Resorption-cycle-dependent polarization of mRNAs for different subunits of V-ATPase in bone-resorbing osteoclasts.

Protein sorting in eukaryotic cells is mainly done by specific targeting of polypeptides. The present evidence from oocytes, neurons, and some other polarized cells suggests that protein sorting can be further facilitated by concentrating mRNAs to their corresponding subcellular areas. However, very little is known about the mechanism(s) involved in mRNA targeting, or how widespread and dynamic such mRNA sorting might be. In this study, we have used an in vitro cell culture system, where large multinucleated osteoclasts undergo continuous structural and functional changes from polarized (resorbing) to a nonpolarized (resting) stage. We demonstrate here, using a nonradioactive in situ hybridization technique and confocal microscopy, that mRNAs for several vacuolar H(+)-ATPase subunits change their localization and polarity in osteoclasts according to the resorption cycle, whereas mRNA for cytoplasmic carbonic anhydrase II is found diffusely located throughout the osteoclast during the whole resorption cycle. Antisense RNA against the 16-kDa or 60-kDa V-ATPase subunit inhibits polarization of the osteoclasts, as determined by cytoskeleton staining. Antisense RNA against carbonic anhydrase II, however, has no such effect.

Dermal patch graft correction of severe chordee secondary to penile corporal body disproportion without urethral division in boys without hypospadias.

Historically, significant ventral penile curvature secondary to corporal body disproportion has been corrected either by dorsal plication or division of the urethral plate. In the rare situations where there is severe chordee in the face of an intact urethra with an orthotopic meatus, division of the urethral plate is commonly performed at the time of grafting the ventral defect created by incising the tunica albuginea. Subsequently, a staged procedure is necessary to reconnect the urethra at a later date. Herein the authors present a novel technique that shows it is possible to perform successful dermal patch orthoplasty without division of the urethra in patients with a normal orthotopic meatus and urethra via urethral mobilization. Three patients over the past 3 years with severe ventral chordee, orthotopic meati and normal urethral anatomy presented for correction. Two patients were 18 years old and one was 10 years old. All three boys were circumcised. The two older boys insisted on dorsal plication as a first approach which worked only temporarily for about 6 months while the younger boy had no prior surgery performed. Each boy underwent a circumcising incision, degloving of the shaft skin, extensive urethral mobilization and dermal patch graft orthoplasty to correct chordee. All surgeries were performed in an outpatient setting. No urinary drainage was used in any patient and a simple bio-occlusive dressing was employed in each case. Follow-up ranged from 11 months to 2 years (mean 1.5 years). All three boys have strong straight erections, full well directed urinary streams and no complications noted to date. Our conclusion based on this experience is that extensive urethral mobilization can allow for correction of severe ventral chordee without urethral division in a single operative setting in boys without hypospadias and a normal urethra. The accompanying movie herein describes the surgical technique.

An immunoreactive 8-azido ATP-labeled protein common to the lysosomal and chromaffin granule membrane.

The H+-ATPases of eukaryotic cell organelles, including the rat liver lysosomal (tritosomal) H+-ATPase and the bovine chromaffin granule ATPase, exhibit similarities in function, substrate requirements, and inhibitor responses. We have explored the possibility that these pumps also exhibit immunological similarities, and that common determinants may be present on polypeptides important to function, such as ATP binding. Toward this end, antibodies were produced in rabbits against a highly purified, detergent-solubilized and fractionated chromaffin granule proton pump preparation. This antibody reacted with a 70-80 kDa protein of the lysosomal membrane on Western blots. We have previously shown that photolysis with 8-azido-ATP inhibits lysosomal N-ethylmaleimide-sensitive, vanadate-, ouabain- and oligomycin-insensitive ATP hydrolysis and H+ transport, with concomitant labeling of a 70-80 kDa membrane protein, amongst others. Here, we report that the photolysis with 8-azido-ATP also leads to inhibition of chromaffin granule H+ pump function and pump-related ATP hydrolysis, with concomitant N-ethylmaleimide-sensitive, ATP-protectable, 8-azido-[alpha-32P]ATP labeling. The anti-chromaffin granule antibody reacts with an approx. 70 kDa protein of the chromaffin granule and the lysosome. This raises the possibility that the 70 kDa 8-azido-ATP-reactive, immunologically similar proteins may play a similar role in pump function such as ATP binding and/or hydrolysis in these organelles.

