FlowCytomix analysis for Toxoplasma gondii infection in pregnant women in central Taiwan.

The study was to determine the seroprevalence of toxoplasmosis in the sera of pregnant women in central Taiwan and to investigate the levels of cytokine in the sera of pregnant women with Toxoplasma gondii infection. The 220 blood samples were collected from pregnant women. The haematological parameters of peripheral blood were analysed by a haematology analyser. Serum samples of the pregnant women were analysed by a commercially available anti-T. gondii IgM/IgG antibody enzyme-linked immunosorbent assay (ELISA) kit and FlowCytomix assays. Six (2.7%) of the sera samples had IgM anti-T. gondii antibodies, and twenty (9.1%) had T. gondii IgG seropositive. All six IgM seropositive samples had low IgG avidity, indicative of acute infection. Total white blood cells and eosinophils were statistically significantly increased (p<0.05) in pregnant women with T. gondii infection, as compared with healthy pregnant women. Th1 cytokines IFN-γ, IL-1ß, IL-2 and IL-12 p70, and Th2 cytokines IL-10 in pregnant women with T. gondii IgM/IgG seropositive were significantly increased (p<0.05), as compared with healthy pregnant women. These results showed that both of Th1 and Th2 cytokines play an important role in the toxoplasmosis of pregnant women.

Induction of plasminogen activators in pregnant women with Toxoplasma gondii infection.

OBJECTIVE: To determine whether plasminogen activators (PAs) are involved in the pathologic process of toxoplasmosis. MATERIALS AND METHODS: Out of 220 pregnant women the study included 26 with a diagnosis of toxoplasmosis: six based on seropositivity for Toxoplasma gondii IgM and 20 based on seropositivity for T. gondii IgG. We measured serum activities and protein levels of PAs by casein zymography and Western blotting, respectively. RESULTS: Serum PAs were higher in healthy pregnant women than in their healthy nonpregnant counterparts. Furthermore, serum PAs were significantly higher in pregnant women infected with T. gondii than in their healthy counterparts. CONCLUSION: PAs participate in the pathogenesis of toxoplasmosis in pregnant women and may be useful markers of T. gondii infection.

Saturated saline enhances the effect of electrochemical therapy.

We conducted this experiment to assess the effect of saline injection in electrochemical therapy. Platinum electrodes using direct current were inserted into egg white or liver parenchyma. Pure water or 0.9%, 3%, or 26% sodium chloride were injected into various objects to compare with the control group (no injection). Power was set at 10 V. In the egg-white experiment, gas bubbles and coagulated protein developed around the electrodes. In ex vivo liver, frothy reddish debris developed around the cathodes, while a hardening and shrunken surface occurred around the anodes. The pH was 14 around the cathodes, 0 around the anodes. The electric current, the amount of coagulated protein, and the severity of tissue damage were all in proportion to the concentrations of the injected saline. The volume destroyed in the 26% saline group was 8.1 times larger than that of the control group. Therefore, injected saline, especially saturated saline, can enhance the effect of electrochemical therapy.

Neurogenic intermittent claudication related to spinal stenosis.

BACKGROUND: The pathophysiology of neurogenic intermittent claudication (NIC) is not very well understood. We examined the theory of "two-level" spinal stenosis as the pathologic cause of NIC. METHODS: Forty-five consecutive patients with NIC were chosen for the study group and another 45 patients with chronic low back pain, but without NIC, were consecutively chosen for the control group. All the patients were examined using several methods that included a personal history survey, physical examination, X-ray studies, computed tomography (CT) and magnetic resonance imaging (MRI). RESULTS: Two-level central canal stenosis was found on MRI in 88.9% of the study group compared to only 37.8% of the control group (Fisher's exact two-tailed test, P = 0.0000). In the study group, 77.8% of the patients had severe or moderate facet joint degeneration on CT. The remaining 22.2% of patients had mild facet joint degeneration. In the control group, 40% of the patients had severe or moderate facet joint degeneration, while it was mild or normal in the remaining 60% (Fisher's exact two-tailed test, P = 0.0005). On X-ray, 42.2% of the study group compared with 17.7% of the control group had spondylolisthesis (Fisher's exact two-tailed test, P = 0.0129). In 35% of the study group compared with 11.1% of the control group, X-ray findings showed scoliosis (Fisher's exact two-tailed test, P = 0.0115). The lumbosacral angle in the study group was 35.8 degrees +/- 6.1 degrees compared to 33.1 degrees +/- 7.5 degrees in the control group (independent Student's two-tailed t-test, P > 0.05). CONCLUSIONS: Our study strongly supports pathophysiology for NIC that is closely related to two-level spinal canal stenosis. Degenerative joint disease, spondylolisthesis and scoliosis of the lumbar spine are predisposing factors of NIC.

