Biomedical subjects
Publications and source records attributed to S C Liu.
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The malaria parasite Plasmodium falciparum undergoes distinct morphologic changes during its 48-h life cycle inside human red blood cells. Parasite proteinases appear to play important roles at all stages of the erythrocytic cycle of human malaria. Proteases involved in erythrocyte rupture and invasion are possibly required to breakdown erythrocyte membrane skeleton. To identify such proteases, soluble cytosolic extract of isolated trophozoites/schizonts was incubated with erythrocyte membrane ghosts or spectrin-actin depleted inside-out vesicles, which were then analyzed by SDS-PAGE. In both cases, a new protein band of 155 kDa was detected. The N-terminal peptide sequencing established that the 155 kDa band represents truncated ankyrin. Immunoblot analysis using defined monoclonal antibodies confirmed that ankyrin was cleaved at the C-terminus. While the enzyme preferentially cleaved ankyrin, degradation of protein 4.1 was also observed at high concentrations of the enzyme. The optimal activity of the purified enzyme, using ankyrin as substrate, was observed at pH 7.0-7.5, and the activity was strongly inhibited by standard inhibitors of cysteine proteinases (cystatin, NEM, leupeptin, E-64 and MDL 28 170), but not by inhibitors of aspartic (pepstatin) or serine (PMSF, DFP) proteinases. Furthermore, we demonstrate that protease digestion of ankyrin substantially reduces its interaction with ankyrin-depleted membrane vesicles. Ektacytometric measurements showed a dramatic increase in the rate of fragmentation of ghosts after treatment with the protease. Although the role of ankyrin cleavage in vivo remains to be determined, based on our findings we postulate that the parasite-derived cysteine protease activity cleaves host ankyrin thus weakening the ankyrin-band 3 binding interactions and destabilizing the erythrocyte membrane skeleton, which, in turn, facilitates parasite release. Further characterization of the enzyme may lead to the development of novel antimalarial drugs.
We have reviewed the recent literature on immunohistochemical markers of cell proliferation in normal oral epithelia and leukoplakias. Most findings, including our own, point to an increased proliferation in oral leukoplakias that correlates with the degree of dysplasia. Although the basal layer of normal oral epithelia showed a very low proliferative activity, oral leukoplakias, even those containing low-grade dysplasia, exhibited a very significant increase in proliferation. High-grade dysplasia could be clearly differentiated from both low-grade dysplasia and normal oral epithelia by the presence of proliferating cells in the superficial cell strata, i.e. above or superficial to the parabasal layer. These changes were detected with several markers including PCNA, Ki-67 (Mib-1), cyclin D1 and CENP-F as well as with procedures using pulse labeling with BrDU, IrDU and tritiated thymidine. Comparison of all methods showed more similarities than discrepancies. Nevertheless, because of its relative simplicity of use and universal acceptance in many other sites, Ki-67 (Mib-1) seems to be the most reliable immunohistochemical marker for future use in cancer prevention and therapeutic clinical trials.
RATIONALE AND OBJECTIVES: To evaluate the attenuation, size, and volume of the pericardial sinuses and recesses by using electrocardiographically triggered, noncontrast-enhanced electron beam tomography (EBT) and to consider its relation with sex, age, and heart volume. METHODS: Findings in 213 consecutive patients without known pericardial disease were studied. The patients underwent EBT scanning of the heart to evaluate coronary artery calcification. Incremental electrocardiographically triggered noncontrast images were obtained with a 100-ms exposure time and a 3-mm slice thickness. The appearance, density, and volume of the pericardial sinuses and recesses were calculated. RESULTS: Among the 213 patients, 97.2% had at least one of the sinuses or recesses visible on EBT. The sinuses or recesses were seen with the following frequency: transverse sinus (93.9%), oblique sinus (71.8%), and superior aortic recess (51.2%). The mean attenuation and volume were 9.9 +/- 7.3 Hounsfield units (HU), 12.6 +/- 8.1 HU, and 12.6 +/- 8.7 HU, and 1.9 +/- 1.3 mL, 1.3 +/- 1.0 mL, and 0.8 +/- 0.8 mL, respectively. The total volume of the pericardial sinuses (3.3 +/- 2.2 mL) had no significant relation with the total heart volume. CONCLUSIONS: Pericardial sinuses and recesses were frequently and well depicted on noncontrast EBT images. In patients without obvious pericardial effusion, physiological fluid collections were observed in the transverse and oblique sinuses or other recesses. Location, attenuation, and volume were helpful in the differentiation of normal pericardial sinuses from pericardial effusions and mediastinal lymph nodes.
