pathologists' measurements with measurements made by an independent examiner. METHODS: Polyps were measured by an independent investigator before and after formalin fixation. The investigator's measurement before fixation (the "gold standard") was compared with the endoscopists' estimates and the pathologists' measurements. RESULTS: Ten endoscopists removed 61 polyps with a snare in 33 patients: 82% were adenomatous and 72% were pedunculated. Mean size was 0.85 +/- 0.6 cm (SD) (range: 0.3 to 3.6 cm, 26% > or = 1 cm). Polyps remained in formalin for a mean of 239 minutes (46 to 1164 minutes). Polyps neither consistently shrank nor enlarged in formalin (maximum change +/- 0.2 cm, r = 0.99 [p < 0.001]). Interobserver agreement between pathologists' and the investigator's post-formalin measurements showed that 55 of 57 polyps (97%) were within +/- 0.3 cm. Endoscopists inaccurately estimated 11 of 56 polyps (20%) (> 0.3 cm difference from the independent examiner). Polyp size was underestimated in three instances (range 0.5 to 0.9 cm) and overestimated in eight (range 0.4 to 0.8 cm). In 5 of 11 instances (46%), this inaccuracy altered polyp size classification across the 1 cm threshold. Results were not dependent on endoscopist, histology, or polyp location. CONCLUSIONS: (1) Polyp size is not significantly affected by formalin fixation; 2) Endoscopists' estimates of polyp size are often unreliable; and, when possible, (3) Pathologists' measurements of polyp size should be used in clinical trials and in clinical practice.

COOH-terminal processing of nascent polypeptides by the glycosylphosphatidylinositol transamidase in the presence of hydrazine is governed by the same parameters as glycosylphosphatidylinositol addition.

Proteins anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) moiety are found in all eukaryotes. After NH2-terminal peptide cleavage of the nascent protein by the signal peptidase, a second COOH-terminal signal peptide is cleaved with the concomitant addition of the GPI unit. The proposed mechanism of the GPI transfer is a transamidation reaction that involves the formation of an activated carbonyl intermediate (enzyme-substrate complex) with the ethanolamine moiety of the preassembled GPI unit serving as a nucleophile. Other nucleophilic acceptors like hydrazine (HDZ) and hydroxylamine have been shown to be possible alternate substrates for GPI. Since GPI has yet to be purified, the use of readily available nucleophilic substitutes such as HDZ and hydroxylamine is a viable alternative to study COOH-terminal processing by the putative transamidase. As a first step in developing a soluble system to study this process, we have examined the amino acid requirements at the COOH terminus for the transamidation reaction using HDZ as the nucleophilic acceptor instead of GPI. The hydrazide-forming reaction shows identical amino acid requirement profiles to that of GPI anchor addition. Additionally, we have studied other parameters relating to the kinetics of the transamidation reaction in the context of rough microsomal membranes. The findings with HDZ provide further evidence for the transamidase nature of the enzyme and also provide a starting point for development of a soluble assay.

A defect in glycosylphosphatidylinositol (GPI) transamidase activity in mutant K cells is responsible for their inability to display GPI surface proteins.

The final step in the pathway that provides for glycosylphosphatidylinositol (GPI) anchoring of cell-surface proteins occurs in the lumen of the endoplasmic reticulum and consists of a transamidation reaction in which fully assembled GPI anchor donors are substituted for specific COOH-terminal signal peptide sequences contained in nascent polypeptides. In previous studies we described a human K562 cell mutant line, designated class K, which assembles all the known intermediates of the GPI pathway but fails to display GPI-anchored proteins on its surface membrane. In the present study, we used mRNA encoding miniPLAP, a truncated form of placental alkaline phosphatase (PLAP), in in vitro assays with rough microsomal membranes (RM) of mutant K cells to further characterize the biosynthetic defect in this line. We found that RM from mutant K cells supported NH2-terminal processing of the nascent translational product, preprominiPLAP, but failed to show any detectable COOH-terminal processing of the resulting prominiPLAP to GPI-anchored miniPLAP. Proteinase K protection assays verified that NH2-terminal processed prominiPLAP was appropriately translocated into the endoplasmic reticulum lumen. The addition of hydrazine or hydroxylamine, which can substitute for GPI donors, to RM from wild-type or mutant cells defective in various intermediate biosynthetic steps in the GPI pathway produced large amounts of the hydrazide or hydroxamate of miniPLAP. In contrast, the addition of these nucleophiles to RM of class K cells yielded neither of these products. These data, taken together, lead us to conclude that mutant K cells are defective in part of the GPI transamidase machinery.

An active carbonyl formed during glycosylphosphatidylinositol addition to a protein is evidence of catalysis by a transamidase.

Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumable the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.

Cleavage without anchor addition accompanies the processing of a nascent protein to its glycosylphosphatidylinositol-anchored form.