An anti-CD45RO immunotoxin eliminates T cells latently infected with HIV-1 in vitro.

Despite the success of highly active antiretroviral therapy (HAART) in lowering circulating HIV-1 to undetectable levels in most infected individuals, several studies have documented the presence of a small reservoir of latently infected cells in HAART patients, the majority of which are CD45RO(+) memory T cells. We previously have demonstrated that latently infected, replication-competent cells can be generated in vitro after eliminating CD25(+) cells with an immunotoxin (IT). The present study was designed to determine whether these latent cells could be eliminated by an anti-CD45RO IT. Our results indicate that the anti-CD45RO IT eliminates >99%, of either M-tropic or T-tropic virus produced by the latently infected cells after mitogen stimulation. This IT also appears to be as effective as the anti-CD25 IT in eliminating the activated, HIV-1-producing cells. In contrast, the anti-CD45RO IT does not kill CD45RA(+) naive cells. Further studies using cells from HIV-1-infected individuals on HAART will be necessary to determine the potential clinical utility of this IT.

RHD gene polymorphisms among RhD-negative Chinese in Taiwan.

BACKGROUND AND OBJECTIVE: The rare occurrence of anti-D-associated hemolytic disease of the newborn among Chinese is attributable in part to the existence of the weak D phenotype Del among apparently RhD-negative individuals. While existing advances in the molecular genetics of the Rh blood group have been noted in recent years, the genomic structure of the Del phenotype has seldom been studied in the literature. We try to explore the genomic structure of the RhD gene among apparently Rh-negative Chinese in Taiwan in this study. METHODS: Genomic DNA from 230 samples of apparently RhD-negative Chinese was studied using four polymerase chain reaction (PCR)-based RhD typing methods. These PCR methods amplified RHD and RHCE genes at exons 4, 5, 7 and 10. All nucleotides responsible for exofacial amino acid differences between RhD and RhCeEe peptides, including amino acids 169, 170, 172, 223, 226, 233, 238, 350, 353, and 354, were contained in these amplified DNA segments. Southern blot analysis using RHD cDNA fragments as probes was performed. RESULTS: According to the serological study, 155 samples (67.4%) were genuinely RhD-negative and 75 samples (32.6%) were of the Del phenotype. Successful amplifications for RHD sequences were possible in all 75 Del samples using four PCR methods. Apparently, all Del individuals carried an intact RHD gene. While 145 individuals of 155 genuinely Rh-negative (63.0% of apparently RhD-negative individuals) had total deletion of their RHD genes, 10 individuals (4.3% of apparently RhD-negative individuals) were shown to have a preserved 3' noncoding region of the RHD exon 10 and a gross deletion of RHD exons 4-10. CONCLUSIONS: Three classes of RhD-negative polymorphisms among Chinese in Taiwan were observed. These included Del with grossly intact RHD and weak RhD expression, genuinely RhD-negative with partial preservation of the RHD gene, and genuinely RhD-negative with total deletion of the RHD gene. A molecular study is warranted to clarify the mechanism responsible for the weak RHD gene expression in Del individuals.

