A 17-bp insertion and a Phe215----Cys missense mutation in the dihydrolipoyl transacylase (E2) mRNA from a thiamine-responsive maple syrup urine disease patient WG-34.

We have amplified the cDNA for the transacylase (E2) subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex from a thiamine-responsive MSUD cell line (WG-34) by the polymerase chain reaction. Sequencing of the amplified WG-34 cDNA showed a 17-bp insertion (AAATACCTTGTTACCAG) apparently resulting from an aberrant splicing of the E2 gene, and a missense (T----G) mutation that changes Phe215 to Cys in the E2 subunit. The existence of these two mutations was confirmed by probing the amplified E2 cDNA or genomic DNA with allele-specific oligonucleotides. The above results support the thesis that the thiamine-responsive MSUD patient (WG-34) is a compound heterozygote at the E2 locus. The implication of the E2 mutations for the thiamine-responsiveness observed in this patient is discussed.     

Expression of epidermal growth factor in the rat kidney

Summary The renal localization and the site of synthesis of epidermal growth factor (EGF) were investigated in the rat kidney by immunohistochemistry and in situ hybridization techniques. EGF was localized in the cells of the thick ascending limb of Henle (TAL) and distal convoluted tubule (DCT). At the ultrastructural level, EGF immunoreactivity was distributed on the apical membrane and trans-Golgi complex of the TAL and DCT cells. These segments of the rat nephron also hybridized to prepro-EGF cRNA probes in a specific manner, indicating that TAL and DCT are the sites of EGF synthesis in the rat kidney.     

Glial fibrillary acidic protein and its mRNA: ultrastructural detection and determination of changes after CNS injury.

We have previously demonstrated that glial fibrillary acidic protein (GFAP) containing intermediate filaments in retinal Müller cells undergo both quantitative induction and subcellular reorganization as a response to long-term retinal detachment (an induced CNS degeneration wherein the Müller cells form a multicellular scar). This study demonstrates by RNA blotting analysis that normal retina expresses a low basal level of GFAP mRNA, which is induced approximately 500% within 3 days of retinal detachment. At the cellular level, electron microscopic in situ hybridization analysis readily detects GFAP mRNA in Müller cells of detached retinas, but not in normal retinas. On the other hand, GFAP mRNA was readily detected in retinal astrocytes (which appear to express GFAP mRNA at high, constitutive levels). In both cell types, the ultrastructural localization of GFAP mRNA was the same. In the nuclei, the GFAP mRNA was associated with amorphous, electron-dense regions within the euchromatin. In the cytoplasm, the GFAP mRNA was associated with intermediate filaments near the nuclear pores, along the filaments when no other structures were apparent, and when the filaments appeared to be associated with ribosomes and polysomes. The ultrastructural location of the GFAP mRNA (especially along the intermediate filaments) may be unique to this mRNA or may represent a more generalized mRNA phenomenon.     

Structural Characteristics of the Short-Tail Fibers of T4 Bacteriophage

The characteristics of pure preparations of short-tail fibers of bacteriophage T4 have been studied in the optical and electron microscope. Three main structures were observed: 1) spheres of 8.1 nm diameter; 2) fibers 43 nm long and 3.8 nm thick; and 3) fibers 54 nm long and 3.2 nm thick. Both types of fibers exhibited a regular beaded appearance. The 43-nm fibers were the most abundant structure. During the process of purification of the short-tail fibers, the formation of aggregates was observed each time the material containing the short-tail fibers was dialyzed against saline solutions. These aggregates became increasingly fibrous (as observed in the optical microscope) as the material used was increasingly enriched in short-tail fibers. Finally, most of the aggregates were of the fibrous type when they were formed from a purified preparation of short-tail fibers. In the electron microscope, it was found that the filamentous aggregates were organized in well-defined bundles. The amino acid composition of the highly purified short-tail fibers was also determined. Among the known fibrous proteins, the ones that most resemble the amino acid composition of the short-tail fibers are actin and fibrinogen. These observations are discussed in relation to the T4 short-tail fiber structure and their localization on the hexagonal baseplate of the T4 tail structure.     

The Receptor-Mediated Activation of Tyrosine Hydroxylation in the Superior Cervical Ganglion of the Rat

The addition of carbachol to superior cervical ganglia causes a rapid increase in tyrosine hydroxylation in situ. The increase occurs in ganglia from both newborn and adult animals, and in ganglia from animals pretreated with reserpine. The increase is not due to increased transport of the substrate. The increase is dependent upon the presence of calcium, and is additive to the stimulation produced by dibutyryl cyclic AMP. The stimulation seems specific for tyrosine hydroxylation; dopamine beta-hydroxylation is not increased. Preincubation experiments suggest that the carbachol-induced stimulation is due to a change in the availability of, or the affinity of the enzyme for, reduced pterin cofactor. The stimulation is inhibited by atropine and also by low concentrations of phenoxybenzamine or haloperidol, which suggests that it is caused by an action of carbachol on the interneurons in the ganglia.     

Multiple linear spiradenomas

A case of eccrine spiradenoma (ESA) is described in a 35-year-old man. During an 18-year period, the original number of tumors grew in size and fused into large groups. Surgical excision of all tumors was performed successfully. The unusual features of the case were a linear distribution and giant multifocal tumors. The histology of the tumors and their appearance at the beginning and 18 years later is described.     

Antigenic differences between Anabaena azollae fresh from the Azolla fern leaf cavity and free-living cyanobacteria

Fluorescent antibody staining indicated differences in surface antigenicity in Anabaena azollae cells fresh from the leaf cavities of the fern, Azolla caroliniana, and algae which were isolated and subcultured from this fern. Such results suggest that either changes in antigenicity occur in this phycobiont during culturing or that isolation selects for an antigenically different mutant strain capable of in vitro growth.Non-Standard Abbreviations FA fluorescent antibody staining - PBS phosphate buffered saline - W microwatt - Anti-F antiserum prepared against fresh cells - Anti-N antiserum prepared against Newton's culture - FTTC fluorescein isothiocyanate To whom offprint requests should be sent