Local recurrence of squamous cell cancer (SCC) causes high morbidity and is often readily accessible, making such patients potential candidates for gene therapy. Cyclin D1 (CD1), critical in the G1-S transition in the cell cycle, is amplified in 20-50% and overexpressed in up to 80% of head and neck SCC. Our earlier studies indicated that CD1 expression increased with progression from low grade to high grade dysplasia, and that treatment of established tumors with antisense cyclin D1 (AS-cyclin D1) led to tumor regression during a one week evaluation period. We hypothesized that: 1) CD1 expression increases with disease progression to advanced SCC, and 2) AS-cyclin D1 therapy would lead to prolonged tumor regression in a xenograft model of human SCC. CD1 expression, evaluated by immunostain in 30 stage III/IV head and neck SCC, increased in the basal layer from normal-dysplasia (P = 0.06) and from dysplasia-carcinoma (P = 0.004). In the germinative layer CD1 expression increased from dysplasia-carcinoma (P = 0.002) but not from normal-dysplasia. Western blotting of eight SCC and two transformed keratinocyte cell lines demonstrated CD1 overexpression in 8/10 (80%) lines. An 11th cell line (A431) had previously been shown to overexpress cyclin D1. 8/9 (89%) cell lines overexpressing CD1 formed tumors in immunodeficient mice, whereas 0/2 cell lines without CD1 overexpression formed a tumor. Three established SCCs, one fast growing, one with moderate growth rate (with CD1 overexpression) and one slow growing (without increased CD1), shrank significantly for 2-4 weeks after AS-cyclin D1 treatment, while tumors transduced with control vector grew. Cyclin D1 expression increases in frequency with disease progression, and antisense cyclin D1 was effective in a xenograft model of human cancer, independent of tumor growth rate.
E2F-1 is the best known ultimate transcription factor in the cyclin/cyclin-dependent kinase/retinoblastoma gene pathway and is probably involved in carcinogenesis and tumor progression. Because E2F-1 can be detected in paraffin sections using immunohistochemical techniques, it could be a useful tumor/proliferation marker. We studied the expression of this gene product in 130 breast tissue specimens from 100 patients and compared it with the expression of Mib-1, the widely used prognostic/proliferative marker, to assess E2F-1 as a new marker of neoplastic proliferation. The percentage of E2F-1-positive cells increased from 1.9% in the normal breast (NB) to 6.3% in ductal carcinoma in situ (DCIS) and to 15.3% in invasive ductal carcinomas (IDC). In addition, higher-grade tumors as well as advanced-stage disease correlated with higher expression of E2F-1. A similar tendency of Mib-1 expression was observed. There was a positive correlation between the E2F-1 and Mib-1 indices. In an in vitro experiment, we found that a similar difference in the expression of E2F-1 existed between a nontumorigenic breast cell line and two widely used breast carcinoma cell lines. The breast carcinoma cell lines T-47D and MCF-7 had more E2F-1-positive cells than the nontumorigenic cell line MCF-10F by immunohistochemistry and Western blot analysis. Because E2F-1 expression was significantly higher in IDC and DCIS than in NB, this study indicates that deregulation of E2F-1 may be involved in the development of breast IDC. In addition, E2F-1 expression could also be involved in tumor progression because the increased E2F-1 index correlated with the known prognostic predictors of breast cancer, such as histological grade, stage, metastasis status, estrogen receptor/progesterone receptor and Mib-1 expression. Thus, E2F-1 is a promising candidate to become a new prognostic/predictive marker of breast cancer.
