Myofibrillogenesis in living cells microinjected with fluorescently labeled alpha-actinin.

Fluorescently labeled alpha-actinin, isolated from chicken gizzards, breast muscle, or calf brains, was microinjected into cultured embryonic myotubes and cardiac myocytes where it was incorporated into the Z-bands of myofibrils. The localization in injected, living cells was confirmed by reacting permeabilized myotubes and cardiac myocytes with fluorescent alpha-actinin. Both living and permeabilized cells incorporated the alpha-actinin regardless of whether the alpha-actinin was isolated from nonmuscle, skeletal, or smooth muscle, or whether it was labeled with different fluorescent dyes. The living muscle cells could beat up to 5 d after injection. Rest-length sarcomeres in beating myotubes and cardiac myocytes were approximately 1.9-2.4 microns long, as measured by the separation of fluorescent bands of alpha-actinin. There were areas in nearly all beating cells, however, where narrow bands of alpha-actinin, spaced 0.3-1.5 micron apart, were arranged in linear arrays giving the appearance of minisarcomeres. In myotubes, alpha-actinin was found exclusively in these closely spaced arrays for the first 2-3 d in culture. When the myotubes became contraction-competent, at approximately day 4 to day 5 in culture, alpha-actinin was localized in Z-bands of fully formed sarcomeres, as well as in minisarcomeres. Video recordings of injected, spontaneously beating myotubes showed contracting myofibrils with 2.3 microns sarcomeres adjacent to noncontracting fibers with finely spaced periodicities of alpha-actinin. Time sequences of the same living myotube over a 24-h period revealed that the spacings between the minisarcomeres increased from 0.9-1.3 to 1.6-2.3 microns. Embryonic cardiac myocytes usually contained contractile networks of fully formed sarcomeres together with noncontractile minisarcomeres in peripheral areas of the cytoplasm. In some cells, individual myofibrils with 1.9-2.3 microns sarcomeres were connected in series with minisarcomeres. Double labeling of cardiac myocytes and myotubes with alpha-actinin and a monoclonal antibody directed against adult chicken skeletal myosin showed that all fibers that contained alpha-actinin also contained skeletal muscle myosin. This was true whether alpha-actinin was present in Z-bands of fully formed sarcomeres or present in the closely spaced beads of minisarcomeres. We propose that the closely spaced beads containing alpha-actinin are nascent Z-bands that grow apart and associate laterally with neighboring arrays containing alpha-actinin to form sarcomeres during myofibrillogenesis.

Realizing the potential of clinical judgment: a real-time strategy for predicting outcomes and cost for medical inpatients.

PURPOSE: We sought to determine whether illness severity and anticipated level of function, as evaluated at the time of admission, were associated with outcomes and costs of care for patients admitted to the medical service. METHODS: All 1,759 patients admitted to the medical service at a large urban academic medical center between July 1, 1997, and September 30, 1997 (excluding those admitted directly to the intensive care units or for protocol chemotherapy), were evaluated and categorized by the admitting intern by illness severity (not ill, mildly ill, moderately ill, severely ill, or moribund) and anticipated level of function at discharge (excellent, good, fair, or poor) as part of their routine sign-out process. Interns' ratings were always available within 24 to 28 hours of admission. In-hospital mortality, length of stay, cost of hospitalization, and anticipated billing revenue were evaluated. RESULTS: Patients who were more severely ill had significantly greater in-hospital mortality. For example, mortality was 1.1% (11 of 972) among those who were not ill or mildly ill, 3.6% (26 of 724) among those who were moderately ill, and 15% (9 of 60) among those who were severely ill. Illness severity (P = 0.003) and anticipated functional status (P < 0.01) were significant predictors of in-hospital mortality. Illness severity and function were also significant predictors of greater length of stay and greater costs of hospitalization (all P < 0.0001). The 389 patients who were moderately ill with fair or poor anticipated function were associated with the largest cumulative losses (about $330,000 during the 3-month period), whereas the 798 mildly ill patients with good or excellent function were associated with the largest cumulative profits ($550,000). CONCLUSION: Physicians' estimates of patients' illness severity and anticipated function at the time of discharge, as made by interns using a system designed to help them sign out to their colleagues, predict outcomes and costs of hospitalization. Such a system may be useful in developing new approaches to management strategies based on prognosis.

Safety and efficacy of two reduced dosing regimens of sodium phosphate tablets for preparation prior to colonoscopy.

