Thomas E. Komorowski

Extraocular muscle regeneration in primates. Local anesthetic-induced lesions.

Retrobulbar administration of several local anesthetics (0.75% bupivacaine, 2.0% mepivacaine or 2.0% lidocaine plus 1:100,000 epinephrine) in monkeys resulted in a low incidence of muscle fiber lesions in the extraocular muscles closest to the site of injection. Most lesions resulted in the degeneration and regeneration of muscle fibers on the surface of the muscles, but occasionally a massive internal lesion was seen. In contrast, large lesions were common in rectus muscles that received direct injections of local anesthetics in both monkeys and humans. The morphology and temporal sequence of muscle fiber degeneration and regeneration was similar to that seen in primate thumb muscles injured by anesthetic agents.

The suprastructure of the saccular macula.

Inclusion of tannic acid in the primary fixative fortuitously preserved the macular suprastructure, from the macular surface to the otoconia, in rat. Results demonstrate that fluid-filled channels around the stereociliary tufts and a slit-like space immediately above the macula are artefacts of tissue preparation. Filaments fill the area between, and show linkage to, the macular surface and the more peripheral, closely knit layer identified here as the true otoconial membrane. The material also passes through the otoconial membrane at various sites to surround the otoconia and extends well beyond the maculas in all directions. Although it may prove later to be endolymph, it is referred to here as supramacular substance. Kinocilia and the tallest stereocilia as well as the otoconia are tethered to the otoconial membrane. Findings were confirmed in decalcified material. Results are discussed in terms of functional and biochemical implications, and by analogy to man-made acceleration sensing devices.

Drug inhibition of Gly-Sar uptake and hPepT1 localization using hPepT1-GFP fusion protein

An hPepT1-GFP fusion construct was made to study drug inhibition of dipeptide uptake and apical, basolateral, or subcellular hPepT1 localization. The hPepT1 stop codon was mutated by polymerase chain reaction and was subsequently cloned into the pEGFP-N1 vector. The hPepT1-GFP fusion construct was then transfected into Caco-2 and HeLa cells, and drug inhibition was studied by inhibiting 3H-Gly-Sar uptake. Western blot analysis was used to determine hPepT1-GFP expression levels and confocal microscopy was used to examine the localization. Both anti-hPepT1 antibody and anti-GFP antibody recognized a 120kd hPepT1-GFP fusion protein in the transfected cells. The 3H-Gly-Sar uptake in transfected HeLa cells was enhanced more than 20 times compared with the control. Valacyclovir (5 mmol/L) was able to completely inhibit 3H-Gly-Sar uptake in these transfected cells. Confocal microscopy showed that the hPepT1-GFP mainly localized in the Caco-2 cell apical membrane, but was present throughout the entire HeLa cell membranes. The hPepT1-GFP fusion protein was not found in either early endosome or lysosome of Caco-2 cells under normal conditions; however, it was detected in some subsets of lysosomes and early endosomes in phorbol 12-myristate 13-acetate (PMA)-treated Caco-2 cells.

Macular Suprastructure, Stereociliary Bonding and Kinociliary/ Stereociliary Coupling in Rat Utricular Macula

This report considers rat utricular macular suprastructure after chemical treatment with the sodium salt of N,N-Naphthaloylhydroxylamine (NHA), used alone or in combination with tannic acid (TA). NHA and TA preserve calcium and complex carbohydrate-protein molecules, respectively. Macromolecules of supramacular substance appear comparable morphologically to material of the external lamina of glycocalyx. Similar material crosslinks stereocilia with approximately equal to 58 nm periodicity and couples parts of kinocilia with specific stereocilia. Particles which occur within kinocilia at certain attachment sites connect to dynein arms of the kinocilia. Interstereociliary connections are aligned with internal linkers to actin. Thus, a basis for dynamic communication between kinocilia and stereociliary actin has been shown. The mechanism would appear to be calcium dependent. The findings support the concept that kinocilia are motile and lead to the stereociliary tuft in vestibular hair cells.