Gene mutations and alternate RNA splicing result in truncated Ig L chains in human gamma H chain disease

The lack of covalently associated L chains features H chain disease proteins produced in some human B cell lymphoproliferative disorders. We cloned and characterized the single rearranged kappa L chain gene from the leukemic lymphocytes of a patient (RIV) affected with gamma 1 H chain disease, to determine the molecular basis for absent L chain. This kappa allele had undergone an effective V-J rearrangement. Extensive somatic mutation focused about the V-J region created a sequence that was only 75% homologous to its germ-line counterpart. Altered acceptor (V kappa) and donor (J kappa) splice sites resulted in an aberrant splice between the leader and C kappa exons and a truncated 850-bp kappa mRNA. RIV leukemic cells as well as myeloma cells transfected with the RIV kappa gene synthesized a truncated protein. Simultaneous defects in H and L chains genes may reflect a hypermutational mechanism for Ig genes in B cells.     

“Unblocking” Serum Activity <Emphasis Type="Italic">in vitro</Emphasis> in the Polyoma System may Correlate with Antitumour Effects of Antiserum <Emphasis Type="Italic">in vivo</Emphasis>

In a variety of tumour systems, individuals carrying progressively growing neoplasms have lymphoid cells with a specific cytotoxic effect on cultured tumour cells from the same individual^1–4. Since the sera of tumour-bearing individuals have been shown to prevent tumour cell destruction by immune lymphocytes in vitro^2,5–8 and since this serum blocking activity appears early in primary and transplant tumour development^5,7, it has been suggested that the appearance of this serum blocking activity might be responsible for the progressive growth of tumours in individuals having cytotoxic lymphocytes. Counteraction of this blocking activity would thus be of primary importance in facilitating the function of an already existing or bolstered cell-mediated immunity. The serum blocking activity might be inhibited in various ways, by preventing the formation of blocking antibody or by interfering with its action (“unblocking”), as demonstrated in Moloney sarcoma regressor sera⁹. This type of serum also has a therapeutic effect on Moloney sarcomas in vivo^10,11, which has been tentatively attributed to its unblocking activity^8,9 or, possibly, to a complement-dependent cytotoxicity¹⁰. Tumour growth in the Moloney sarcoma system, however, might be due in part to continuous recruitment of neoplastic cells by virus-induced transformation and so the therapeutic effect could be due to a virus-neutralizing serum activity^9,10.     

Genetic transfer and expression of reconstructed yeast artificial chromosomes containing normal and translocated BCL2 proto-oncogenes.

The goal of this study was to determine whether it will be feasible to study the expression of a large, human gene, such as the BCL2 proto-oncogene, by DNA transfection. The BCL2 proto-oncogene is 230 kb in size and is deregulated in tumor cells by translocation into the immunoglobulin heavy-chain locus. Yeast artificial chromosomes (YACs) containing the human BCL2 gene were altered by homologous recombination in Saccharomyces cerevisiae to yield replicas of the normal and translocated alleles. Constructions containing either allele and ranging in size from 360 to 800 kb were integrated stably into a mouse tumor line. Fifty-eight percent of the clones contained a copy of the entire YAC insert. Over 50% of these clones expressed appropriate levels of human BCL2 RNA and protein. These studies suggested that the expression of large human genes and their pathologic rearrangements can be studied by transfection techniques employing YACs propagated in S. cerevisiae.     

On the mechanism of ATP-induced shape changes in the human erythrocyte membranes: the role of ATP

In the preceding paper (Sheetz, M. and S.J. Singer. 1977. J Cell Biol. 73:638-646) it was shown that erythrocyte ghosts undergo pronounced shape changes in the presence of mg-ATP. The biochemical effects of the action of ATP are herein examined. The biochemical effects of the action of ATP are herein examined. Phosphorylation by ATP of spectrin component 2 of the erythrocyte membrane is known to occur. We have shown that it is only membrane protein that is significantly phosphorylated under the conditions where the shape changes are produced. The extent of this phosphorylation rises with increasing ATP concentration, reaching nearly 1 mol phosphoryle group per mole of component 2 at 8mM ATP. Most of this phosphorylation appears to occur at a single site on the protein molecule, according to cyanogen bromide peptide cleavage experiments. The degree of phosphorylation of component 2 is apparently also regulated by a membrane-bound protein phosphatase. This activity can be demonstrated in erythrocyte ghosts prepared from intact cells prelabeled with [(32)P]phosphate. In addition to the phosphorylation of component 2, some phosphorylation of lipids, mainly of phosphatidylinositol, is also known to occur. The ghost shape changes are, however, shown to be correlated with the degree of phosphorylation of component 2. In such experiment, the incorporation of exogenous phosphatases into ghosts reversed the shape changes produced by ATP, or by the membrane-intercalating drug chlorpromazine. The results obtained in this and the preceding paper are consistent with the proposal that the erythrocyte membrane possesses kinase and phosphates activities which produce phosphorylation and dephosphorylation of a specific site on spectrin component 2 molecules; the steady-state level of this phosphorylation regulates the structural state of the spectrin complex on the cytoplasmic surface of the membrane, which in turn exerts an important control on the shape of the cell.     