The dose-effect of adaptive response of thymocyte apoptosis and cell cycle progression induced by whole-body X-ray irradiation (WBI) was studied in male Kunming mice. The inductive doses (D1) were 25, 50, 75, 100 or 200 mGy 6 h before the challenging doses (D2) of 1.0, 1.5 or 2.0 Gy. The changes in the percentages of the thymocyte apoptotic bodies (TAB) and the cells in different phases of cell cycle were measured with flow cytometry. The percentages of TAB decreased, the arrests of G1 and G2 + M phases diminished, and the cells of DNA synthesis of S phase increased when the D1 + D2 groups was compared with the D2 groups. When D1 was 200 mGy, the adaptive response of thymocyte apoptosis and cell cycle progression were no longer induced by low dose radiation (LDR). In addition, the extracellular fluid from the splenocytes were cultured with Con A for 48 h in vitro 24 h after 75 mGy WBI was placed in the murine thymocyte suspension from mice irradiated with 2.0 Gy WBI and co-incubated. The thymocyte apoptosis decreased. Especially, noteworthy was that the percentages of TAB after the incubation for 72 h were significantly lower than those in 2.0 Gy irradiated thymocytes (P < 0.05). These results indicate that when the mice were irradiated with 25-100 mGy (D1, 12.5 mGy/min) 6 h before 1.0-2.0 Gy (D2, 0.287 Gy/min) exposure, an adaptive response of thymocyte apoptosis and cell cycle progression may be induced under the condition of WBI, and LDR (75 mGy) may change the microenvironment of immune cells and decrease the thymocyte apoptosis.
The effect of atmospheric aerosols and regional haze from air pollution on the yields of rice and winter wheat grown in China is assessed. The assessment is based on estimates of aerosol optical depths over China, the effect of these optical depths on the solar irradiance reaching the earth's surface, and the response of rice and winter wheat grown in Nanjing to the change in solar irradiance. Two sets of aerosol optical depths are presented: one based on a coupled, regional climate/air quality model simulation and the other inferred from solar radiation measurements made over a 12-year period at meteorological stations in China. The model-estimated optical depths are significantly smaller than those derived from observations, perhaps because of errors in one or both sets of optical depths or because the data from the meteorological stations has been affected by local pollution. Radiative transfer calculations using the smaller, model-estimated aerosol optical depths indicate that the so-called "direct effect" of regional haze results in an approximately 5-30% reduction in the solar irradiance reaching some of China's most productive agricultural regions. Crop-response model simulations suggest an approximately 1:1 relationship between a percentage increase (decrease) in total surface solar irradiance and a percentage increase (decrease) in the yields of rice and wheat. Collectively, these calculations suggest that regional haze in China is currently depressing optimal yields of approximately 70% of the crops grown in China by at least 5-30%. Reducing the severity of regional haze in China through air pollution control could potentially result in a significant increase in crop yields and help the nation meet its growing food demands in the coming decades.
The insulin receptor substrates (IRS) 1 and 2 are required for normal growth and glucose homeostasis in mice. To determine whether IRS-3, a recently cloned member of the IRS family, is also involved in the regulation of these, we have generated mice with a targeted disruption of the IRS-3 gene and characterized them. Compared with wild-type mice, the IRS-3-null mice showed normal body weight throughout development, normal blood glucose levels in the fed and fasted state and following an oral glucose bolus, and normal fed and fasted plasma insulin levels. IRS-3 is most abundant in adipocytes and is tyrosine-phosphorylated in response to insulin in these cells. Therefore, isolated adipocytes were analyzed for changes in insulin effects. Insulin-stimulated glucose transport in the adipocytes from the IRS-3-null mice was the same as in wild-type cells. The extent of tyrosine phosphorylation of IRS-1/2 following insulin stimulation was similar in adipocytes from IRS-3-null and wild-type mice, and the insulin-induced association of tyrosine-phosphorylated IRS-1/2 with phosphatidylinositol 3-kinase and SHP-2 was not detectably increased by IRS-3 deficiency. Thus, IRS-3 was not essential for normal growth, glucose homeostasis, and glucose transport in adipocytes, and in its absence no significant compensatory augmentation of insulin signaling through IRS-1/2 was evident.