Saccharomyces cerevisiae expression of exogenous vacuolar ATPase subunits B.

The precise function of subunit B of the vacuolar H(+)-ATPase class is unknown, but it is essential for proton pumping. We have previously reported the DNA sequence and predicted protein sequence of the vacuolar ATPase subunit B for Candida tropicalis (Gu, H.H., Gallagher, M.J., Rupkey, S. and Dean, G.E. (1990) Nucleic Acids Res. 18, 7446). When the Candida gene was expressed in a Saccharomyce cerevisiae delta vat2 mutant from which the homologous gene had been deleted, viability and vacuolar acidification was restored to apparently wild-type levels. The predicted identity between these two proteins is 90%. We have searched for vacuolar ATPase subunits B from other species that might show a difference in function, when expressed in yeast, relative to the endogenous gene. We have cloned an apparently full-length 1.8-kb bovine subunit B cDNA from adrenal medulla that is about 1 kb shorter than the previously reported bovine brain cDNA (Puopolo, K., Kumamoto, C., Adachi, I., Magner, R. and Forgac, M. (1992) J. Biol. Chem. 267, 3696-3706; Nelson, R.D., Guo, X.L., Masood, K., Brown, D., Kalkbrenner, M. and Gluck, S. (1992) Proc. Natl. Acad. Sci. USA 89, 3541-3545), but nearly identical throughout the coding nucleotide and protein sequences; it is only 74% identical to the Saccharomyces subunit B protein sequence. Upon expression of this cDNA in two different delta vat2 deletion strains, the bovine cDNA restored function only partially, as judged by both viability at high pH and vacuolar acidification. Current work is aimed at determining which regions of the bovine protein require alteration in order to fully restore the delta vat2 strain to wild-type acidification, with the eventual goal of identifying interactive residues between subunit B and other proteins required for pump function.

An institutional commitment to pain management.

PURPOSE: To share the development, implementation, and evaluation of a program called "An Institutional Commitment to Pain Management," which is based on the philosophy of organizational influence on pain management. METHODS: A tested pain education model was disseminated to 32 physician/nurse teams in settings throughout California, after which the 64 professionals returned to their institutions to serve as role models and catalysts to change the practice of pain management. Each team member completed a 39-item survey about knowledge and attitudes related to pain, which was developed by B.R.F. and colleagues, and also identified three goals for the implementation of course information. Precourse data also included administration of the knowledge and attitudes survey to participating physicians' and nurses' colleagues (10 physicians and 20 nurses per institution). Each team completed five chart audits using the pain audit tool (PAT), which was developed by B.R.F. and colleagues at the City of Hope National Medical Center. The PAT identifies how pain is managed currently at the institutional level. Final course evaluation 8 months after course completion included a summary of activities implemented by the teams as well as the factors that served as barriers and benefits to improve the quality of pain management. RESULTS: Two hundred seventy-two physicians and 629 nurses completed the survey about knowledge and attitudes related to pain, and 154 PATs were submitted. These results, as well as evaluation at the completion of the course, are discussed. CONCLUSION: The Institutional Commitment to Pain Management program is an evolving model that was developed to overcome barriers to pain relief by obtaining the commitment from institutions to improve the management of pain for their patients.

Active specific immunotherapy of metastatic melanoma with an antiidiotype vaccine: a phase I/II trial of I-Mel-2 plus SAF-m.

