Sodium-calcium exchangers have long been considered inert with respect to monovalent cations such as lithium, choline, and N-methyl-D-glucamine. A key question that has remained unsolved is how despite this, Li ؉ catalyzes calcium exchange in mammalian tissues. Here we report that a Na ؉ /Ca 2؉ exchanger, NCLX cloned from human cells (known as FLJ22233), is distinct from both known forms of the exchanger, NCX and NCKX in structure and kinetics. Surprisingly, NCLX catalyzes active Li ؉ /Ca 2؉ exchange, thereby explaining the exchange of these ions in mammalian tissues. The NCLX protein, detected as both 70-and 55-KDa polypeptides, is highly expressed in rat pancreas, skeletal muscle, and stomach. We demonstrate, moreover, that NCLX is a K ؉ -independent exchanger that catalyzes Ca 2؉ flux at a rate comparable with NCX1 but without promoting Na ؉ /Ba 2؉ exchange. The activity of NCLX is strongly inhibited by zinc, although it does not transport this cation. NCLX activity is only partially inhibited by the NCX inhibitor, KB-R7943. Our results provide a cogent explanation for a fundamental question. How can Li ؉ promote Ca 2؉ exchange whereas the known exchangers are inert to Li ؉ ions? Identification of this novel member of the Na ؉ / Ca 2؉ superfamily, with distinct characteristics, including the ability to transport Li ؉ , may provide an explanation for this phenomenon.Plasma membrane Na ϩ /Ca 2ϩ exchange is an important element in cellular Ca 2ϩ homeostasis. It has been extensively investigated in mammals, especially in cardiac and neuronal tissues (1), where this mechanism is essential for regulation of Ca 2ϩ homeostasis. Na ϩ /Ca 2ϩ exchange also plays a key role in many other organs and tissues by modulating the intracellular Ca 2ϩ response (2). The Na ϩ /Ca 2ϩ exchangers described to date are members of four major families (1). Of these, only NCX1-3 and NCKX1-4 are found in mammalian cells, whereas the other two are expressed in plants, yeast, and bacteria.The stoichiometry of NCX exchangers is based on 3-4Na ϩ / 1Ca 2ϩ , and the protein is structurally organized as nine transmembrane helixes divided by a large cytoplasmic loop (3). NCKX family proteins have a stoichiometry of 4Na/1Ca/1K, and their topology is thought to consist of two sets of five membrane helixes divided by a cytoplasmic loop and NH 2 -terminal extracellular domain (4). The two families share several important functional and structural motifs: a structural hallmark of the Na ϩ /Ca 2ϩ exchanger superfamily is the presence of two regions of sequence similarity, called ␣1 and ␣2, which are considered necessary and sufficient for generating exchange activity (1, 5). Functionally, these exchangers are considered catalytically inert to inorganic and organic monovalent cations such as Li ϩ , NMG ϩ , 1 and choline ϩ , which are often used in "sodium-free" solutions to reverse Na ϩ /Ca 2ϩ exchange activity (2).Although Li ϩ ions are not transported by either NCX or NCKX, numerous physiological studies have reported a significant effect of this ion ...

No abstract

No abstract

Phase imaging with a tapping mode atomic force microscope (AFM) has many advantages for imaging moving DNA and DNA-enzyme complexes in aqueous buffers at molecular resolution. In phase images molecules can be resolved at higher scan rates and lower forces than in height images from the AFM. Higher scan rates make it possible to image faster processes. At lower forces the molecules are imaged more gently. Moving DNA molecules are also resolved more clearly in phase images than in height images. Phase images in tapping mode AFM show the phase difference between oscillation of the piezoelectric crystal that drives the cantilever and oscillation of the cantilever as it interacts with the sample surface. Phase images presented here show moving DNA molecules that have been replicated with Sequenase in the AFM and DNA molecules tethered in complexes with Escherichia coli RNA polymerase.

Exposure of Leishmania promastigotes to temperatures typical of mammals result in a stress response, which is accompanied by an increase in the steady state level of heat shock transcripts and their translation. Accumulation of the heat shock protein (hsp83) mRNA occurs due to differential decay rates at the altered temperatures, while transcription is unaffected. A similar pattern of post-transcriptional regulation was observed for a transfected chloramphenicol acetyltransferase (CAT) gene, which was flanked at both ends by intergenic regions (IR) of hsp83. Shortening the 5' untranslated region (UTR) by 100 nts produced an active CAT enzyme, but abolished the temperature-dependent regulation of the CAT-hsp83 mRNA turn-over. The 3' UTR is also involved in the temperature-dependent degradation of hsp83 mRNA, since exchange of the hsp83 3' UTR with a parallel fragment from a non-heat shock gene abolished the differential turn-over of CAT mRNA. Thus, the regulated decay of hsp83 mRNA is controlled by sequence or conformational elements present in both upstream and downstream UTRs. Like the endogenous hsp83, translation of CAT mRNA which contained hsp83 UTRs was higher at 35 degrees C. This was observed only with transcripts in which stability increased at elevated temperatures. Modifications which abolished the temperature dependence of CAT mRNA decay, eliminated its elevated translation at the higher temperatures. The correlation suggests a mechanistic link between the translational machinery and mRNA stability.