Analysis of the 3D microstructure of experimental cathode films for lithium-ion batteries under increasing compaction.

It is well known that the microstructure of electrodes in lithium-ion batteries has an immense impact on their overall performance. The compaction load during the calendering process mainly determines the resulting morphology of the electrode. Therefore, NCM-based cathode films from uncompacted (0 MPa) to most highly compacted (1000 MPa) were manufactured, which corresponds to global porosities ranging from about 50% to 18%. All samples have been imaged using synchrotron tomography. These image data allow an extensive analysis of the 3D cathode microstructure with respect to increasing compaction. In addition, the numerous microstructural changes can be quantified using several characteristics describing the morphology of cathode samples. Three characteristics, namely global porosity, global volume fraction of active material and mean cathode thickness, are compared to experimental results. In addition, the microstructural analysis by means of 3D image data and image processing techniques allows the investigation of characteristics which are hard or impossible to ascertain by experiments, for example the continuous pore size distribution and the sphericity distribution of NCM-particles. Finally, the dependency of microstructural characteristics on compaction load is described by the help of parametric probability distributions. This approach can be used, for example, to predict the distribution of a certain characteristic for an 'unknown' compaction load, which is a valuable information with regard to the optimization and development process of NCM-cathodes in lithium-ion batteries. LAY DESCRIPTION: It is well known that the microstructure of electrodes in lithium-ion batteries has an immense impact on their overall performance. The manufacturing of the batteries includes the so-called calendering, where the electrodes are compressed with a certain pressure, which is called compaction load. This process step mainly determines the resulting morphology of the electrode and thus the properties of the battery. Therefore, eight cathodes with different compaction loads were manufactured and imaged by synchrotron tomography, which leads to 3D images containing detailed information about the inner structure of the cathode. This image data allows an extensive analysis of the 3D cathode microstructure with respect to increasing compaction. In order to quantify the microstructural changes we use several characteristics describing diverse properties of the morphology. Furthermore, the 3D image data can be used for the computation of characteristics which can not be determined by experiments. Therefore, 3D image data allows us to understand how the microstructure of cathodes is influenced by the compaction load. Finally, we are able to predict the distribution of a certain characteristic for arbitrary compaction loads. This information is valuable with regard to the development of improved lithium-ion batteries.

Positions and numbers of FKS mutations in Candida albicans selectively influence in vitro and in vivo susceptibilities to echinocandin treatment.

Candidemia is the fourth most common kind of microbial bloodstream infection, with Candida albicans being the most common causative species. Echinocandins are employed as the first-line treatment for invasive candidiasis until the fungal species is determined and confirmed by clinical diagnosis. Echinocandins block the FKS glucan synthases responsible for embedding ß-(1,3)-d-glucan in the cell wall. The increasing use of these drugs has led to the emergence of antifungal resistance, and elevated MICs have been associated with single-residue substitutions in specific hot spot regions of FKS1 and FKS2. Here, we show for the first time the caspofungin-mediated in vivo selection of a double mutation within one allele of the FKS1 hot spot 1 in a clinical isolate. We created a set of isogenic mutants and used a hematogenous murine model to evaluate the in vivo outcomes of echinocandin treatment. Heterozygous and homozygous double mutations significantly enhance the in vivo resistance of C. albicans compared with the resistance seen with heterozygous single mutations. The various FKS1 hot spot mutations differ in the degree of their MIC increase, substance-dependent in vivo response, and impact on virulence. Our results demonstrate that echinocandin EUCAST breakpoint definitions correlate with the in vivo response when a standard dosing regimen is used but cannot predict the in vivo response after a dose escalation. Moreover, patients colonized by a C. albicans strain with multiple mutations in FKS1 have a higher risk for therapeutic failure.

Candida glabrata persistence in mice does not depend on host immunosuppression and is unaffected by fungal amino acid auxotrophy.

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

Unusual routes of protein secretion: the easy way out.