OBJECTIVES: To evaluate the safety and efficacy of two reduced dosing regimens of sodium phosphate tablets (Visicol, InKine Pharmaceutical Co. Inc., Blue Bell, PA, USA) for colon cleansing prior to colonoscopy. METHODS: In a randomized, multicentre, endoscopist-blinded clinical study, adults undergoing colonoscopy received either 28 tablets (42 g) or 32 tablets (48 g) of sodium phosphate for colon cleansing. The endoscopist used a validated four-point scale to rate the overall quality of colon cleansing, as well as cleansing in the ascending colon. Adverse events were collected and evaluated. RESULTS: The quality of overall colon cleansing was 'excellent' or 'good' in 84% or more of both groups, with no significant difference between the two doses. No patient had a preparation rated as 'inadequate' or required a repeat procedure. All patients were able to complete the assigned dose of tablets, and there were no deaths, serious adverse events or dropouts from the study. CONCLUSIONS: A reduced tablet regimen for sodium phosphate tablets, using either 28 or 32 tablets, is well tolerated and effective for colon cleansing prior to colonoscopy.

The effect of probiotics on Clostridium difficile diarrhea.

Clostridium difficile is the leading cause of nosocomially acquired intestinal infection in the United States, affecting virtually all cases of pseudomembranous colitis and up to 20% of cases of antibiotic-associated diarrhea. Even after receiving antibiotic treatment with either metronidazole or vancomycin, 20% of patients will have recurrent Clostridium difficile diarrhea. An innovative approach to the problem involves the introduction of competing, nonpathogenic (probiotic) organisms into the intestinal tract to restore microbial balance. The theoretical premise behind this approach is that the protective intestinal microflora is damaged by antibiotic treatment; the initial antibiotic exposure thus leaves the host susceptible to colonization and subsequent infection by Clostridium difficile. A so-called "second-hit" to the intestinal microflora occurs when the infected host is treated with flagyl or vancomycin, further destroying susceptible bacterial flora. Probiotic agents, such as Lactobacillus GG and Saccharomyces boulardii, have been studied for the treatment of Clostridium difficile. We are currently running a prospective, randomized, placebo-controlled trial of Lactobacillus GG in combination with standard antibiotics for the treatment of Clostridium difficile infection. Although it is too early to draw statistically significant conclusions, two patterns seem to be emerging: Lactobacillus GG is effective in reducing the 3-wk recurrence rate of Clostridium difficile, and patients feel better when taking Lactobacillus GG, as compared with the placebo, with early disappearance of abdominal cramps and diarrhea. In conclusion, the use of probiotics for the treatment of primary and recurrent Clostridium difficile diarrhea looks promising. Patients seem to have less recurrent Clostridium difficile diarrhea and early symptomatic improvement when using the probiotic Lactobacillus GG.

Enteroclysis: a multidisciplinary approach.

We developed a method for passing the enteroclysis catheter at endoscopy in patients requiring upper endoscopy and enteroclysis. This method reduced patient discomfort, the time needed for fluoroscopic tube placement, and overall radiation doses. We conclude that endoscopy and enteroclysis performed together is practical in these patients.

Treatment of complicated sarcoidosis with infliximab anti-tumor necrosis factor-alpha therapy.

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) may have an important role in the clinical exacerbation of sarcoidosis. OBJECTIVE: To treat sarcoidosis with infliximab, a chimeric human-murine anti-human TNF-alpha monoclonal antibody. DESIGN: Case report. SETTING: U.S. academic medical center. PATIENT: A 72-year-old woman with sarcoidosis presenting with severe protein-losing enteropathy, hypoalbuminemia, and proximal myopathy who had not responded adequately to corticosteroid therapy and whose clinical course was further complicated by acute tubular necrosis and renal failure requiring long-term hemodialysis. INTERVENTION: Intravenous infusion of infliximab, 5 mg/kg of ideal body weight; infusion was repeated at 2 and 6 weeks. MEASUREMENTS: Clinical response of enteropathic and myopathic symptoms and serum albumin level. RESULTS: Enteropathic and myopathic symptoms resolved after infliximab therapy, and the serum albumin level also improved. However, the clinical course was complicated by the development of a hypercoagulable state associated with circulating anticardiolipin antibodies, which prompted discontinuation of infliximab therapy. CONCLUSIONS: Infliximab therapy was successful in a patient with sarcoidosis. Tumor necrosis factor-alpha may be an important mediator of clinical disease in sarcoidosis and could be an attractive target for therapeutic intervention. However, infliximab may cause adverse effects associated with cytokine cascade manipulation.

