Secondary mAb--vcMMAE conjugates are highly sensitive reporters of antibody internalization via the lysosome pathway

Monoclonal antibodies (mAb) selectively recognizing tumor surface antigens are an important and evolving approach to targeted cancer therapy. One application of therapeutic mAbs is drug targeting via mAb-drug conjugate (ADC) technology. Identification of mAbs capable of internalizing following antigen binding has been accomplished by tracking decline of surface-bound mAb or by internalization of a secondary mAb linked to a toxin. These methods may not be sufficiently sensitive for screening nor wholly predictive of the mAbs' capacity for a specific drug delivery. We have developed a highly selective and sensitive method to detect mAbs for cell internalization and drug delivery. This system uses secondary anti-human or anti-murine mAbs conjugated to the high-potency drug monomethyl auristatin E (MMAE) via a highly stable, enzymatically cleavable linker. Prior studies of this drug linker technology demonstrated internalization of a primary ADC leads to trafficking to lysosomes, drug release by lysosomal cathepsin B, and ensuing cell death. A secondary antibody--drug conjugate (2 degrees ADC) capable of binding primary mAbs bound to the surface of antigen-positive cells has comparable drug delivery capability. The system is sufficiently sensitive to detect internalizing mAbs in nonclonal hybridoma supernatants and is predictive of the activity of subsequently produced primary ADC. Because of their high extracellular stability, the noninternalized 2 degrees ADC are 100--1000-fold less toxic to cells over extended periods of time, permitting an assay in which components can be added without need for separate wash steps. This homogeneous screening system is amenable to medium-throughput screening applications and enables the early identification of mAbs capable of intracellular trafficking for drug delivery and release.     

Recombinant human serum amyloid P component from Pichia pastoris: production and characterization

Human serum amyloid P component (SAP) was expressed in the methylotrophic yeast Pichia pastoris. SAP cDNA was placed under control of regulatory sequences derived from the alcohol oxidase gene (AOX1), and its protein product was secreted using the Saccharomyces cerevisiae alpha-mating factor signal sequence. Recombinant SAP (r-SAP) was produced in a bioreactor with computer controlled fed-batch mode and purified by use of a C-terminal histidine tag. The yield of purified r-SAP was 3-4mg from 1L supernatant and 5-6mg from 1L cell paste, indicating that the majority of the produced SAP was not secreted. Treatment of the cell paste with EDTA increased the yield further by about 30%. The N-terminal of r-SAP purified from the supernatant showed non-complete cleavage of the alpha-mating factor signal sequence. Purified r-SAP, analyzed under native conditions, was shown to be a decamer, like purified human SAP (h-SAP), with monomers of 27kDa. Each monomer had one N-glycosylation site, positioned at the same site as for h-SAP. r-SAP bound to antibodies produced against h-SAP. Furthermore, r-SAP bound to ds DNA and influenza A virus subunits in a Ca(2+)-dependent manner and inhibited influenza A virus hemagglutination. These results indicate that r-SAP produced in P. pastoris has the same biological activity as purified h-SAP.     

Evidence that exogenous substances can be phagocytized by alveolar epithelial cells and transported into blood capillaries

Since the ability of alveolar epithelial cells to ingest inhaled fine particles has not been characterized in detail, the present study seeks to evaluate this physiological activity. We used a 0.2% suspension of intact or lecithin-coated polystyrene latex beads (240 nm in diameter). A 5-ml suspension of intact or lecithin-coated latex beads was intratracheally administered to rats using a compressor nebulizer. Thereafter, the lungs were perfused intratracheally with glutaraldehyde solution and cut into small pieces. The samples were postfixed with osmium tetroxide, embedded in epoxy resin and examined under an electron microscope. Both lecithin-coated and uncoated beads were incorporated into alveolar macrophages. Some of the ingested beads in the alveolar macrophages were sequestered within lysosomes. Types I and II alveolar epithelial cells selectively incorporated only lecithin-coated beads, which were also observed within the cytoplasm of monocytes in the capillary lumen. These findings suggest that alveolar epithelial cells can incorporate exogenous particles, which are then transferred from the alveoli to intravascular spaces by transcytosis.     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T₀-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T₂-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T₂-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.     

Microbial colonization drives lymphocyte accumulation and differentiation in the follicle-associated epithelium of Peyer's patches

Peyer's patches (PPs) are lined by follicle-associated epithelium (FAE) with Ag-transporting M cells. To investigate the spatial relationships of B cells, T cells, and dendritic cells (DCs) in PPs during microbial colonization, their in situ redistribution was examined in germfree (GF) rats exposed to a conventional pathogen-free microflora (conventionalized, CV). Although occasional B and T cells occurred in the FAE of GF rats, it contained mainly immature DCs (CD4(+)CD86(-)), whereas mature DCs (CD86(high)) were seen in the interfollicular zones even under GF conditions. In CV rats, DCs had disappeared from the FAE, which instead contained clusters by B and T cells associated with induction of putative M cell pockets. CD86 was seen neither in the FAE nor in the follicles under GF conditions, but it became apparent on intraepithelial B cells 5 wk after colonization. The level of CD86 on these B cells was comparable to that on germinal center B cells, although the B cell follicles did not show direct contact with the M cell areas. B cells in the follicular mantles acquired Bcl-2 after 12 wk in CV rats, whereas B cells in the FAE did not express Bcl-2 at a substantial level throughout the experimental period. The cellular redistribution patterns and phenotypic characteristics observed after colonization suggested that immature DCs, but not B cells, are involved in Ag presentation during primary immune responses against intestinal bacteria. However, the spatial cellular relationships sequentially being established among DCs, B cells, and T cells in PPs, are most likely important for the induction of post-germinal center B cells subsequently residing within the M cell pockets.     