Increasing numbers of polypeptides are being discovered that lack a cleavable hydrophobic signal sequence and are released from cells without passing through the classical secretory pathway. This article reviews the current knowledge of these alternative secretion pathways in prokaryotes and eukaryotes and discusses whether the mechanisms described in bacteria and yeast can be used as paradigms to explain unusual secretory phenomena in animal cells.

The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

Plasma membrane translocation of fluorescent-labeled phosphatidylethanolamine is controlled by transcription regulators, PDR1 and PDR3.

The transcription regulators, PDR1 and PDR3, have been shown to activate the transcription of numerous genes involved in a wide range of functions, including resistance to physical and chemical stress, membrane transport, and organelle function in Saccharomyces cerevisiae. We report here that PDR1 and PDR3 also regulate the transcription of one or more undetermined genes that translocate endogenous and fluorescent-labeled (M-C6-NBD-PE) phosphatidylethanolamine across the plasma membrane. A combination of fluorescence microscopy, fluorometry, and quantitative analysis demonstrated that M-C6-NBD-PE can be translocated both inward and outward across the plasma membrane of yeast cells. Mutants, defective in the accumulation of M-C6-NBD-PE, were isolated by selectively photokilling normal cells that accumulated the fluorescent phospholipid. This led to the isolation of numerous trafficking in phosphatidylethanolamine (tpe) mutants that were defective in intracellular accumulation of M-C6-NBD-PE. Complementation cloning and linkage analysis led to the identification of the dominant mutation TPE1-1 as a new allele of PDR1 and the semidominant mutation tpe2-1 as a new allele of PDR3. The amount of endogenous phosphatidylethanolamine exposed to the outer leaflet of the plasma membrane was measured by covalent labeling with the impermeant amino reagent, trinitrobenzenesulfonic acid. The amount of outer leaflet phosphatidylethanolamine in both mutant strains increased four- to fivefold relative to the parent Tpe+ strain, indicating that the net inward flux of endogenous phosphatidylethanolamine as well as M-C6-NBD-PE was decreased. Targeted deletions of PDR1 in the new allele, PDR1-11, and PDR3 in the new allele, pdr3-11, resulted in normal M-C6-NBD-PE accumulation, confirming that PDR1-11 and pdr3-11 were gain-of-function mutations in PDR1 and PDR3, respectively. Both mutant alleles resulted in resistance to the drugs cycloheximide, oligomycin, and 4-nitroquinoline N-oxide (4-NQO). However, a previously identified drug-resistant allele, pdr3-2, accumulated normal amounts of M-C6-NBD-PE, indicating allele specificity for the loss of M-C6-NBD-PE accumulation. These data demonstrated that PDR1 and PDR3 regulate the net rate of M-C6-NBD-PE translocation (flip-flop) and the steady-state distribution of endogenous phosphatidylethanolamine across the plasma membrane.

Klebsiella pneumoniae prevents spore germination and hyphal development of Aspergillus species.

Different bacteria and fungi live as commensal organisms as part of the human microbiota, but shifts to a pathogenic state potentially leading to septic infections commonly occur in immunocompromised individuals. Several studies have reported synergistic or antagonistic interactions between individual bacteria and fungi which might be of clinical relevance. Here, we present first evidence for the interaction between Klebsiella pneumoniae and several Aspergillus species including A. fumigatus, A. terreus, A. niger and A. flavus which cohabit in the lungs and the intestines. Microbiological and molecular methods were employed to investigate the interaction in vitro, and the results indicate that Klebsiella pneumoniae is able to prevent Aspergillus spp. spore germination and hyphal development. The inhibitory effect is reversible, as demonstrated by growth recovery of Aspergillus spp. upon inhibition or elimination of the bacteria, and is apparently dependent on the physical interaction with metabolically active bacteria. Molecular analysis of Klebsiella-Aspergillus interaction has shown upregulation of Aspergillus cell wall-related genes and downregulation of hyphae-related genes, suggesting that Klebsiella induces cell wall stress response mechanisms and suppresses filamentous growth. Characterization of polymicrobial interactions may provide the basis for improved clinical management of mixed infections by setting the stage for appropriate diagnostics and ultimately for optimized treatment strategies.

