[Lichen ruber hypertrophicus with verrucous carcinoma]. A 51-year-old patient presented with aggregated papules and with a central, partly erosive plaque on the left lower leg. Histological examination revealed hypertrophic lichen planus peripherally and a verrucous carcinoma within the centre. The lesion was removed surgically. Shortly after wards an atrophic lichen planus developed exclusively within the skin graft, which was effectively treated with topical steroid cream. The patient remained disease-free for 2 years. The literature relating to malignant transformation of cutaneous lichen planus was reviewed. In most cases, as in the present one, the hypertrophic form of lichen planus and the verrucous type of carcinoma (Ackerman) are reported. Recent investigations have revealed human papilloma virus types 6 and 11 within such lesions.


[Hypertrichosis lanuginosa acquisita in ulcerative colitis with colon cancer]. Hypertrichosis lanuginosa acquisita is regarded as an obligatory cutaneous paraneoplasm and is defined as the sudden and excessive appearance of lanugo hairs on the entire integument associated with malignant neoplasm of internal organs. We observed such a case of hypertrichosis in a 30-year-old man with a long history of ulcerative colitis who developed carcinoma of the caecum with lymph node metastasis.


[Cutis marmorata teleangiectatica congenita (Van Lohuizen syndrome)]. The 2-year spontaneous course of a case of cutis marmorata teleangiectatica congenita in a girl is reported. The child had typical cutis marmorata, with teleangiectasis, phlebectasias and ulceration particularly on parts of the left leg and in the anogenital region, with no further anomalies. During symptomatic therapy over 2 years the lesions faded spontaneously.


Fibrous dysplasia.

Fibrous dysplasia. The controversy is this case centers around the management of this lesion, not the differential diagnosis. All the consultants agreed that the history, physical, and CT findings were consistent with various fibroosseous lesions, the most likely being fibrous dysplasia. The need for additional tests varied with a bone scan (Dr. Kearns), a bone scan and CT scan (Dr. McGill), and MRI, MRI angiogram, bone survey, BUN, creatinine, calcium, and phosphorus (Dr. Potsic). Cosmetic and functional changes were considered priorities for the consultants, with orbital compression, malocclusion, tooth eruption, nasal obstruction, and sinusitis (Dr. Kearns), nasolacrimal duct obstruction and orbital compression (Drs. McGill and Potsic) being the concerns. Because this lesion is benign and slow-growing, the consensus is that surgery should be reserved for functional or cosmetic compromise. But how aggressive should one be and what approach should be used? The approaches varied with midface degloving or lateral rhinotomy (Dr. Kearns), midface degloving (Dr. McGill), or a Caldwell-luc and lateral rhinotomy (Dr. Potsic). Assessment of this tumor postoperatively should be with patient examinations and serial CT scans. None of the consultants worried about sarcomatous changes in this tumor.


Primary prevention of haemophilus influenzae type b. Routine immunisation of infants against Haemophilus influenzae type b (Hib) has the potential to prevent up to 1200 cases of serious infection and 60 deaths every year in the United Kingdom. Sara Hodgson reports the findings of a study in the Oxford region to determine the safety and immunogenicity of the Hib conjugate vaccine PRP-T when given to British infants in concurrence with primary immunisations, according to the accelerated schedule of two, three and four months of age.


HIB. Educating parents and professionals. A totally new immunisation (Hib) offers protection against the commonest cause of bacterial meningitis in the under fives. Judith Moreton describes the development and implementation of health education programmes in Oxfordshire to achieve a 90 per cent take-up in the first year, even among traveller gypsies, who are recognised as generally having poor immunisation uptake.


HIB. Assessing information needs.

HIB. Assessing information needs. Planning the launch of the new Haemophilus influenzae b vaccine in October of this year has challenged health educators involved with child immunisation, writes Christine McGuire of the Health Education Authority (HEA). Here she outlines the findings from initial research commissioned to explore parental and professional information needs.


Marketing: Part 1 of 4. Promoting the health visiting profession. Despite its long history, health visiting still suffers a low public and professional profile; few people know what health visitors do. In the first of a four-part series LIZ DAY argues that health visitors should adopt commercial marketing strategies to 'sell' their services to the public, to NHS managers and to primary health care colleagues. She suggests ways to adapt these commercial sales techniques to an NHS context.


Continuing your education ... with CATS. MIKE McMANUS and ANNE DEAN discuss the credit accumulation and transfer scheme (CATS) and consider many of the common questions regarding the system: how to accredit current professional qualifications; use of credited learning, and potential continuing education pathways open to health visitors. Finally, the authors discuss the validity of such awards.


Are groups for parents of children with sleep problems effective? Sleep groups are increasingly being used to support parents who have children with sleep problems. JANINA SZYNDLER and GEORGINA BELL present a model of sleep groups developed by clinical psychologists in Kingston and Esher and examine the effectiveness of this approach.


White Paper. I. Crossing the health divide. The health of the nation white paper has been widely welcomed as a clear shift in NHS policy towards health promotion. But will the strategy achieve real improvements in the health of those who need help most? What part can health visitors and school nurses expect to play?


GP fundholders. Guidance a 'mixed blessing'. The Government has taken further steps along the road of making GP fundholders the main gatekeepers of primary care with guidance on how to 'buy in' health visiting and other community services from units or trusts.


Comparing need with resource allocation. In a study of health visitor caseloads in Bristol and Weston health authority, 16,000 families were assessed against 26 criteria known by health visitors to affect long term health. Considerable variations were found in the long term risk to the health of individual families. Families with high levels of health risk were concentrated in the caseloads of some health visitors and in a few geographical localities. The results suggest that there is a serious misallocation of health visiting resources and that steps should be taken to adjust resources to reflect more closely the patterns of need.


Marketing: Part 2 of 4. Shaping and selling the health visiting image. Integral to the marketing process is the image projected to the consumer of the product s/he is being asked to buy. In the second of this series of articles on marketing health visiting, ANNE KELLY examines past and present images of health visiting and suggests ways to market a new image in keeping with clients' needs and health policies.


Home care nursing orientation model: content and strategies. Orientation for the home healthcare nurse consists of specific content areas that are organized into a comprehensive program. The teaching strategies used to deliver the content are just as important as the information included in the orientation program.


Comprehensive discharge planning: a hospital-home healthcare partnership. Shorter hospital stays place new demands on the discharge planning process. A research study that evaluated discharge planning outcomes reveals a critical need for comprehensive discharge planning involving both hospital and home healthcare agencies.


Deceptive documentation in home healthcare nursing. Home healthcare nurses are sometimes faced with a dilemma that is unique in nursing. Should they document client needs and health status so that reimbursement for nursing care is assured, even when the client does not meet all criteria for reimbursement? The discussion that follows examines this question by applying traditional principles of healthcare ethics to an individual case and offers one resolution to a dilemma that has not been addressed previously in the nursing literature.


When orientation is not enough: a group approach for new home healthcare nurses. New home healthcare nurses have learning needs that do not become evident until after formal orientation is completed. This agency designed a group support and education program to meet these needs.


The documentation dilemma: an integrated solution. Every home healthcare manager must contend with the demands of documentation. One approach to ensure quality and address staff concerns is presented.


A comparison of Foley catheter types in home care use. Quality assurance activities routinely track problem-prone areas of patient care. Foley catheter management may be such an area for many agencies.


Outcomes of care from the professional's perspective. Patient satisfaction is considered an important indicator of the quality of care provided by a home healthcare agency. Increased emphasis is now placed on patient outcomes of care by certification and accreditation standards. This detailed analysis of the outcomes of care by the patient, the family, and the professionals through the use of unsolicited patient letters of commendation and the clinical record did not reveal any specific pattern. The visiting nurse association's home care services are available on a nondiscriminatory basis. This fact is noted in the agency's literature and policy manual. The data analysis for this project supports this policy statement. Patients, family members, and a physician wrote letters of commendation regardless of the LOS, payer source, total charges, time spent with the patient, and personnel who provided the care.


Cochinmicins, novel and potent cyclodepsipeptide endothelin antagonists from a Microbispora sp. I. Production, isolation, and characterization. Cochinmicins I, II, III are novel peptolides produced in submerged-fermentation cultures of Microbispora sp. ATCC 55140. These closely related compounds are separated by HPLC and are novel competitive endothelin antagonists. Cochinmicins II and III are stereoisomeric to each other. Cochinmicin I is the deschloro analog of cochinmicin III.


Cochinmicins, novel and potent cyclodepsipeptide endothelin antagonists from a Microbispora sp. II. Structure determination. Cochinmicins I, II, and III are competitive endothelin antagonists produced by Microbispora sp. ATCC 55140. The cochinmicins are cyclic depsipeptides containing six alpha-amino acids and a pyrrolecarboxylic acid. Based upon MS, 1D and 2D NMR, and LC data, the structures and absolute stereochemistries of the cochinmicins have been assigned. All three components have the same basic sequence and contain one equivalent each of D-allo-threonine, D-alanine, L-phenylalanine, D-phenylalanine, 5-chloropyrrole-2-carboxylic acid (or pyrrole-2-carboxylic acid in cochinmicin I), plus two equivalents of 3,5-dihydroxyphenylglycine (DHPG). The phenylalanine residues were differentiated via a methanolysis product which contained only one of the phenylalanine residues. Both DHPG residues have the D configuration in the more active cochinmicins I and III. Cochinmicin II contains both D- and L-DHPG and these residues have been differentiated in the sequence based upon 1H NMR data.