Glucocorticoids modulate human gonadotrophin releasing hormone upstream promoter activity in transfected human placental cells (JEG-3).

A human gonadotrophin releasing hormone (GnRH) upstream promoter/luciferase reporter gene construct (H2 construct) was generated by inserting a 1.7 kb XbaI/AflII fragment containing the human GnRH upstream promoter region only into a promoter-less luciferase reporter vector. When JEG-3 cells were transiently transfected with this construct and treated with cortisol or its synthetic analogue dexamethasone, a stimulatory effect on the upstream promoter activity was observed. This stimulation was dependent on the cotransfection of a glucocorticoid receptor (GR) cDNA expression vector due to the low level of GR in JEG-3 cells and could be completely abolished by RU486, a glucocorticoid antagonist. Moreover, the cortisol actions could be modulated to a different extent by oestradiol. Thus, since the human placenta contains GRs and the increase in cortisol metabolism near term is regulated by oestrogen, the current findings suggest that cortisol may be physiologically involved in the regulation of GnRH gene expression in the human placenta.

Highly purified CD25- resting T cells cannot be infected de novo with HIV-1.

Previous studies have demonstrated that the expression of CD25 can distinguish CD25- latently infected cells from CD25+ cells actively producing virus. Our studies were designed to characterize the nature and stability of the viral genome in CD25- quiescent HIV-1-infected cells and to determine whether these cells could be infected de novo with HIV-1. Our results show that: (i) When unfractionated peripheral blood mononuclear cells are first infected with HIV-1 and the CD25- cells then isolated, the latter contain only incomplete DNA transcripts and no full-length DNA or 2-LTR circles. Phytohemagglutinin activation of these CD25- cells results in the generation of full-length viral DNA and p24 production. (ii) When CD25- CD4+ cells are first purified from peripheral blood mononuclear cells and then incubated with HIV-1, viral DNA cannot be detected, suggesting that these purified cells cannot be infected. Furthermore, CD25-adherent cells do not facilitate the infection of CD4+ CD25- T cells when they were present at the time of incubation with HIV-1. Taken together, these studies suggest either that (i) the CD25- cells containing incomplete DNA transcripts are derived from infected-activated CD25+ cells, which subsequently become CD25- or (ii) the presence of CD25+ cells is required for the infection of CD25- cells in vitro.

The use of cyclosporine, FK506, and SDZ NIM811 to prevent CD25- quiescent peripheral blood mononuclear cells from producing human immunodeficiency virus.

It has been shown that the combined use of two pharmacologic agents can inhibit human immunodeficiency virus (HIV) production by peripheral blood mononuclear cells in vitro. One, an anti-CD25 immunotoxin (IT), kills activated T cells that produce virus; the other, the immunosuppressive drug cyclosporine, prevents the quiescent cells, which harbor HIV, from becoming activated. The present study compares the antiviral activities of two agents, SDZ NIM811 and FK506, to that of cyclosporine. In combination with the anti-CD25 IT, these drugs significantly suppressed virus production. In the absence of prior addition of the IT, the ability of the drugs to inhibit virus production was much lower, suggesting that they work effectively in latently infected cells. In the case of SDZ NIM811, the inhibition of virus production was accompanied by a modest inhibition of cell proliferation. In contrast, FK506 exerted strong antiproliferative activity. Cyclosporine was both moderately antiproliferative and a potent antiviral agent.

Expression of CD25 defines peripheral blood mononuclear cells with productive versus latent HIV infection.