Major evolutionary events in the origin and diversification of the fern genus Polystichum (Dryopteridaceae)

Recent advances in molecular systematics of the ferns make it possible to address long-standing questions about classification of the major fern genera, such as the worldwide genus Polystichum (Dryopteridaceae), comprising at least 200 species. In this study we examined rbcL sequences and morphological characters from 55 fern taxa: 34 were from Polystichum and 21 were from other genera in the Dryopteridaceae. We found that Phanerophlebia, possibly including Polystichopsis, is the sister group to Polystichum sensu lato (s.l.), including Cyrtomium. Polystichum as commonly recognized is paraphyletic. Our results lead us to suggest recognizing the clade of earliest diverging Polystichum species as a distinct genus (Cyrtomidictyum) and to continue to recognize Cyrtomium as a separate genus, leaving a monophyletic Polystichum sensu stricto (s.s.). We resolved a tropical American clade and an African clade within Polystichum s.s. However, the resemblance between the once-pinnate, bulb-bearing calciphilic species found in Asia and the West Indies appears to be the result of convergent evolution. Optimizing our morphological character transformations onto the combined phylogeny suggests that the common ancestor of Polystichum s.l. and Phanerophlebia had evolved the common features of the alliance, including ciliate petiole-base scales, once-pinnate fronds, ultimate segments with scarious tips, peltate indusia, and microscales.     

Distinct effects on heparan sulfate structure by different active site mutations in NDST-1

Heparan sulfate polymerization and modification take place in the Golgi compartment. The modification reactions are initiated by glucosaminyl N-deacetylase/N-sulfotransferase (NDST), a bifunctional enzyme that removes N-acetyl groups from selected N-acetyl-d-glucosamine units followed by N-sulfation of the generated free amino groups. Four isoforms of NDST have been identified. NDST-1 and -2 have a wide and largely overlapping tissue distribution, but it is not known if they can act on the same heparan sulfate chain. We have introduced point mutations into NDST-1 cDNA, which selectively destroy the N-deacetylase or N-sulfotransferase activity of the enzyme [Wei, Z., and Swiedler, S. J. (1999) J. Biol. Chem. 274, 1966-70 and Sueyoshi, T., et al. (1998) FEBS Lett. 433, 211-4]. Stable 293 cell lines expressing the NDST-1 mutants were then generated. Structural analyses of heparan sulfate synthesized by these cells and by cells overexpressing wild-type NDST-1 demonstrate that the N-deacetylation step is not only prerequisite but also rate-limiting, determining the degree of N-sulfation. Transfection of mutant NDST-1 lacking N-deacetylase activity had no effect on heparan sulfate sulfation, while cells expressing wild-type enzyme or NDST-1 lacking N-sulfotransferase activity both resulted in the production of oversulfated heparan sulfate. Since no increase in the amount of N-unsubstituted glucosamine residues was seen after transfection of the mutant lacking N-sulfotransferase activity, the results also suggest that two different enzyme molecules can act on the same glucosamine unit. In addition, we show that oversulfation of heparan sulfate produced by cells tranfected with wild-type NDST-1 or the mutant lacking N-sulfotranferase activity results in decreased sulfation of chondroitin sulfate.     

The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.     

Sequential opening of IP(3)-sensitive Ca(2+) channels and SOC during alpha-adrenergic activation of rabbit vena cava

Alpha(1)-aderenoceptor-mediated constriction of rabbit inferior vena cava (IVC) is signaled by asynchronous wavelike Ca(2+) oscillations in the in situ smooth muscle. We have shown previously that a putative nonselective cationic channel (NSCC) is required for these oscillations. In this report, we show that the application of 2-aminoethoxyphenyl borate (2-APB) to antagonize inositol 1,4,5-trisphosphate (InsP(3))-sensitive Ca(2+) release channels (IP(3)R channels) can prevent the initiation and abolish ongoing alpha(1)-aderenoceptor-mediated tonic constriction of the venous smooth muscle by inhibiting the generation of these intracellular Ca(2+) concentration ([Ca(2+)](i)) oscillations. The observed effects of 2-APB can only be attributed to its selective inhibition on the IP(3)R channels, not to its slight inhibition of the L-type voltage-gated Ca(2+) channel and the sarco(endo)plasmic reticulum Ca(2+) ATPase. Furthermore, 2-APB had no effect on the ryanodine-sensitive Ca(2+) release channel and the store-operated channel (SOC) in the IVC. These results indicate that the putative NSCC involved in refilling the sarcoplasmic reticulum (SR) and maintaining the tonic contraction is most likely an SOC-type channel because it appears to be activated by IP(3)R-channel-mediated SR Ca(2+) release or store depletion. This is in accordance with its sensitivity to Ni(2+) and La(3+) (SOC blockers). More interestingly, RT-PCR analysis indicates that transient receptor potential (Trp1) mRNA is strongly expressed in the rabbit IVC. The Trp1 gene is known to encode a component of the store-operated NSCC. These new data suggest that the activation of both the IP(3)R channels and the SOC are required for PE-mediated [Ca(2+)](i) oscillations and constriction of the rabbit IVC.