PURPOSE: To determine the toxicity and immunologic activity of an antiidiotype melanoma vaccine that consists of monoclonal antibody I-Mel-2 (MELIMMUNE-2, IDEC Pharmaceuticals, La Jolla, CA) and an immunologic adjuvant SAF-m. PATIENTS AND METHODS: Twenty-six patients with metastatic melanoma, 17 of whom had previously received chemotherapy, were given 2 mg of I-Mel-2 and either 100 micrograms (n = 6) or 250 micrograms (n = 20) of SAF-m. Antiidiotype vaccine was given intramuscularly (IM) biweekly for 4 weeks, and then bimonthly until disease progression. Human antimurine antibodies (HAMA), anti-I-Mel-2 antibodies, and specific antibody (Ab)3 against the melanoma epitope mimicked by the vaccine were titrated before treatment, biweekly from weeks 4 to 12, and every 4 to 8 weeks thereafter. Computed tomographic (CT) scans of the chest, abdomen, and pelvis and magnetic resonance imaging (MRI) of the brain were obtained before and bimonthly during treatment to evaluate responses. RESULTS: Elevated titers of human antimouse antibodies and anti-I-Mel-2 antibodies were associated with clinical antitumor effect (P = .02 and P = .05, respectively). Ab3 was absent in most patients, but was found in the best clinical responder. Fever, myalgias/arthralgias, fatigue, nausea, and headaches were the most common toxicities. Grade III myalgias/arthralgias and headaches required dose reduction of SAF-m in eight patients at the 250-microgram dose. No treatment-related death occurred. Six patients had an antitumor effect: one complete response in liver and lung, two minor responses, and three stable disease. The patient with a complete response has survived nearly 5 years. CONCLUSION: I-Mel-2 antiidiotype vaccine was safe, tolerated best at the 100-microgram dose of SAF-m, and had immunologic and clinical activity.

Cloning and sequence analysis of an H(+)-ATPase-encoding gene from the human dimorphic pathogen Histoplasma capsulatum.

A gene related to the PMA1 gene from Saccharomyces cerevisiae was isolated from the pathogenic human dimorphic fungus, Histoplasma capsulatum, using fungal-specific oligodeoxyribonucleotide (oligo) probes. This gene has been given the name Hc-PMA1. The structural organization of Hc-PMA1 consists of three exons (375, 2329 and 44 bp) and two introns (115 and 116 bp). The nucleotide sequence predicts an H(+)-ATPase-related protein of 916 amino acids (aa). Comparison of the deduced aa sequence to that of Neurospora crassa and S. cerevisiae (PMA1) plasma membrane H(+)-ATPases showed a greater similarity to that from N. crassa (85% identity). Furthermore, the two introns in the Hc-PMA1 gene interrupt the coding region in the precise locations determined for two of the four N. crassa Nc-PMA introns. H. capsulatum intron 1 contains two repeat motifs, d(TA)16 and d(TG)10, each potentially capable of forming non-B DNA structures. Northern analysis of H. capsulatum total RNA indicated that the Hc-PMA1-specific mRNA is approx. 3.3 kb in size, in agreement with the predicted size of the gene.

Structure and expression of subunit A from the bovine chromaffin cell vacuolar ATPase.

Subunit A of the vacuolar H(+)-ATPase class is thought to be responsible for the ATP hydrolysis which drives proton-pumping. We report here the cloning and sequence determination of the first mammalian cDNA encoding a bovine vacuolar ATPase subunit A from an adrenal medulla cDNA library. Northern blots of bovine adrenal medulla RNA reveal a message of approximately 3.8 kb. The predicted peptide sequence, consisting of 618 amino acids with a calculated molecular weight of 68397 daltons, is similar to the sequences of the three known subunit A proteins. beta-Galactosidase-subunit A fusion proteins were immuno-decorated by an antiserum raised to the subunit A protein from corn coleoptile vacuoles.

Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules.

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b-VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N-terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin > dopamine = norepinephrine > epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N-terminus sequence of the purified vesicular amine transporter [Stern-Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730-9733].

Complications of urinary tract reconstruction.

Urinary tract reconstruction has benefited a vast number of patients with dysfunctional lower urinary tracts caused by congenital abnormalities, previous surgery, or both. Reconstructive efforts have been innovative and continue to evolve. With this evolution, new complications continue to appear, and in order to minimize the risk to the patient, we must recognize our previous lessons. Appropriate patient selection is essential in achieving a successful outcome in this group. The patient's neurologic status, urologic anatomy, renal function, and motivation are also important factors in choosing the appropriate patient and correct surgical approach. Close follow-up remains the single most important element in assuring long-term well-being for most of these patients.

