Intracellular Ca2+ and pacemaking within the rabbit sinoatrial node: heterogeneity of role and control

Recent studies have proposed that release of calcium from the sarcoplasmic reticulum (SR) modulates the spontaneous activity of the sinoatrial node (SAN). Previously we have shown that several calcium regulatory proteins are expressed at a lower level in the centre of the SAN compared with the periphery. Such differences may produce heterogeneity of intracellular calcium handling and pacemaker activity across the SAN. Selective isolations showed that the centre of the SAN is composed of smaller cells than the periphery. Measurements of cytosolic calcium in spontaneously beating cells showed that diastolic calcium, systolic calcium, the calcium transient amplitude and spontaneous rate were greater in larger (likely to be peripheral) cells compared with smaller (likely to be central) SAN cells. The SR calcium content was greater in larger cells, although SR recruitment was more efficient in smaller cells. The sodium–calcium exchanger and sarcolemmal calcium ATPase had a lower activity and the exchanger was responsible for a larger proportion of sarcolemmal calcium extrusion in smaller cells compared with larger cells. Ryanodine had a greater effect on the spontaneous calcium transient in larger cells compared with smaller cells, and slowed pacemaker activity in larger cells but not smaller cells, thus abolishing the difference in cycle length. This study shows heterogeneity of intracellular calcium regulation within the SAN and this contributes to differences in pacemaker activity between cells from across the SAN. The smallest central cells of the leading pacemaker region of the SAN do not require SR calcium for spontaneous activity nor does disruption of the SR alter pacemaking in these primary pacemaker cells.     

NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

Why are human newborns so fat? Relationship between fatness and brain size at birth [retracted article]

The plumpness of the human newborn has long been recognized as a trait in need of explanation among researchers. Using a linear regression analysis, we find that head circumference is significantly and positively associated with BMI at birth, after gestational age and birthlength were controlled for, in a sample of 1,069 healthy liveborn routinely delivered at the University Hospital of Coimbra (partial correlation r = 0.409, P < 0.0001). This significant association is consistent with the idea that newborn fatness is related to the higher need of lipids in newborn humans as an energetic and plastic substrate during its accelerated brain growth period. As birthweight and birth head size are associated with head size and cognitive abilities in childhood and adult life, it could be postulated that these cognitive abilities could have acted as selective pressure responsible for the newborn fatness increase in our lineage. Am. J. Hum. Biol. 16:24–30, 2004. © 2003 Wiley‐Liss, Inc.     

SIGN-R1, a novel C-type lectin expressed by marginal zone macrophages in spleen,mediates uptake of the polysaccharide dextran

The marginal zone macrophages of the spleen are implicated in the clearance of polysaccharides, but underlying mechanisms need to be pinpointed. SIGN-R1 is one of five recently identified mouse genes that are homologous to human DC-SIGN and encode a single, external, C-terminal C-type lectin domain. We find that a polyclonal antibody to a specific SIGN-R1 peptide reacts primarily and strongly with a subset of macrophages in the marginal zone of spleen and lymph node medulla. In both sites, SIGN-R1 exists primarily in an aggregated form, resistant to dissociation into monomers upon boiling in SDS under reducing conditions. Upon transfection into three different cell lines, high-mol.-wt forms bearing SIGN-R1 are expressed, as well as reactivity with ER-TR9, a mAb previously described to react selectively with marginal zone macrophages. SIGN-R1-expressing macrophages preferentially sequester dextrans following i.v. injection. Likewise, when phagocytic cells are enriched from spleen and tested in culture, dextran is selectively endocytosed by a subset of very large SIGN-R1(+) cells representing approximately 5% of total released macrophages. Uptake of FITC-dextran by these macrophages in vivo and in vitro is blocked by ER-TR9 and polyclonal anti-SIGN-R1 antibodies. Following transfection with SIGN-R1, cell lines become competent to endocytose dextrans. The dextran localizes primarily to compartments lacking transferrin receptor and the LAMP-1 CD107a panlysosomal antigen. Therefore, SIGN-R1 mediates the uptake of dextran polysaccharides, and it is predominantly expressed in the macrophages of the splenic marginal zone and lymph node medulla.     