Rough microsomal membranes from most mammalian cells, in the presence of a translation system, process nascent proteins with appropriate COOH-terminal signal peptides to their mature glycosylphosphatidylinositol (GPI)-linked forms. The present study, using preprominiplacental alkaline phosphatase as substrate, shows that as much as 10% of the mature product is cleaved correctly but is not linked to GPI. Some of the factors that influence the relative proportions of GPI linked to free mini-placental alkaline phosphatase are the amounts of GPI in the cells and the amino acid substituent at the omega site of the nascent protein. A mechanism for explaining cleavage both with and without GPI addition is presented, which supports a transamidase type of enzyme as the catalyst.

Characterization of the promoter region of the gene for the rat neutral and basic amino acid transporter and chromosomal localization of the human gene.

The promoter region of the rat kidney neutral and basic amino acid transporter (NBAT) gene has been isolated and sequenced. The major transcription initiation site was mapped by primer extension. The entire promoter region and a set of 5' deletions within it were expressed at a high level in LLC-PK1 cells using the luciferase indicator gene. Positive and negative regulatory elements in the promoter region were observed. A human genomic clone of the transporter was also obtained and was used to localize the NBAT gene at the p21 region of chromosome 2.

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment.

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell-free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.

Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide.

Secreted proteins are processed from a nascent form that contains an NH2-terminal signal peptide. During processing, the latter is cleaved by a specific NH2-terminal signal peptidase. The nascent form of phosphatidylinositol glycan (PI-G) tailed proteins contain both an NH2- and a COOH-terminal signal peptide. The two signal peptides have much in common, such as size and hydrophobicity. The COOH-terminal peptide is also cleaved during processing. We propose that the amino acid in a nascent protein that ultimately combines with the PI-G moiety be designated the omega site. Amino acids adjacent and COOH-terminal to the omega site would then be omega + 1, omega + 2, etc. In previous studies, we showed that allowable substitutions at the omega site of an engineered form of placental alkaline phosphatase (miniPLAP) are limited to 6 small amino acids. In the present study, mutations were made at the omega + 1 and omega + 2 sites. At the omega + 1 site, processing to varying degrees was observed with 8 of the 9 amino acids substituted for alanine, the normal constituent. Only the proline mutant showed no processing. By contrast, the only substituents permitted at the omega + 2 site were glycine and alanine, with only trace activity observed with serine and cysteine. Thus, just as there is a -1, -3 rule for predicting cleavage by NH2-terminal signal peptidase, there appears to be a comparable omega, omega + 2 rule for predicting cleavage/PI-G addition by COOH-terminal signal transamidase.

Selectivity at the cleavage/attachment site of phosphatidylinositol-glycan anchored membrane proteins is enzymatically determined.

Nascent precursors of phosphatidylinositol-glycan (PI-G)-linked membrane proteins contain a hydrophobic COOH-terminal sequence of 15-30 residues that is eliminated during processing to yield a newly exposed COOH terminus to which the PI-G moiety is added. There is no consensus as to the primary structure of the terminal peptide but there is a specific requirement for the amino acid destined to become the COOH terminus. In nascent human placental alkaline phosphatase (PLAP), the PI-G tail is attached to Asp-484. Site-directed mutants with glycine, alanine, cysteine, serine, or asparagine (category I) at residue 484 become PI-G tailed, appear in the plasma membrane, and are enzymatically active when expressed in COS cells. Although mutants with glutamic acid, glutamine, proline, tryptophan, leucine, valine, phenylalanine, threonine, methionine, and tyrosine (category II) are expressed equally well, only small amounts appear on the plasma membrane. Furthermore, they are not PI-G tailed and have little alkaline phosphatase activity. Studies with truncated PLAP-489 rule out nonspecific conformational changes in category II mutant proteins as a reason for their failure to be processed in COS cells and point to a specific COOH-terminal processing enzyme. Direct evidence that the selectivity for category I amino acids is enzymatically determined was obtained in a cell-free translation/processing system by using rabbit reticulocyte lysate and CHO cell rough microsomal membranes. In this in vitro system, both category I and category II mutants of PLAP-513 were translated, glycosylated, and cleaved by NH2-terminal signal peptidase. However, an additional and selective cleavage at residue 484 was observed only with category I mutants.

Selectivity of the cleavage/attachment site of phosphatidylinositol-glycan-anchored membrane proteins determined by site-specific mutagenesis at Asp-484 of placental alkaline phosphatase.