The present studies were designed to further determine whether the CD25 marker could distinguish between cells productively and latently infected with HIV. This was accomplished by combining immunotoxin (IT)-mediated killing of CD25+ cells, highly sensitive indirect immunofluorescence to detect remaining CD25+ cells, and PCR-mediated amplification of proviral DNA in immunotoxin-treated vs untreated HIV-infected cells. Our results demonstrate that: 1) By direct immunofluorescence 3 to 8% of PBMCs are CD25+, whereas by indirect immunofluorescence 30% are CD25+. The increased number of CD25+ cells is due to their detection by the highly sensitive indirect immunofluorescence assay. Up to 60% of the CD25+ cells are CD4+ and 12% are CD8+. 2) Treatment of HIV-infected PBMCs with an anti-CD25 IT for 6 days eliminated both CD25high and CD25low cells and decreased the production of p24 by 99%. 3) Differences in the HIV proviral genome were detected in the unfractionated PBMCs vs PBMCs from which CD25+ cells had been eliminated by IT treatment. Hence, PBMCs containing both CD25+ and CD25- cells express all intermediate proviral species and full-length double-stranded proviral DNA. In contrast, CD25- quiescent cells contain predominantly intermediate species. These results confirm and extend our previous observations that expression of CD25 can distinguish latently infected cells from cells producing virus.

Expression of IL-2 and IL-4 in T lymphocytes transformed by herpesvirus saimiri.

Herpesvirus saimiri (H. saimiri) is a highly oncogenic lymphotropic herpesvirus which can immortalize T lymphocytes and cause tumors in rabbits and New World monkeys. T cells infected with strain 484-77 of group C express four viral U-like small RNAs (HSUR1-4) and a 1.2-kb mRNA which encodes open reading frames ORF-1 and ORF-2. ORF-1 encodes a collagen-like oncoprotein. Deletion mutation analysis showed that ORF-1 and ORF-2 are essential for IL-2 independent growth of human T cells infected with H. saimiri. An earlier study also demonstrated that H. saimiri-immortalized cells carry functional IL-2 receptors. The work presented in this report investigated whether IL-2 and IL-4 is produced by H. saimiri-immortalized T lymphocytes. Both IL-2 mRNA and IL-4 mRNA were detected in various monkey T cells as well as human peripheral blood lymphocytes infected with wild-type H. saimiri. Secretion of IL-2 was suggested by cyclosporin A inhibition. IL-4 secretion by monkey T cell cultures was demonstrated by a bioassay and inhibition of bioactivity by an antibody to IL-4. The data also show that recombinant IL-4 stimulate H. saimiri-immortalized T cells; thus, IL-4 receptors are expressed. However, antibodies to human IL-4, IL-4 receptor, or soluble IL-4 receptor did not curtail growth of transformed cells. T cells infected with ORF-1 and ORF-2 deletion mutants expressed no detectable IL-2 mRNA. ORF-1, ORF-2, HSUR1, and HSUR2, were all essential for expression of IL-4 mRNA. These data are consistent with the hypothesis that H. saimiri-immortalized monkey and human T lymphocytes proliferate through autocrine secretion of IL-2 and that ORF-1, ORF-2, and HSUR sequences of the virus are involved in expression of lymphokines.

Induction of a herpesvirus saimiri small RNA AU binding factor (AUBF70) activity and lymphokine mRNAs by T cell mitogens.

Herpesvirus saimiri (H. saimiri) can transform T lymphocytes and cause lymphoid tumors in rabbits and New World monkeys. H. saimiri-immortalized T cells express IL-2 and IL-4. The putative oncogenes of a group C strain of H. saimiri have been mapped to a region of the unique L-DNA which includes genes encoding four U-like small nuclear RNAs (HSUR1-HSUR4). Jurkat T cells express a 70 kD RNA binding factor (AUBF70) which binds HSUR2. Here we examined AUBF70 expression in resting and mitogen-stimulated human peripheral blood T cells and its sequence specificity and subcellular distribution. Band-shift assays demonstrated that resting human T cells express low amounts of AUBF70 which is induced by mitogen treatment. IL-2 and IL-4 mRNAs were co-induced with AUBF70 suggesting that AUBF70 is a positive regulator of lymphokine gene expression. Normal resting, mitogen-stimulated, and leukemic Jurkat T cells all express AUBF70 with virtually identical V8 proteolytic enzyme digestion patterns. Northern blots demonstrated that HSUR1 and HSUR2 are localized both in the nucleus and cytoplasm. HSUR2 accumulate in the cytoplasm in the presence of actinomycin D, which is consistent with re-transport of HSURs to the nucleus by (an) unstable factor(s). We hypothesize that HSUR1 and 2 transport AUBF70 from the cytoplasm to the nucleus; in the nucleus, AUBF70 binds and stabilizes lymphokine transcripts. Increased stability of lymphokine mRNAs could contribute to oncogenic transformation induced by H. saimiri.