BACKGROUND: Both proliferating cell nuclear antigen (PCNA) and Ki-67 are proliferative markers known to correlate with the cell proliferative state. The aim of this study was to evaluate the usefulness of PCNA and Ki-67 immunoreactivity in the assessment of clinicopathologic features and prognosis in patients with malignant ovarian germ cell tumors. METHODS: Thirty-one patients with surgically resected malignant ovarian germ cell tumors were investigated. The clinicopathologic features and survival data of these patients were recorded. Immunohistochemical staining with monoclonal antibodies (PC 10 for PCNA, and MIB-1 for Ki-67) were performed on paraffin embedded tissue from each patient. The correlation of the immunoreactivity of these two markers with the clinicopathologic features and prognosis were studied. RESULTS: All of the tumors were positive for PCNA and Ki-67, but the intensity of expression varied widely. The immunoreactivity in each tumor was also heterogeneous. The scoring of PCNA and Ki-67 was determined by a semiquantitative method. Both advanced tumor stage (stages III and IV) and high PCNA score (scores 3 and 4) indicated a poorer prognosis for survival than did early stage (stages I and II) and a low PCNA score (scores 1 and 2) (p = 0.017 and p = 0.008, respectively). In addition, the proportion of tumor relapse and tumor-induced death was more accurately predicted by PCNA and Ki-67 scoring than by tumor staging (chi 2 = 0.3159, chi 2 = 0.7186 and chi 2 = 1.9689, respectively). CONCLUSIONS: PCNA and Ki-67 proliferative markers appear promising to differentiate patients into low- and high-risk groups. In the presence of a high PCNA or Ki-67 score, aggressive postoperative chemotherapy is warranted, even if the disease is in a very early stage.
OBJECTIVE: The expression of p53 and cyclin D1 proteins was analyzed by image analysis in oral premalignant lesions and normal oral mucosa. STUDY DESIGN: Punch biopsies from the normal oral mucosa were obtained from 20 normal donors and 41 patients with oral dysplastic leukoplakias. After controlled formaldehyde fixation and paraffin embedding, immunohistochemistry was used to detect cyclin D1 and p53. Image analysis was performed using stain intensity levels established by determining color thresholds (nuclear score) in the basal and parabasal layers. RESULTS: Analysis of sections showed a similar pattern: only two normal donors had a few intensely positive p53 cells in the basal layer of the floor of the mouth and the tongue epithelia. Similarly, only three donors had intensely positive cyclin D1 cells in the normal epithelia of the same sites. Most cells fell in the range of negative or marginal stain (lower quartiles or terciles of nuclear score). These data on normal mucosa were compared with low grade oral leukoplakias (LGD) with mild to moderate dysplasia and with high grade leukoplakias (HGD) with severe dysplasia. Both markers were differentially expressed in precursor lesions versus normal epithelia. Statistical analysis of our data shows that the intensity of the immunohistochemical stain, as reflected in the nuclear scores of p53, is a reliable parameter that can differentiate between LGD and HGD of the oral mucosa. This was especially true when higher nuclear scores were compared. In contrast, low nuclear scores are more effective in differentiating normal epithelia from dysplastic epithelia. Although cyclin D1 immunohistochemistry does not stain as intensely as p53 stain, similar conclusions can be derived from those data. CONCLUSION: Image analysis of these two markers proved useful in distinguishing normal oral epithelia from low grade and high grade leukoplakias. With further developments in this field it is hoped that image analysis procedures could be used in different types of studies in which variations of protein expression in tissue sections could have prognostic implications or could be useful to determine subtle effects of curative or preventive treatment.
