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We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH ([pH.sub.i]) and extracellular pH ([pH.sub.o]). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte [pH.sub.i]: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, N[H.sup.+.sub.4]. We find that both N[H.sub.2]-terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the N[H.sub.2]-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336-347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic [Ca.sup.2+] ([Ca.sup.2+.sub.i]) diminished or abolished AE2 stimulation by N[H.sup.+.sub.4] and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of [Ca.sup.2+] signaling. Unlike AE2 stimulation by N[H.sup.+.sub.4] and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain. band 3; Xenopus oocyte; isotopic flux; site-directed mutagenesis; chloride-bicarbonate exchange; chloride-bicarbonate exchanger; intracellular calcium ion

For complex diseases in which multiple mediators contribute to overall disease pathogenesis by distinct or redundant mechanisms, simultaneous blockade of multiple targets may yield better therapeutic efficacy than inhibition of a single target. However, developing two separate monoclonal antibodies for clinical use as combination therapy is impractical, owing to regulatory hurdles and cost. Multi-specific, antibody-based molecules have been investigated; however, their therapeutic use has been hampered by poor pharmacokinetics, stability and manufacturing feasibility. Here, we describe a generally applicable model of a dual-specific, tetravalent immunoglobulin G (IgG)-like molecule—termed dual-variable-domain immunoglobulin (DVD-Ig)—that can be engineered from any two monoclonal antibodies while preserving activities of the parental antibodies. This molecule can be efficiently produced from mammalian cells and exhibits good physicochemical and pharmacokinetic properties. Preclinical studies of a DVD-Ig protein in an animal disease model demonstrate its potential for therapeutic application in human diseases. [ABSTRACT FROM AUTHOR]

Genes predicted to be associated with the putative proteasome of Mycobacterium tuberculosis (Mtb) play a critical role in defence of the bacillus against nitrosative stress. However, proteasomes are uncommon in eubacteria and it remains to be established whether Mtb's prcBA genes in fact encode a proteasome. We found that coexpression of recombinant PrcB and PrcA in Escherichia coli over a prolonged period at 37°C allowed formation of an α7β7β7α7, 750 kDa cylindrical stack of four rings in which all 14 β-subunits were proteolytically processed to expose the active site threonine. In contrast to another Actinomycete, Rhodococcus erythropolis, Mtb's β-chain propeptide was not required for particle assembly. Peptidolytic activity of the 750 kDa particle towards a hydrophobic oligopeptide was nearly two orders of magnitude less than that of the Rhodococcus 20S proteasome, and unlike eukaryotic and archaeal proteasomes, activity of the Mtb 750 kDa particle could not be stimulated by SDS, Mg2+ or Ca2+. Electron microscopy revealed what appeared to be obstructed α-rings in the Mtb 750 kDa particle. Deletion of the N-terminal octapeptide from Mtb's α-chain led to disappearance of the apparent obstruction and a marked increase of peptidolytic activity. Unlike proteasomes isolated from other Actinomycetes, the open-gate Mtb mutant 750 kDa particle cleaved oligopeptides not only after hydrophobic residues but also after basic, acidic and small, neutral amino acids. Thus, Mtb encodes a broadly active, gated proteasome that may work in concert with an endogenous activator. [ABSTRACT FROM AUTHOR]

We showed previously that the nonerythroid anion exchanger AE2 and the erythroid anion exchanger AE1 differ greatly in their regulation by acute changes in intracellular pH (pH[sub i]) and extracellular pH (pH[sub o]). We have now examined how AE2, but not AE1, is activated by two stimuli with opposing effects on oocyte pH[sub i]: an alkalinizing stimulus, hypertonicity, and an acidifying stimulus, NH[sub 4, sup +]. We find that both NH[sub 2]terminal cytoplasmic and COOH-terminal transmembrane domains of AE2 are required for activation by either stimulus. Directed by initial deletion mutagenesis studies of the NH[sub 2]-terminal cytoplasmic domain, an alanine scan of AE2 amino acids 336-347 identified residues whose individual mutation abolished or severely attenuated sensitivity to both or only one activating stimulus. Chelation of cytoplasmic Ca[sup 2+] (Ca[sup 2+, sub i]) diminished or abolished AE2 stimulation by NH[sub 4, sup +] and by hypertonicity. Calmidazolium inhibited AE2 activity, but not that of AE1. AE2 was insensitive to many other modifiers of Ca[sup 2+] signaling. Unlike AE2 stimulation by NH[sub 4, sup +] and by hypertonicity, AE2 inhibition by calmidazolium required only AE2's COOH-terminal transmembrane domain. [ABSTRACT FROM AUTHOR]