Angiopoietin-1 protects the adult vasculature against plasma leakage.

Pathological increases in vascular leakage lead to edema and swelling, causing serious problems in brain tumors, in diabetic retinopathy, after strokes, during sepsis and also in inflammatory conditions such as rheumatoid arthritis and asthma. Although many agents and disease processes increase vascular leakage, no known agent specifically makes vessels resistant to leaking. Vascular endothelial growth factor (VEGF) and the angiopoietins function together during vascular development, with VEGF acting early during vessel formation, and angiopoietin-1 acting later during vessel remodeling, maturation and stabilization. Although VEGF was initially called vascular permeability factor, there has been less focus on its permeability actions and more effort devoted to its involvement in vessel growth and applications in ischemia and cancer. Recent transgenic approaches have confirmed the profound permeability effects of VEGF (refs. 12-14), and have shown that transgenic angiopoietin-1 acts reciprocally as an anti-permeability factor when provided chronically during vessel formation, although it also profoundly affects vascular morphology when thus delivered. To be useful clinically, angiopoietin-1 would have to inhibit leakage when acutely administered to adult vessels, and this action would have to be uncoupled from its profound angiogenic capabilities. Here we show that acute administration of angiopoietin-1 does indeed protect adult vasculature from leaking, countering the potentially lethal actions of VEGF and inflammatory agents.

Chromatographic purification of recombinant adenoviral and adeno-associated viral vectors: methods and implications.

In recent years, recombinant adenoviral and adeno-associated viral (AAV) vectors have been exploited in a number of gene delivery approaches. The use of these vectors in clinical gene transfer has increased the demand for their characterization, production and purification. Although the classical method of adenovirus or AAV purification by density gradient centrifugation is effective on a small scale, chromatographic separation is the most versatile and powerful method for large-scale production of recombinant adenovirus or AAV. This review describes different chromatographic modes for adenovirus or AAV purification and process development, as well as the utility of different purification steps for virus production. Advances in the development of viral vectors for gene therapy, such as the discovery of new AAV serotypes, adenoviral and AAV retargeting and improved production of helper-dependent adenoviral vectors, require further development of efficient purification methods.

Glycosyltransferase mutants: key to new insights in glycobiology.

Glycosyltransferases (Glyc-T's) catalyze the synthesis of the carbohydrate portions of glycoproteins, glycolipids, and proteoglycans. Most Glyc-T's transfer one sugar in one linkage and are encoded by a unique gene. Thus, synthesis of a branched carbohydrate may require expression of at least 30 Glyc-T genes. Mutations that alter Glyc-T activity therefore provide an approach to identifying functions for carbohydrates. These fall into two general categories: 1) intramolecular functions pertaining to the physical and biochemical properties of a glycoconjugate; and 2) intermolecular functions that involve recognition of sugar (or sugars) by another molecule, such as the selectins that bind to specific sugar sequences at the cell surface. In this review, the origin, nature, and uses of Glyc-T mutants that have been used to study both types of carbohydrate function will be summarized. Many Glyc-T genes are now cloned, so transgenic and gene disruption techniques are the latest strategies for identifying new functions for carbohydrates. It is already apparent that the consequences of altering the spectrum of carbohydrates expressed by a cell or an organism range from "none" to "death." A key challenge for the future is to identify molecular bases for the complex phenotypes created by glycosylation engineering. Equally important will be to understand factors that regulate Glyc-T genes, a new class of genes whose study may reveal novel mechanisms of biological regulation.

Mice lacking N-acetylglucosaminyltransferase I activity die at mid-gestation, revealing an essential role for complex or hybrid N-linked carbohydrates.

Eukaryotic cells require N-linked carbohydrates for survival. However, the biosynthetic intermediate Man5GlcNAc2Asn, in place of mature N-linked structures, allows glycoprotein synthesis and somatic cell growth to proceed normally. To determine whether the same would be true in a complex biological situation, the gene Mgat-1 was disrupted by homologous recombination in embryonic stem cells and transmitted to the germ line. The Mgat-1 gene encodes N-acetylglucosaminyltransferase I [GlcNAc-TI; alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase; UDP-N-acetyl-D-glucosamine:glycoprotein (N-acetyl-D-glucosamine to alpha-D-mannosyl-1,3-(R1)-beta-D-mannosyl-R2) beta-1,2-N-acetyl-D-glucosaminyltransferase, EC 2.4.1.101], the transferase that initiates synthesis of hybrid and complex N-linked carbohydrates from Man5GlcNAc2Asn. Mice lacking GlcNAc-TI activity did not survive to term. Biochemical and morphological analyses of embryos from 8.5 to 13.5 days of gestation showed that Mgat-1-/-embryos are developmentally retarded, most noticeably in neural tissue, and die between 9.5 and 10.5 days of development.

Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.

Complex N-glycans in Mgat1 null preimplantation embryos arise from maternal Mgat1 RNA.