Secretion of peptides and proteins lacking hydrophobic signal sequences: the role of adenosine triphosphate-driven membrane translocators.

In this review, we will emphasize the role of ATP-dependent membrane transporters in protein export and intracellular protein trafficking in prokaryotic and eukaryotic cells. ATP-binding-cassette (ABC)-transport proteins, also termed "traffic ATPases," belong to a superfamily of ubiquitous ATP-driven membrane transporters that share extensive sequence similarity and highly conserved domain organization. They are implicated in a remarkable variety of transmembrane transport processes, including the transport of ions, heavy metals, sugars, anticancer drugs, amino acids, oligopeptides, and proteins. Bacterial ABC-proteins include the well-characterized periplasmic permeases involved in nutrient uptake, but also include protein secretion systems, such as the exporter for the Escherichia coli enterotoxin hemolysin A. Prominent eukaryotic members of this superfamily include the human P-glycoprotein (which is associated with the phenomenon of multiple drug resistance in tumor cells), the product of the cystic fibrosis gene (CFTR), the gene (pfmdr) implicated in chloroquine resistance of the malarial parasite, putative peptide transporters encoded at the locus for the class II major histocompatibility complex (MHC), and the yeast Ste6 transporter which mediates export of a peptide hormone that lacks a classical hydrophobic signal peptide. The well-established function of prokaryotic ABC-transporters in the secretion of proteins without typical signal sequences, and the example set by the Ste6 transporter, have led to the reasonable hypothesis that certain ABC-proteins in animal cells may be operating by a similar mechanism to mediate the export of a new class of secretory proteins, those lacking a classical hydrophobic signal peptide.

Endocytosis and vacuolar degradation of the plasma membrane-localized Pdr5 ATP-binding cassette multidrug transporter in Saccharomyces cerevisiae.

Multidrug resistance (MDR) to different cytotoxic compounds in the yeast Saccharomyces cerevisiae can arise from overexpression of the Pdr5 (Sts1, Ydr1, or Lem1) ATP-binding cassette (ABC) multidrug transporter. We have raised polyclonal antibodies recognizing the yeast Pdr5 ABC transporter to study its biogenesis and to analyze the molecular mechanisms underlying MDR development. Subcellular fractionation and indirect immunofluorescence experiments showed that Pdr5 is localized in the plasma membrane. In addition, pulse-chase radiolabeling of cells and immunoprecipitation indicated that Pdr5 is a short-lived membrane protein with a half-life of about 60 to 90 min. A dramatic metabolic stabilization of Pdr5 was observed in delta pep4 mutant cells defective in vacuolar proteinases, and indirect immunofluorescence showed that Pdr5 accumulates in vacuoles of stationary-phase delta pep4 mutant cells, demonstrating that Pdr5 turnover requires vacuolar proteolysis. However, Pdr5 turnover does not require a functional proteasome, since the half-life of Pdr5 was unaffected in either pre1-1 or pre1-1 pre2-1 mutants defective in the multicatalytic cytoplasmic proteasome that is essential for cytoplasmic protein degradation. Immunofluorescence analysis revealed that vacuolar delivery of Pdr5 is blocked in conditional end4 endocytosis mutants at the restrictive temperature, showing that endocytosis delivers Pdr5 from the plasma membrane to the vacuole.

Identification of mouse histone deacetylase 1 as a growth factor-inducible gene.

Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression, and developmental events. The acetylation state of histones is controlled by the activities of acetylating and deacetylating enzymes. By using differential mRNA display, we have identified a mouse histone deacetylase gene, HD1, as an interleukin-2-inducible gene in murine T cells. Sequence alignments revealed that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator. Indirect immunofluorescence microscopy proved that mouse HD1 is a nuclear protein. When expressed in yeast, murine HD1 was also detected in the nucleus, although it failed to complement the rpd3delta deletion phenotype. HD1 mRNA expression was low in G0 mouse cells but increased when the cells crossed the G1/S boundary after growth stimulation. Immunoprecipitation experiments and functional in vitro assays showed that HD1 protein is associated with histone deacetylase activity. Both HD1 protein levels and total histone deacetylase activity increased upon interleukin-2 stimulation of resting B6.1 cells. When coexpressed with a luciferase reporter construct, HD1 acted as a negative regulator of the Rous sarcoma virus enhancer/promoter. HD1 overexpression in stably transfected Swiss 3T3 cells caused a severe delay during the G2/M phases of the cell cycle. Our results indicate that balanced histone acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells.

Genetic separation of FK506 susceptibility and drug transport in the yeast Pdr5 ATP-binding cassette multidrug resistance transporter.

Overexpression of the yeast Pdr5 ATP-binding cassette transporter leads to pleiotropic drug resistance to a variety of structurally unrelated cytotoxic compounds. To identify Pdr5 residues involved in substrate recognition and/or drug transport, we used a combination of random in vitro mutagenesis and phenotypic screening to isolate novel mutant Pdr5 transporters with altered substrate specificity. A plasmid library containing randomly mutagenized PDR5 genes was transformed into appropriate drug-sensitive yeast cells followed by phenotypic selection of Pdr5 mutants. Selected mutant Pdr5 transporters were analyzed with respect to their expression levels, subcellular localization, drug resistance profiles to cycloheximide, rhodamines, antifungal azoles, steroids, and sensitivity to the inhibitor FK506. DNA sequencing of six PDR5 mutant genes identified amino acids important for substrate recognition, drug transport, and specific inhibition of the Pdr5 transporter. Mutations were found in each nucleotide-binding domain, the transmembrane domain 10, and, most surprisingly, even in predicted extracellular hydrophilic loops. At least some point mutations identified appear to influence folding of Pdr5, suggesting that the folded structure is a major substrate specificity determinant. Surprisingly, a S1360F exchange in transmembrane domain 10 not only caused limited substrate specificity, but also abolished Pdr5 susceptibility to inhibition by the immunosuppressant FK506. This is the first report of a mutation in a yeast ATP-binding cassette transporter that allows for the functional separation of substrate transport and inhibitor susceptibility.

Inventory and function of yeast ABC proteins: about sex, stress, pleiotropic drug and heavy metal resistance.

Saccharomyces cerevisiae was the first eukaryotic organism whose complete genome sequence has been determined, uncovering the existence of numerous genes encoding proteins of the ATP-binding cassette (ABC) family. Fungal ABC proteins are implicated in a variety of cellular functions, ranging from clinical drug resistance development, pheromone secretion, mitochondrial function, peroxisome biogenesis, translation elongation, stress response to cellular detoxification. Moreover, some yeast ABC proteins are orthologues of human disease genes, which makes yeast an excellent model system to study the molecular mechanisms of ABC protein-mediated disease. This review provides a comprehensive discussion and update on the function and transcriptional regulation of all known ABC genes from yeasts, including those discovered in fungal pathogens.

The yeast multidrug transporter Pdr5 of the plasma membrane is ubiquitinated prior to endocytosis and degradation in the vacuole.