Ankyrin mutations and combined spectrin and ankyrin deficiency are prominent features of red blood cells (RBCs) in patients with hereditary spherocytosis (HS). Band 3 is the most abundant integral protein in the human RBC membrane. Previous studies have shown that the lateral mobility, but not the rotational mobility, of band 3 is increased in RBCs from patients with severe autosomal recessive HS and selective spectrin deficiency. These observations are consistent with the steric hindrance model of lateral mobility restriction. Here we use the fluorescence photobleaching recovery and polarized fluorescence depletion techniques to measure the lateral and rotational mobility of band 3 in intact RBCs from six patients with HS, ankyrin mutations, and combined spectrin and ankyrin deficiency. As predicted by the steric hindrance model, the lateral diffusion rate of band 3 is greater in spectrin- and ankyrin-deficient RBCs than in control cells, and the magnitude of the increase correlates with the degree of spectrin deficiency. Unlike RBCs from patients with HS and selective spectrin deficiency, however, HS RBCs with ankyrin mutations exhibit a marked increase in band 3 rotational diffusion. The magnitude of the increase correlates inversely with the ankyrin/band 3 ratio and with the fraction of band 3 retained in the membrane skeleton following detergent extraction. These data suggest that ankyrin deficiency relaxes rotational constraints on the major (slowly rotating) population of band 3 molecules. Increases in band 3 rotation could be due to release of band 3 from low-affinity binding sites on ankyrin.
Only 5% to 10% of band 3 null mice survive the neonatal period. To determine the cause of death, 3 adult and 11 newborn band 3 null mice were submitted for histopathologic examination. All but 1 pup showed evidence of thrombosis including: (1) large thrombotic lesions in the heart, which were partially organized, calcified in some fields, and endothelialized, indicating a process that developed premortem (3 of 3 adults and 6 of 11 pups). (2) Subcapsular necrotic areas in the liver suggestive of premortem ischemic events caused by arteriolar occlusions (8 of 11 pups). (3) Large vein thrombi (4 of 11 pups). To investigate the etiology of this hypercoagulable state, we have used the Russell's viper venom test (RVV) to show that red blood cells (RBCs) from band 3 null mice significantly shorten the RVV clotting time of normal plasma in a dose-dependent fashion, whereas RBCs from normal mice have no effect, suggesting that the membrane of band 3 null RBCs provides a suitable surface for activation of the prothrombinase complex. Using flow cytometry, we have examined the phosphatidylserine (PS)-specific binding of fluorescein isothiocyanate (FITC)-annexin V to normal and band 3 null RBCs. A subpopulation of cells (3% to 5% of RBCs) with increased FITC-annexin V binding was detected in band 3 null RBCs as compared with normal RBCs. Furthermore, the entire cell population of band 3 null RBCs shows a measurable increase in the mean fluorescence intensity, suggesting that band 3 null RBCs may have increased PS exposure on the outer membrane leaflet. These findings are further supported by direct fluorescence microscopy of normal and band 3 null RBCs labeled with FITC-annexin V. Based on these observations, we postulate that the high mortality of band 3 null mice may be related to a hypercoagulable state, which appears to originate from changes in the phospholipid composition of the membrane leading to PS exposure on the outer leaflet.
Chicken GABA(A) receptor beta4L and beta4S subunits were expressed in Xenopus oocytes by cRNA injection. Oocytes expressing either beta4 subunit alone or in combination with the chicken alpha1 subunit were studied using the two-electrode voltage-clamp technique. Both the beta4L and beta4S subunits form homomeric GABA-gated Cl- channels with similar efficiencies. In comparison, oocytes expressing either the chicken alpha1 or beta2S polypeptide show no or barely detectable GABA responses, as reported by others for most single-subunit vertebrate GABA(A) receptors. The GABA-gated currents due to the beta4L-subunit homomer were not affected by the presence of actinomycin D during cRNA expression, indicating that nascent oocyte polypeptides are not required for channel formation. The homomeric beta4L-subunit receptors show high affinity for GABA with an EC50 value of 4.3 +/- 0.4 microM and a Hill coefficient of 1.1 +/- 0.1 (n = 6). In response to GABA application at the EC25 value, currents elicited from the beta4L-subunit receptor are enhanced by 50 microM pentobarbital (110 +/- 10%, n = 3) and 10 microM loreclezole (60 +/- 3%, n = 3), inhibited by 10 microM picrotoxinin (93 +/- 3%, n = 3), but not affected by 1 microM diazepam. These properties are similar to those found for oocytes expressing heteromeric chicken alpha1beta4L and alpha1beta2S receptors. Since the beta subunits of GABA(A) receptors provide essential determinants for receptor assembly and subcellular localization, homomeric beta4-subunit receptors are a useful model system for further study of the structure and function of GABA(A) receptors.