Mice with a null mutation in the Mgat1 gene lack N-acetyl-glucosaminyltransferase I (GlcNAc-TI; EC 2.4.1.101), and die at mid-gestation. This result suggested that development of Mgat1-/- blastocysts and their implantation could occur in the absence of complex and hybrid N-glycans. However, inner cell mass of all blastocysts from several Mgat1 +/- heterozygous crosses bind the lectin E-PHA, indicating that Mgat1 null mutant blastocysts are able to synthesize complex N-glycans (Campbell et al., (1995) Glycobiology, 5, 535-543). In order to directly test this hypothesis, Mgat1-/- blastocysts were positively identified by polymerase chain reaction (PCR) of genomic DNA. Reverse transcriptase PCR (RT-PCR) of RNA isolated from the same blastocysts, and restriction analysis of the PCR products, revealed that Mgat1 null blastocysts contained Mgat1 RNA derived from the wild-type Mgat1 gene. Consistent with this, all 3.5 day blastocysts from five heterozygous crosses bound the lectin L-PHA, a lectin previously shown not to bind to E8.5 or E9.5 Mgat1-/- embryos that lack complex N-glycans (Ioffe and Stanley (1994) Proc. Natl. Acad. Sci., USA, 91, 728-732). Blastocysts of 4.5 days postcoitum (dpc) obtained by culturing 3.5 dpc blastocysts also bound L-PHA. However, mutant embryos that did not bind L-PHA were present among progeny from E5.5 onward. Therefore, the effects of the Mgat1 null mutation are not operative until sometime between implantation and E5.5, due to the continued presence of maternally derived Mgat1 mRNA in preimplantation embryos.

Abnormal regulation of the leptin gene in the pathogenesis of obesity.

A subset of obese humans has relatively low plasma levels of leptin. This finding has suggested that in some cases abnormal regulation of the leptin gene in adipose tissue is etiologic in the pathogenesis of the obese state. The possibility that a relative decrease in leptin production can lead to obesity was tested by mating animals carrying a weakly expressed adipocyte specific aP2-human leptin transgene to C57BL/6J ob/ob mice (which do not express leptin). The transgene does not contain the regulatory elements of the leptin gene and is analogous to a circumstance in which the cis elements and/or trans factors regulating leptin RNA production are abnormal. The ob/ob mice carrying the transgene had a plasma leptin level of 1. 78 ng/ml, which is approximately one-half that found in normal, nontransgenic mice (3.72 ng/ml, P < 0.01). The ob/ob animals expressing the leptin transgene were markedly obese though not as obese as ob/ob mice without the transgene. The infertility as well as several of the endocrine abnormalities generally evident in ob/ob mice were normalized in the ob/ob transgenic mice. However, the ob/ob transgenic mice had an abnormal response when placed at an ambient temperature of 4 degreesC, suggesting that different thresholds exist for the different biologic effects of leptin. Leptin treatment of the ob/ob transgenic mice resulted in marked weight loss with efficacy similar to that seen after treatment of wild-type mice. In aggregate these data suggest that dysregulation of leptin gene can result in obesity with relatively normal levels of leptin and that this form of obesity is responsive to leptin treatment.

WW6: an embryonic stem cell line with an inert genetic marker that can be traced in chimeras.

Mutant mice produced by gene targeting in embryonic stem (ES) cells often have a complex or embryonic lethal phenotype. In these cases, it would be helpful to identify tissues and cell types first affected in mutant embryos by following the contribution to chimeras of ES cells homozygous for the mutant allele. Although a number of strategies for following ES cell development in vivo have been reported, each has limitations that preclude its general application. In this paper, we describe ES cell lines that can be tracked to every nucleated cell type in chimeras at all developmental stages. These lines were derived from blastocysts of mice that carry an 11-Mb beta-globin transgene on chromosome 3. The transgene is readily detected by DNA in situ hybridization, providing an inert, nuclear-localized marker whose presence is not affected by transcriptional or translational controls. The "WW" series of ES lines possess the essential features of previously described ES lines, including giving rise to a preponderance of male chimeras, all of which have to date exhibited germ-line transmission. In addition, clones selected for single or double targeting events form strong chimeras, demonstrating the feasibility of using WW6 cells to identify phenotypes associated with the creation of a null mutant.

Absence of soluble leptin receptor in plasma from dbPas/dbPas and other db/db mice.

The leptin receptor (Ob-R) is alternatively spliced into at least five different RNAs designated Ob-R(a-e). Ob-R(a-d) predict receptors with a single transmembrane domain, and Ob-Re predicts a secreted form of the receptor. The presence of an approximately 120-kDa soluble leptin receptor in mouse plasma was confirmed by precipitation with leptin-Sepharose beads followed by immunobloting with anti-leptin receptor antibodies. The soluble leptin receptor is larger than that predicted by the primary sequence. Deglycosylation of the receptor with peptide N:glycosidase F results in a decrease in molecular mass to a size consistent with that of the primary sequence. The secreted receptor was present in plasma from wild type mice but was truncated in plasma from db3J/db3J and absent in dbPas/dbPas plasma. Although db3J/db3J mice are known to have a frameshift mutation at amino acid 625, the basis for the mutation in dbPas/dbPas mice was not known. Further studies indicated that dbPas/dbPas mice carry a duplication of exons 4 and 5 of Ob-R. This mutation introduces a premature stop codon into the protein at amino acid 281. The absence of Ob-R in db3J/db3J and dbPas/dbPas mice confirm the identify of the 120-kDa plasma protein as Ob-Re.