Identification of microtubule-associated protein tau isoforms in Alzheimer's paired helical filaments.

AD66 proteins derived from sodium dodecylsulfate (SDS) insoluble paired helical filaments (PHF) were isolated from Alzheimer's brain using a purification procedure developed previously in this laboratory, and characterized by immunologic and chemical cleavage methods. AD66 proteins were immunoreactive with antibodies that recognize the amino terminal, tubulin-binding, and carboxy terminal domains of microtubule-associated protein tau indicating the presence of the entire tau sequence in AD66 proteins. These proteins were reactive with antibody 423 that binds to PHF but not human adult tau. Immunologic and chemical cleavage studies indicated that only two of the six tau isoforms were present in these proteins. AD66 proteins were comprised of tau proteins containing only three tubulin binding domains with either a 29 amino acid insert or no amino terminal insert. For comparative purposes, SDS soluble PHF-tau (A68 proteins) was purified from Alzheimer's brains and normal adult tau purified from control brains. Antibody Alz-50 was immunoreactive with PHF-tau or normal tau regardless of alkaline phosphatase treatment while immunoreactivity was only observed with dephosphorylated AD66 proteins. A second phosphorylated epitope on AD66 proteins but not PHF-tau or normal tau proteins was demonstrated with antibody PHF9. These data suggest that AD66 proteins represent a more phosphorylated form of tau than PHF-tau or normal tau proteins. Two-dimensional gel electrophoresis demonstrated that AD66 proteins have higher apparent molecular weights and lower pI values than normal tau, differences possibly due to the greater phosphorylation observed in these proteins.

Alzheimer's paired helical filaments: amyloid precursor protein epitope mapping.

Paired helical filaments (PHF) were electrophoretically purified and solubilized from Alzheimer's neurofibrillary tangles and consisted of a primary 66 kDa protein on SDS-PAGE analysis. A panel of antibodies raised against restricted regions of the beta-amyloid precursor protein (APP) were employed for epitope mapping studies of this 66 kDa PHF protein. Western blot studies revealed that C-terminal APP antibodies were immunoreactive with the 66 kDa PHF protein. Further analysis revealed that only antisera raised against peptides that include the beta/A4-amyloid region within the C-terminal portion of APP were immunoreactive with PHF proteins. These data complement previous immunocytochemical studies which indicated that C-terminal APP antibodies preferentially label PHF-containing neurofibrillary tangles in Alzheimer's brain. The present data suggest a similarity of secondary or tertiary structure between beta/A4-amyloid and PHF which accounts for the cross-reactivity of beta/A4-amyloid antibodies with PHF proteins.

Predicting the leakage performance of small bodyworn disposable incontinence pads using laboratory tests.

An international multi-centre project has been run to create an international standard for measuring the leakage performance of small, disposable incontinence pads for lightly incontinent women. One hundred and thirteen women tested batches of nine different incontinence pads of widely differing designs and noted the severity with which each individual used pad had leaked so that leakage performance could be determined as a function of urine weight. In addition, testers rated the overall leakage performance of each of the nine products on a five-point scale. These clinical data were compared with laboratory data from 153 different pad measurements, each of which was evaluated by seeing how well the data it yielded correlated with the clinical test data. A wetback test emerged as the clear winner. It usually predicted the clinical leakage performance of pads to an accuracy of +/- 10%. It involved applying 25 ml of 1% w/v saline to a pad and measuring how much escaped into a filter paper held against the wet pad for 1 min under a pressure of 1.5 kPa. Pads which released the least test fluid into the filter paper leaked least in the user tests. The method will be published as an ISO standard during 1997.

Disposable bedpads for incontinence: predicting their clinical leakage properties using laboratory tests.