We have recently demonstrated that the Pdr5 ATP binding cassette multidrug transporter is a short-lived protein, whose biogenesis involves cell surface targeting followed by endocytosis and delivery to the vacuole for proteolytic turnover [Egner, R., Mahé, Y., Pandjaitan, R., and Kuchler, K. (1995) Mol. Cell. Biol. 15, 5879-5887]. Using c-myc epitope-tagged ubiquitin, we now have shown that Pdr5 is a ubiquitinated plasma membrane protein in vivo. Ubiquitination of Pdr5 was detected in both wild type and conditional end mutants defective in endocytic vesicle formation. Likewise, the Ste6 a-factor pheromone transporter, which represents another short-lived ABC transporter whose turnover requires vacuolar proteolysis, was also found to be ubiquitinated, and ubiquitin-modified Ste6 massively accumulated in end4 mutants at the restrictive temperature. By contrast, the plasma membrane ATPase Pma1, a long-lived and metabolically very stable protein, was found not to be ubiquitinated. Our results imply a novel function for ubiquitin in protein trafficking and suggest that ubiquitination of certain short-lived plasma membrane proteins may trigger their endocytic delivery to the vacuole for proteolytic turnover.

A cDNA from brain of Xenopus laevis coding for a new precursor of thyrotropin-releasing hormone.

Thyrotropin-releasing hormone (TRH) is found in large amounts in the skin of Xenopus laevis. In this tissue, 3 TRH precursor mRNAs can be detected of which the 2 more expressed encode almost identical proteins. However, Northern blot analysis of TRH precursor mRNAs in the brain of X. laevis revealed the existence of a new mRNA of about 1200 nucleotides which was present along with the larger TRH precursor mRNA identified in the skin. A cloned cDNA of a TRH precursor, corresponding in size to this new mRNA, was isolated and sequenced from a Xenopus brain lambda gt11 library. It encodes a precursor polypeptide which also contains 7 copies of TRH. However, at the amino acid level it differs by about 16% from the corresponding prepro-TRHs from skin. We have also attempted to characterize the gene encoding this prepro-TRH from Xenopus brain. Only the first and part of the second exon could be detected which are separated by an intron containing more than 8000 base pairs. Interestingly, the 5'-flanking region of this gene does not contain the characteristic promoter elements of the mammalian TRH genes suggesting marked differences in the regulation of their expression.

The yeast ATP binding cassette (ABC) protein genes PDR10 and PDR15 are novel targets for the Pdr1 and Pdr3 transcriptional regulators.

The yeast transcription factors Pdr1 and Pdr3 control pleiotropic drug resistance (PDR) development, since they regulate expression of ATP-binding cassette (ABC) drug efflux pumps through binding to cis-acting sites known as PDREs (PDR responsive elements). In this report, we show by Northern blotting, gel shift mobility assays and DNase I footprinting that transcription of the ABC genes PDR10 and PDR15 is also controlled by Pdr1 and Pdr3. In addition, in vitro band shift assays demonstrate that a GST-Pdr1 fusion protein can bind to the PDREs of PDR10 and PDR15. DNase I footprinting allowed the identification of the precise PDRE binding motifs, indicating the presence of a novel slightly degenerate PDRE motif in the PDR15 promoter. Finally, PDR10 and PDR15 mRNA levels vary dramatically in abundance in isogenic yeast strains carrying either deltapdr1, deltapdr3 and deltapdr1 deltapdr3 deletions or pdr1-3 and pdr3-2 gain-of-function mutations, demonstrating that both PDR10 and PDR15 are new members of the yeast PDR network.

Human Dermo-1 has attributes similar to twist in early bone development.

Basic helix-loop-helix (bHLH) transcription factors are implicated in cell lineage determination and differentiation. Dermo-1 encodes a bHLH transcription factor that shares extensive homology with another bHLH transcription factor, Twist. We have cloned and characterized human Dermo-1 from two different bone cytoplasmic DNA (cDNA) libraries. Dermo-1 mRNA and protein expression were examined in human embryo and adult tissue sections. Dermo-1 is expressed in a subset of mesodermally and ectodermally derived tissues. We further examined expression of Dermo-1/Twist in human tissues and cell lines. In addition, we observed Dermo-1 expression in response to basic fibroblast growth factor in osteoblastic cell lines. To evaluate the functionality of the human Dermo-1 transcription factor in osteoblast metabolism, we made stable osteoblastic cell lines that over- and underexpress human Dermo-1. These cell lines were analyzed and compared with previously published data of similar cell lines transfected with Twist. Our results demonstrate that Dermo-1 caused changes similar to Twist in the osteogenic properties of osteoblastic cells, such as morphology, bone marker gene expression, and biochemical response to cytokines. However, Dermo-1 expression also has unique effects in regulating the mechanism of proliferation, on alkaline phosphatase enzyme activity, and in temporal expression patterns. We speculate that expression of Twist and Dermo-1 maintains cells in an osteoprogenitor or preosteoblast-like state, respectively, and prevents premature or ectopic osteoblast differentiation. Therefore, Twist and Dermo-1 must be sequentially downregulated in order to initiate the cascade of events responsible for osteogenic cell differentiation. These results indicate that, during osteoblast development, Dermo-1 may inhibit osteoblast maturation and maintain cells in a preosteoblast phenotype by utilizing mechanisms similar but not identical to those utilized by Twist.