Many proteins are now known to be anchored to the plasma membrane by a phosphatidylinositol-glycan (PI-G) moiety that is attached to their COOH termini. Placental alkaline phosphatase (PLAP) has been used as a model for investigating mechanisms involved in the COOH-terminal processing of PI-G-tailed proteins. The COOH-terminal domain of pre-pro-PLAP provides a signal for processing during which a largely hydrophobic 29-residue COOH-terminal peptide is removed, and the PI-G moiety is added to the newly exposed Asp-484 terminus. This cleavage/attachment site was subjected to an almost saturation mutagenesis, and the enzymatic activities, COOH-terminal processing, and cellular localizations of the various mutant PLAP forms were determined. Substitution of Asp-484 by glycine, alanine, cysteine, asparagine, or serine (category I) resulted in PI-G-tailed and enzymatically active proteins. However, not all category I mutant proteins were PI-G tailed to the same extent. Pre-pro-PLAP with other substituents at position 484 (threonine, proline, methionine, valine, leucine, tyrosine, tryptophan, lysine, glutamic acid, and glutamine; category II) were expressed, as well as the category I amino acids, but there was little or no processing to the PI-G-tailed form, and this latter group exhibited very low enzyme activity. The bulk of the PLAP protein produced by category II mutants and some produced by category I mutants were sequestered within the cell, apparently in the endoplasmic reticulum (ER). Most likely, certain amino acids at residue 484 are preferred because they yield better substrates for the putative "transamidating" enzyme. In transfected COS cells, at least, posttranslational PI-G-tail processing does not go to completion even for preferred substrates. Apparently PI-G tailing is a requisite for transport from the ER and for PLAP enzyme activity. Proteins that are not transamidated are apparently retained in the ER in an inactive conformation.

Scintillation proximity radioimmunoassay utilizing 125I-labeled ligands.

A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an 125I-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the 125I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications of the principle to study the kinetics of interaction between receptors and ligands are discussed.

Specific polyclonal antibodies to the carboxyl terminus of [Met]enkephalin-Arg6-Gly7-Leu8.

The octapeptide Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu was recently isolated from bovine adrenal chromaffin granules and serves as a marker for proenkephalin from which it is derived. Polyclonal antisera which are highly specific for the carboxyl terminus have been raised against the synthetic peptide. The only significant cross-reactivity was with the 18.2-k Da and 5.3-k Da enkephalin-containing peptides (EC peptides) which contain the octapeptide at their carboxyl termini and the [des-Tyr] and [des-Tyr-Gly] congeners of the octapeptide. Extracts of bovine adrenal medulla and rat spinal cord were shown to contain significant amounts of the octapeptide, the two larger EC peptides, and the two smaller congeners.

Marked increases in large enkephalin-containing polypeptides in the rat adrenal gland following denervation.

When rat adrenal glands are denervated, large increases in the amounts of enkephalin and enkephalin-containing polypeptides appear. In the normal gland, only trace amounts occur. One of the larger polypeptides (approximately 22,000 daltons) increases rapidly and by 48 hr following denervation, attains 20 times its original level. At this time, the levels of free enkephalins are essentially unchanged. By 96 hr, the 22,000-dalton polypeptide begins to decrease as free enkephalins and intermediate-sized enkephalin-containing polypeptides increase. This series of events is consistent with a precursor (22,000-dalton polypeptide)/product (enkephalin) relationship.

Isolation of the opioid heptapeptide Met-enkephalin [Arg6,Phe7] from bovine adrenal medullary granules and striatum.

Bovine adrenal chromaffin granules have been shown to contain, in addition to Met-enkephalin and Leu-enkephalin, at least three small peptides with opiate receptor activity. One of these adrenal peptides has been purified to homogeneity and its sequence was shown to be Met-enkephalin-[Arg6,Phe&]. This heptapeptide was also found in beef striatal extracts in amounts comparable to those of Leu-enkephalin.

Purification to homogeneity of camel pituitary pro-opiocortin, the common precursor of opioid peptides and corticotropin.

Pro-opiocortin was purified from camel pituitaries by procedures including high-performance liquid chromatography. The precursor relationship of the pure protein to the opioid peptides and to corticotropin was confirmed. Partial chemical analysis consisting of amino acid analysis and tryptic peptide mapping was carried out with the aid of sensitive fluorescence detection.

On betaH-Leu5-endorphin and schizophrenia.

A previously unknown peptide, betaH-Leu5-endorphin, has been reported in the dialysates of schizophrenic patients. Accordingly, hemofiltrates from two schizophrenic and two control patients were examined for the presence of betaH-Leu5-endorphin. The opioid peptides were detected by a radioreceptor assay after separation and identification by gel filtration and high-performance liquid chromatography. With a detection limit of 30 pmole/L of hemofiltrate, no betaH-Leu5-endorphin or Met5-endorphin was found in controls or in patients. Whatever the possible involvement of endorphins in schizophrenic behavior, they are not present at detectable levels in the hemofiltrates of two well-characterized schizophrenic patients, thereby casting doubt on a general relationship of Leu-endorphin and schizophrenia.

High molecular weight opioid-containing proteins in striatum.

Striatal extracts of guinea pigs, rats, and cattle were found to contain two large proteins (greater than 40,000 and greater than 100,000 daltons) that on treatment with trypsin yield opioid peptides differing chromatographically from the opioid nonapeptide generated by trypsin digestion of endorphins, beta-lipotropin, or pro-opiocortin. Furthermore, the large opioid proteins found in the pituitary do not appear to be present in the striatum. These and other findings indicate that the striatal enkephalins are produced via a pathway from the one deduced from studies on the pituitary.