Matlystatins, new inhibitors of typeIV collagenases from Actinomadura atramentaria. I. Taxonomy, fermentation, isolation, and physico-chemical properties of matlystatin-group compounds. During the course of a screening for inhibitors of typeIV collagenases, new metabolites, designated matlystatins, have been isolated from an actinomycete strain, which was identified as a strain of Actinomadura atramentaria. Matlystatins were composed of five congeners, which were separated and purified by n-butanol extraction and chromatography.


Antifungal anthracycline antibiotics, spartanamicins A and B from Micromonospora spp. Spartanamicins A and B, two antifungal antibiotics, were produced by a culture of Micromonospora spp. strain No. MSU-43097 (ATCC 53803), isolated from a potted soil containing asparagus (Asparagus officinalis L.) plants. The antibiotics were isolated from the mycelial cake using organic solvents. The structures of spartanamicins A and B were determined by spectral and chemical means. Spartanamicin B is more active as an antifungal compound than it's analogue, A. The minimum inhibitory concentration for spartanamicin B on Candida albicans and Aspergillus, Cladosporium, Cryptococcus, Rhodotorula and Staphylococcus spp. ranged from 0.2 to 1 microgram/ml. It was not active against Staphylococcus aureus, Escherichia coli and Citrobacter spp. but some strains of S. aureus were sensitive.


Epoxomicin, a new antitumor agent of microbial origin. An actinomycete strain No. Q996-17 produced a novel compound, epoxomicin, which exhibited in vivo antitumor activity against B16 melanoma. Structural studies indicated that it is a new member of the epoxy-beta-aminoketone group, and is closely related to eponemycin.


APHE-1 and APHE-2, two new antimicrobial and cytotoxic antibiotics. I. Taxonomy, fermentation, isolation and biological activity. APHE-1 and APHE-2 are two new antibiotics produced by Streptoverticillium griseocarneum NCIMB 40447. They exhibited a remarkable cytotoxic activity against several tumor cell lines from different origin. Furthermore, they showed weak antimicrobial activity against Gram-positive bacteria, filamentous fungi and yeasts.


APHE-1 and APHE-2, two new antimicrobial and cytotoxic antibiotics. II. Physico-chemical properties and structure elucidation. Two new pyrazolo-isoquinolinone antibiotics, APHE-1 and APHE-2, have been isolated from the culture filtrate and mycelia of Streptoverticillium griseocarneum NCIMB 40447. Molecular formulae were established as C13H12N2O for APHE-1 and C14H14N2O for APHE-2, by elemental analysis, NMR and mass spectra. 2D NMR techniques (1H-1H COSY-45 and 1H-13C correlated spectroscopy) have been applied to establish their structures.


Production of bellenamine and new metabolites in a synthetic medium. A streptomyces antibiotic, bellenamine has been produced in a simple synthetic medium consisting of D-galactose, dextrin, ammonium sulfate and calcium carbonate. Three new minor metabolites, N-(aminomethyl)succinamic acid, 1'-N-acetylbellenamine and D-beta-lysinamide have been isolated from the synthetic medium culture. They showed no antibiotic activity.


Biosynthesis of benastatin A.

Biosynthesis of benastatin A. The biosynthesis of benastatin A, produced by Streptomyces sp. MI384-DF12, has been studied by feeding experiments with 14C- and 13C-labeled compounds followed by measurement of radioactivity and 13C NMR analysis. The results indicate that benastatin A is derived from two methionine units and fourteen acetate units, condensed in the "head-to-tail" fashion of typical polyketide biosynthesis.


Isolation and identification of 3-propylidene-delta 1-pyrroline-5-carboxylic acid, a biosynthetic precursor of lincomycin. An accumulated lincomycin intermediate in UC 8292, a lincomycin nonproducing strain of Streptomyces lincolnensis, has been isolated and purified by employing an assay system based on complementation of UC 11066, another lincomycin nonproducing strain of S. lincolnensis. The structure of the purified intermediate is shown to be 3-propylidene-delta 1-pyrroline-5-carboxylic acid, or 1, 2, 3, 6-tetradehydro-propylproline by mass spectrometry and NMR spectroscopic studies. Based on the structure of this newly found intermediate, a biosynthetic pathway for propylproline is proposed as tyrosine-->L-3-hydroxytyrosine (Dopa)-->-->-->-->3-propylidene-delta 1-pyrroline-5-carboxylic acid-->3-propyl-delta 2-pyrroline-5-carboxylic acid-->propylproline.


Enhancement by ubenimex (bestatin) of host resistance to Candida albicans infection. Ubenimex is a low molecular weight microbial metabolite which has been demonstrated to have antitumor and immunomodulatory activities. In this study, the protective effect of ubenimex on Candida albicans infection was investigated in normal and immunosuppressed mice. In normal mice, treatment with ubenimex at 0.5, 5 and 25 mg/kg for 5 days prior to infection prolonged survival time in a dose-dependent manner. In immunosuppressed mice treated with a single dose of cyclophosphamide 4 days prior to infection, ubenimex treatment at 5 mg/kg for 5 days significantly increased the number of survivors. Ubenimex-treated mice had a significant increase in number of peritoneal exudate cells with neutrophils as well as enhanced functions, including phagocytosis and active oxygen production. These results suggest the potential usefulness of ubenimex as a prophylactic agent for the management of patients with opportunistic fungal infections.


Gender and the relationship between perceived fairness of pay or promotion and job satisfaction. Brockner and Adsit (1986) found that satisfaction with an exchange relationship was more strongly related to perceptions of equity among men than women. Kahn (1972) reported that men were more likely than women to distribute outcomes to individuals in direct proportion to their input. We evaluated potential gender differences among 12,979 personnel in 30 different organizational systems in (a) correlations between fairness and job satisfaction scores and (b) standardized group differences in the perceived amounts of pay and promotion fairness and expressed levels of facet and global job satisfaction. The fairness-satisfaction relationship was not higher for men, and there were no practical differences in fairness perceptions and job satisfaction between men and women.


Comparing traditional and integrative learning methods in organizational training programs. Previous research and anecdotal reports have suggested that when certain teaching approaches are used, students not only learn more but also experience greater satisfaction with the training process. This study examined the effects of integrative-learning-based (IL) training in relation to lecture-based training. Employees enrolled in a 3-day manufacturing resource planning training course were randomly assigned to either IL or traditional training. Subjects reacted more favorably to IL-based training. Trained subjects performed significantly better than those in a no-treatment control group, but no differences were noted between training interventions.


Development of physical ability tests for police officers: a construct validation approach. A construct validation approach was followed to affirm that 8 physical ability test events were significantly related to two important constructs underlying the job performance of police officers: strength and endurance. A sample of 115 incumbent police officers took 8 physical ability tests and were rated by supervisors on their physical performances in their job. LISREL methods were used to test the model specified, and a reasonable fit was achieved. Portions of the model were tested on an independent sample of 161 applicants; the fit of the model was again acceptable. A nomological network of relationships, in which strength and endurance factors correlated in expected directions with other physiological and demographic variables, was hypothesized and tested. Finally, the data were examined for potential gender differences and bias. Considerable differences were shown between men and women on both test and performance variables, and women would be overpredicted if a common regression line were used for selection purposes.


Developing a support system for teaching and assessing clinical competence. Primary care medical school faculty, in partnership with the faculty and staff of a Department of Biomedical Communications (Office of Medical Education) developed a teaching and logistical support system using standardized patients. The patients are used to teach history and physical examination skills to students in an introductory clinical medicine course. Having both clinical assessment team members, who are skilled biomedical communicators, and designated clinic rooms for standardized patients provides the foundation necessary for this growing area in medical education. Improved student performance, as measured by an Objective Structured Clinical Examination (OSCE), and students' positive ratings and comments in the evaluation of the course demonstrated the efficacy of using standardized patients in teaching and assessing clinical performance.


Multimedia for clinical education in myocardial perfusion imaging. Interactive multimedia has many advantages as a presentation medium for diagnostic imaging case studies, particularly for teaching digital imaging techniques such as myocardial perfusion imaging. Multimedia documents may incorporate interactive audiovisual presentations, sophisticated diagnostic images, anatomical illustrations, and text in medical instructional materials. The article describes a multimedia teaching atlas of SPECT and planar myocardial perfusion imaging delivered on Apple Macintosh II microcomputers, and discusses some of the advantages of delivering medical educational materials with interactive multimedia techniques.


Satellite system addresses rural health problems. The University of Nebraska Medical Center uses the recently established NEB*SAT state-owned satellite television system to transmit several types of educational presentations to rural Nebraska. The system represents the latest in a series of pioneering two-way television activities which have opened educational opportunities to students and professionals off-campus. Compressed video, transmitted via the NEB*SAT system, allows us to share credit courses in nursing and medical technology, administrative meetings, case conferences, and continuing education programs.


Ernest W. Beck: 1991 Association of Medical Illustrators Lifetime Achievement Award. On August 15, 1991, the Association of Medical Illustrators bestowed its highest honor, the Lifetime Achievement Award, on Ernest W. Beck. Ernie has been a pioneer in the profession, who has selflessly contributed his time and energy toward the advancement of the AMI. Ernie serves as an inspiration and example for the young illustrators today who seek to find their way in the profession, maintaining the highest level of professional conduct and ethical business practice. It is altogether fitting and proper that the Association of Medical Illustrators has recognized Ernie's unselfish contributions to its success.