Herpesvirus saimiri small RNA and interleukin-4 mRNA AUUUA repeats compete for sequence-specific factors including a novel 70K protein.

A highly oncogenic strain of the lymphotropic tumour virus herpesvirus saimiri (HVS; strain 484-77) expresses four small RNAs (HSUR1 to 4) in high copy numbers in transformed T cells. In HSUR1 and HSUR2 the 5' terminal regions contain conserved AUUUA sequence repeats. The same AUUUA repeats occur in the 3' non-coding regions of growth factor, lymphokine and protooncogene mRNAs, and the sequence is involved in rapid mRNA degradation. We report here that by using a highly specific u.v. cross-linking method we identified a novel 70K binding factor with AUUUA sequence specificity. Non-radiolabelled competition and V8 protease analysis show that the protein can form a complex with the 3' non-coding region of interleukin-4 mRNA and bind the AUUUA repeats of a HVS small RNA. We also detected an AUUUA-specific minor 32K human protein with the same electrophoretic mobility as a marmoset factor implicated in growth factor mRNA destabilization. The findings are consistent with the hypothesis that the viral small RNAs can compete for factors involved in rapid degradation of growth factor mRNAs and may contribute to viral oncogenesis.

Stress fractures of the ribs in amateur golf players.

BACKGROUND: Stress fractures of the ribs are sometimes seen at the Outpatient Department in patients with a history of playing golf enthusiastically. Many are diagnosed as "muscle strain" or "myofascial pain" and patients are simply advised to take some rest or are treated with analgesics and local injection. This case study investigated 11 amateur golfers whose chief complaint was anterior, posterior or lateral chest pain. After X-ray and bone scan evaluation, "stress fracture of the ribs" was diagnosed. A questionnaire presented to them trying to find the possible mechanisms of these stress fractures. Biomechanical analysis showed that the bending force of the ribs was located at posterolateral segments where fractures tend to occur. Overuse, poor technique and inadequate stretch in beginners are postulated as causes for apparent increased susceptibility to these skeletal injuries. METHODS: Questionnaires inquired about (1) warm-up time, (2) number of strikes, (3) fracture sites, (4) pain patterns, (5) combined injuries. RESULTS: All 11 patients were beginners with right side hand dominance who had begun to play golf within the year. Right side ribs fracture occurred in six cases; left side ribs fracture occurred in eight cases including three patients with two fracture sites. Localized pain was reported in six cases and there were five cases with radiating pain along costal margins. All the golfers had spent no more than 10 minutes in warm up them. Seven patients suffered from multiple injuries after they had played. Five were diagnosed by X-ray and six showed positive finding after Tc-99m MDP bone scan. All lesions were located at the posterolateral segments of the ribs. CONCLUSIONS: Stress fractures of the ribs in amateur golfers are certainly not uncommon. Predominant muscle forces are generated by forced coupling of scapular retraction and protraction, acting through the serratus anterior. With early diagnosis and relative rest for four to eight weeks, the pain will improve. Overuse, poor technique and inadequate stretch will probably lead to stress fracture of the rib.

Functional approach to treatment of back pain in primary care: a preliminary report.

