Preclinical in vivo characterization of new polymeric drug conjugate candidates is crucial for understanding the effects of certain chemical modifications on distribution and elimination of these carrier systems, which is the basis for rational drug design. In our study we synthesized dual fluorescent HPMA copolymers of different architectures and molecular weights, containing one fluorescent dye coupled via a stable hydrazide bond functioning as the carrier label and the other one modeling the drug bound to a carrier via a pH-sensitive hydrolytically cleavable hydrazone bond. Thus, it was possible to track the in vivo fate, namely distribution, elimination and tumor accumulation, of the polymer drug carrier and a cleavable model drug simultaneously and noninvasively in nude mice using multispectral optical imaging. We confirmed our in vivo results by more detailed ex vivo characterization (imaging and microscopy) of autopsied organs and tumors. There was no significant difference in relative biodistribution in the body between the 30 KDa linear and 200 KDa star-like polymer, but the star-like polymer circulated much longer. We observed a moderate accumulation of the polymeric carriers in the tumors. The accumulation of the pH-sensitive releasable model drug was even higher compared to the polymer accumulation. Additionally, we were able to follow the long-term in vivo fate and to prove a time-dependent tumor accumulation of HPMA copolymers over several days.

We investigated the role of the pef operon, containing the genes for plasmid-encoded (PE) fimbriae of Salmonella typhimurium, in adhesion to the murine small intestine. In an organ culture model, a mutant of S. typhimurium carrying a tetracycline resistance cassette inserted in pefC was found to be associated in lower numbers with murine small intestine than the wild type. Similarly, heterologous expression of PE fimbriae in Escherichia coli increased the bacterial numbers recovered from the intestine in the organ culture model. Adhesion to villous intestine mediated by PE fimbriae was further demonstrated by binding of an E. coli strain expressing PE fimbriae to thin sections of mouse small intestine. The contribution of pef-mediated adhesion on fluid accumulation was investigated in infant mice. Intragastric injection of S. typhimurium 14028 and SR-11 caused fluid accumulation in infant mice. In contrast, pefC mutants of S. typhimurium 14028 and SR-11 were negative in the infant mouse assay. Introduction of a plasmid containing pefBACD and orf5, the first five genes of the pef operon, into the pefC mutant complemented for fluid accumulation in the infant mouse assay. However, heterologous expression of PE fimbriae in E. coli did not result in fluid accumulation in the infant mouse, suggesting that factors other than fimbriae are involved in causing fluid accumulation. Salmonella typhimurium is the most common cause of acute gastroenteritis in humans in the United States. However, the mechanism by which S. typhimurium causes diarrhea in humans is not well defined. Although at least three different toxic activities of S. typhimurium have been found in several animal and cell culture models, their contribution to the generation of diarrhea in humans has never been conclusively demonstrated (3, 10, 13, 23, 24, 31, 32, 37-39, 51). In fact, salmonellosis appears to be a complex, multifactorial process (43), and the ability of S. typhimurium to multiply in the lamina propria and cause inflammation may contribute significantly to diarrheal disease (8, 9, 11). Bacterial adhesins are known to support colonization of the host's alimentary tract, thereby increasing the bacterial load in proximity to the epithelial lining. As a consequence, fimbriae of enterotoxigenic Escherichia coli and Vibrio cholerae are necessary for diarrhea (5, 14, 18, 42, 45, 46). Although several fimbrial adhesins have been found in S. typhimurium (1), fimbriae have so far not been implicated in fluid accumulation in animal models. In this report, we present evidence that plasmid-encoded (PE) fimbriae of S. typhimurium mediate adhesion to mouse small intestine and are necessary for fluid accumulation in the infant mouse assay. MATERIALS AND METHODS Bacterial strains, cell lines, and growth conditions. Bacterial strains used in this study are listed in Table 1. All bacteria were cultured in Luria-Bertani broth (LB; 5 g of yeast extract, 10 g of tryptone, and 10 g of NaCl per liter) or on plates (LB broth containing 15 g of agar per liter) at 37ЊC. A...