Selective inhibition of prostaglandin biosynthesis by gold salts and phenylbutazone

Gold salts and phenylbutazone selectively inhibit the synthesis of PGF_2α and PGE₂ respectively. Lowered production of one prostaglandin species is accompanied by an increased production of the other. Selective inhibition by these drugs was observed in the presence of adrenaline, reduced glutathione and copper sulphate under conditions when most anti-inflammatory compounds inhibited PGE₂ and PGF_2α syntheses equally. It is postulated that selective inhibitors may have a different mode of action and beneficial effects may be related to the endogenous ratio of PGE to PGF required for normal function.     

Phosphorylation of BCL-2 regulates ER Ca2+ homeostasis and apoptosis

Phosphorylation of BCL-2 within an unstructured loop inhibits its antiapoptotic effect. We found that phosphorylated BCL-2 predominantly localized to the endoplasmic reticulum (ER) and tested whether phosphorylation would control its activity at this organelle, where Ca(2+) dynamics serve as a critical control point for apoptosis. Phosphorylation greatly inhibits the ability of BCL-2 to lower [Ca(2+)](er) and protect against Ca(2+)-dependent death stimuli. Cells expressing nonphosphorylatable BCL-2(AAA) exhibited increased leak of Ca(2+) from the ER and further diminished steady-state [Ca(2+)](er) stores when compared to cells expressing BCL-2(wt). Consequently, when BCL-2 is phosphorylated, Ca(2+) discharge from the ER is increased, with a secondary increase in mitochondrial Ca(2+) uptake. We also demonstrate that phosphorylation of BCL-2 inhibits its binding to proapoptotic family members. This inhibitory mechanism manifested at the ER, where phosphorylated BCL-2 was unable to bind proapoptotic members. [Ca(2+)](er) proved coordinate with the capacity of BCL-2 to bind proapoptotic BH3-only members, further integrating the apoptotic pathway and Ca(2+) modulation. Unexpectedly, the regulation of ER Ca(2+) dynamics is a principal avenue whereby BCL-2 phosphorylation alters susceptibility to apoptosis.     

c-Myc functionally cooperates with Bax to induce apoptosis

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.     

Bax-dependent spermatogonia apoptosis is required for testicular development and spermatogenesis

Bax is a multidomain, proapoptotic member of the Bcl-2 family that is required for normal spermatogenesis in mice. Despite its proapoptotic function, previous results found that Bax-deficient mature male mice demonstrate increased cell death and dramatic testicular atrophy. The present study examined the role of Bax during the normal development of the testis to determine whether the increased cell death in mature mice could be explained by decreased apoptosis earlier in development. Consistent with this hypothesis, testicular atrophy is preceded by increased testicular weight and hypercellular tubules in immature Bax-deficient mice. TUNEL staining at Postnatal Day (P) 7 and morphological quantitation between P5 and P15 demonstrates decreased germ cell apoptosis in Bax-deficient mice. By P15, increased numbers of type A spermatogonia, and at P12 and P15, an increase in intermediate type spermatogonia were noted in Bax-deficient animals. By P25, the number of basal compartment cells was greatly increased in Bax-deficient animals compared with controls such that four or five layers of preleptotene spermatocytes were routinely present within the basal compartment of the testis. Although the Sertoli cell barrier was significantly removed from the basement membrane, it appeared intact as judged by the hypertonic fixation test. During late pubertal development, massive degeneration of germ cells took place, including many of those cell types that previously survived in the first wave of spermatogenesis. The data indicate that Bax is required for normal developmental germ cell death in the type A spermatogonia, specifically dividing (A(2), A(3), and A(4)) spermatogonia, at a time at which the number of spermatogonia is regulated in a density-dependent manner. The massive hyperplasia that occurs in Bax-deficient mice subsequently results in Bax independent cell death that may be triggered by overcrowding of the seminiferous epithelium.