Fungal ABC proteins: pleiotropic drug resistance, stress response and cellular detoxification.

A number of prominent genetic diseases are caused by mutations in genes encoding ATP-binding cassette (ABC) proteins (Ambudkar, Gottesmann, 1998). Moreover, several mammalian ABC proteins such as P-glycoprotein (P-gp) (Gottesman et al., 1995) and multidrug-resistance-associated proteins (MRPs) (Cole, Deeley, 1998) have been implicated in multidrug resistance (MDR) phenotypes of tumor cells highly resistant to many different anticancer drugs. The characteristics of MDR phenomena include the initial resistance to a single anticancer drug, followed by the development of cross-resistance to many structurally and functionally unrelated drugs. Similar mechanisms of MDR exist in pathogenic fungi, including Candida and Aspergillus (Vanden Bossche et al., 1998), and also in parasites such as Plasmodium and Leishmania (Ambudkar, Gottesmann, 1998), as well as in many bacterial pathogens (Nikaido, 1998). To dissect the mechanisms of MDR development and to elucidate the physiological functions of ABC proteins, many efforts have been made during the past decade. Importantly, yeast orthologues of mammalian disease genes made this unicellular eukaryote an invaluable model system for studies on the molecular mechanisms of ABC proteins, in order to better understand and perhaps improve treatment of ABC gene-related disease. In this review, we provide an overview of ABC proteins and pleiotropic drug resistance in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. Furthermore, we discuss the role of ABC proteins in clinical drug resistance development of certain fungal pathogens.

Retinol in avian oogenesis: molecular properties of the carrier protein.

Normal embryo development in oviparous (egg-laying) species requires the coordinated targeting to growing oocytes of nutrients and regulatory molecules such as retinol, the precursor of active retinoids. Serum retinol-binding protein (RBP) is the major carrier protein for retinol in the circulatory system of vertebrates. In oviparous animals, RBP is thought to function in the delivery of retinol to yolk, in analogy to other important nutrients and vitamins known to accumulate within the oocyte. Here, immunoelectron microscopy revealed that RBP indeed accumulates in yolk, in particular in the electron-lucent phase of yolk organelles known to harbor other serum-derived yolk proteins and their receptors. To gain understanding of the RBP-mediated serum-to-yolk transport of retinol, we have characterized the chicken carrier protein at the molecular level. The essential function of RBPs is emphasized by the first known avian RBP structure, which confirms that these vitamin carriers are among the most highly conserved serum proteins known. Interestingly, by analysis of RBP hepatic RNA and serum protein levels, we identified a unique property of chicken RBP relative to other known RBPs and yolk precursors, i.e., the absence of estrogen induction. One cause of the observed reduction in RBP RNA is an estrogen-dependent decrease of RBP gene transcription. Furthermore, Northern blot analysis of tissues of the hen demonstrated a lack of RBP synthesis by the oocyte or other ovarian cells, confirming the exogenous (hepatic) origin of yolk RBP. These results provide strong evidence that chicken RBP is an essential serum-to-yolk vitamin carrier with certain properties different from those of other such transporters.