Vascularized bone transfer.

Vascularized bone transfer. We evaluated the results of reconstruction of a skeletal defect with use of a vascularized bone graft from the iliac crest or fibula in 160 patients who had been managed consecutively between 1979 and 1989. The indications for the procedure were a skeletal defect including non-union, resulting from resection of a tumor; traumatic bone loss; osteomyelitis; or a congenital anomaly. The average duration of follow-up was forty-two months (range, twelve to 112 months). For the entire series, the rate of union after the primary procedure was 61 per cent and the over-all rate at the latest follow-up examination (including the patients who had a secondary procedure) was 81 per cent. In a subgroup of seventy-six patients who had union after the primary procedure and did not have additional treatment, the average interval until union was six months and the average interval until full activity was sixteen months. The results were more favorable for the patients who had had reconstruction for resection of a tumor (of sixty-nine patients, fifty-six had union), for a congenital anomaly (of six patients, five had union), or for a non-union without infection (of twenty-five patients, twenty-three had union). The results were less satisfactory for patients who had had the reconstruction for bone loss due to osteomyelitis (of sixty patients, forty-six had union). Our data suggest that vascularized bone transfer for the reconstruction of large skeletal defects is a valuable procedure in appropriately selected patients.


Pudendal nerve palsy complicating intramedullary nailing of the femur. A prospective study of 106 patients who had static interlocking nailing of the shaft of the femur was performed to determine the relationship between the duration and magnitude of intraoperative traction and the development of a pudendal nerve palsy. A strain-gauge, mounted in the countertraction post, measured the magnitude of the perineal pressure over time. All nailings were performed with the patient in the supine position. Postoperatively, the patients were interviewed by one of us, who had been blinded from the results of the recordings of intraoperative pressure, for a history of erectile dysfunction and changes in labial, scrotal, or penile sensation. A light-touch sensory examination of the genitalia was performed on all patients. Ten patients (six men and four women) had a pudendal nerve palsy: nine had sensory changes only, and one complained of erectile dysfunction. The symptoms had resolved at the three-month follow-up evaluation in all patients except one man who complained of dysesthesia six months postoperatively. The patients in whom a palsy did not develop had been positioned on the fracture-table and the perineal post for an average of 2.6 hours (range, 1.4 to 5.2 hours) compared with an average of 2.8 hours (range, 2.0 to 4.3 hours) for those in whom a palsy did not develop (p = 0.15). The magnitude of the total traction forces averaged 34.9 kilogram-hours for the patients who did not have a palsy compared with 73.3 kilogram-hours for those who did (p < 0.03). Adduction of the hip, as well as manipulations for reduction of the fracture, significantly increased the traction forces.(ABSTRACT TRUNCATED AT 250 WORDS)


The effect of a composite of polyorthoester and demineralized bone on the healing of large segmental defects of the radius in rats. The effect of a composite of demineralized bone mixed with polyorthoester on the healing of large segmental defects in the rat radius was studied. Sixty male Wistar rats were divided into four groups, A through D, and an osteoperiosteal diaphyseal defect of 50 per cent of the length of the bone was made in the right radius of each rat. In Group A, the defect was filled with polyorthoester and demineralized bone; in Group B, demineralized bone; and in Group C, polyorthoester. No material was implanted in the defects in the Group-D rats. The rats were killed fifty days postoperatively. The formation of bone in the defects was quantified with computer-assisted measurements of the area on radiographs. The host-tissue response was evaluated with light microscopy. Defects that had been filled with the composite of polyorthoester and demineralized bone or with demineralized bone alone showed regeneration of bone corresponding to 93.6 and 77.6 per cent of the area of the defect, respectively. Defects that had no implant or that had been filled with polyorthoester alone showed significantly less formation of bone. No inflammation was seen with light microscopy, and only traces of the polyorthoester could be detected in the defects that had been filled with the composite or with polyorthoester alone.


The effects of controlled mechanical loading on group-II, III, and IV afferent units from the lumbar facet joint and surrounding tissue. An in vitro study. An in vitro model was developed to investigate the responses of afferent units in the lumbar spine to controlled loading as measured by a load-cell. The neuronal discharge was recorded simultaneously with loading. Three types of neuronal responses were observed. The first type of response involved phasic-type mechanoreceptors, which responded to movement, regardless of direction or initial position. The response did not outlast the movement phase of loading. These units may serve as velocity detectors. The second type of response was seen in slowly adapting low-threshold mechanoreceptors, which tended to respond to loading in the 0.3 to 0.5-kilogram range with an immediate and sustained increase in the rate of firing. This type of response appears to be associated with the activation of low-threshold group-II and group-III fibers, which were located in muscles and tendons inserting into the facet joint. The third type of response involved slowly adapting high-threshold mechanoreceptors, which could not be activated until a threshold of three to five kilograms had been exceeded. It appears that this type of response is at least partially due to the activation of high-threshold group-III and group-IV capsular afferent units, which may signal noxious mechanical stimulation.


The transverse carpal ligament. An important component of the digital flexor pulley system. The effect of release of the transverse carpal ligament on the mechanics of the flexor tendons was investigated with use of a fresh-frozen cadaver model. A 25 per cent increase in the amount of excursion of the tendons that was consumed by motion of the wrist was demonstrated for the digitorum profundus tendons after transection of the transverse carpal ligament, and a 20 per cent increase was noted for the digitorum superficialis tendons (p < 0.001). These values represent 5.4 +/- 1.2 and 5.5 +/- 1.3 millimeters of bow-stringing for each group of tendons.


Radio-ulnar dissociation. A review of twenty cases. The results of treatment were reviewed for twenty patients who had sustained concomitant injuries of the lateral compartment of the radiohumeral joint and the ipsilateral distal radio-ulnar joint. The ages of the patients ranged from eight to seventy-four years (average, thirty-five years) and the duration of follow-up ranged from four months to twenty-seven years (average, 113 months). In fifteen patients, the injury of the wrist was diagnosed after a mean delay of seven years and eleven months (range, one month to twenty-six years). In all fifteen, the radial head injury was treated by excision, either initially or after some delay. After excision of the radial head, all fifteen patients complained of severe pain at the distal radio-ulnar joint. The results, on the basis of elbow and wrist scores of fair or better without complications, were satisfactory in only three patients. In the remaining five patients, in whom the injuries of both the elbow and the wrist had been identified at the initial evaluation, the radial head was either preserved or replaced. The results, on the basis of elbow and wrist scores of fair or better, were graded as satisfactory in four of these patients. Our data show that any injury to the lateral side of the elbow should prompt a careful evaluation of the ipsilateral distal radio-ulnar joint for associated instability.


Fractures adjacent to humeral prostheses. In a review of records and radiographs from 1974 through 1988, we identified seven patients who had a humeral fracture after either a total shoulder replacement or a shoulder hemiarthroplasty. All seven patients had complications after the fracture, and five fractures did not unite until an operation was done. All of the fractures that were treated operatively healed without sequelae. Four patients who were managed operatively had satisfactory relief of pain and one had fair relief. One patient who had a non-union refused further treatment for medical reasons. The one fracture that united without operative treatment healed with the tip of the prosthesis outside of the humeral shaft, but persistent pain led to a revision total shoulder replacement. The average time to union after the operation was approximately five months (range, four to seven months). There was a permanent decrease in the motion of the shoulder from preinjury levels in five of the six patients who had union of the fracture.


Operative treatment of failed repairs of the rotator cuff. Thirty-one patients had a repeat repair after a failure of a previously performed repair of the rotator cuff, and the causes of the original failure and the results of the repeat repair were analyzed. The clinical situation was complex, with multiple factors contributing to the failures. A large or massive tear of the cuff in thirty patients (97 per cent) and persistent subacromial impingement in twenty-eight patients (90 per cent) were the most common factors associated with failure. The over-all result of the repeat repair was satisfactory in sixteen patients (52 per cent) and unsatisfactory in fifteen (48 per cent). Twenty-five patients (81 per cent) had satisfactory relief of pain. However, fourteen patients (45 per cent) had persistent weakness that led to an unsatisfactory result. The factors that were associated with a better result were an intact acromion, an intact origin of the deltoid muscle, and the finding that the remaining tissue of the rotator cuff was of good quality. The factors that were associated with an inferior result were a previous lateral acromionectomy, a previously detached origin of the deltoid muscle, and the finding that the tissue of the rotator cuff that was available at the time of the repeat operation was of poor quality. Persistent pain is the primary indication for a repeat repair. The functional results are not as predictable, especially when the tissue of the cuff is poor and the deltoid origin has been detached previously.


