Model for mammalian metallothionein structure.

The results of physicochemical studies of mammalian metallothioneins are summarized and used to propose a model of the protein. The primary structures of all mammalian metallothioneins are very homologous; there are 38 invariant residues and 20 of them are cysteines. The results of UV and CD optical studies indicated that all 20 cysteines are involved in the ligation of 7 mol of metal per mol of metallothionein and that the protein does not contain any alpha-helix structure. A theoretical analysis by the Chou-Fasman method has predicted 11 beta-bends, each one involving at least one cysteine residue. The most significant structural data, provided by 113Cd NMR, demonstrated that the 7 mol of bound Cd2+ are arranged in two separate metal clusters, one containing four metal ions and the other containing three, with all Cd2+ tetrahedrally coordinated to cysteine thiolate ligands. The 11 cysteine residues of the carboxyl-terminal portion of the metallothionein chain (residues 30-61) are ligated to the 4-metal cluster as shown by 113Cd NMR of this enzymatically cleaved fragment. The remaining cysteine residues from the amino-terminal polypeptide portion (residues 1-29) form the 3-metal cluster. Such a division of the chain is consistent with the presence of an intron in the mouse metallothionein-1 gene corresponding to residue 32 in the polypeptide chain. A two-domain molecular model has been constructed based on an analysis of all the available data and is described in detail. The accuracy of this model was tested by 1H NMR at 500 MHz and the data are in agreement with our proposed structure.     

Long-term follow-up of children breast-fed by mothers receiving depot-medroxyprogesterone acetate

A long-term follow-up study compared development and health of 128 breast-fed children whose mothers had received depotmedroxyprogesterone acetate (depot-MPA) while lactating and 142 control children whose mothers had used mechanical contraceptives or no contraceptives or had undergone sterilization. The children, who were approximately 4-1/2 years old at follow-up, showed no ill effects on their growth and development and health status from exposure to depot-MPA. Depot-MPA-treated mothers lactated significantly longer than controls and also had greater parity than controls. These factors apparently contributed to a difference in weight at follow-up. Compared with the SempePedron standard, more of the depot-MPA group were underweight and more controls were overweight.     

Progressive and regressive changes in the nucleus pulposus. Part I. The neonate

Magnetic resonance (MR) imaging, correlated with anatomic sections, was used to characterize the progressive and regressive changes in the nucleus pulposus in neonates. The spines of five fetuses and five full-term infants between 16 and 40 weeks old were studied. In anatomic sections, the nucleus pulposus was sharply demarcated from the anulus fibrosus, Sharpey fibers were conspicuous, and a plate of primitive notochord was evident in the equator of the disk. On long repetition time (TR)/long echo time (TE) or long TR/short TE MR images, Sharpey fibers (low signal intensity) and notochord (low signal intensity) could be differentiated from the high-signal-intensity nucleus pulposus and anulus fibrosus. The major differences between the fetal and infant spines were the amount of notochord in the disk and ossification in the vertebral body.     

Discovery of a potent inhibitor of the antiapoptotic protein Bcl-xL from NMR and parallel synthesis

The antiapoptotic proteins Bcl-x(L) and Bcl-2 play key roles in the maintenance of normal cellular homeostasis. However, their overexpression can lead to oncogenic transformation and is responsible for drug resistance in certain types of cancer. This makes Bcl-x(L) and Bcl-2 attractive targets for the development of potential anticancer agents. Here we describe the structure-based discovery of a potent Bcl-x(L) inhibitor directed at a hydrophobic groove on the surface of the protein. This groove represents the binding site for BH3 peptides from proapoptotic Bcl-2 family members such as Bak and Bad. Application of NMR-based screening yielded an initial biaryl acid with an affinity (K(d)) of approximately 300 microM for the protein. Following the classical "SAR by NMR" approach, a second-site ligand was identified that bound proximal to the first-site ligand in the hydrophobic groove. From NMR-based structural studies and parallel synthesis, a potent ligand was obtained, which binds to Bcl-x(L) with an inhibition constant (K(i)) of 36 +/- 2 nM.     

A small-molecule inhibitor of Bcl-XL potentiates the activity of cytotoxic drugs in vitro and in vivo

Inhibition of the prosurvival members of the Bcl-2 family of proteins represents an attractive strategy for the treatment of cancer. We have previously reported the activity of ABT-737, a potent inhibitor of Bcl-2, Bcl-X(L), and Bcl-w, which exhibits monotherapy efficacy in xenograft models of small-cell lung cancer and lymphoma and potentiates the activity of numerous cytotoxic agents. Here we describe the biological activity of A-385358, a small molecule with relative selectivity for binding to Bcl-X(L) versus Bcl-2 (K(i)'s of 0.80 and 67 nmol/L for Bcl-X(L) and Bcl-2, respectively). This compound efficiently enters cells and co-localizes with the mitochondrial membrane. Although A-385358 shows relatively modest single-agent cytotoxic activity against most tumor cell lines, it has an EC(50) of <500 nmol/L in cells dependent on Bcl-X(L) for survival. In addition, A-385358 enhances the in vitro cytotoxic activity of numerous chemotherapeutic agents (paclitaxel, etoposide, cisplatin, and doxorubicin) in several tumor cell lines. In A549 non-small-cell lung cancer cells, A-385358 potentiates the activity of paclitaxel by as much as 25-fold. Importantly, A-385358 also potentiated the activity of paclitaxel in vivo. Significant inhibition of tumor growth was observed when A-385358 was added to maximally tolerated or half maximally tolerated doses of paclitaxel in the A549 xenograft model. In tumors, the combination therapy also resulted in a significant increase in mitotic arrest followed by apoptosis relative to paclitaxel monotherapy.