Microinjection of Lucifer yellow CH into sea urchin eggs and embryos.

Eggs and embryos of Arbacia punctulata were microinjected with the fluorescent dye, Lucifer yellow CH, using a simple pressure injection system. When injected into eggs that were subsequently fertilized, the dye was distributed throughout all cells of the developing embryo. If one cell of a two-cell embryo was injected, dye did not diffuse into the uninjected blastomere. During subsequent development, all progeny of the injected cell contained dye resulting in an embryo that was half-fluorescent. Blue light irradiation of a two-cell embryo, one cell of which had been injected with Lucifer yellow, caused the injected blastomere to stop further divisions while the uninjected blastomere developed normally and was free of dye. These results indicate that the first two blastomeres of Arbacia embryos are not electrically coupled, nor up to the time of hatching, is there any coupling between cells in one half of the first cleavage plane and cells in the other half.

Midbody sealing after cytokinesis in embryos of the sea urchin Arabacia punctulata.

Cytokinesis consists of a contractile phase followed by sealing of the connecting midbody to form two separated cells. To determine how soon the midbody sealed after cleavage furrow contraction, the fluorescent dye Lucifer Yellow CH(457.3 M.W.) was microinjected into cells at various intervals after cleavage had begun. Mitotic PtK2 cells were recorded with video-microscopy so that daughter cells in the epithelial sheet could be identified for several hours after cell division. One daughter cell of each pair followed was microinjected to determine whether the dye diffused into the other daughter cell. For intervals up to four hours after the beginning of cytokinesis, diffusion took place between daughter cells. After this time the dye did not spread between daughter cells. In sea urchin blastomeres of the first, second and third divisions, Lucifer Yellow passed between daughter blastomeres only during the first 15 min after cytokinesis. If one cell of a two-cell, four-cell or eight-cell embryo was microinjected more than 15 min after the last cleavage, the dye remained in the injected cell and was distributed to all progeny of that cell, resulting in blastulae that were eigher one-half, one-quarter or one-eighth fluorescent, respectively. Thus, although cleavage furrow contraction takes approximately the same amount of time in sea urchin blastomeres and PtK2 cells, the time of midbody sealing differs dramatically in the two cell types. Our results also indicate the importance of knowing the mitotic history of cells when injecting dyes into interphase cells for the purpose of detecting gap junctions.

Observations of microfilament bundles in living cells microinjected with fluorescently labelled contractile proteins.

Fluorescently labelled contractile proteins (alpha-actinin and filamin) were used to study the dynamic nature of three types of microfilament bundles: myofibrils, stress fibres and polygonal networks. Cultured muscle and non-muscle cells that were microinjected with fluorescent alpha-actinin rapidly incorporated the labelled protein into Z-bands, stress fibre densities and the polygonal foci. Living, injected cells were then observed for varying periods of time, and changes in orientation and periodicity of the myofibrils, stress fibres and polygonal networks were recorded. Permeabilized cells were also reacted with fluorescently labelled proteins and with contractile protein antibodies in order to analyse further the changes taking place in the myofibrils and stress fibres. In both living cardiac myocytes and living skeletal muscle myotubes, contractile myofibrils were present in the same cell with non-contractile nascent myofibrils. The periodicities of small Z-bodies in the nascent non-contractile myofibrils were shorter than the Z-band spacings in the contractile myofibrils, yet both types of myofibrils contained muscle myosin. Over a period of 24 h, a nascent myofibril in a living, microinjected myotube was observed to grow from Z-body spacings of 0.9-1.3 micron to full sarcomere spacings (2.3 microns). During the same time, nascent myofibrils appeared de novo and Z-band alignment became more ordered in the fully formed myofibrils. Stress fibres were not observed to undergo the predictable type of growth seen in myofibrils, but stress fibre periodicities did change in some fibres; some shortened while others lengthened. The orientation of fibres shifted in cytoplasm of both mobile cells and stationary cells. Attachment plaques and foci also changed position and in some cases subdivided and/or disappeared. Models of stress fibres and polygonal networks are presented that suggest that the changes in the periodicities of the dense bodies in stress fibres and the distances between polygonal foci are related to the movement of the interdigitating actin and myosin filaments.

Fecal occult blood testing in hospitalized patients.