The inferior capsular-shift procedure for multidirectional instability of the shoulder. Thirty-eight patients (forty-three shoulders) who had disabling multidirectional instability of the shoulder were managed with an inferior capsular-shift procedure through an anterior approach. All of the patients were followed for a minimum of two years. The postoperative range of motion of the shoulders was well maintained. The mean forward elevation was 172 degrees; external rotation, 77 degrees; and internal rotation, to the level of the eighth thoracic vertebra. Four patients (four shoulders) had recurrence of symptomatic and disabling multidirectional instability, but thirty-nine (91 per cent) of the shoulders continued to function well with no instability. Nine patients (24 per cent) continued to have episodes of apprehension, which correlated with the residual inferior and posterior translations found at the postoperative physical examination. Thirty-four patients (thirty-nine shoulders) stated that they were subjectively satisfied with the status of the shoulder, but four patients, in whom the instability had recurred, were not satisfied. Thirty-seven (86 per cent) of the shoulders were judged to have been improved by the procedure, the initial postoperative stability had been maintained, and the result had not deteriorated with time. Six shoulders, however, including the four with recurrent instability, were thought by the patient to have deteriorated with the increased duration of follow-up. It was our experience that if non-operative treatment of multidirectional instability of the shoulder failed, the inferior capsular-shift procedure provided satisfactory objective and subjective results. Failures and recurrences of symptomatic instability occurred early in the postoperative period. There appeared to be no deterioration of the results with follow-up to seventy-one months.


Treatment of slipped capital femoral epiphysis with a spica cast. Thirteen patients who had seventeen slipped capital femoral epiphyses were managed with a spica cast between 1984 and 1986. The average time in the plaster cast was three months. Complications were noted in fourteen of the hips. Three pressure sores developed in two patients. Further slipping developed in three hips once the cast had been removed, and chondrolysis developed in one of these hips. Chondrolysis developed in eight additional hips, and the lesion was transient in four of them. Degenerative changes developed in all nine hips with chondrolysis, regardless of whether the chondrolysis was transient or permanent. The degenerative changes were Iowa Grade I in three of these hips, Grade II in two hips, and Grade III in four. Chondrolysis developed in six of the eight black patients and in four of the five black boys. Chondrolysis developed in six of the nine hips that had a Grade-II or III slip. These findings have led us to abandon the use of a spica cast as a mode of treatment for slipped capital femoral epiphysis.


Geographic variations in the rates of elective total hip and knee arthroplasties among Medicare beneficiaries in the United States. We analyzed the variations in the rates of elective total hip and total knee arthroplasties for 1988 in the United States to determine whether the rates correlated with the numbers of surgeons. There were 56,204 total hip arthroplasties and 68,491 total knee arthroplasties, performed in the home states of the patients among all of the Medicare beneficiaries. Medicare beneficiaries include most people who are more than sixty-five years old in the United States and a small proportion of younger people who are eligible for Medicare for other reasons. Seventy-nine per cent of the patients who had had a total hip arthroplasty and 89 per cent of those who had had a total knee arthroplasty had been managed with the operation because of osteoarthrosis. Both operations were most common in the seventy to seventy-four-year age-group. We calculated the rate of operations per 100 beneficiaries for each state and age-adjusted the results. Across all of the states, bilateral procedures constituted 1.6 per cent of the total hip arthroplasties and 4.8 per cent of the total knee arthroplasties. The in-hospital rates of mortality were 0.72 per cent for total hip arthroplasties and 0.45 per cent for total knee arthroplasties. The destinations after discharge from the hospital were similar for the two groups of patients, with more than 65 per cent of the patients being discharged directly to their homes. There were no significant differences among states in terms of the length of stay in the hospital or reimbursement of the hospital.(ABSTRACT TRUNCATED AT 250 WORDS)


Serum calcitonin may be a marker for inhalation injury in burns. One of the principal causes of death from burns is inhalation injury. The pulmonary neuroendocrine cell contains and secretes immunoreactive calcitonin (iCT), and, under the influence of various irritative stimuli, can be induced to secrete iCT in excess. A prospective study of serum iCT levels in 41 patients with burns was undertaken. Mean serum iCT levels were four times normal values at the time of admission and reached 31 times normal values by 24 hours after injury. These levels did not correlate specifically with burn size. However, serum iCT had a very strong positive correlation with mortality, and in addition, was highest in patients who died early after injury compared with those who died late after injury. Patients who were clinically suspected to have pulmonary injury and who died had markedly higher levels of iCT than those who survived. In addition, serum iCT correlated positively with the need for mechanical ventilation and the amount of pulmonary shunting. Although other factors may also play a role in hypercalcitoninemia, serum iCT may be an important marker for the presence of inhalation injury, as well as a prognostic indicator.


Effects of rat interleukin-2 and rat interferon on the natural killer cell activity of rat spleen cells after thermal injury. The natural killer cell activity of splenocytes from rats with scald injury was observed to be significantly suppressed at 7 days after injury compared with that of normal nonburned controls. Incubation of splenocytes from normal rats or rats with burn injury with either rat interleukin-2 or rat interferon (IFN-alpha and IFN-beta) significantly increased the natural killer cell activity. Addition of a rabbit anti-rat interferon antibody to spleen cells incubated with interleukin-2 did not produce any significant alteration in interleukin-2-related enhancement of natural killer cell activity. These results suggest that enhancement of natural killer cell activity after incubation of splenocytes with interleukin-2 is not due to interferon production but is an independent event. Preincubation of spleen cells with a mouse monoclonal antibody to rat interleukin-2 receptor was observed to abolish the interleukin-2-related enhancement of natural killer cell activity completely, whereas it partially blocked the interferon-related enhancement. These results were also confirmed by enhancement of natural killer cell activity of burned rats after in vivo administration of interleukin-2. Our studies thus indicate that after thermal injury, the observed decrease of natural killer cell activity can be enhanced by both interleukin-2 and interferon independently of each other. The decreased natural killer cell activity may be due to a decrease in interleukin-2 production or availability and not to an interleukin-2 receptor defect. These studies thus point toward a potential therapeutic significance of interleukin-2 in enhancing immune function after thermal injury.


Carbon monoxide and pulmonary circulation in an ovine model. The direct pulmonary vasoconstrictive effects of inhaled carbon monoxide were evaluated in chronically instrumented and anesthetized sheep (1.7% halothane in air) (n = 8). The response to carbon monoxide (2%), which was applied for 8 minutes through a double-lumen tube alternately to the left or right lung of each animal, was compared with baseline values. The induced carboxyhemoglobin level (65%) led to increases in cardiac output, pulmonary arterial pressure, stroke volume index, and heart rate. Systemic vascular resistance decreased, and pulmonary vascular resistance and mean arterial pressure were unchanged. The changes in pressure and flow were equivalent no matter which lung was exposed to carbon monoxide. No diversion of blood from one lung to the other was observed during the test period. We conclude that carbon monoxide does not have a direct pulmonary vasoconstrictive effect. The increase in pulmonary arterial pressure is a result of the decrease in mixed venous oxygen content (stimulus for hypoxic pulmonary vasoconstriction) and the increase in cardiac output.


An in vitro model for studying the effect of the subcutaneous tunnel and the skin exit site on the accuracy of central venous catheter tip cultures. The diagnosis of central venous catheter-related sepsis depends on a positive culture obtained from the distal intravascular portion of the catheter. The effects of the subcutaneous tunnel and the skin exit site on the accuracy of cultures obtained from the catheter are unknown. We have developed an in vitro model to study the effect of these variables. By inoculating polyethylene catheters embedded in agar with Pseudomonas aeruginosa, we were able to show that: (1) capillary action occurs along catheters in an agar tunnel, (2) organisms that are growing on the distal segment (tip) of the catheter can be dislodged from the surface of the catheter when it is pulled through the agar tunnel, and (3) pulling a catheter through a contaminated area results in distal contamination.


Core body temperature responses immediately after cutaneous thermal injury in rats. Internal temperatures were continuously measured in rats that received 20% or 40% body surface area cutaneous scald injuries in 25 degrees C and 38.5 degrees C environments. In animals that received thermal injuries in the 25 degrees C environment, intraperitoneal, intraesophageal, and intrarectal temperatures rapidly rose to 40 degrees C within 5 minutes but returned to normal values within 15 minutes after injury. When 20% body surface area injuries were induced in a 25 degrees C environment, all of the animals survived. In the 25 degrees C environment, neither acute core body temperature elevations nor use of fluid resuscitation predicted survival. In contrast, in a 38.5 degrees C environment core body temperatures rapidly exceeded 41 degrees C with the 20% injury, and all of the animals died within minutes in spite of fluid resuscitation. These studies suggest that the ambient environmental temperature may significantly influence a thermally injured animal's ability to rapidly eliminate absorbed heat of injury and result in an elevated core body temperature, which may contribute to the immediate lethality of the injury.


Diagnosis of local infection of a burn by semiquantitative culture of the eschar surface. The purpose of this study was to determine whether semiquantitative surface cultures of the burn eschar are as reliable or useful as the classic invasive biopsy culture method. We used eschars from patients with burns in an in vitro system. Lyophilized pigskin was used to validate our methodology. Because of its simplicity and high degree of sensitivity and specificity as compared with quantitative biopsy culture, semiquantitative surface culture has a place in burn wound surveillance.


Advances in burn care management: role of the speech-language pathologist. Because the inclusion of the speech-language pathologist in a burn management team is not widely practiced, we discuss our successes as members of a burn team. We also review speech-language evaluation and treatment strategies and present two patients with head and neck burns who gained from our intervention.


A new compound curved needle for skin and skin-graft closure. A new compound curved needle has been developed for skin and skin-graft closure from a unique stainless steel alloy, American Society for Testing Materials (ASTM) 45500. This needle has a short, straight, sharpened point with a reverse cutting edge and a curved distal section. In spite of its geometry, it exhibits sharpness and resistance to bending and breakage similar to that of a needle with a single radius of curvature that is manufactured from the same alloy. The design of this new needle enables the surgeon to pass it through the skin with greater accuracy to a controlled depth and length of bite.


