A high proportion of mutations in the BRCA1 gene in German breast/ovarian cancer families with clustering of mutations in the 3′ third of the gene

We have analyzed 61 German breast and breast/ovarian cancer families for BRCA1 mutations using single-strand conformation polymorphism analysis (SSCP) followed by sequencing. Forty-seven of the families had at least three cases (at least two under 60 years) and 14 families had only two cases of breast/ovarian cancer (at least one under 50 years). Twenty-eight families were breast/ovarian and 33 were breast cancer-only families. Eighteen mutations in BRCA1 were detected in 11/28 breast/ovarian cancer families and 7/33 breast cancer families and none in the families with only two cases. We identified 17 truncation mutations (8 frameshift, 7 nonsense and 2 splice variants) and one missense mutation. Seven of these are novel and two, the 5382insC and 5622C→T mutations, occurred in two apparently unrelated families. The genotype of the two families with the 5382insC mutation is compatible with the rare haplotype segregating with the 5382insC mutation in different populations, further supporting its European origin. One unclassified missense alteration, R841W, was found in one family but did not segregate with the disease, suggesting that it is more likely a polymorphism. We also report and discuss the sequence of several new unclassified single-nucleotide changes first identified by SSCP. Of the 18 mutations, 13 occurred in the 3′ third of the gene (end of exon 11–24) and ovarian cancers were found in eight of these families. Received: 5 February 1998 / Accepted: 7 April 1998     

Malate dehydrogenase of spinach: Substrate kinetics of different forms

Sephadex G-200 gel filtration of an ammonium sulfate fraction, containing the bulk of NAD-dependent malate dehydrogenase, yields forms of differing MW. Both Mg²⁺ and NADH stabilize the 127000 daltons MW form. K⁺, or incubation with dithioerythritol, cause splitting and partial reaggregation, resulting in MWs ranging between 35000 and 180000 daltons. Chromatography in the presence of dithioerythritol and NADH results in an enzyme with a non-linear reaction rate at low substrate concentrations. Plots of initial velocity vs substrate and cofactor concentration respectively are characterized by two slopes of positive cooperativity separated by an intermediary plateau of negative cooperativity. Gel chromatography in the presence of Mg²⁺ or K⁺ or drastic dilution of the enzyme results in an enzyme with linear reaction rates also at low substrate concentration. Its kinetics are consistent with the view that the enzyme undergoes conformational changes when the substrate concentration is varied.     

Über die Zusammensetzung des Zikadenschaumes

Ohne ZusammenfassungDer Deutschen Forschungsgemeinschaft sind wir für ihre Unterstützung, Herrn Prof. Dr. A. Koch für Literaturhinweise zu Dank verpflichtet.     

Structural biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology.     

Activation of the sodium/hydrogen exchanger via the fibronectin-integrin pathway results in hematopoietic stimulation

The proliferative response of hematopoietic cells is regulated by many factors, including the presence and type of growth factors, the cellular microenvironment, and the physiochemical conditions prevailing in the tissue milieu. A process fundamental to all cells is the regulation of the intracellular acid-base conditions. One of the mechanisms by which intracellular pH (pH_i) is regulated is through the sodium/hydrogen exchanger, a ubiquitous membrane protein which exploits the intra- and extracellular sodium ion gradient to drive hydrogen ions out of the cell. However, activation of the exchanger via mitogenic and nonmitogenic signals leads to an increase in pH_i which, in turn, may directly or indirectly result in a proliferative response. It has been shown that interaction of fibronectin with its integrin receptor subunits α₄ and α₅ can result in activation of the Na⁺/H⁺ exchanger. In this report, we demonstrate that when mouse bone marrow cells are physically brought together in a preculture system we designate as high cell density culture (HCDC), in a small volume and at the same cellularity as that in the marrow, hematopoietic stem and progenitor cell populations are stimulated with no additional stimulation in the presence of growth factors. Neutralizing antibodies to the growth factors added to HCDC had little, if any, effect on the degree of stimulation. However, when antibodies to fibronectin or the α₄ integrin subunit were added to HCDC, inhibition was observed, indicating that the observed hematopoietic stimulation occurred via the fibronectin-integrin pathway. Addition of 5 μM 5-(N,N-hexamethylene) amiloride (5-HMA), a specific inhibitor of the Na⁺/H⁺ exchanger, also resulted in inhibition of in vitro hematopoiesis. Since the exchanger was implicated, we then measured the pH_i of normal and HCDC-treated bone marrow cells in the absence and presence of 5-HMA by flow cytometry using the fluorescent pH-sensitive indicator, carboxy SNARF-1 AM. It was found that cells subjected to HCDC exhibited a higher pH_i than normal fresh cells. In each case, the pH_i was lowered in the presence of 5-HMA. Furthermore, addition of antibodies to fibronectin or the α₄ integrin subunit to HCDC also reduced the pH_i to a similar level to that found for 5-HMA. Our results demonstrate, for the first time, that a hematopoietic stem and progenitor cell proliferative response can be initiated by activation of the Na⁺/H⁺ exchanger, leading to an increase in pH_i, via cell-cell interaction through the fibronectin-integrin pathway. This pathway could, therefore, be significant not only in normal hematopoietic regulation, but also under pathophysiological conditions. J. Cell. Physiol. 177:109–122, 1998. © 1998 Wiley-Liss, Inc.