Idiopathic macular holes and direction of vitreomacular traction: structural changes and surgical outcomes.

PurposeTo compare the structural changes, clinical course, and treatment outcomes of vertical and horizontal vitreomacular traction (VMT) induced impending macular holes (IMHs) and full-thickness macular holes (FTMHs).MethodsIn this retrospective study, 23 and 32 cases of IMHs and FTMHs, respectively, were analyzed. The IMH cases were divided into two subgroups: IMH with and without foveal detachment. Vitreofoveal traction angles (TAs) between the inner retinal surface and posterior hyaloid were measured from horizontal and vertical optical coherence tomography (OCT) images by using the trigonometric function (the angle equals the arctangent of the height over the base) after adjustments for magnification factors. The largest angle was defined as the vitreomacular TA for the examined case. The critical angle-the TA differentiating cases with (vertical traction) or without (horizontal traction) foveal detachment (vertical traction)-was determined using regression analysis. Pretreatment and posttreatment OCT images, clinical courses, and treatment outcomes were compared between the two groups.ResultsThe critical angle was 27.2°. Cases of vertical traction had higher foveal height in the IMH group and wider bases in the FTMH group (P<0.05 respectively). IMHs with vertical traction had greater VM attachment than those with horizontal traction. In the FTMH group, postoperative visual improvement was lower (P=0.002); in the vertical traction group, inner segment:outer segment defects persisted longer (P=0.02).ConclusionsThe critical angle separating vertical from horizontal traction was 27.2°. Vertical VMT results in greater foveal structural changes in IMHs and possibly less favorable surgical outcomes in FTMHs.

Small-molecule inhibition of CBP/catenin interactions eliminates drug-resistant clones in acute lymphoblastic leukemia.

Drug resistance in acute lymphoblastic leukemia (ALL) remains a major problem warranting new treatment strategies. Wnt/catenin signaling is critical for the self-renewal of normal hematopoietic progenitor cells. Deregulated Wnt signaling is evident in chronic and acute myeloid leukemia; however, little is known about ALL. Differential interaction of catenin with either the Kat3 coactivator CREBBP (CREB-binding protein (CBP)) or the highly homologous EP300 (p300) is critical to determine divergent cellular responses and provides a rationale for the regulation of both proliferation and differentiation by the Wnt signaling pathway. Usage of the coactivator CBP by catenin leads to transcriptional activation of cassettes of genes that are involved in maintenance of progenitor cell self-renewal. However, the use of the coactivator p300 leads to activation of genes involved in the initiation of differentiation. ICG-001 is a novel small-molecule modulator of Wnt/catenin signaling, which specifically binds to the N-terminus of CBP and not p300, within amino acids 1-110, thereby disrupting the interaction between CBP and catenin. Here, we report that selective disruption of the CBP/ß- and γ-catenin interactions using ICG-001 leads to differentiation of pre-B ALL cells and loss of self-renewal capacity. Survivin, an inhibitor-of-apoptosis protein, was also downregulated in primary ALL after treatment with ICG-001. Using chromatin immunoprecipitation assay, we demonstrate occupancy of the survivin promoter by CBP that is decreased by ICG-001 in primary ALL. CBP mutations have been recently identified in a significant percentage of ALL patients, however, almost all of the identified mutations reported occur C-terminal to the binding site for ICG-001. Importantly, ICG-001, regardless of CBP mutational status and chromosomal aberration, leads to eradication of drug-resistant primary leukemia in combination with conventional therapy in vitro and significantly prolongs the survival of NOD/SCID mice engrafted with primary ALL. Therefore, specifically inhibiting CBP/catenin transcription represents a novel approach to overcome relapse in ALL.

Widespread white matter alterations in patients with end-stage renal disease: a voxelwise diffusion tensor imaging study.