The status of burn rehabilitation services in the United States: results of a national survey. A survey to determine the status of burn rehabilitation services in the United States was developed and sent to 186 burn treatment facilities. The facilities were divided into four groups based upon number of admissions per year (0 to 80, 81 to 120, 121 to 200, and 200+). Completed surveys were received from 114 facilities. Results indicated that burn facilities of different sizes were consistent with respect to the severity of burn injuries treated, the length of hospitalization for acute injuries, and the duration of physician follow-up after discharge. Burn facilities with more admissions were more likely to report (1) organized outpatient burn rehabilitation programs, (2) available specialized burn rehabilitation personnel, (3) regular interdisciplinary inpatient staffing conferences and outpatient clinics, and (4) structured educational activities for staff and Full-time equivalent burn rehabilitation personnel were equally represented across facilities of different sizes. The overall results suggest that there have been substantial improvements in the comprehensiveness of burn rehabilitation care since a previous survey in 1983. Minimum guidelines for burn rehabilitation are suggested based upon the relative consistency between burn facilities indicated by the survey results.


Technical considerations in the use of external condom catheter systems. Since the advent of rehabilitation engineering new advances have been made that have revolutionized condom catheter drainage systems (CCDS). An innovative CCDS has been designed that ensures unobstructed urine flow. Its condom catheter has several unique design features. It has a double row of convolutions near the catheter tip that prevent kinking and twisting so that the pathway remains open. The condom catheter features a unique inner flap that fits snugly on the glans to prevent backflow of urine on the shaft. This condom catheter is connected to a vented leg bag that eliminates the development of a partial vacuum in the connecting tube. This vacuum can create siphoning, which in turn interferes with urine flow into the leg bag. In addition, a new rechargeable battery-operated clipper has been developed that makes nick-free hair removal from the genitals exceptionally easy. This atraumatic hair removal eliminates the pubic hair that becomes trapped under the condom catheter. The clinical impact of these new advances in CCDS requires further investigation.


A pressure prevention device for burned ears. Full-thickness burns to the external ear can produce loss of skin and cartilage and can result in severe cosmetic deformity. Even partial-thickness burns render the ear vulnerable to tissue loss if the helix is subjected to pressure from pillows, dressings, or straps that are used to secure endotracheal tubes. Because of the incidence of burned ear deformities and the difficulty in reconstructing the external ear, an ear protection device has been designed. The bilateral ear protection device, referred to as "headgear", is fitted to all patients in the burn center who require intubation for an inhalation injury; it is worn continuously until extubation. During a 15-month period 39 consecutive critically burned patients were fitted with the headgear because of the need for ventilator support and/or for protection of the burned ear(s). Pressure necrosis of ear tissue was prevented in all 33 survivors.


A nasal trumpet orthosis to maintain nares openings and respiratory function for patients with facial burns: a case report. Management of facial burns is a challenge to the burn team because it may lead to functional and cosmetic compromise. Severe scarring of the nares may lead to nasal occlusion. This article introduces a method of maintaining nasal patency that allows respiratory exchange through the use of a custom-fabricated, semirigid tubular orthosis. The technique for fabrication is reviewed, and the use of the device is addressed through a case report. This inexpensive, readily available device is useful in preventing nasal occlusion that results from scar formation.


Caloric and nitrogen balances as predictors of nutritional outcome in patients with burns. Measurement of energy expenditure with indirect calorimetry allows determination of caloric balance. The present study was done to determine the predictive value of caloric and nitrogen balances for nutritional outcome. Energy expenditure was obtained weekly and interpolated between measurements for daily caloric balance. Nitrogen balance was obtained weekly. Because nitrogen output fluctuated, interpolation of daily values was not possible. Nutritional outcome was defined by whether body weight was lost or maintained and by levels of visceral proteins (albumin, prealbumin, and transferrin). The study group included 12 patients with 7% to 82.5% total body surface area burns. Eleven patients survived their burn injuries, and one died of congestive heart failure at 38 days, after her burn wounds had healed. Nine patients had good nutritional outcomes (group 1) and three had poor nutritional outcomes (group 2) (including the one who died). Nitrogen balance was 1.3 +/- 1.0 gm/day in group 1 and 4.5 +/- 1.7 gm/day in group 2 (mean +/- SEM; p > 0.10). Caloric balance was 515 +/- 130 kcal/day in group 1 and -667 +/- 140 in group 2 (p < 0.001). Caloric balance was significantly different between the two groups, whereas nitrogen balance was not. Caloric intake correlated positively with nitrogen intake (r = 0.92). Nitrogen intake was 16% of total caloric intake. Nitrogen intake from blood products was appreciable and averaged 15% of total nitrogen intake (range, 0% to 47%); 11.3 +/- 1.6 gm/day in group 1 and 14.8 +/- 3 gm/day in group 2 (p > 0.10).(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of a burn prevention program in a public school system. The "Learn Not to Burn" prevention program is a burn prevention curriculum sponsored by the North Carolina Jaycee Burn Center, the State Department of Public Instruction, and the North Carolina Department of Insurance Fire and Rescue Division. The goal of the program is to reduce burn-related deaths and injuries in North Carolina through burn prevention education by making the "Learn Not to Burn" curriculum available to primary school children across the state at no cost to the schools. The curriculum instrument is a reusable notebook that provides a means for teachers to integrate burn prevention into regular class subject areas. At the time of initiation of this study approximately 70% of the school systems in North Carolina had been provided with the "Learn Not to Burn" curriculum.


Psychiatric referral pattern in a burn center. Patients with burn injuries have unique psychologic problems that frequently require psychiatric care. However, little research has been done on specific patterns of psychiatric referral in burn units. This article addresses the pattern of psychiatric referral for 28 patients over a period of 1 year in a regional burn treatment center. Demographics and characteristics of the patient population are summarized, and reasons for psychiatric referral are noted.


Expression of c-fos and AP-1 activity in senescent human fibroblasts is not sufficient for DNA synthesis. Human fibroblasts have a limited replicative life span when maintained in culture after which they become unresponsive to treatment with mitogens, a phenomenon most commonly called senescence. Experiments indicating that serum does not induce expression of the c-fos proto-oncogene in senescent fibroblasts raised the issue of a potential central role for c-fos in the phenotype of sustained growth arrest. This was directly tested by microinjection of oncogenic c-Ha-ras protein into senescent fibroblasts. While ras injection was found to induce marked nuclear c-fos expression and functional AP-1 transcription activity, this did not lead to DNA synthesis. These results suggest that the senescence phenotype cannot be solely attributed to the absence of c-fos expression and that the proliferative block in these cells is either independent of AP-1 transcriptional activity, downstream of it, or involves multiple molecular mechanisms.


A human centromere protein, CENP-B, has a DNA binding domain containing four potential alpha helices at the NH2 terminus, which is separable from dimerizing activity. The alphoid DNA-CENP-B (centromere protein B) complex is the first sequence-specific DNA/protein complex detected in the centromeric region of human chromosomes. In the reaction, CENP-B recognizes a 17-bp sequence (CENP-B box) and assembles two alphoid DNA molecules into a complex, which is designated complex A (Muro, Y., H. Masumoto, K. Yoda, N. Nozaki, M. Ohashi, and T. Okazaki. 1992. J. Cell Biol. 116:585-596). Since CENP-B gene is conserved in mammalian species and CENP-B boxes are found also in mouse centromere satellite DNA (minor satellite), this sequence-specific DNA-protein interaction may be important for some kind of common centromere function. In this study we have characterized the structure of CENP-B and CENP-B-alphoid DNA complex. We have shown by chemical cross-linking that CENP-B formed a dimer, and have estimated by molecular weight determination the composition of complex A to be a CENP-B dimer and two molecules of alphoid DNA. The DNA binding domain has been delimited within the NH2-terminal 125-amino acid region containing four potential alpha-helices using truncated CENP-B made in Escherichia coli cells. We have shown that CENP-B had sites highly sensitive to proteases and that the DNA binding domain was separable from the dimerizing activity by the proteolytic cleavage at 20 kD from the COOH terminus of the molecule. Thus, CENP-B may organize a higher order structure in the centromere by juxtaposing two CENP-B boxes in the alphoid DNA repeat through both the DNA-protein and protein-protein interactions.


High resolution scanning electron microscopy of the nuclear envelope: demonstration of a new, regular, fibrous lattice attached to the baskets of the nucleoplasmic face of the nuclear pores. The nuclear envelope (NE) of amphibian oocytes can be readily isolated in relatively structurally intact and pure form and has been used extensively for structural studies. Using high resolution scanning electron microscopy (HRSEM), both surfaces of the NE can be visualized in detail. Here, we demonstrate the use of HRSEM to obtain high resolution information of NE structure, confirming previous data and providing some new information. NEs, manually isolated from Triturus cristatus oocytes, have been mounted on conductive silicon chips, fixed, critical point dried and coated with a thin, continuous film of chromium or tantalum and viewed at relatively high accelerating voltage in a field emission scanning electron microscope with the sample within the objective lens. Both nucleoplasmic and cytoplasmic surfaces of the nuclear pore complexes (NPC) have been visualized, revealing the cytoplasmic coaxial ring, associated particles, central plug/transporter and spokes. The nucleoplasmic face is dominated by the previously described basketlike structure attached to the nucleoplasmic coaxial ring. In Triturus, a novel, highly regular flat sheet of fibers, termed the NE lattice (NEL) has been observed attached to the distal ring of the NPC basket. The NEL appears to be distinct from the nuclear lamina. Evidence for the NEL is also presented in thin TEM sections from Triturus oocytes and GVs and in spread NEs from Xenopus. A model is presented for NEL structure and its interaction with the NPCs is discussed.


Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane. We have investigated the localization of Kex1p, a type I transmembrane carboxypeptidase involved in precursor processing within the yeast secretory pathway. Indirect immunofluorescence demonstrated the presence of Kex1p in a punctate organelle resembling the yeast Golgi apparatus as identified by Kex2p and Sec7p (Franzusoff, A., K. Redding, J. Crosby, R. S. Fuller, and R. Schekman. 1991. J. Cell Biol. 112:27-37). Glycosylation studies of Kex1p were consistent with a Golgi location, as Kex1p was progressively N-glycosylated in an MNN1-dependent manner. To address the basis of Kex1p targeting to the Golgi apparatus, we examined the cellular location of a series of carboxy-terminal truncations of the protein. The results indicate that a cytoplasmically exposed carboxy-terminal domain is required for retention of this membrane protein within the Golgi apparatus. Deletions of the retention region or overproduction of wild-type Kex1p led to mislocalization of Kex1p to the vacuolar membrane. This unexpected finding is discussed in terms of models involving either the vacuole as a default destination for membrane proteins, or by endocytosis to the vacuole following their default localization to the plasma membrane.


Trafficking of malarial proteins to the host cell cytoplasm and erythrocyte surface membrane involves multiple pathways. During the asexual stage of malaria infection, the intracellular parasite exports membranes into the erythrocyte cytoplasm and lipids and proteins to the host cell membrane, essentially "transforming" the erythrocyte. To investigate lipid and protein trafficking pathways within Plasmodium falciparum-infected erythrocytes, synchronous cultures are temporally analyzed by confocal fluorescence imaging microscopy for the production, location and morphology of exported membranes (vesicles) and parasite proteins. Highly mobile vesicles are observed as early as 4 h postinvasion in the erythrocyte cytoplasm of infected erythrocytes incubated in vitro with C6-NBD-labeled phospholipids. These vesicles are most prevalent in the trophozoite stage. An immunofluorescence technique is developed to simultaneously determine the morphology and distribution of the fluorescent membranes and a number of parasite proteins within a single parasitized erythrocyte. Parasite proteins are visualized with FITC- or Texas red-labeled monoclonal antibodies. Double-label immunofluorescence reveals that of the five parasite antigens examined, only one was predominantly associated with membranes in the erythrocyte cytoplasm. Two other parasite antigens localized only in part to these vesicles, with the majority of the exported antigens present in lipid-free aggregates in the host cell cytoplasm. Another parasite antigen transported into the erythrocyte cytoplasm is localized exclusively in lipid-free aggregates. A parasite plasma membrane (PPM) and/or parasitophorous vacuolar membrane (PVM) antigen which is not exported always colocalizes with fluorescent lipids in the PPM/PVM. Visualization of two parasite proteins simultaneously using FITC- and Texas red-labeled 2 degrees antibodies reveals that some parasite proteins are constitutively transported in the same vesicles, whereas other are segregated before export. Of the four exported antigens, only one appears to cross the barriers of the PPM and PVM through membrane-mediated events, whereas the others are exported across the PPM/PVM to the host cell cytoplasm and surface membrane through lipid (vesicle)-independent pathways.


Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila. We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.


Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)


Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells. Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)


Zyxin and cCRP: two interactive LIM domain proteins associated with the cytoskeleton. Interaction with extracellular matrix can trigger a variety of responses by cells including changes in specific gene expression and cell differentiation. The mechanism by which cell surface events are coupled to the transcriptional machinery is not understood, however, proteins localized at sites of cell-substratum contact are likely to function as signal transducers. We have recently purified and characterized a low abundance adhesion plaque protein called zyxin (Crawford, A. W., and M. C. Beckerle. 1991. J. Biol. Chem. 266:5847-5853; Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. 1992. J. Cell Biol. 116:1381-1393). We have now isolated and sequenced zyxin cDNA and we report here that zyxin exhibits an unusual proline-rich NH2-terminus followed by three tandemly arrayed LIM domains. LIM domains have previously been identified in proteins that play important roles in transcriptional regulation and cellular differentiation. LIM domains have been proposed to coordinate metal ions and we have demonstrated by atomic absorption spectroscopy that purified zyxin binds zinc, a result consistent with the idea that zyxin has zinc fingers. In addition, we have discovered that zyxin interacts in vitro with a 23-kD protein that also exhibits LIM domains. Microsequence analysis has revealed that the 23-kD protein (or cCRP) is the chicken homologue of the human cysteine-rich protein (hCRP). By double-label indirect immunofluorescence, we found that zyxin and cCRP are extensively colocalized in chicken embryo fibroblasts, consistent with the idea that they interact in vivo. We conclude that LIM domains are zinc-binding sequences that may be involved in protein-protein interactions. The demonstration that two cytoskeletal proteins, zyxin and cCRP, share a sequence motif with proteins important for transcriptional regulation raises the possibility that zyxin and cCRP are components of a signal transduction pathway that mediates adhesion-stimulated changes in gene expression.


Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins. Kinesin and dynein are motor proteins that move in opposite directions along microtubules. In this study, we examine the consequences of having kinesin and dynein (ciliary outer arm or cytoplasmic) bound to glass surfaces interacting with the same microtubule in vitro. Although one might expect a balance of opposing forces to produce little or no net movement, we find instead that microtubules move unidirectionally for several microns (corresponding to hundreds of ATPase cycles by a motor) but continually switch between kinesin-directed and dynein-directed transport. The velocities in the plus-end (0.2-0.3 microns/s) and minus-end (3.5-4 microns/s) directions were approximately half those produced by kinesin (0.5 microns/s) and ciliary dynein (6.7 microns/s) alone, indicating that the motors not contributing to movement can interact with and impose a drag upon the microtubule. By comparing two dyneins with different duty ratios (percentage of time spent in a strongly bound state during the ATPase cycle) and varying the nucleotide conditions, we show that the microtubule attachment times of the two opposing motors as well as their relative numbers determine which motor predominates in this assay. Together, these findings are consistent with a model in which kinesin-induced movement of a microtubule induces a negative strain in attached dyneins which causes them to dissociate before entering a force-generating state (and vice versa); reversals in the direction of transport may require the temporary dissociation of the transporting motor from the microtubule. The bidirectional movements described here are also remarkably similar to the back-and-forth movements of chromosomes during mitosis and membrane vesicles in fibroblasts. These results suggest that the underlying mechanical properties of motor proteins, at least in part, may be responsible for reversals in microtubule-based transport observed in cells.


Dynein from Dictyostelium: primary structure comparisons between a cytoplasmic motor enzyme and flagellar dynein. We report here the cloning and sequencing of a cytoplasmic dynein heavy chain gene from the cellular slime mold Dictyostelium discoideum. Using a combination of approaches, we have isolated 14,318 bp of DNA sequence which contains an open-reading frame of 4,725 amino acids. The deduced molecular weight of the polypeptide predicted by this reading frame is 538,482 D. Overall, the polypeptide sequence is 51% similar and 28% identical to the recently published sequences of the beta-dynein heavy chain from sea urchin flagella (Gibbons, I. R., B. H. Gibbons, G. Mocz, and D. J. Asai. 1991. Nature (Lond.). 352: 640-643; Ogawa, K. 1991. Nature (Lond.). 352:643-645). It contains four GXXXXGKT/S motifs that form part of a consensus sequence for ATP-binding domains; these motifs are clustered near the middle of the polypeptide. The distribution of the regions sharing sequence similarity between the Dictyostelium and sea urchin heavy chain polypeptides suggests that the amino termini of dyneins may contain domains that specify axonemal or cytoplasmic functions.


Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity. Yeast calmodulin (CaM) is required for the progression of nuclear division (Ohya, Y. and Y. Anraku. 1989. Curr. Genet. 15:113-120), although the precise mechanism and physiological role of CaM in this process are unclear. In this paper we have characterized the phenotype caused by a temperature-sensitive lethal mutation (cmdl-101) in the yeast CaM. The cmdl-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubation of the cmdl-101 cells at a nonpermissive temperature causes a severe defect in chromosome segregation. The rate of chromosome loss in the cmdl-101 mutant is higher than wild-type cell even at permissive temperature. The primary visible defect observed by immunofluorescence and electron microscopic analyses is that the organization of spindle microtubules is abnormal in the cmdl-101 cells grown at nonpermissive temperature. Majority of budded cells arrested at the high temperature contain only one spindle pole body (SPB), which forms monopolar spindle, whereas the budded cells of the same strain incubated at permissive temperature all contain two SPBs. Using the freeze-substituted fixation method, we found that the integrity of the nuclear morphology of the cmdl-101 mutant cell is significantly disturbed. The nucleus in wild-type cells is round with smooth contours of nuclear envelope. However, the nuclear envelope in the mutant cells appears to be very flexible and forms irregular projections and invaginations that are never seen in wild-type cells. The deformation of the nuclear becomes much more severe as the incubation at nonpermissive temperature continues. The single SPB frequently localizes on the projections or the invaginations of the nuclear envelope. These observations suggest that CaM is required for the functions of SPB and spindle, and the integrity of nucleus.