The major immediate-early gene of human cytomegalovirus encodes several isoforms of an immediate-early protein which has distinct transcriptional regulatory properties. The IE86 isoform autorepresses the major immediate-early promoter by directly binding the cis repression signal element located between the TATA box and the mRNA cap site. In addition to this activity, IE86 stimulates other viral and cellular promoters. One mechanism by which eukaryotic regulatory proteins are thought to stimulate transcription is by contacting one or more general transcription factors. We show that the IE86 protein physically interacts with the DNA-binding subunit (TATA-binding protein) human transcription factor IID via the TATA-binding protein-contacting domain in the N terminus of IE86. In a mobility shift assay, IE86 was also observed to stabilize the binding of TATA-binding protein to promoter DNA. The domains within IE86 responsible for mediating transactivation and repression functioned independently. These experiments thus demonstrate the elegant ability of human cytomegalovirus to join different protein domains to produce distinct multifunctional proteins. The immediate-early (IE) genes of human cytomegalovirus (HCMV) encode transcriptional regulatory factors which, together with host cell proteins, temporally regulate subsequent viral gene expression (4, 10, 40, 41, 43-45, 47). We are interested in understanding the role of viral and cellular proteins involved in coordinating RNA polymerase II activity associated with HCMV gene regulation. Three predominant IE protein isoforms originate from an abundant region of IE expression which is controlled by the major IE promoter (MIEP) (5, 42, 44, 45). These isoforms are generally distinguished by their apparent molecular masses of 72 (IE72), 86 (IE86), and 55 (IE55) kDa. Since these IE protein isoforms are all derived from a single precursor mRNA by differential splicing and polyadenylation site usage, the virus can join various domains to create proteins with similar and distinct functions. Recently, IE86 was shown bind the cis repression signal (crs) element located between the TATA box and the mRNA cap site of the MIEP (21, 24). The IE86 protein possesses a domain between amino acid residues 365 and 519 which contains a putative zinc finger and a leucine-rich region (Fig. IB) that was shown to be critical for this interaction (21, 32). The crs element is responsible for conferring negative repression on the MIEP by IE86 (7, 16, 30, 36, 38). Although IE86 and the TATA-binding protein (TBP) were observed to bind DNA simultaneously, binding of one protein impaired the binding of the other (21). Although IE86 may effect the binding of transcription factor IID (TFIID), the multisubunit complex containing TBP, to the crs element, the main block in transcription is likely at the level of TFIIB recruitment (21). The IE55 protein, however, lacks the domain in IE86 between amino acid residues 365 and 519 and conse-* Corresponding author.

The human cytomegalovirus (HCMV) major immediate-early promoter (MIEP) is one of the first promoters to activate upon infection. To examine HCMV MIEP tissue-specific expression, transgenic mice were established containing the lacZ gene regulated by the MIEP (nucleotides ؊670 to ؉54). In the transgenic mice, lacZ expression was demonstrated in 19 of 29 tissues tested by histochemical and immunochemical analyses. These tissues included brain, eye, spinal cord, esophagus, stomach, pancreas, kidney, bladder, testis, ovary, spleen, salivary gland, thymus, bone marrow, skin, cartilage, and cardiac, striated and smooth muscles. Although expression was observed in multiple organs, promoter activity was restricted to specific cell types. The cell types which demonstrated HCMV MIEP expression included retinal cells of the eye, ductile cells of the salivary gland, exocrine cells of the pancreas, mucosal cells of the stomach and intestine, neuronal cells of the brain, muscle fibers, thecal cells of the corpus luteum, and Leydig and sperm cells of the testis. These observations indicate that the HCMV MIEP is not a pan-specific promoter and that the majority of expressing tissues correlate with tissues naturally infected by the virus in the human host.

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The human cytomegalovirus major immediate-early gene encodes several protein isoforms which autoregulate the major immediate-early promoter (MIEP). One of these isoforms, the IE86 protein, represses the MIEP through a DNA sequence located between the TATA box and the transcription initiation site, designated the cis repression signal (crs). Through mutational analysis, amino acid domains within IE86 responsible for binding the crs element were located at the C terminus. Mutation of the putative zinc finger domain, which precluded IE86 from binding DNA, converted the protein from a repressor of MIEP transcription into an activator. DNase I protection analysis demonstrated that the IE86 footprint overlapped the sequence protected by the TATA-binding protein (TBP). Investigation of whether IE86 was able to displace TBP from DNA revealed that both proteins could bind DNA simultaneously. However, higher concentrations of IE86 were required to obtain protection of the crs element in the presence of prebound TBP. Similarly, higher concentrations of TBP were required to obtain protection in the presence of prebound IE86. These observations indicate that steric hinderance impairs but does not prevent both proteins from binding DNA synchronously.

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Microangiopathy with subsequent organ damage represents a major complication in several diseases. The mechanisms leading to microvascular occlusion include von Willebrand factor (VWF), notably the formation of ultra-large von Willebrand factor fibers (ULVWFs) and platelet aggregation. To date, the contribution of erythrocytes to vascular occlusion is incompletely clarified. We investigated the platelet-independent interaction between stressed erythrocytes and ULVWFs and its consequences for microcirculation and organ function under dynamic conditions. In response to shear stress, erythrocytes interacted strongly with VWF to initiate the formation of ULVWF/erythrocyte aggregates via the binding of Annexin V to the VWF A1 domain. VWF-erythrocyte adhesion was attenuated by heparin and the VWF-specific protease ADAMTS13. In an in vivo model of renal ischemia/reperfusion injury, erythrocytes adhered to capillaries of wild-type but not VWF-deficient mice and later resulted in less renal damage. In vivo imaging in mice confirmed the adhesion of stressed erythrocytes to the vessel wall. Moreover, enhanced eryptosis rates and increased VWF binding were detected in blood samples from patients with chronic renal failure. Our study demonstrates that stressed erythrocytes have a pronounced binding affinity to ULVWFs. The discovered mechanisms suggest that erythrocytes are essential for the pathogenesis of microangiopathies and renal damage by actively binding to ULVWFs.