BACKGROUND: Back pain is one of the most common problems encountered in primary care clinics and causes great loss of work time, economy and expenditure of medical care. Knowledge of the cause of low back pain and effective treatment are rudimentary. We studied the functional approach to treatment of back pain of two acute (M:F = 1:1) and thirty chronic (M:F = 12:18) patients with back pain seen at a rural primary care clinic in September and October 1992. METHODS: Patients were included after completing an agreement form regarding the treatment plan (emphasizing therapeutic exercise, health education and as little medication as possible) in the first interview. A primary care physician specialised in family medicine personally gave treatment to the patients with free attendance to the clinic. A functional approach was adopted in four categories-therapeutic exercise, static and dynamic body biomechanics, awareness of proprioception, and maintenance of relaxed and balanced muscle tone. The patients were encouraged to perform daily activities rather than rest. RESULTS: Two patients with acute back pain gained complete relief without medication, modalities, equipment or other treatment one and two weeks after the first visit, respectively. Among the 30 chronic patients, after eight weeks of treatment, 12/30 (40%) gained complete relief from pain, 4/30 (13.3%) obtained more than 3/4 and less than complete relief, 7/30 (23.3%) had more than 1/2 and less than 3/4 relief, 2/30 (6.7%) gained less than 1/2 pain relief, 1/30 (3.3%) had no improvement, 1/30 (3.3%) deteriorated, and 3/30 (10%) were lost during surveillance. The patients reported no loss of work time attributed to back pain. Two acute patients visited the clinic four times and three times, respectively. The former received three prescriptions for an upper respiratory infection one month after complete remission of back pain, and the latter received no prescriptions for medication. On average, each chronic patient visited the clinic 4.83 times in 8 weeks. In only 25 of 145 (17.2%) visits was medication prescribed for other indications, and in 8 of 145 (5.5%) visits vitamin B complex or Alinamin-F for placebo was prescribed. We used neither modalities nor equipment. Only 7/30 (23.3%) patients received concurrent treatment from other medical institutions. Regarding compliance with the programmed exercise at home, two acute patients complied well. Among the 30 chronic patients, 22 (73.3%) had good compliance, 3 (10%) had poor compliance, 2 (6.7%) failed to comply and 3 (10%) were lost in follow up. CONCLUSIONS: Treating patients with back pain in primary care clinics with a functional approach by physicians appropriately trained in primary care was a good, efficacious, economic, and practical way, although neglected elsewhere. Further research and practice in our society might be worth while.

Production and characterization of monoclonal antibodies identifying breast tumor-associated antigens.

We have generated a mouse monoclonal antibody (H23) against the retrovirus-like particles (human mammary tumor virus) released in vitro by the human breast adenocarcinoma cell line T47D. This antibody reacts specifically with a glycoprotein with an apparent molecular mass of 68 kDa (gp68) that is detected in the growth medium of T47D cells as well as in pleural effusion fluids from breast adenocarcinoma patients. No detectable levels of this antigen could be observed in pleural effusions of patients with cancers other than of breast origin. The H23-related antigen was localized in the cytoplasm of breast tumor cells as well as on the cell surface of both T47D cells and metastatic cells from breast cancer patients. A survey of tissue from 812 patients was performed by using H23 in an indirect immunoperoxidase assay. The results showed that the antigen was detectable in 91% of all breast tumors tested. No cytoplasmic staining was observed in either normal tissues or nonbreast carcinomas. Only one of the benign breast tissues tested (out of a total of 56 samples of tissue) was positive for this antigen. Given the ability of this antibody to specifically detect breast tumor cells, H23 may be of importance in diagnosis and in clinical follow-up of patients for the detection of metastatic lesions by imaging and for therapy.

Three-phase bone scintigraphy in Pellegrini-Stieda disease.

In a patient with Pellegrini-Stieda disease, radiographs of the knees were unremarkable at the time the three-phase bone scintigraphy was abnormal. The results of follow-up radiographs three months later remained normal in the left knee, where local steroid injection was given, but revealed typical positive results in the right knee with no treatment. The three-phase bone scintigraphic pattern is rather typical and antedates the radiographic changes. Thus, the radionuclide technique would provide a useful procedure for the early diagnosis and treatment of Pellegrini-Stieda disease.