Phorbol esters alter cell fate during development of sea urchin embryos. Protein kinase C (PKC) has been implicated as important in controlling cell differentiation during embryonic development. We have examined the ability of 12-O-tetradecanoyl phorbol-13-acetate (TPA), an activator of PKC, to alter the differentiation of cells during sea urchin development. Addition of TPA to embryos for 10-15 min during early cleavage caused dramatic changes in their development during gastrulation. Using tissue-specific antibodies, we have shown that TPA causes the number of cells that differentiate as endoderm and mesoderm to increase relative to the number that differentiate as ectoderm. cDNA probes show that treatment with TPA causes an increase in accumulation of RNAs specific to endoderm and mesoderm with a concomitant decrease in RNAs specific to ectoderm. Treatment of isolated prospective ectodermal cells with TPA causes them to differentiate into endoderm and mesoderm. The critical period for TPA to alter development is during early to mid cleavage, and treatment of embryos with TPA after that time has little effect. These results indicate that PKC may play a key role in determining the fate of cells during sea urchin development.


Cell surface acetylcholinesterase molecules on multinucleated myotubes are clustered over the nucleus of origin. Multinucleated skeletal muscle fibers are compartmentalized with respect to the expression and organization of several intracellular and cell surface proteins including acetylcholinesterase (AChE). Mosaic muscle fibers formed from homozygous myoblasts expressing two allelic variants of AChE preferentially translate and assemble the polypeptides in the vicinity of the nucleus encoding the mRNA (Rotundo, R. L. 1990. J. Cell Biol. 110:715-719). To determine whether the locally synthesized AChE molecules are targeted to specific regions of the myotube surface, primary quail myoblasts were mixed with mononucleated cells of the mouse muscle C2/C12 cell line and allowed to fuse, forming heterospecific mosaic myotubes. Cell surface enzyme was localized by immunofluorescence using an avian AChE-specific monoclonal antibody. HOECHST 33342 was used to distinguish between quail and mouse nuclei in myotubes. Over 80% of the quail nuclei exhibited clusters of cell surface AChE in mosaic quail-mouse myotubes, whereas only 4% of the mouse nuclei had adjacent quail AChE-positive regions of membrane, all of which were located next to a quail nucleus. In contrast, membrane proteins such as Na+/K+ ATPase, which are not restricted to specific regions of the myotube surface, are free to diffuse over the entire length of the fiber. These studies indicate that the AChE molecules expressed in multinucleated muscle fibers are preferentially transported and localized to regions of surface membrane overlying the nucleus of origin. This targeting could play an important role in establishing and maintaining specialized cell surface domains such as the neuromuscular and myotendinous junctions.


Nerve growth factor withdrawal-induced cell death in neuronal PC12 cells resembles that in sympathetic neurons. Previous studies have shown that in neuronal cells the developmental phenomenon of programmed cell death is an active process, requiring synthesis of both RNA and protein. This presumably reflects a requirement for novel gene products to effect cell death. It is shown here that the death of nerve growth factor-deprived neuronal PC12 cells occurs at the same rate as that of rat sympathetic neurons and, like rat sympathetic neurons, involves new transcription and translation. In nerve growth factor-deprived neuronal PC12 cells, a decline in metabolic activity, assessed by uptake of [3H]2-deoxyglucose, precedes the decline in cell number, assessed by counts of trypan blue-excluding cells. Both declines are prevented by actinomycin D and anisomycin. In contrast, the death of nonneuronal (chromaffin-like) PC12 cells is not inhibited by transcription or translation inhibitors and thus does not require new protein synthesis. DNA fragmentation by internucleosomal cleavage does not appear to be a consistent or significant aspect of cell death in sympathetic neurons, neuronal PC12 cells, or nonneuronal PC12 cells, notwithstanding that the putative nuclease inhibitor aurintricarboxylic acid protects sympathetic neurons, as well as neuronal and nonneuronal PC12 cells, from death induced by trophic factor removal. Both phenotypic classes of PC12 cells respond to aurintricarboxylic acid with similar dose-response characteristics. Our results indicate that programmed cell death in neuronal PC12 cells, but not in nonneuronal PC12 cells, resembles programmed cell death in sympathetic neurons in significant mechanistic aspects: time course, role of new protein synthesis, and lack of a significant degree of DNA fragmentation.


Restricted tissue distribution of a 37-kD possible adherens junction protein. A major polypeptide of M(r) 37,000 was purified from a desmosome-enriched citric acid-insoluble pellet of pig tongue epithelium. The polypeptide was solubilized from the 4-M urea-insoluble pellet with 9 M urea, and extracts were separated by carboxymethyl cellulose and gel filtration chromatography. The 37-kD protein was obtained in milligram quantities as a single band on two-dimensional gels in 30% yield after 21-fold purification from the citric acid-insoluble fraction. The protein is not glycosylated and has a pI of approximately 8.7. Although isolated from a fraction rich in desmosomes, the 37-kD protein is not a desmosomal protein. Indirect immunofluorescence analysis of frozen sections of tongue and other tissues demonstrated that antibodies raised to the 37-kD protein bound only to suprabasal cell layers at punctate regions of the periphery of the cell and was absent from most regions of epidermis, whereas antibodies to desmoplakins I and II, desmosomal proteins, bound similarly but in all epidermal layers. Immunoelectron microscopy localized the 37-kD protein to the cell periphery in regions between, but never in, desmosomes. By immunofluorescence, the 37-kD protein colocalized with actin as well as with vinculin and uvomorulin in oral tissues. Like the 37-kD protein, vinculin and uvomorulin were absent from the basal layer. Based on its appearance, localization, and solubility properties, the 37-kD protein is probably a component of adherens junctions; its restriction to suprabasal cells and exclusion from the epidermis are unique.


A23187-induced translocation of 5-lipoxygenase in osteosarcoma cells. In a previous study, osteosarcoma cells expressing both 5-lipoxygenase (5-LO) and 5 lipoxygenase-activating protein (FLAP) synthesized leukotrienes upon A23187 stimulation (Dixon, R. A. F., R. E. Diehl, E. Opas, E. Rands, P. J. Vickers, J. F. Evans, J. W. Gillard, and D. K. Miller. 1990. Nature (Lond.). 343:282-284). Osteosarcoma cells expressing 5-LO but not expressing FLAP were unable to synthesize leukotrienes. Thus, it was determined that FLAP was required for the cellular synthesis of leukotrienes. To examine the role of FLAP in A23187-induced translocation of 5-LO to a membrane fraction, we have studied the A23187-stimulated translocation of 5-LO in osteosarcoma cells expressing both 5-LO and FLAP, and in osteosarcoma cells expressing 5-LO only. We demonstrate that in cells expressing both 5-LO and FLAP, 5-LO translocates to membranes in response to A23187 stimulation. This 5-LO translocation is inhibited when cells are stimulated in the presence of MK-886. In osteosarcoma cells expressing 5-LO but not expressing FLAP, 5-LO is able to associate with membranes following A23187 stimulation. In contrast to the cells containing both 5-LO and FLAP, MK-886 is unable to prevent 5-LO membrane association in cells transfected with 5-LO alone. Therefore, we have demonstrated that in this cell system, 5-LO membrane association and activation can be separated into at least two distinct steps: (1) calcium-dependent movement of 5-LO to membranes without product formation, which can occur in the absence of FLAP (membrane association), and (2) activation of 5-LO with product formation, which is FLAP dependent and inhibited by MK-886 (enzyme activation).


Molecular isoforms of murine CD44 and evidence that the membrane proximal domain is not critical for hyaluronate recognition. We previously found that the CD44 glycoprotein on some lymphocytes can mediate adhesion to hyaluronate (HA) bearing cells. However, many questions remain about the molecular heterogeneity of CD44 and mechanisms which control its recognition of this ligand. In vitro mutagenesis and DNA sequencing have now been used to investigate the importance of the membrane proximal region of murine CD44 for recognition of soluble or cell surface HA. CD44 with an 83 amino acid deletion in this region mediated binding to soluble ligand and the apparent avidity increased markedly in the presence of a particular antibody to CD44, IRAWB14. The shortened CD44 was however inefficient in mediating adhesion of transfected cells to HA immobilized on cell surfaces. Four new murine isoforms of CD44 were isolated from a carcinoma line by use of the polymerase chain reaction. Only two of them correspond to ones recently discovered in rat and human cells. The longest variant nearly doubled the length of the extracellular portion of the molecule and introduced an additional 20 potential sites for glycosylation. When expressed on T lymphoma cells, all four of the new murine CD44 isoforms were capable of mediating adhesion to HA bearing cells. This result contrasts with a report that a related human CD44 isoform lacks this ability when expressed on B lineage lymphoma cells. The new murine isoforms also conferred the ability to recognize soluble HA and were very responsive to the IRAWB14 antibody. A brief survey of normal murine cell lines and tissues revealed that the hemopoietic isoform was the most abundant species. These findings indicate that the NH2-terminal portion of CD44 is sufficient for HA recognition and that this function is not necessarily abrogated by variations which occur in the membrane proximal domain. They add to the known molecular diversity of CD44 and provide another experimental model in which isoform specific functions can be investigated.