BACKGROUND AND PURPOSE: ESRD results in excessive accumulation of urea and toxic metabolites. Hemodialysis is usually performed to maintain health in patients with ESRD; however, it may cause silent white matter alterations in the earlier stages. Hence, this study aimed to perform voxelwise diffusion tensor analysis for global detection of subtle white matter alterations in patients with ESRD. MATERIALS AND METHODS: Twenty-eight patients with ESRD and 25 age-matched control subjects were enrolled in this study. Each subject underwent CASI assessment and DTI. After spatial normalization of DTI images, voxelwise statistical analyses were performed to compare DTI parameters between the 2 groups. RESULTS: In patients with ESRD, AD, RD, and MD values were significantly increased, whereas the FA value was significantly decreased, mostly in the corpus callosum, bilateral sagittal stratum, and pons. Multiple regression analysis further revealed that both RD and MD were positively correlated with the duration of hemodialysis in the pons; however, no significant correlation was observed with FA. Negative correlations of RD and MD and a positive correlation of FA with the CASI score were observed in the corona radiata. CONCLUSIONS: We concluded that voxelwise DTI analysis is helpful in the detection of white matter alterations caused by hemodialysis.

Differential expression of vascular endothelial growth factor, placenta growth factor and their receptors in placentae from pregnancies complicated by placenta accreta.

Placenta accreta is a pregnancy complication characterized by the presence of life-threatening uteroplacental neovascularization. The factors involving its development are unknown. Vascular endothelial growth factor (VEGF), placenta growth factor (PlGF) and their receptors (VEGFR) have important roles in vascular remodeling. We have investigated the differential expression of these proteins in placentae from placenta accreta (cases) and normal placentation (controls). Immunohistochemically, the expression of VEGFR-2 in the syncytiotrophoblast was significantly lower in cases than in controls during both the second and third trimesters (P = 0.005 and 0.002, respectively). However, VEGFR-2 expression in the cytotrophoblastic and extravillous trophoblastic cells and VEGFR-1, -3 and Ki-67 in the trophoblast populations were not significantly different between controls and cases (P > 0.05). Ki-67 immunostaining also showed that endothelial cells of the larger vessels were stained weaker in normal placenta than in placenta accreta. The majority of VEGFR-2 expression, as demonstrated by Western blot or reverse transcription polymerase chain reaction, was consistent with the immunohistochemical findings in the syncytiotrophoblast. Furthermore, enzyme-linked immunosorbent assay in the placental lysates showed that the women with placenta accreta demonstrated significantly higher VEGF (P = 0.001) and lower soluble VEGFR-2 (P = 0.015) concentrations than did women with normal pregnancy. PlGF and soluble VEGFR-1 levels did not show any significance in study groups (P > 0.05). These observations suggest that the participation of up-regulated VEGF and down-regulated VEGFR-2 (both membrane-bound and soluble forms) may be associated with the development of placenta accreta.

The clinical study of congenital looped/coiled peripapillary retinal vessels.

PURPOSE: To present clinical manifestations of eyes with peculiar looped/coiled peripapillary retinal vessels. METHODS: Seven patients with looped/coiled retinal vessels on or near the optic disc were enrolled. All patients went through detailed ophthalmologic examinations and fluorescein angiography (FAG). RESULTS: There were two men and five women. Patients' age ranged from 15 to 71 years (mean: 39 years). The follow-up period ranged from 3 to 74 months (average: 27 months). One patient had bilateral involvement. Five of the seven patients noticed sudden onset of floaters in one eye. The colour fundus photography revealed looped/coiled retinal vessels on or near the optic disc, and most of the vessels were arteries. Accompanied retinal, preretinal, or vitreous haemorrhage was noted in all five patients who had sudden onset of floaters. Fluorescein angiography showed no leakage from the looped/coiled retinal vessels. No specific underlying diseases were noted in any patients. Follow-up examination revealed reabsorption of haemorrhage, and no change of the abnormal vessel patterns in any eyes. CONCLUSIONS: The peculiar fundus lesion of looped/coiled peripapillary retinal vessels is likely a benign congenital retinal vascular anomaly that does not progress. Floaters secondary to preretinal or vitreous haemorrhage is the most frequent complaint. The prognosis is excellent.

KAI1 metastasis suppressor gene is frequently down-regulated in cervical carcinoma.

