Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

Spectrophotometric determination of de novo hemozoin/beta-hematin formation in an in vitro assay

Formation of hemozoin in the malaria parasite, due to its unique nature, is an attractive molecular target. Several laboratories have been trying to unravel the molecular mechanism of hemozoin biosynthesis within the parasite digestive vacuoles. Use of different assay protocols for in vitro beta-hematin (synthetic identical to hemozoin) formation by these laboratories has led to inconsistent and often contradictory findings. Much of the difficulty may be attributed to oligomeric heme aggregates, which may be indistinguishable in some detection approaches if adequate separation of beta-hemtin is not achieved. Therefore, there is an urgent need for a widely accepted protocol for in vitro beta-hematin formation. We describe here a spectrophotometric assay for in vitro beta-hematin formation. The assay has been validated with the Plasmodium falciparum lysate, the parasite lipid extracts, and some commercially available fatty acids, which are known to initiate/catalyze beta-hematin formation in vitro. The necessity for multiple wash steps for accurate quantification of de novo hemozoin/beta-hematin formation was verified experimentally. It was necessary to wash the pellet, which contains beta-hematin and heme aggregates, sequentially with Tris/SDS buffer and alkaline bicarbonate solution for complete removal of monomeric heme and heme aggregates and accurate quantification of beta-hematin formed during the assay. The pellets and side products in the supernatant were characterized by infrared spectroscopy. No beta-hematin formation occurred in the absence of a catalytic/initiating factor. Based on these findings, a filtration-based assay that uses 96-well microplates, and which has important application in in vitro screening and identification of novel inhibitors of hemozoin formation as potential blood schizontocidal antimalarials, has been developed.     

The isolation,characterization and sequence of two divergent β-tubulin genes from soybean (Glycine max L.)

Two divergent -tubulin genes (designated S-1 and S-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii -tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different -tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of -tubulin genes (thus far undetected) exist in the soybean genome. The S-1 and S-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode -tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with -tubulins from several organisms showed that they are most homologous to Chlamydomonas -tubulin (85–87%), with lesser degrees of homology to -tubulins of vertebrate species (79–83%), Trypanosoma brucei (80–81%) and Saccharomyces cerevisiae (66–68%). The amino acid sequences of S-1 and S-2 are as divergent from each other as they are from the Chlamydomonas -tubulin. The amino acids at the diverged positions in S-2 are nearly all conservative substitutions while in S-1, 18 of the 69 substitutions were non-conservative. Both soybean -tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas -tubulin genes. Codon usage in the two soybean -tubulins is remarkably similar (D ²=0.87), but differs from codon usage in other soybean genes.     

The chemosystematics of Egyptian Trigonella species

Flavonoid glycosides of eight Trigonella species belonging to four sections of the Leguminosae [Gamal El Din, E.M. and Hassan, A.E. (1995) Taeckholmia, in press] were investigated. Fifteen glycosides were found based on the aglycones kaempferol, quercetin and the less common 7,4′-dihydroxyflavone and 7,3′,4′-trihydroxyflavone. One isoflavone (formononetin) was found in all Trigonella species. Chemosystematic relationships are discussed.     

Fluoride Toxicity and Status of Serum Thyroid Hormones, Brain Histopathology, and Learning Memory in Rats: A Multigenerational Assessment

High-fluoride (100 and 200?ppm) water was administered to rats orally to study the fluoride-induced changes on the thyroid hormone status, the histopathology of discrete brain regions, the acetylcholine esterase activity, and the learning and memory abilities in multigeneration rats. Significant decrease in the serum-free thyroxine (FT4) and free triiodothyronine (FT3) levels and decrease in acetylcholine esterase activity in fluoride-treated group were observed. Presence of eosinophilic Purkinje cells, degenerating neurons, decreased granular cells, and vacuolations were noted in discrete brain regions of the fluoride-treated group. In the T-maze experiments, the fluoride-treated group showed poor acquisition and retention and higher latency when compared with the control. The alterations were more profound in the third generation when compared with the first- and second-generation fluoride-treated group. Changes in the thyroid hormone levels in the present study might have imbalanced the oxidant/antioxidant system, which further led to a reduction in learning memory ability. Hence, presence of generational or cumulative effects of fluoride on the development of the offspring when it is ingested continuously through multiple generations is evident from the present study.     

Leaf sheath cuticular waxes on bloomless and sparse-bloom mutants of Sorghum bicolor

Leaf sheath cuticular waxes on wild-type Sorghum bicolor were approximately 96% free fatty acids, with the C28 and C30 acids being 77 and 20% of these acids, respectively. Twelve mutants with markedly reduced wax load were characterized for chemical composition. In all of the 12 mutants, reduction in the amount of C28 and C30 acids accounted for essentially all of the reduction in total wax load relative to wildtype. The bm2 mutation caused a 99% reduction in total waxes. The bm4, bm5, bm6, bm7 and h10 mutations caused more than 91% reduction in total waxes, whereas the remaining six mutants, bm9, bm11, h7, h11, h12 and h13, caused between 35 and 78% reduction in total wax load. Relative to wild-type, bm4 caused a large increase in the absolute amount of C22, C24 and C26 acids, and reduction in the C28 and longer acids, suggesting that bm4 may suppress elongation of C26, acyl-CoA primarily. The h10 mutation increased the absolute amounts of the longest chain length acids, but reduced shorter acids, suggesting that h10 may suppress termination of acyl-CoA elongation. The bm6, bm9, bm11, h7, h11, h12 and h13 mutations increased the relative amounts, but not absolute amounts, of longer chain acids. Based on chemical composition alone, it is still uncertain which genes and their products were altered by these mutations. Nevertheless, these Sorghum cuticular wax mutants should provide a valuable resource for future studies to elucidate gene involvement in the biosynthesis of cuticular waxes, in particular, the very-long-chain fatty acids.