Identification and preliminary characterization of mouse Adam33

BACKGROUND: The metalloprotease-disintegrin family, or ADAM, proteins, are implicated in cell-cell interactions, cell fusion, and cell signaling, and are widely distributed among metazoan phyla. Orthologous relationships have been defined for a few ADAM proteins including ADAM10 (Kuzbanian), and ADAM17 (TACE), but evolutionary relationships are not clear for the majority of family members. Human ADAM33 refers to a testis cDNA clone that does not contain a complete open reading frame, but portions of the predicted protein are similar to Xenopus laevis ADAM13. RESULTS: In a 48 kb region of mouse DNA adjacent to the Attractin gene on mouse chromosome 2, we identified sequences very similar to human ADAM33. A full-length mouse cDNA was identified by a combination of gene prediction programs and RT-PCR, and the probable full-length human cDNA was identified by comparison to human genomic sequence in the homologous region on chromosome 20p13. Mouse ADAM33 is 44% identical to Xenopus laevis ADAM13, however a phylogenetic alignment and consideration of functional domains suggests that the two genes are not orthologous. Mouse Adam33 is widely expressed, most highly in the adult brain, heart, kidney, lung and testis. CONCLUSIONS: While mouse ADAM33 is similar to Xenopus ADAM13 in sequence, further examination of its embryonic expression pattern, catalytic activity and protein interactions will be required to assess the functional relationship between these two proteins. Adam33 is expressed in the mouse adult brain and could play a role in complex processes that require cell-cell communication

The effect of parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHRP) on Na+H+ exchanger activity and analysis of signal transduction mechanisms

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na$sp+$/H$sp+$ exchanger (NHE) activity in various types of cells such as osteoblastic cells and renal proximal tubule OK cells. Na$sp+$/H$sp+$ exchangers are plasma membrane transporters catalyzing the electroneutral exchange of 1 H$sp+$ for 1 Na$sp+.$ Several mammalian isoforms of NHE have been so far identified with each mediating a variety of specific functions. Parathyroid hormone, playing an essential role in the physiology of blood Ca$sp{2+}$ and phosphate homeostasis, inhibits renal proximal tubular bicarbonate reabsorption by inhibiting the apically located Na$sp+$/H$sp+$ exchanger. However, which specific isoform of NHE mediated this effect and the specific signaling components involved were unknown. In our studies we determined that Na$sp+$/H$sp+$ exchanger NHE-3 isoform is expressed in the renal proximal tubule OK cells and that N-terminal PTH and PTHRP analogues upon binding to their receptor stimulate both the PKA and the PKC pathways, each of which can independently lead to inhibition of this exchanger activity. NHEs also play an important role in the regulation of intracellular pH which is subject to fluctuation occurring during the process of hormone stimulated bone formation and bone remodeling. Again the specific NHE isoform(s) mediating this effect and the signaling pathways involved were unidentified. It is determined by our studies that NHE type 1 is expressed in osteoblastic cell line, UMR-106 cells, and that PTH and PTHRP stimulate this exchanger via a cAMP-dependent pathway exclusively. It was believed that motivation of NHE-1 in the UMR-106 cells and inhibition of NHE-3 in the OK cells by N-terminal analogues of PTH and PTHRP involves binding of these analogues to a common G protein-coupled receptor called the "classical" PTH/PTHRP receptor. However, with the recent discovery of other PTH and/or PTHRP receptor, this hypothesis is no longer clea

DNA Sequencing Using Rolling Circle Amplification and Precision Glass Syringes in a High-Throughput Liquid Handling System

An automated high-throughput method that employs rolling circle amplification (RCA) to generate template for large-scale DNA sequencing has been developed using liquid handling systems equipped with precision glass syringes. A protocol was designed to perform the sequencing analysis from template preparation to thermal cycle sequencing within the same vessel, thus minimizing the amount of liquid handling and transfer. The amplified DNA was directly used for cycle sequencing with no need for any purification procedures. Total RCA reaction volumes as low as 500 nL generated sufficient templates for successful sequencing. Reducing the RCA total reaction volumes by a 40-fold factor, from a total of 20 μL to 500 nL, resulted in a significant reduction in cost, from $1.25/reaction to less than$0.04/reaction. Additionally, the volume of the sequencing reactions was reduced from a total of 20 to 10 μL, thus generating a further cost advantage. This high-throughput DNA sequencing protocol maximizes the speed and precision of processing while significantly reducing the cost of amplification