Faculty Bibliography

In analyzing the sequence tags for mutant mouse embryonic stem (ES) cell lines in BayGenomics (a mouse gene-trapping resource), we identified a novel gene, 1-acylglycerol-3-phosphate O-acyltransferase (Agpat6), with sequence similarities to previously characterized glycerolipid acyltransferases. Agpat6's closest family member is another novel gene that we have provisionally designated Agpat8. Both Agpat6 and Agpat8 are conserved from plants, nematodes, and flies to mammals. AGPAT6, which is predicted to contain multiple membrane-spanning helices, is found exclusively within the endoplasmic reticulum (ER) in mammalian cells. To gain insights into the in vivo importance of Agpat6, we used the Agpat6 ES cell line from BayGenomics to create Agpat6-deficient (Agpat6-/-) mice. Agpat6-/- mice lacked full-length Agpat6 transcripts, as judged by northern blots. One of the most striking phenotypes of Agpat6-/- mice was a defect in lactation. Pups nursed by Agpat6-/- mothers die perinatally. Normally, Agpat6 is expressed at high levels in the mammary epithelium of breast tissue, but not in the surrounding adipose tissue. Histological studies revealed that the aveoli and ducts of Agpat6-/- lactating mammary glands were underdeveloped, and there was a dramatic decrease in the size and number of lipid droplets within mammary epithelial cells and ducts. Also, the milk from Agpat6-/- mice was markedly depleted in diacylglycerols and triacylglycerols. Thus, we identified a novel glycerolipid acyltransferase of the ER, AGPAT6, which is crucial for the production of milk fat by the mammary gland

Growth of cancer in rodent models and in patients elicits immune responses directed toward various antigens expressed by the transformed cell. Clearly though, as most tumors grow, unmanipulated antitumor immune responses are incapable of eliminating cancer. Over the past approximately 15 years, antitumor immunoglobulin and T cells have been used to identify tumor antigens, which in turn, have served as the basis for therapeutic vaccine trials. However, experimental cancer vaccines, although in some patients result in elimination of large tumor burdens, have a low frequency of long-term cancer remission in most patients, ca. <5%. Therefore, as tumors express antigens that distinguish themselves from nontransformed cells in immunological terms (i.e., elicit immune responses to growth of primary tumor and can target tumor cells in vivo), and tumor vaccines prime unsuccessful antitumor immune responses in patients, it is likely that growth of cancer induces immune tolerance to tumor cells. Although there are several types of T cell tolerance, mature, antigen-specific CD8+ T cells isolated from tumors are lytic-defective, implying that the tumor microenvironment inactivates the antitumor effector phase. The nature of the functional local tolerance to antitumor immune response is the subject of this review

OBJECTIVES: To illustrate the progressive loss of cross-echinocandin activity on Candida albicans isolates with strong clonal homology from a patient with advanced HIV infection and chronic oesophagitis progressively resistant to uninterrupted micafungin treatment. METHODS: Antifungal susceptibility profiles for different antifungal agents were determined against serial C. albicans isolates retrieved before and during therapy. Multilocus sequencing typing (MLST) was performed on each of the isolates. FKS1 mutations conferring reduced susceptibility to echinocandin drugs were determined by DNA sequence analysis. RESULTS: Four C. albicans isolates showing identical allelic homology were retrieved from the patient at the initiation and during therapy with micafungin. The progressive lack of clinical response to micafungin therapy was associated with increased MICs of all three echinocandin drugs (caspofungin, micafungin and anidulafungin) in association with the acquisition of mutations in the FKS1 gene. CONCLUSIONS: This report documents for the first time a progressive loss of activity of all three echinocandin drugs against clonally related C. albicans isolates following long-term clinical exposure to this new class of antifungal agents.

