The assessment and prediction of lung absorption and disposition are an increasingly essential preclinical task for successful discovery and product development of inhaled drugs for both local and systemic delivery. Hence, in vitro, ex vivo and in vivo preclinical methods of lung absorption continue to evolve with several technical, methodological and analytical refinements. As in vitro lung epithelial cell monolayer models, the air-liquid interface (ALI)-cultured Calu-3 cells have most frequently been used, but the NCI-H441 and hAELVi cells have now been proposed as the first immortalized human alveolar epithelial cells capable of forming highly-restricted monolayers. The primary ALI-cultured three-dimensional (3D) human lung cell barriers have also become available; efforts to incorporate aerosol drug deposition into the in vitro lung cell models continue; and stem cell-derived lung epithelial cells and "lung-on-a-chip" technology are emerging. The ex vivo isolated perfused rat lung (IPRL) methods have increasing been used, as they enable the kinetic determination of tissue/organ-level diffusive and membrane protein-mediated absorption and competing non-absorptive loss; the assessment of "pre-epithelial" aerosol biopharmaceutical events in the lung, such as dissolution and release; and the ex vivo-to-in vivo extrapolation and prediction. Even so, in vivo small rodent-based methods have been of mainstay use, while large animal-based methods find an additional opportunity to study region-dependent lung absorption and disposition. It is also exciting that human pharmacokinetic (PK) profiles and systemic exposures for inhaled drugs/molecules may be able to be predicted from these in vivo rodent PK data following lung delivery using kinetic modeling approach with allometric scaling. Overall, the value of these preclinical assessments appears to have shifted more to their translational capability of predicting local lung and systemic exposure in humans, in addition to rationalizing optimal inhaled dosage form and delivery system for drugs/molecules in question. It is critically important therefore to make appropriate selection and timely exploitation of the best models at each stage of drug discovery and development program for efficient progress toward product approval and clinical use.

We have previously identified in the human EST sequence data base four overlapping clones that could be aligned with both a predicted protein sequence, deduced from the C. elegans genomic sequence, and partial amino acid sequences, obtained for a protein from canine pancreatic microsomes. We suggested that these proteins are homologs of yeast microsomal and DnaJ-like protein Scj1p and termed them ERj3p. Here we verified the predicted protein sequence of human ERj3p by sequence analysis of the corresponding cDNA. Multiple alignment of related sequences identified these proteins as true homologs of yeast Scj1p. Biochemical analysis of the canine protein characterized ERj3p as a soluble glycoprotein of the pancreatic endoplasmic reticulum. This pancreatic DnaJ-like protein was shown to interact with lumenal DnaK-like proteins, such as BiP. Furthermore, we found that ERj3p interacts with SDF2L1 protein that may be involved in protein Oglycosylation. We propose that ERj3p represents a cochaperone of DnaK-like chaperones of the mammalian endoplasmic reticulum and is involved in folding and maturation of newly synthesized proteins.

The purpose of this paper is to review the history of using lectins to target and deliver drugs to their site of action. The hour of birth of "lectinology" may be defined as the description of the agglutinating properties of ricin, by Herrmann Stillmark in 1888, however, the modern era of lectinology began almost 100 years later in 1972 with the purification of different plant lectins by Sharon and Lis. The idea to use lectins for drug delivery came in 1988 from Woodley and Naisbett, who proposed the use of tomato lectin (TL) to target the luminal surface of the small intestine. Besides the targeting to specific cells, the lectin-sugar interaction can also been used to trigger vesicular transport into or across epithelial cells. The concept of bioadhesion via lectins may be applied not only for the GI tract but also for other biological barriers like the nasal mucosa, the lung, the buccal cavity, the eye and the blood-brain barrier.