Downregulation of major histocompatibility complex antigens in invading glioma cells: stealth invasion of the brain

Invasion into surrounding brain tissue is a fundamental feature of gliomas and the major reason for treatment failure. The process of brain invasion in gliomas is not well understood. Differences in gene expression and/or gene products between invading and noninvading glioma cells may identify potential targets for new therapies. To look for genes associated with glioma invasion, we first employed Affymetrix microarray Genechip technology to identify genes differentially expressed in migrating glioma cells in vitro and in invading glioma cells in vivo using laser capture microdissection. We observed upregulation of a variety of genes, previously reported to be linked to glioma cell migration and invasion. Remarkably, major histocompatiblity complex (MHC) class I and II genes were significantly downregulated in migrating cells in vitro and in invading cells in vivo. Decreased MHC expression was confirmed in migrating glioma cells in vitro using RT-PCR and in invading glioma cells in vivo by immunohistochemical staining of human and murine glioblastomas for beta2 microglobulin, a marker of MHC class I protein expression. To the best of our knowledge, this report is the first to describe the downregulation of MHC class I and II antigens in migrating and invading glioma cells, in vitro and in vivo, respectively. These results suggest that the very process of tumor invasion is associated with decreased expression of MHC antigens allowing glioma cells to invade the surrounding brain in a 'stealth'-like manner.     

Psychological and social effects of orthodontic treatment

Adolescents with commonly occurring forms of malocclusion often are presumed to be at risk for negative self-esteem and social maladjustment. A randomized control group design was used to assess the psychosocial effects of orthodontic treatment for esthetic impairment. Ninety-three participants, 11 to 14 years old, with mild to moderate malocclusions, were randomly assigned to receive orthodontic treatment immediately or after serving as delayed controls. A battery of psychological and social measures was administered before treatment, during treatment, and three times after completion of treatment, the last occurring one year after termination. Repeated measures analyses of variance assessed group differences at the five time points. Parent-, peer-, and self-evaluations of dental-facial attractiveness significantly improved after treatment, but treatment did not affect parent- and self-reported social competency or social goals, nor subjects' self-esteem. In summary, dental-specific evaluations appear to be influenced by treatment, while more general psychosocial responses are not.This research was supported by Grant NIH-NIDR-R01-DE06154 from the National Institute of Dental Research.     

IgG anti-tetanus toxoid antibody synthesis by human bone marrow. I. Two distinct populations of marrow B cells and functional differences between marrow and peripheral blood B cells

This investigation uses a system for inducing and detecting anti-tetanus toxoid antibody (anti-TT) synthesis to study specific antibody (Ab) synthesis by bone marrow mononuclear cells (MC). We measured the amounts of anti-TT secreted and the number of B cells secreting antibody (Ab). The ELISA plaque detects single B cells secreting specific Ab. The results show that (1) spontaneous anti-TT secretion by MC is higher than spontaneous anti-TT secretion by peripheral blood lymphocytes (PBL) using an ELISA plaque (P<0.01); (2) spontaneous anti-TT production by MC correlated with the serum anti-TT titers as measured by an ELISA (r=0.75,P=0.005); (3) two types of marrow B cells were identified—one that spontaneously secretes anti-TT and another that produces anti-TT after TT-stimulation; (4) the frequency of anti-TT-secreting B cells is higher in MC than in PBL; (5) the amount of Ab secreted per marrow B cell is not different from that secreted by a peripheral B cell; and (6) marrow B cells could be induced to produce anti-TTin vitro up to 10 months without added cytokines. These results show that bone marrow is a major repository for differentiated B cells that spontaneously produce Abs to maintain circulating Abs titers and for memory B cells that can be induced to produce specific Ab.     

The effects of chronic papillary necrosis on acid excretion

Complete papillary necrosis in rats can be induced within 1 month following a single injection of 2-bromoethylamine hydrobromide (BEA) (50 mg, i.v.). Utilizing a combination of clearance and balance techniques the effects of complete absence of the papilla was examined as regards urinary acidification, whole kidney glomerular filtration rate (GFR), single nephron GFR, and morphology. Whole kidney GFR was not different from control, however, the percent filtering juxtamedullary nephrons was markedly diminished (87.2±2.1 vs. 31.5±3.6% filtering, control vs. BEA, respectively,P<0.001) and significantly reduced in the superficial nephrons (80.6±3.6 vs. 62.2±6.1% filtering, control vs. BEA, respectively,P<0.05). There was a significant decrease in juxtamedullary single nephron GFR and an increase in the superficial single nephron GFR as assessed by the quantitative Hanssen's technique in the animals with chronic papillary necrosis. Complete papillary necrosis was associated with normal arterial bicarbonate concentration, pH, and plasma electrolyte concentrations. At the same degree of acidemia (induced by NH₄Cl administration) minimal urinary pH, ammonium excretion, and titratable acid excretion were not different than seen in age matched controls. The response to Na₂SO₄ infusion and phosphate infusion was the same in both groups of animals. The urineblood (U-B)pCO₂, an index of urinary acidification, was identical in BEA and control animals. Scanning electron microscopy showed scarring of the juxtamedullary glomeruli one month after BEA. The papilla was sloughed and lying free in the renal pelvis in every experimental animal. These data demonstrate that complete papillary necrosis is not associated with acidosis nor a defect in urinary acidification.