Alteration of gap junctions and connexins in the right atrial appendage during cardiopulmonary bypass

OBJECTIVES: We investigated the influence of cardiopulmonary bypass on cardiomyocyte gap junctions and connexins. METHODS: Samples were collected at intervals during operation from the right atrial appendage in 21 patients (mean [+/- SD] age 55 +/- 21 years). Immunodetection of connexins was conducted by Western blotting and confocal microscopy with parallel electron microscopic examination of gap junctions. RESULTS: Downregulation of connexin 43 during the course of operation occurred in more than half of the patients. The mean densitometric value of connexin 43 decreased by 23%, with samples from patients with coronary artery disease showing a greater reduction than seen in those from patients with other diseases (31% +/- 22% vs 10% +/- 24%, P =.04). Such alterations were confirmed by confocal microscopy, which also demonstrated reduced connexin 45 immunolabeling in most patients. Electron microscopy revealed a reduction in the dimensions of cell membrane-located gap junctions and more frequent intracytoplasmic gap junctional membrane in samples from later time points (P =.04). CONCLUSIONS: Downregulation of connexins accompanied by a reduction in gap junctions is common in the cardiomyocytes of the right atrial appendage during cardiopulmonary bypass. The association of a marked reduction in connexin 43 with coronary artery disease may imply inadequate intraoperative cardiac protection in patients with this disease.     

Aortic wall alterations after use of gelatin-resorcinol-formalin glue

We report the case of a patient who underwent reoperation 8 years after aortic valve replacement because of aneurysmal dilatation of the aortic root. During the initial intervention, gelatin-resorcinol-formalin glue had been applied on the outside of the aortic root. Perioperative examination revealed a necrotic appearance of the right coronary sinus, with contained ruptures at two different sites. Histologic analysis showed major destruction of the aortic root media, leading to vascular wall thinning and rupture. The use of gelatin-resorcinolformalin glue may expose patients to major alterations of the aortic wall.     

Double partial monosomies (10p- and Xp-) in a female baby with choanal atresia

Chromosomal abnormalities involving double partial monosomies are very rare. A female infant with non-mosaic monosomy 10p13-->10pter along with monosomy Xp11.4-->Xpter which arose de novo is described. The clinical manifestations of this patient included microcephaly, mild synophrys, short and down-slanted palpebral fissures, ptosis of the left eye, long eyelashes, a depressed nasal bridge, dysplastic ears, micrognathia, a short neck. sensorineural hearing impairment, and severe growth retardation. Left choanal atresia and laryngomalacia were detected by flexible fibroscopy. No signs of hypoparathyroidism or defective cellular immunity could be found. Fluorescence in situ hybridization (FISH) with whole-chromosome painting probes for chromosomes 10 and X was performed, which excluded the possibility of cryptic translocations of the involved chromosome segments. No submicroscopic chromosome 22q11 deletion could be found by FISH. Thus this very rare coexistence of double independent partial monosomies was confirmed. There are no previous reports of such concurrent double partial monosomies.     

Radioiodinated styrylbenzene derivatives as potential SPECT imaging agents for amyloid plaque detection in Alzheimer's disease

Development of probes for β-amyloid (Aβ) plaques, a critical factor associated with Alzheimer’s disease (AD), provides important tools for studying their role in AD. Previously, we reported [¹²⁵I]IMSB and [¹²⁵I]ISB as excellent probes for Aβ plaque labeling. Despite their exquisite in vitro binding characteristics, low brain uptakes (likely due to two ionizable carboxylic acid groups) limited their potential as in vivo imaging agents. To improve brain penetration, we have successfully prepared a neutral radioiodinated probe [¹²⁵I]3. The improved probe displayed good binding affinity for Aβ aggregates (K_i=2.0 ± 0.2 using Aβ40 aggregates). In addition, the brominated counterpart displayed fluorescent-staining properties of Aβ plaques in postmortem AD brain sections similar to BSB, a fluoroscent probe reported previously. [¹²⁵I]3 gave excellent plaque labeling by film autoradiography of AD brain sections. Unlike [¹²⁵I]IMSB (which preferentially detects Aβ40 plaques), the improved radioioinated probe, [¹²⁵I]3, can readily detect plaques containing aggregates of both Aβ40 and Aβ42. The initial brain uptake of [¹²⁵I]3 in normal mice at 2 min p.i. was moderate (0.18% ID) and displayed a very slow washout from the brain (0.11 %.ID at 4 h p.i). Taken together, these data suggest that [¹²⁵I]3 is useful for in vitro plaque detection, it may not be suitable for in vivo monitoring of Aβ progression and deposition.     