Tropomyosin isoforms in chicken embryo fibroblasts: purification, characterization, and changes in Rous sarcoma virus-transformed cells.

Seven polypeptides (a, b, c, 1, 2, 3a, and 3b) have been previously identified as tropomyosin isoforms in chicken embryo fibroblasts (CEF) (Lin, J. J.-C., Matsumura, F., and Yamashiro-Matsumura, S., 1984, J. Cell. Biol., 98:116-127). Spots a and c had identical mobility on two-dimensional gels with the slow-migrating and fast-migrating components, respectively, of chicken gizzard tropomyosin. However, the remaining isoforms of CEF tropomyosin were distinct from chicken skeletal and cardiac tropomyosins on two-dimensional gels. The mixture of CEF tropomyosin has been isolated by the combination of Triton/glycerol extraction of monolayer cells, heat treatment, and ammonium sulfate fractionation. The yield of tropomyosin was estimated to be 1.4% of total CEF proteins. The identical set of tropomyosin isoforms could be found in the antitropomyosin immunoprecipitates after the cell-free translation products of total poly(A)+ RNAs isolated from CEF cells. This suggested that at least seven mRNAs coding for these tropomyosin isoforms existed in the cell. Purified tropomyosins (particularly 1, 2, and 3) showed different actin-binding abilities in the presence of 100 mM KCl and no divalent cation. Under this condition, the binding of tropomyosin 3 (3a + 3b) to actin filaments was significantly weaker than that of tropomyosin 1 or 2. CEF tropomyosin 1, and probably 3, could be cross-linked to form homodimers by treatment with 5,5'-dithiobis-(2-nitrobenzoate), whereas tropomyosin a and c formed a heterodimer. These dimer species may reflect the in vivo assembly of tropomyosin isoforms, since dimer formation occurred not only with purified tropomyosin but also with microfilament-associated tropomyosin. The expression of these tropomyosin isoforms in Rous sarcoma virus-transformed CEF cells has also been investigated. In agreement with the previous report by Hendricks and Weintraub (Proc. Natl. Acad. Sci. USA., 78:5633-5637), we found that major tropomyosin 1 was greatly reduced in transformed cells. We have also found that the relative amounts of tropomyosin 3a and 3b were increased in both the total cell lysate and the microfilament fraction of transformed cells. Because of the different actin-binding properties observed for CEF tropomyosins, changes in the expression of these isoforms may, in part, be responsible for the reduction of actin cables and the alteration of cell shape found in transformed cells.

Monoclonal antibodies against chicken tropomyosin isoforms: production, characterization, and application.

Eight mouse monoclonal antibodies, CH1, CH106, CH291, CL2, CG1, CG3, CG beta 2 and CG beta 6, against chicken tropomyosin isoforms have been prepared and characterized. The antigens recognized by these isoform-specific monoclonal antibodies were identified by both solid-phase radioimmunoassay and protein immunoblotting. To some extent, most antibodies showed isoform-specific, but one (CG3) recognized all isoforms of tropomyosin from chicken materials. The effects of monoclonal antibodies on the binding of cardiac tropomyosin to F-actin were investigated. Antibodies CH1, CH106, and CH291 had the ability to interfere with the binding of tropomyosin to F-actin, whereas others appeared to have no effect. Monoclonal antibody CL2 was able to distinguish the skeletal muscle tropomyosin-enriched microfilaments from the fibroblastic tropomyosin-enriched microfilaments of differentiating muscle cells. This antibody will be most useful for studying the compartmentalization of microfilaments and microfilament-associated proteins, particularly actin and tropomyosin isoforms during muscle differentiation. Immunofluorescence microscopy with CG1 antibody which recognized CEF tropomyosin isoforms 1 and 3 revealed the continuous staining of stress fibers in some populations of CEF cells. On the other hand, both periodic fluorescent staining and continuous staining of stress fibers were observed with CG3 antibody in all CEF cells.