Identification of a candidate human spectrin Src homology 3 domain-binding protein suggests a general mechanism of association of tyrosine kinases with the spectrin-based membrane skeleton.

Spectrin is a widely expressed protein with specific isoforms found in erythroid and nonerythroid cells. Spectrin contains an Src homology 3 (SH3) domain of unknown function. A cDNA encoding a candidate spectrin SH3 domain-binding protein was identified by interaction screening of a human brain expression library using the human erythroid spectrin (alphaI) SH3 domain as a bait. Five isoforms of the alphaI SH3 domain-binding protein mRNA were identified in human brain. Mapping of SH3 binding regions revealed the presence of two alphaI SH3 domain binding regions and one Abl-SH3 domain binding region. The gene encoding the candidate spectrin SH3 domain-binding protein has been located to human chromosome 10p11.2 --> p12. The gene belongs to a recently identified family of tyrosine kinase-binding proteins, and one of its isoforms is identical to e3B1, an eps8-binding protein (Biesova, Z., Piccoli, C., and Wong, W. T. (1997)Oncogene 14, 233-241). Overexpression of the green fluorescent protein fusion of the SH3 domain-binding protein in NIH3T3 cells resulted in cytoplasmic punctate fluorescence characteristic of the reticulovesicular system. This fluorescence pattern was similar to that obtained with the anti-human erythroid spectrin alphaI SigmaI/betaI SigmaI antibody in untransfected NIH3T3 cells; in addition, the anti-alphaI SigmaI/betaI SigmaI antibody also stained Golgi apparatus. Immunofluorescence obtained using antibodies against alphaI SigmaI/++betaI SigmaI spectrin and Abl tyrosine kinase but not against alphaII/betaII spectrin colocalized with the overexpressed green fluorescent protein-SH3-binding protein. Based on the conservation of the spectrin SH3 binding site within members of this protein family and published interactions, a general mechanism of interactions of tyrosine kinases with the spectrin-based membrane skeleton is proposed.

Light and electron microscopic immunocytochemical localization of two major proteins in garlic bulb.

Garlic is known as a potent spice and a medicine with broad therapeutic properties ranging from antibacterial to anticancer, antidiabetic, and anticoagulant. Two major proteins of 40 KD and 14 KD constituting approximately 96% of total garlic proteins have been recently purified at our Institute. This immunocytochemical and ultrastructural study revealed that the 40 KD protein was localized in the parenchyma sheath cells (PSC) of garlic bulbs, whereas the 14 KD protein was present in the cortical cells (CC). Immunogold electron microscopy study indicated that the 40 KD protein was specifically localized in the globular granules of the cytoplasmic area of PSC. Each globular granule was amorphous and homogenous with membrane limiting its outermost layer. The yellowish color of PSC in freshly cut slices of garlic bulb suggested that PSC may have sulfur-containing compounds such as allicin, the primary contributor of the pungency and medicinal properties of garlic. Ellman's reagent test quantitatively revealed that there were 17.8 n moles sulfhydryl (SH)/ml of 40 KD garlic protein. Microtubule tubulin in mitotic figures from PHA-stimulated human short-term whole blood cultures reacted strongly with antitubulin antibody but reacted negatively with anti-40 KD garlic protein antibodies and therefore was not related to the 40 KD garlic protein immunocytochemically.

Dialyzed fetal bovine serum increases cytogenetic fragile X expression.

UNLABELLED: Short-term whole blood cultures from 9 unrelated male individuals with the fragile X [fra(X)] syndrome were exposed to 5-fluorodeoxyuridine (FUdR). The fra(X) frequency was higher in 8 of 9 cases where the complete medium contained dialyzed fetal bovine serum (DFBS). In 3 of the cases, the fra(X) frequency nearly tripled (e.g., 12/100 to 33/100) while in 2 others, it nearly doubled (e.g., 15/100 to 29/100). When DFBS cultures from 2 other fra(X) individuals were exposed to increasing folic acid concentrations ranging from 2 to 4,000 x 10(-6) M, there was virtually no change in fra(X) expression. In 6 of 9 DFBS cultures, the mitotic index decreased, and it increased in 3. Therefore, although the fra(X) frequency increased, in most DFBS cultures the mitotic index decreased. Whether the reduction in mitotic index indicates an inverse correlation between reduced mitotic index and increased fra(X) expression, at least in cultures from some individuals, will be determined by additional studies. IN CONCLUSION: (1) medium supplementation with dialyzed fetal bovine serum should be considered when using FUdR for fra(X) identification in order to avoid potentially false negative results; (2) there appears to be no direct correlation between increased mitotic index and increased fra(X) expression in whole blood cultures; (3) increased folic acid concentrations do not affect fra(X) expression when FUdR fra(X) induction is employed; therefore requesting people to refrain from taking vitamins, including folic acid, before fra(X) testing (a practice that still persists in some places) appears unnecessary.

Folic acid treatment of fragile X males: a further study.

Investigations of the effect of high dose folic acid treatment of fragile X syndrome in males has produced mixed results. However, no study had examined the possible drug effects of folic acid on non-fragile X control males. Therefore, we examined the effect of folic acid on fragile X males using non-fragile X control males. Subjects were assigned randomly to an ABA or BAB design. Duration of either folic acid or placebo condition was 4 months. Folic acid or placebo was given in a double-blind fashion. At the end of each condition, the subjects' behavior was assessed. At the end of the study, parents were asked to complete a questionnaire. Using parents' responses, we examined 22 items on the Autistic Descriptors Checklist and two subscales from the Vineland Adaptive Behavior Scale which corresponded to areas of behavior parents' noted to have shown improvement. We did not find significant differences between fragile X males and control males, within subjects, nor across folic acid and placebo conditions. Thus, our follow-up study confirms and extends our original findings, as well as those of other researchers: namely, that no dramatic changes in behavior result from high dose folic acid. Moreover, subtle improvements observed in earlier investigations were not confirmed.

High dose folic acid treatment of fragile (X) males.

We conducted an experimental trial of high-dose folic acid given to five males, ages 8 to 26 years, with the fra(X) syndrome. In this double blind study, each subject received 250 mg per day of folic acid for 3 months, followed by placebo for 3 months, and folic acid again for an additional three months. Based on IQ tests, behavior ratings, the Autistic Descriptors Checklist, and parental ratings, there was little evidence to suggest any positive effects seen during the administration of high-dose folic acid. Therefore, this study has provided little support for a hypothesis of benefit of high-dose folic acid in the treatment of the fra(X) syndrome.

Folic acid therapy in the fragile X syndrome.

Two brothers with fra(X) positive X-linked mental retardation (XLMR) were treated with folic acid. Initially a double blind cross-over design was employed followed by a long-term high dose trial. A decrease in the frequency of fra(X) positive cells was observed when low folic acid culture medium was used but not when an FUdR induction system was employed. Selected behavioral characteristics improved in both while receiving folic acid. Decreased hyperactivity, greater attention span, increased motor coordination, increased quantity and quality of speech were noted. Improvement in Leiter mental age and regression after cessation of treatment was seen in one subject but not in the other. Further controlled trials with larger numbers of subjects using high doses of folic acid over longer periods of time are needed to assess the possible benefits of this experimental form of treatment.