Previous studies have shown that the rough endoplasmic reticulum (ER) contains nascent precursor polypeptide gated channels. Circumstantial evidence suggests that these channels are formed by the Sec61p complex. We reconstituted the purified Sec61p complex in a lipid bilayer and characterized its dynamics and regulation. The Sec61p complex is sufficient to form the precursor polypeptide activated channel under co- and posttranslational transport conditions. Activity of the Sec61p channel in both transport modes is induced by direct interaction with precursor protein. The Sec61p complex comprises a highly dynamic pore covering conductances corresponding to channel openings from ∼6 to 60 Å. Its properties are indistinguishable from those we observed with native ER channels, directly demonstrating that these channels are formed by the Sec61p complex.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human Hsp70/Hsp90-organizing protein (Hop). Guided by Hop structural data and sequence alignment analyses, we have used site-directed mutagenesis, co-precipitation assays, circular dichroism spectroscopy, steady-state fluorescence, and surface plasmon resonance spectroscopy to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal tetratricopeptide repeat domain (TPR1) of mSTI1 to bind to heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. We have shown that substitutions in the first TPR motif of Lys(8) or Asn(12) did not affect binding of mSTI1 to Hsc70, whereas double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn(43) lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys(8) or Asn(12) reduced but did not abrogate binding. These results suggest that mSTI1-Hsc70 interaction requires a network of interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70 but also outside the TPR domain. We propose that the electrostatic interactions in the first TPR motif made by Lys(8) or Asn(12) define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, we substituted residues on TPR1 potentially involved in hydrophobic contacts with Hsc70. The modified protein had reduced binding to Hsc70 but now showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on TPR2A. Our results suggest that binding of Hsc70 to TPR1 is more specific than binding of Hsp90 to TPR2A with serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in vivo.

Recently, the homolog of yeast protein Sec63p was identified in dog pancreas microsomes. This pancreatic DnaJ‐like protein was shown to be an abundant protein, interacting with both the Sec61p complex and lumenal DnaK‐like proteins, such as BiP. The pancreatic endoplasmic reticulum contains a second DnaJ‐like membrane protein, which had been termed Mtj1p in mouse. Mtj1p is present in pancreatic microsomes at a lower concentration than Sec63p but has a higher affinity for BiP. In addition to a lumenal J‐domain, Mtj1p contains a single transmembrane domain and a cytosolic domain which is in close contact with translating ribosomes and appears to have the ability to modulate translation. The interaction with ribosomes involves a highly charged region within the cytosolic domain of Mtj1p. We propose that Mtj1p represents a novel type of co‐chaperone, mediating transmembrane recruitment of DnaK‐like chaperones to ribosomes and, possibly, transmembrane signaling between ribosomes and DnaK‐like chaperones of the endoplasmic reticulum.

Cotranslational protein transport into dog pancreas microsomes involves the Sec61p complex plus a luminal heat shock protein 70. Posttranslational protein transport into the yeast endoplasmic reticulum (ER) involves the so-called Sec complex in the membrane, comprising a similar Sec61p subcomplex, the putative signal peptide receptor subcomplex, and the heat shock protein 40-type subunit, Sec63p, plus a luminal heat shock protein 70. Recently, human homologs of yeast proteins Sec62p and Sec63p were discovered. Here we determined the concentrations of these two membrane proteins in dog pancreas microsomes and observed that the canine homologs of yeast proteins Sec62p and Sec63p are abundant proteins, present in almost equimolar concentrations as compared with Sec61alphap monomers. Furthermore, we detected fractions of these two proteins in association with each other as well as with the Sec61p complex. The J domain of the human Sec63p was shown to interact with immunoglobulin heavy chain binding protein. Thus, the membrane of the mammalian ER contains components, known from the posttranslationally operating protein translocase in yeast. We suggest that these components are required for efficient cotranslational protein transport into the mammalian ER as well as for other transport processes.

Dog pancreas microsomes represent the key components of the established model system for the analysis of protein transport into the mammalian endoplasmic reticulum. More recently, these microsomes were also employed in cell-free systems which address questions related to protein folding and protein degradation in the mammalian endoplasmic reticulum. In order to get at a complete picture of these undoubtedly related processes in the in vitro system we need to know all the proteins we are dealing with, and their respective stoichiometries. Here we give a progress report on our attempts to identify and to quantify the soluble molecular chaperones and folding catalysts which are present in the lumen of dog pancreas microsomes. Eventually, we will need to know how the in vitro system compares with the situation in intact pancreatic cells as well as in other cells.