KAI1 is a metastasis suppressor gene located on human chromosome 11p11.2. It belongs to a structurally distinct family of cell surface glycoproteins. Decreased KAI1 expression has been observed in several common solid epithelial tumors, including prostatic, pancreatic, lung, hepatic, colorectal, ovarian, and esophageal cancers. A recent study also observed frequent loss of KAI1 expression in a number of squamous cell carcinomas of the cervix by immunohistochemical technique. To further confirm whether this gene is altered in this malignancy, we analyzed KAI1 expression in various stages of cervical carcinoma by a molecular method. Total cellular RNA was extracted from 84 primary invasive cervical carcinomas and 6 metastatic or recurrent lesions. cDNA was synthesized and was used for real-time quantitative polymerase chain reaction analysis. The level of KAI1 expression was obtained as the value of threshold cycle (Ct) and was quantitated with a comparative Ct method. In addition, paraffin blocks of the tumors were selected and prepared for immunohistochemical study with an anti-KAI1 polyclonal antibody, C-16. Both the real-time quantitative polymerase chain reaction method and immunohistochemical study revealed a frequent decrease in KAI1 expression in invasive cervical cancers and metastatic or recurrent lesions. However, the reduction in KAI1 was not related to progression of the disease. When tumor cell differentiation was analyzed, poorly differentiated tumors showed a greater decrease in KAI1 expression than well or moderately differentiated tumors (P < 0.001). Histologically, KAI1 loss was observed equally in both squamous cell carcinoma and adeno-/adenosquamous carcinoma. Since down-regulation of KAI1 occurs in both early and late stages of cervical cancer, we suggest that its involvement in the progression of this malignancy is an early event.

FHIT (fragile histidine triad) gene analysis in cervical intraepithelial neoplasia.

OBJECTIVE: Recently a candidate tumor suppressor gene, FHIT (fragile histidine triad), was identified at chromosome 3p14.2. Abnormality of this gene has been observed in a variety of human tumors. Although aberrant FHIT transcripts in a substantial percentage of cervical cancer cell lines and primary cervical tumors were also noted, some other studies revealed different results. Therefore, its association with the development of cervical cancer is still debatable. Because allelic loss in chromosome 3p is also a frequent finding in cervical intraepithelial neoplasia (CIN), we compared the transcription pattern and expression of FHIT in the preinvasive cervical lesions and normal cervical epithelia to investigate its possible role in cervical carcinogenesis. METHODS: Thirty-five consecutive CIN lesions taken from conization specimens and 33 normal cervical epithelial tissues taken from hysterectomy for benign diseases were included in this study. Total RNA was extracted from the pathology-confirmed tissue samples and first-strand cDNA was synthesized. It was amplified using a nested reverse transcription polymerase chain reaction (RT-PCR) method. The PCR products were then subjected to subcloned sequence analysis. Paraffin blocks from all of the samples were selected and prepared for immunohistochemical study with an anti-FHIT polyclonal antibody. RESULTS: All the cDNAs of CIN and normal cervical epithelial tissues showed the expected size of RT-PCR product. However, 7 of the 35 (20%) CIN lesions and 5 of the 33 (15%) normal cervical epithelia also presented aberrant transcripts in addition to the normal-sized transcript of FHIT. Deletion of the cDNA segment covering exon 4 to exon 8 was the most frequent finding in the cases that showed abnormal FHIT transcripts. FHIT protein was intermediately or strongly expressed in most of the CIN lesions and normal squamous epithelia. However, reduced or absent FHIT expression was observed heterogeneously in the 7 CIN lesions and 5 normal cervices in which aberrant FHIT transcripts were detected. CONCLUSION: Because the normal-sized FHIT transcript was present robustly in all of the CIN lesions and the abnormal FHIT transcripts occurred with similar frequency and pattern in the CIN lesions and normal cervical tissues, we suggest that abnormal FHIT transcription might not be causal in the early process of cervical carcinogenesis.

p53 mutation is infrequent in clear cell carcinoma of the ovary.