Recombinant human bone morphogenetic protein 2B stimulates PC12 cell differentiation: potentiation and binding to type IV collagen. Bone morphogenetic protein 2B (BMP 2B, also known as BMP 4) induces cartilage and bone morphogenesis in ectopic extraskeletal sites. BMP 2B is one of several bone morphogenetic proteins which along with activins and inhibins are members of the transforming growth factor-beta (TGF-beta) family. Both BMP 2B and activin A, but not TGF-beta 1, induce rat pheochromocytoma PC12 neuronal cell differentiation and expression of VGF, a nervous system-specific mRNA. PC12 cells exhibited approximately 2,500 receptors per cell for BMP 2B with an apparent dissociation constant of 19 pM. Extracellular matrix components, including fibronectin, laminin, and collagen type IV potentiated the activity of BMP and activin A, with the latter being the most active. Direct experiments demonstrated that radioiodinated BMP 2B bound to collagen type IV better than to either laminin or fibronectin. These data demonstrate a common neurotrophic activity of both BMP 2B and activin A, and suggest that these regulatory molecules alone and in conjunction with extracellular matrix components may play a role in both the development and repair of nervous tissue.


Cancer-related urinary proteinase inhibitor, EDC1: a new method for its isolation and evidence for multiple forms. During the past several years, numerous laboratories have reported isolation and purification of proteinase inhibitors from human urine. Many of these molecules were incompletely characterized and some of them may have been artifacts in part because of harsh procedures used for their isolation. Consequently, there is disagreement and confusion regarding the biochemical characteristics of these inhibitors. We previously reported the isolation of a proteinase inhibitor, EDC1, from the urine of a leukemic patient. This molecule, M(r) 30 kDa, was antigenically related to plasma inter-alpha-trypsin inhibitor (IATI) and inhibited the growth of a virally transformed B cell line. Immunoreactive EDC1 was also the major component of low molecular weight proteinuria observed in cancer patients. We now report a new method for the isolation of EDC1 from urine of patients with adenocarcinomas of colon and lung and melanoma and compare its partial amino acid sequence with that of HI 30, a proteinase inhibitor previously isolated from pooled normal urine by Hochstrasser et al. [Hoppe-Seyler's Z Physiol Chem 357:153-162, 1976]. Our method involves i) a batchwise cation exchange, ii) gel filtration chromatography, iii) anion exchange chromatography on FPLC, and iv) reverse phase C18 chromatography on HPLC. This method is mild and results in an overall yield of 0.4 to 1.2 mg of EDC1/liter urine. On the basis of the partial N-terminal amino acid sequence of its N terminal (38 residues) and middle regions (29 residues), EDC1 appears to be identical with HI30. Surprisingly, during this isolation procedure, another proteinase inhibitor, M(r) 22 kDa, which cross-reacted with antisera to EDC1 and IATI, was also isolated. The 22 kDa molecule was a major component of the IATI related urinary molecules and was identical with the 30 kDa EDC1 in which first the 15 N terminal residues were clipped. The lower M(r) inhibitor was not an artifact formed during storage or isolation procedure because the Western blot analysis of fresh cancer and normal urine revealed the 30 and 22 kDa molecules. Thus, both the 30 kDa EDC1 (or HI30) and its clipped variant, the 22 kDa molecule, are physiologic components of IATI related urinary proteinase inhibitors and excretion of both forms may be increased in patients with advanced cancer.


Phosphorylation of vinculin in human platelets spreading on a solid surface. Vinculin is a cytoskeletal protein believed to be involved in linking microfilaments to the cell membrane. It is a substrate for the Ca(2+)- and phospholipid-dependent protein kinase C. We show here that when human platelets attach and spread on a solid surface, the alpha isoforms of vinculin become phosphorylated at serine and/or threonine residues. Phosphorylation is dependent on adhesion to a surface, since suspended, unattached platelets can produce filopodia but no phosphorylation of vinculin. Phosphorylation is also dependent on actin polymerization, as it does not occur when platelets had been pretreated with cytochalasin B. Most likely, protein kinase C is responsible for the phosphorylation of vinculin, since phosphorylation also occurs when platelets are treated with a phorbol ester, which activates protein kinase C, and is blocked by treatment with a staurosporine derivative which inhibits this enzyme. These results suggest that phosphorylation plays a role in anchoring vinculin at sites of microfilament-membrane interaction.


Growth-regulated expression of vimentin in hamster fibroblasts is a result of increased transcription. We have previously shown that vimentin is a growth-regulated gene whose mRNA levels increase after serum stimulation of quiescent hamster fibroblasts. In this study, the control of the growth-regulated expression of vimentin was determined in ts13 cells induced to proliferate by serum. Both transcriptional and post-transcriptional mechanisms of regulation were examined by determining transcriptional rates, cytoplasmic transcript abundance, transcript stability, and protein abundance. We observed a fourfold increase in vimentin transcripts in the cytoplasm of serum-stimulated ts13 cells. Since transcripts are stable in both quiescent and stimulated cells, this induction of vimentin expression is a result of a fivefold increase in vimentin-specific transcriptional activity. As a result of this increased transcript availability, the abundance of polymerized vimentin protein increased following serum stimulation of quiescent fibroblasts. Overall, the induction of vimentin expression in fibroblasts by serum is a consequence of increased vimentin-specific transcriptional activity. The significance of this with regard to cytoskeletal organization and cell division is discussed.


Density-dependent modulation of vascular smooth muscle alpha-actin biosynthetic processing in differentiated BC3H1 myogenic cells. The expression of vascular smooth muscle (VSM) alpha-actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non-proteolytic dissociation of cell-cell and cell-matrix contacts in post-confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA-dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal alpha-actin mRNA. Muscle alpha-actin mRNA levels in EDTA-dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse-labeling techniques using L-[35S]cysteine to identify muscle actin biosynthetic intermediates revealed that EDTA-dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle alpha-actin polypeptide chains. However EDTA-dispersed myocytes were less efficient in the post-translational processing of immature VSM alpha-actin compared to non-dispersed myocytes. Simple cell-to-cell contact may mediate VSM alpha-actin processing efficiency since high-density preparations of EDTA-dispersed myocytes processed more VSM alpha-actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mechanisms capable of discriminating between different isoform classes of nascent actin polypeptide chains.


HMG-like chromosomal proteins in Trypanosoma cruzi. HMG-like chromosomal proteins from Trypanosoma cruzi were studied. Four HMG-like proteins, designated HMG A, HMG-B, HMG-C, and HMG-E, were isolated and found to have molecular weights of 35.5 kd, 27.5 kd, 21.8 kd and 10.4 kd, respectively. Immunological relatedness was demonstrated between the mammalian HMG 1,2 and the HMG-A and HMG-B from T. cruzi. The relative amounts of HMG-C and HMG-E proteins vary in T. cruzi depending to the proliferative stage of the cells. HMG-E protein is increased in proliferating cells when compared to its level in non-proliferating cells. HMG-C is increased in the non-proliferating cells. Probably, the shifts observed in the relative amounts of HMG-like proteins are related to the proliferating cells of this flagellate. The results are consistent with those described for other lower eukaryotes where the HMG-like proteins isolated are similar but not identical to HMG proteins from vertebrates.


Transforming growth factor-beta 1 induces expression of statin during differentiation of human promonocytic leukemia cells. Transforming growth factor-Beta (TGF-beta) is a potent growth inhibitor for several cell types including epithelial cells and hematopoietic progenitor cells. Using a human promonocytic leukemia cell line, THP-1, we have shown that TGF-beta inhibits their proliferation and promotes differentiation into cells exhibiting macrophage-like properties. Therefore, a key question is whether TGF-beta influences the expression of genes associated with proliferation and/or growth inhibition. TGF-beta treatment of THP-1 cells results in downregulation of expression of c-myc. We also observe that TGF-beta 1-treated cells express reduced levels of the cell cycle regulated histone, H2B, but express elevated levels of an RNA splicing variant of this histone that has been observed to be upregulated in growth inhibited and terminally differentiated cells. In addition, a nuclear protein associated with senescence and withdrawal of cells from the cell cycle, statin, is also expressed by THP-1 cells in response to TGF-beta 1 treatment. These results suggest that TGF-beta 1 is capable of inducing expression of specific nuclear proteins associated with differentiation and/or cessation of proliferation that may result in changes in nuclear organization and altered gene expression. Such changes in nuclear organization may be incompatible with continued proliferation of the cells.


Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large-cell lymphoma cells possess transcription activities and regulatory properties in vitro. Intact nuclei derived from poorly or highly liver-metastatic murine large-cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes with Msp-I. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase-I and subsequent isolation by two-dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two-dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence of v-abl, p53, c-neu, c-H-ras, beta-casein, 18s rDNA, and mu-chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associated p53 DNA, whereas six contained c- or v-abl, four contained mu-chain gene, two contained c-H-ras, one contained dot-blot beta-casein, two contained 18s rDNA, and c-neu was found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS-PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back-hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from approximately 80 kbp to approximately 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of M(r) approximately 140,000, isoelectric point (pl) approximately 5.8 synthesized a high molecular weight RNA in vitro that hybridized with beta-casein cDNA, but beta-casein is not expressed in RAW117 cells, suggesting that the silencing of the beta-casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of beta-casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expression or regulation of RNA polymerases in vitro.