In Xenopus, Wnt signals and their transcriptional effector beta-catenin are required for the development of dorsal axial structures. In zebrafish, previous loss-of-function studies have not identified an essential role for beta-catenin in dorsal axis formation, but the maternal-effect mutation ichabod disrupts beta-catenin accumulation in dorsal nuclei and leads to a reduction of dorsoanterior derivatives. We have identified and characterized a second zebrafish beta-catenin gene, beta-catenin-2, located on a different linkage group from the previously studied beta-catenin-1, but situated close to the ichabod mutation on LG19. Although the ichabod mutation does not functionally alter the beta-catenin-2 reading frame, the level of maternal beta-catenin-2, but not beta-catenin-1, transcript is substantially lower in ichabod, compared with wild-type, embryos. Reduction of beta-catenin-2 function in wild-type embryos by injection of morpholino antisense oligonucleotides (MOs) specific for this gene (MO2) results in the same ventralized phenotypes as seen in ichabod embryos, and administration of MO2 to ichabod embryos increases the extent of ventralization. MOs directed against beta-catenin-1 (MO1), by contrast, had no ventralizing effect on wild-type embryos. beta-catenin-2 is thus specifically required for organizer formation and this function is apparently required maternally, because the ichabod mutation causes a reduction in maternal transcription of the gene and a reduced level of beta-catenin-2 protein in the early embryo. A redundant role of beta-catenins in suppressing formation of neurectoderm is revealed when both beta-catenin genes are inhibited. Using a combination of MO1 and MO2 in wild-type embryos, or by injecting solely MO1 in ichabod embryos, we obtain expression of a wide spectrum of neural markers in apparently appropriate anteroposterior pattern. We propose that the early, dorsal-promoting function of beta-catenin-2 is essential to counteract a later, dorsal- and neurectoderm-repressing function that is shared by both beta-catenin genes.

OBJECTIVE: To evaluate in vitro effects of arsenite and of arsenite plus N-acetylcysteine on mouse oocyte meiosis. DESIGN: Morphological study using mouse oocytes submitted to in vitro maturation (IVM). SETTING: Laboratory of reproductive biology. ANIMAL(S): Six-week-old CD-1 mice superovulated with pregnant mare serum gonadotropin. INTERVENTION(S): During IVM, mouse oocytes were exposed to arsenite alone or to arsenite plus N-acetylcysteine. MAIN OUTCOME MEASURE(S): Meiotic anomalies were assessed using immunofluorescence microscopy and PolScope (Cambridge Research and Instrumentation, Boston, MA) imaging. RESULT(S): In vitro arsenite administration produced dose-dependent and time-dependent meiotic anomalies, characterized by spindle disruption or chromosome misalignment. After 12-14 hours of IVM, exposure to 2 microg/mL of arsenite for 12-14 hours or to 8 microg/mL of arsenite for 2 hours arrested oocyte maturation at the germinal vesicle or germinal-vesicle breakdown stage. Exposure to 4 microg/mL of arsenite for 2 hours arrested oocyte maturation at metaphase I stage in 95% of exposed oocytes (80% exhibiting abnormalities) after 12-14 hours in IVM. After 12-14 hours in IVM, of the oocytes exposed to 2 microg/mL of arsenite for 2 hours, only 15% reached the meiosis II stage (5% exhibiting abnormalities). After 15-17 hours in IVM, however, of the oocytes exposed to 2 microg/mL of arsenite for 2 hours, 65.2% reached the meiosis II stage (43.5% exhibiting abnormalities). Co-administration of N-acetylcysteine prevented the arsenite-induced meiotic abnormalities and the delayed IVM. CONCLUSION(S): In vitro arsenite exposure caused meiotic abnormalities that were prevented by co-administration of N-acetylcysteine, suggesting that arsenite-induced meiotic aberrations are mediated by reactive oxygen species

Oocytes are complex cells comprising many cellular systems, each of which is essential for proper oocyte function. Methods to assess the diverse and critical cellular systems in oocytes derived from in vitro maturation are badly needed

A semibiological molecular machine with an implemented "AND" logic gate was developed, which was capable of controlling the folding process of proteins in response to ATP and light as input stimuli. The molecular design made use of a genetically engineered chaperonin GroEL bearing, at both entrance parts of its cylindrical cavity, cysteine residues, which were functionalized by an azobenzene derivative to construct photoresponsive mechanical gates (azo-GroEL). This engineered chaperonin trapped denatured green fluorescent protein (GFP(denat)) and prohibited its refolding. However, when hosting azo-GroEL detected ATP (input stimulus 1) and UV light (input stimulus 2) at the same time, it quickly released GFP(denat) to allow its refolding. In contrast, reception of either input stimulus 1 or 2 resulted in only very slow or no substantial refolding of GFP(denat). Implementation of such "AND" logic gate mechanisms in mechanically driven biomolecular systems is an important step toward the design of secured drug delivery systems.