OBJECTIVE: p53 gene alteration has been extensively studied in epithelial ovarian cancer. However, its occurrence in clear cell carcinoma, an infrequent histologic subtype of epithelial ovarian cancer, is rarely reported. The aim of this study is to determine the status of p53 gene alteration in this distinct type of ovarian carcinoma. METHODS: Paraffin blocks of tumors from 38 patients with primary or recurrent ovarian clear cell carcinoma were studied for p53 alteration. All these tumors were subjected to immunohistochemical and molecular analysis. Two monoclonal antibodies (DO-7 and PAb 1801) were used for immunohistochemical staining. Genomic DNAs extracted from paraffin blocks of the 38 tumors were subscribed for a nested polymerase chain reaction/single-strand conformation polymorphism (PCR/SSCP) analysis. Tumors showing band shift on SSCP were further prepared for DNA sequencing to determine the site of mutation. RESULTS: Overexpression of p53 was observed in only one stage III clear cell carcinoma. However, focal positive p53 staining was noted in another five tumors. Of the six tumors showing positive immunohistochemistry, p53 alterations were noted in four tumors. Three tumors revealed a missense point mutation: two were in exon 7 (TCT(227) --> TTT and GGC(245) --> AGC) and one was in exon 5 (CGC(156) --> CAC). Another tumor revealed a 12-bp deletion in two possible ways: it might involve the last four codons at the 3' end of exon 4 (nucleotides 12,288-12,299) or it might cross over the splice junction between exon 4 and intron 4 (nucleotides 12,290-12,301). The former would result in a predicted protein product of 389 amino acids whereas the latter would cause a frameshift in the gene sequence and would result in a truncated protein. CONCLUSION: Mutations in p53 appear to be much less frequent in clear cell carcinoma than in other histologic types of epithelial ovarian cancer. We suggest that p53 alterations may not play an important role in the development of clear cell carcinoma.

Frequent down-regulation and lack of mutation of the KAI1 metastasis suppressor gene in epithelial ovarian carcinoma.

OBJECTIVE: KAI1 is a recently identified metastasis suppressor gene on human chromosome 11p11.2. It belongs to a structurally distinct family of cell surface glycoproteins. Decreased KAI1 expression seems to be involved in the progression of human prostate, lung, pancreatic, and possibly breast cancer, and recently a reduced KAI1 protein expression has been demonstrated in several ovarian carcinoma cell lines. The aim of this study is to determine whether the KAI1 gene is altered in human epithelial ovarian carcinomas. In addition, its prognostic significance in this tumor is also evaluated. METHODS: To detect KAI1 expression, 102 tumor samples from benign, borderline, primary invasive, metastatic, and recurrent epithelial ovarian tumors were prepared for immunohistochemical study with C-16, an anti-KAI1 polyclonal antibody. In addition, cellular RNA from 24 primary invasive and 7 recurrent tumors was also analyzed for KAI1 expression by using a reverse transcriptase PCR (RT-PCR) technique. The PCR single-strand conformation polymorphism method and direct DNA sequencing were used to detect KAI1 mutation in the 44 primary invasive and 8 recurrent ovarian carcinomas. RESULTS: In immunohistochemical study, decrease of KAI1 protein expression was associated with the progression of ovarian tumor. However, it had no relation to the stage of primary invasive cancers because of its frequent occurrence in early stage tumors. KAI1 expression was also frequently down-regulated in primary invasive and recurrent tumors in RT-PCR analysis. Except for a missense change at codon 241 (ATC to GTC), which causes the substitution of a valine for an isoleucine in the amino acid sequence and occurs in both normal and tumor tissues, no mutation of the KAI1 gene was found in any of the 52 carcinomas. Although there was a trend for deteriorating survival from patients with KAI1-preserved tumors to those with KAI1-decreased and -negative tumors, statistically it was not significant (P = 0.079). CONCLUSION: KAI1 may play a role in the malignant progression of epithelial ovarian carcinoma through the down-regulation of expression rather than gene mutation. Since the decreased expression presented frequently in early stage tumors, it may be an early event in the progression of this tumor and its prognostic significance needs further investigation with a larger number of cases.

Immunostimulation of tiger shrimp (Penaeus monodon) hemocytes for generation of microbicidal substances: analysis of reactive oxygen species.