The FHIT gene has been implicated as a tumor suppressor gene in human malignancies. To determine if FHIT alterations play a role in human squamous cell carcinogenesis of the head and neck (HNSCC), we examined the gene and its product by RT-PCR, SSCP, Northern, Southern, and Western blot analysis in primary HNSCC and/or HNSCC cell lines. Three of 32 tumor samples lacked detectable expression of FHIT by RT-PCR but showed amplification of a control gene of similar size. One of 29 primary tumors and 2/9 HNSCC cell lines exhibited aberrant transcripts generated by RT-PCR methods using one set of 40 cycles of amplification. FHIT mRNA expression was absent in seven HNSCC cell lines but detectable in primary keratinocytes by Northern analysis. Using specific polyclonal antiserum to the full-length FHIT protein in immunoblot analyses, 4/9 cell lines analysed showed no expression of pFhit, two exhibited low levels of expression, and three expressed a putative truncated pFhit. One of 15 tumors analysed also exhibited an overexpressed truncated protein. PCR/SSCP and Southern analysis of one cell line DNA that expressed a truncated protein indicated that it sustained homozygous loss of FHIT exon 5. Our results suggest that alterations in FHIT at the DNA, RNA, and protein levels exist at a low but significant frequency in HNSCCs. Further studies regarding the potential biological activity of FHIT are needed to clarify the role of this gene in HNSCC tumorigenesis.
Band 3, the anion transport protein of the erythrocyte membrane, exists in the membrane as a mixture of dimers (B3D) and tetramers (B3T). The dimers are not linked to the skeleton and constitute the free mobile band 3 fraction. The tetramers are linked to the skeleton by their interaction with ankyrin. In this report we have examined the temporal synthesis and assembly of band 3 oligomers into the plasma membrane during red cell maturation. The oligomeric state of newly synthesized band 3 in early and late erythroblasts was analyzed by size-exclusion high-pressure liquid chromatography of band 3 extracts derived by mild extraction of plasma membranes with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether). This analysis revealed that at the early erythroblast stage, the newly synthesized band 3 is present predominantly as tetramers, whereas at the late stages of erythroid maturation, it is present exclusively as dimers. To examine whether the dimers and tetramers exist in the membrane as preformed stable species or whether they are interconvertible, the fate of band 3 species synthesized during erythroblast maturation was examined by pulse-chase analysis. We showed that the newly synthesized band 3 dimers and tetramers are stable and that there is no interconversion between these species in erythroblast membranes. Pulse-chase analysis followed by cellular fractionation showed that, in early erythroblasts, the newly synthesized band 3 tetramers are initially present in the microsomal fraction and later incorporated stably into the plasma membrane fraction. In contrast, in late erythroblasts the newly synthesized band 3 dimers move rapidly to the plasma membrane fraction but then recycle between the plasma membrane and microsomal fractions. Fluorescence photobleaching recovery studies showed that significant fractions of B3T and B3D are laterally mobile in early and late erythroblast plasma membranes, respectively, suggesting that many B3T-ankyrin complexes are unattached to the membrane skeleton in early erythroblasts and that the membrane skeleton has yet to become tightly organized in late erythroblasts. We postulate that in early erythroblasts, band 3 tetramers are transported through microsomes and stably incorporated into the plasma membrane. However, when ankyrin synthesis is downregulated in late erythroblasts, it appears that B3D are rapidly transported to the plasma membrane but then recycled between the plasma membrane and microsomal compartments. These observations may suggest novel roles for membrane skeletal proteins in stabilizing integral membrane protein oligomers at the plasma membrane and in regulating the endocytosis of such proteins.
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The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.