Further data on the selective expression of Ly-5 isoforms

Ly-5 (CD45) glycoproteins of the mouse, expressed by all or most hematopoietic cell lineages and specified by a single Ly-5 gene, range in size from isoform T200 of T cells (the smallest), in which exons 4, 5, and 6 are not represented, to isoform B220 of B cells (the largest), in which all three of these optional exons are represented. The main purpose of the present study, utilizing the polymerase chain reaction (PCR), was to ascertain whether known isoforms of intermediate size are generated by single or dual usage of optional exons 4, 5, and 6. Transcripts representing all eight isoforms predictable from varied use of three exons were observed among a diverse panel of nine B-cell tumors in culture, but there was no evident concordance with known contrasting differential features that distinguish members of the B-cell tumor panel. No two B tumors exhibited the same variety of transcripts and the relative quantities of transcripts expressed varied greatly from tumor to tumor. Cloning of B-cell tumors did not alter their distinctive transcript patterns. Separation methods (sodium dodecyl sulfate polyacrylamide gel electrophoresis; SDS-PAGE) did not suffice to segregate all corresponding expressed isoforms but did establish that transcripts representing usage of a single optional exon and of two optional exons were actually translated, which supports a provisional inference that all eight isoforms exist. The considerable diversity of B-cell transcript phenotypes was not seen among seven T-cell leukemias, two cytolytic T-cell lines, and three Th 1 helper T-cell lines, all of which displayed a uniform phenotype comprising major expression of the T200 transcript (no optional exon) and minor expression of a transcript employing exon 5. However, a panel of five cloned Th2 T-cell lines, which represent a second and functionally different branch of the helper/inducer T-cell category, exhibited a characteristic transcript pattern which distinguished them from a panel of three Th1 T-cell lines. The major transcript in the Th2 lines was also T200, but the Th2 lines showed higher representation of transcripts containing optional exons. A single Th2 clone expressed an unusual transcript suggesting a potential isoform not compounded simply by varied inclusion of the three identified optional exons. After activation of the helper T-cell lines with concanavalin A (Con A), expression of transcripts containing optional exons appeared to decrease

Different effects on triacylglycerol packaging to oil bodies in transgenic rice seeds by specifically eliminating one of their two oleosin isoforms

Expression of OLE16 and OLE18, two oleosin isoforms in oil bodies of rice seeds, was suppressed by RNA interference. Electron microscopy revealed a few large, irregular oil clusters in 35S::ole16i transgenic seed cells, whereas accumulated oil bodies in 35S::ole18i transgenic seed cells were comparable to or slightly larger than those in wild-type seed cells. Large and irregular oil clusters were observed in cells of double mutant seeds. These unexpected differences observed in oil bodies of 35S::ole16i and 35S::ole18i transgenic seeds were further analyzed. In comparison to wild-type plants, OLE18 levels were reduced to approximately 40% when OLE16 was completely eliminated in 35S::ole16i transgenic plants. In contrast, OLE16 was reduced to only 80% of wild-type levels when OLE18 was completely eliminated in 35S::ole18i transgenic plants. While the triacylglycerol content of crude seed extracts of 35S::ole16i and 35S::ole18i transgenic seeds was reduced to approximately 60% and 80%, respectively, triacylglycerol in isolated oil bodies was respectively reduced to 45% and 80% in accordance with the reduction of their oleosin contents. Oil bodies isolated from both 35S::ole16i and 35S::ole18i transgenic seeds were found to be of comparable size and stability to those isolated from wild-type rice seeds, although they were merely sheltered by a single oleosin isoform. The drastic difference between the triacylglycerol contents of crude seed extracts and isolated oil bodies from 35S::ole16i transgenic plants could be attributed to the presence of large, unstable oil clusters that were sheltered by insufficient amounts of oleosin and therefore could not be isolated together with stable oil bodies

Porcine Lactoferrin Expression in Transgenic Rice and Its Effects as a Feed Additive on Early Weaned Piglets

The purpose of this study is to determine the growth performance and immune characteristics of early weaned piglets receiving rice bran expressing porcine lactoferrin as a feed additive. Full-length cDNA encoding porcine lactoferrin (LF) driven by a rice actin promoter was transformed into rice plants, and its integration into the rice genome was verified by Southern blot analysis. The expression of recombinant LF (rLF) in whole grains and rice bran was also confirmed, and the amount of rLF accumulated in rice bran was estimated by immunoblot assay to be approximately 0.1% of rice bran weight. An iron-binding assay showed that the rLF retained iron-binding activity and the binding capacity of 1 mg/mL rLF would be saturated by 100 μM of FeCl3. Thirty-six early weaned piglets at 21 days old were randomly selected into two groups and fed a diet containing 5% transgenic rice bran containing 50 mg/kg rLF (rLF group) and 5% rice bran (control group) to investigate the piglets’ growth performance and immune characteristics. The results showed no significant difference in growth performance between the groups during the feeding period. However, the aerobic bacteria, anaerobic bacteria, and coliform counts in the cecal contents of the rLF-fed group were significantly lower than those of the control group. Additional immune characteristics such as the IgG concentration in the rLF group was higher than the control group at the 28th day, but leukocyte counts and the peripheral lymphocyte ratio remained similar. In summary, porcine LF expressed in rice bran, a byproduct of rice, can be used as a functional additive to improve antimicrobial capabilities and IgG concentration of early weaned piglets