Acceleration of G(1) cooperates with core binding factor beta-smooth muscle myosin heavy chain to induce acute leukemia in mice

The genes encoding the AML1 (RUNX1) or CBFbeta subunits of core binding factor (CBF) are commonly altered by translocation or mutation in human leukemias. Because CBF oncoproteins slow G(1), we sought to determine whether mutations that accelerate G(1) potentiate their ability to induce transformation. Wild-type or p16(INK4a)p19(ARF) (-/-) marrow cells transduced with CBFbeta-smooth muscle myosin heavy chain (SMMHC) were transplanted into wild-type, syngeneic recipients. CBFbeta-SMMHC significantly increased the development of acute leukemias from marrow lacking the overlapping p16p19 genes, based on analysis of Kaplan-Meier event-time distributions. Wild-type marrow was also transduced with vectors expressing either E7 alone or both E7 and CBFbeta-SMMHC. Combining oncogenes again increased leukemia formation. Exposing mice transplanted with CBFbeta-SMMHC-transduced cells to a mutagen, ethylnitrosourea, markedly accelerated leukemogenesis compared to expressing CBFbeta-SMMHC with loss of p16p19, indicating the need for multiple "hits" for transformation. The INV/p16p19 and INV/E7 leukemias were lymphoid and were clonal and retransplantable. Overall, these findings indicate that CBF mutations cooperate with genetic alterations that accelerate G(1) to induce acute leukemia.     

Culture of neural cells on silicon wafers with nano-scale surface topograph

Optical imaging of neuronal network activity yields information of spatial dynamics which generally is analyzed visually. The transient appearance of spatial activity patterns is difficult to gauge in a quantifiable manner, or may even altogether escape detection. Here, we employ geometric shape matching using Fréchet distances or straight skeletons to search for pre-selected patterns in optical imaging data with adjustable degrees of tolerance. Data were sampled from fluorescence changes of a voltage-sensitive dye recorded with a 464-photodiode array. Fluorescence was monitored in a neuronal network in vitro. Neuronal activity prompting fluorescence fluctuations consisted of spontaneous epileptiform discharges in neocortical slices from patients undergoing epilepsy surgery. The experiments show that: (a) spatial activity patterns can be detected in optical imaging data; (b) shapes such as "mini-foci" appear in close correlation to bioelectric discharges monitored with field potential electrodes in a reproducible manner; (c) Fréchet distances yield more conservative matches regarding rectangular, and less conservative hits with respect to radially symmetric shapes than the straight skeleton approach; and (d) tolerances of 0.03-0.1 are suited to detect faithful images of pre-selected shapes, whereas values >0.8 will report matches with any polygonal pattern. In conclusion, the methods reported here are suited to detect and analyze spatial, geometric dynamics in optical imaging data.     

Analysis of the temporal expression of nestin in human fetal brain derived neuronal and glial progenitor cells

Nestin expression in the developing human brain was examined with the use of unique human specific anti-nestin antibodies. Double immunostaining of cell cultures and tissue sections derived from first and second trimester human fetal brain (HFB) examined the co-expression of nestin with other cell type specific phenotypic markers. The immunocytochemical analysis shows that from first to second trimester, the majority of developing glial cells exhibited a transitional state marked by co-expression of nestin and GFAP. However, the corresponding transitional state for developing neuronal cells, co-expressing nestin and MAP-2, was rarely detected. These results imply different temporal patterns of nestin expression in cells of glial and neuronal lineages. Confocal microscopy of HFB tissue section staining also revealed a similar pattern of nestin co-expression with glial and neuronal markers. Our results suggest that nestin expression alone may not identify an undifferentiated stem cell, and that progenitor cells in glial and neuronal lineages express nestin in different temporal patterns.     

Production and secretion of calcitonin gene-related peptide from human lymphocytes

Programmed cell death (apoptosis) is critical for the normal development and homeostasis of the immune system. There is increasing evidence that dysregulations of apoptotic pathways are associated with autoimmune disease, including multiple sclerosis (MS). Cellular commitment to apoptosis is partly regulated by the Bcl-2 family proteins, which includes the death antagonists Bcl-2 and Bcl-X(L), and death agonists Bax and Bad. Since the role of these proteins in the pathogenesis of MS is currently unknown, we analyzed their expression profile in peripheral and intrathecal lymphocytes from MS patients and appropriate controls. We observed a significant reduction in the expression ratios of pro-apoptotic to anti-apoptotic Bcl-2 members in both peripheral and intrathecal lymphocytes from MS patients when compared to corresponding ratios in patients with inflammatory or noninflammatory neurologic controls, or healthy individuals. The relative coexpression ratios of these pro- and anti-apoptotic Bcl-2 family proteins in MS were more significant than the expression of individual members. The low cellular expression ratios of pro-apoptotic proteins in MS were confirmed in vitro activated T lymphocytes. Cellular expression of Bcl-2, Bcl-X(L), Bax or Bad in MS patients was independent of the expression of other apoptotic regulatory molecules, such as Fas receptor protein or FLIP. Our findings suggest that the abnormal expression patterns of Bcl-2 family proteins in MS may promote apoptotic resistance of potentially pathogenic, autoreactive lymphocytes, and may allow for continuing cellular proliferation and tissue destruction within the central nervous system.