We have evaluated the diagnostic value of the fecal occult blood test (FOBT) in hospitalized patients. We reviewed the medical records of patients who had a positive FOBT not associated with a large gastrointestinal bleed, and who had a subsequent complete evaluation of their gastrointestinal (GI) tract. Of the 50 subjects who met the study criteria, 21 had various GI symptoms and 13 reported weight loss. Patients taking medications that may influence the FOBT result were distributed as follows: 15 were taking nonsteroidal antiinflammatory drugs, eight were taking iron supplementation, three were using steroid drugs, and three were taking anticoagulant drugs. Nonneoplastic lesions were found in 47 patients. Neoplastic lesions were discovered in 11 patients: seven had adenomatous polyps, two had colorectal cancer, one had gastric cancer, and one had duodenal cancer. Only two of seven patients with adenomatous polyps had lesions > 1 cm. In the study population, the positive predictive value of FOBT for finding colonic neoplasms was 18% and for any GI neoplasm it was 22%. Our data indicate that in hospitalized patients (a) the yield of colonic neoplasms from FOBT is approximately 50% less than that in healthy outpatients, and (b) a positive FOBT test is unlikely to lead to the detection of GI malignancy in the absence of suggestive clinical findings.

Stress fiber and cleavage furrow formation in living cells microinjected with fluorescently labeled alpha-actinin.

alpha-Actinins, isolated from muscle and nonmuscle sources and labeled with various fluorescent dyes, were microinjected into living PtK2 cells during interphase to observe the reformation of stress fibers following cell division. Fluorescently labeled ovalbumin and bovine serum albumin were also injected as control proteins. alpha-Actinin was incorporated into stress fibers within 5 minutes after injection and remained present in the fibers for up to 11 days. The pattern of incorporation was the same regardless of whether the alpha-actinin was isolated from muscle or nonmuscle tissues or whether it was labeled with fluorescein, Lucifer Yellow, or rhodamine dyes. In contrast, neither labeled ovalbumin nor bovine serum albumin were incorporated into stress fibers. When the injected cells entered prophase, all stress fibers disassembled, resulting in a distribution of the fluorescent alpha-actinin throughout the cytoplasm. During cytokinesis, the fluorescent alpha-actinin was concentrated in the broad area between the separated chromosomes and along the edge of the cell in the cleavage area. Within 10 minutes after the completion of cleavage, the first fluorescent stress fibers reformed parallel to the spreading edges of the daughter cells and in close association with the midbody with a concomitant loss of alpha-actinin in the former cleavage furrow. Additional fibers formed adjacent to these first stress fibers. In some cases, new stress fibers formed between two existing stress fibers and some stress fibers moved up to 4 micron apart from one another in the course of 2 hours. Thus, fluorescent alpha-actinin, injected into living cells, undergoes the same cyclical changes in distribution as endogenous alpha-actinin during the cell cycle: from stress fibers to cleavage furrow and back to stress fibers.

Incorporation of fluorescently labeled actin and tropomyosin into muscle cells.

The two major proteins in the I-bands of skeletal muscle, actin and tropomyosin, were each labeled with fluorescent dyes and microinjected into cultured cardiac myocytes and skeletal muscle myotubes. Actin was incorporated along the entire length of the I-band in both types of muscle cells. In the myotubes, the incorporation was uniform, whereas in cardiac myocytes twice as much actin was incorporated in the Z-bands as in any other area of the I-band. Labeled tropomyosin that had been prepared from skeletal or smooth muscle was incorporated in a doublet in the I-band with an absence of incorporation in the Z-band. Tropomyosin prepared from brain was incorporated in a similar pattern in the I-bands of cardiac myocytes but was not incorporated in myotubes. These results in living muscle cells contrast with the patterns obtained when labeled actin and tropomyosin are added to isolated myofibrils. Labeled tropomyosins do not bind to any region of the isolated myofibrils, and labeled actin binds to A-bands. Thus, only living skeletal and cardiac muscle cells incorporate exogenous actin and tropomyosin in patterns expected from their known myofibrillar localization. These experiments demonstrate that in contrast to the isolated myofibrils, myofibrils in living cells are dynamic structures that are able to exchange actin and tropomyosin molecules for corresponding labeled molecules. The known overlap of actin filaments in cardiac Z-bands but not in skeletal muscle Z-bands accounts for the different patterns of actin incorporation in these cells. The ability of cardiac myocytes and non-muscle cells but not skeletal myotubes to incorporate brain tropomyosin may reflect differences in the relative actin-binding affinities of non-muscle tropomyosin and the respective native tropomyosins. The implications of these results for myofibrillogenesis are presented.