A multi-centre project has been run to identify laboratory tests capable of predicting the leakage performance of disposable incontinence bedpads. Each of 95 subjects tested each of six products for a week in turn and reported whether or not they and/or their carers found the leakage performance of each product acceptable. In addition, carers noted the severity with which individual used bedpads had leaked so that, when they had been weighed, their leakage performance could be determined as a function of urine weight. These clinical data were compared with results from the 16 different laboratory tests used routinely for bedpad evaluation in three hospital laboratories. Each test was evaluated by seeing how well the data it yielded correlated with the clinical test data. No individual test was very successful at predicting the performance of bedpads when used as sole protection but a combination of an absorption capacity test and an absorption time test predicted the percentage of users/carers finding leakage performance acceptable, accurate to within +/- eight percentage points for all six test products. A different absorption capacity test proved most successful for bedpads used as back-up to body-worn products. It predicted the percentage of users/carers finding leakage performance acceptable, accurate to +/- five percentage points for all six products.

Ethical issues in pain management at home.

This review of several studies from the authors' program of research supports the notion that the experience of pain, particularly in the home, has a profound effect not only on the patient, but on family and health care providers as well. Family caregivers revealed their private grief and burden when describing the physical suffering they witnessed. They faced the daily responsibility of making decisions regarding a loved one's pain, and the decisions often resulted in ethical conflicts. Caregivers generally felt ill-prepared for the administration of pain medication. They struggled to balance the need for medications to relieve pain against the perceived unwanted side effects and fear of addiction. These studies also demonstrate that a concerted effort must be made by health care providers to support the pain management role of family caregivers. By listening, being available, making pain management a priority, and reinforcing appropriate pain management principles, health care professionals help sustain family caregivers in carrying out their anguishing and unrelenting task.

Role of registered nurses in error prevention, discovery and correction.

BACKGROUND: Registered nurses have a vital role in discovering and correcting medical error. OBJECTIVE: To describe the type and frequency of errors detected by American critical care nurses, and to ascertain who made the errors discovered by study participants. METHODS: Daily logbooks were used to collect information about errors discovered by a random sample of 502 critical care nurses during a 28-day period. RESULTS: Although the majority of errors discovered and corrected by critical care nurses involved medications (163/367), procedural errors were common (n = 115). Charting and transcription errors were less frequently discovered. The errors discovered by participants were attributed to a wide variety of staff members including nurses, doctors, pharmacists, technicians and unit secretaries. CONCLUSIONS: Given the importance of nurses in maintaining patient safety, future studies should identify factors that enhance their effectiveness to prevent, intercept and correct healthcare errors.

Vaginal replacement in children.

Between 1980 and 1990, 17 patients underwent total vaginal replacement at our hospital. The majority of these patients presented with müllerian failure or gender reassignment for intersex. Colon vaginal replacement was done in 15 patients and small bowel was used in 2. Complications included prolapse in 2 patients and stenosis in 2. Of the 17 patients 4 are married, 10 are sexually active, only 1 reports dyspareunia and 1 requires home self-dilation.

Monoclonal antibody PHF-9 recognizes phosphorylated serine 404 of tau protein and labels paired helical filaments.

Paired helical filaments (PHFs) purified from alzheimer's brain consist of hyperphosphorylated microtubule-associated protein tau. In PHF, phosphorylation occurs at ser/thr tau residues. Several of these ser/thr phosphorylation sites lie immediately C-terminal to the tau tubulin binding domain. The C-terminal ser396 to thr413 tau region contains two or more phosphorylated residues and eight possible ser/thr phosphorylation sites. Immunologic studies and mass spectroscopy have identified ser396 as one of the phosphorylation sites but identification of more C-terminal phosphorylated residues has been hampered by the lack of monoclonal antibodies (Mabs) that recognize defined epitopes in this region. We have raised Mabs against PHF purified from Alzheimer's brain. One of these Mabs, PHF-9, showed phosphorylation-dependent binding to purified PHF and recognized a phosphorylated epitope in the C-terminal portion of cyanogen bromide-digested PHF. Epitope mapping studies employing synthetic tau phosphopeptides indicated that PHF-9 labeled a 13-mer tau peptide phosphorylated at ser404 but not the corresponding non-phosphorylated peptide. PHF-9 demonstrated no immunoreactivity with a synthetic peptide phosphorylated at ser396 indicating that the PHF-9 epitope is C-terminal to ser396. In conclusion, the present study describes a Mab, PHF-9, which recognizes phosphorylated ser404 of tau independently of phosphorylated ser396 and indicates that tau ser404 is phosphorylated in PHF.