NBT staining was used to determine the presence of superoxide anions (O2-) produced by tiger shrimp (Penaeus monodon) hemocytes attached to a coverslip. When cells were treated with beta-glucan, blue granules were observed in 41% of studied hemocyte cytoplasm. For zymosan-treated, PMA-treated, and control cells, the percentages of hemocytes showing similar blue granules were 31, 9, and 5%, respectively. A comparison of stimulative effects on 15 hemocyte suspensions, each collected from a single tiger shrimp, showed that beta-glucan had the strongest effect on intracellular O2- generation, followed by zymosan and PMA (2.5, 2, and 1.3 times greater than the O2- generated by the control group, respectively). After oxidizing phenol red to measure the amounts of hydrogen peroxide (H2O2) produced by the hemocytes, we found that beta-glucan had the strongest stimulative effect (12.2 nmol/mg protein), followed by zymosan and PMA (7.2 and 2.6 nmol/mg, respectively). However, a luminol-enhanced chemiluminescence analysis of hypochlorites (OCl-) produced by the experimental hemocytes showed that neither zymosan nor beta-glucan had a stimulative effect on OCl- production. However, following PMA stimulation, hemocyte chemiluminescence was detected although only at 1.7 mV. Using H2O2 as substrate and guaiacol as an electron acceptor, the enzyme activity of crude enzyme extract derived from broken hemocytes was analyzed; enzyme activity similar to that of human myeloperoxidase (MPO) (0.104 U/mg protein) was observed. The data showed that only PMA had any stimulative effect on MPO-like enzyme activity (2.23 times that of the control group); zymosan and beta-glucan did not have any observable effects on this specific enzyme activity. This is the first documented demonstration of a respiratory burst in shrimp hemocytes.

Identification of the ATP.Mg-dependent protein phosphatase activator (FA) as a synapsin I kinase that inhibits cross-linking of synapsin I with brain microtubules.

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified and purified to near homogeneity from brain. In this report, as evidenced on SDS-polyacrylamide gel electrophoresis followed by autoradiography, factor FA has further been identified as a cAMP and Ca(2+)-independent brain kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton, and is believed to be involved in the modulation of neurotransmission. Kinetic study further indicated that factor FA could phosphorylate synapsin I with a low Km value of about 2 microM and with a molar ratio of 1 mol of phosphate per mole of protein. Peptide mapping analysis revealed that factor FA specifically phosphorylated the tail region of synapsin I but on a unique site distinct from those phosphorylated by Ca2+/calmodulin-dependent protein kinase II and cAMP-dependent protein kinase, the two well-established synapsin I kinases. Functional study further revealed that factor FA could phosphorylate this unique specific site on the tail region of synapsin I and thereby inhibit cross-linking of synapsin I with microtubules. The results further suggest the possible involvement of factor FA as a synapsin I kinase in the regulation of axonal transport process of synaptic vesicles via the promotion of vesicles motility during neurotransmission.

Cyclic inhibition-potentiation of the crosslinking of synapsin I with brain microtubules by protein kinase FA (an activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent type-1 protein phosphatase activating factor (FA) was identified as a protein kinase that could phosphorylate synapsin I, a neuronal protein that coats synaptic vesicles, binds to cytoskeleton and is believed to be involved in the modulation of neurotransmission. More importantly, more than 90% of the phosphates in 32P-synapsin I phosphorylated by FA could be removed by the activated ATP.Mg-dependent type-1 protein phosphatase and the synapsin I phosphatase activity was found to be strictly FA-dependent. Functional study further revealed that as a synapsin I kinase, factor FA could phosphorylate synapsin I and thereby inhibits crosslinking of synapsin I with tubulin, while as a synapsin I phosphatase activator, FA could promote the crosslinking copolymerization of synapsin I with tubulin. Taken together, the results provide initial evidence that a cyclic modulation of the crosslinking copolymerization of synapsin I with brain microtubules can be controlled by factor FA, representing an efficient cyclic cascade control mechanism for the regulation of axonal transport process during neurotransmission.

Selective modulation of the two antagonistic activities of protein kinase FA (the activator of ATP.Mg-dependent protein phosphatase).

The ATP.Mg-dependent protein phosphatase activating factor (FA) has been identified as a protein kinase. The results are unexpected since factor FA possesses two activities which are antagonistic. As a kinase, factor FA catalyzes protein phosphorylation, while as a phosphatase activator, it catalyzes protein dephosphorylation. In this report, we found that the two opposing activities of factor FA could be selectively modulated. For instance, heparin at concentrations of 0.1-0.3 mg/ml could stimulate FA to work preferentially as a kinase towards phosphorylation of proteins but simultaneously inhibit it to work as a phosphatase activator towards dephosphorylation of the same proteins. In a similar manner, alkaline pH could stimulate FA to work as a kinase but block it to work as a phosphatase activator. This is the first report providing initial evidence that the two opposing activities of factor FA can be selectively modulated in a reciprocal manner by various triggers, suggesting that a simultaneous coordinate control mechanism may well be involved in regulating the activities of factor FA in the cell.