High-purity isolation of bullfrog hair bundles and subcellular and topological localization of constituent proteins.

The small number of hair cells in auditory and vestibular organs severely impedes the biochemical characterization of the proteins involved in mechano-electrical transduction. By developing an efficient and clean "twist-off" method of hair bundle isolation, and by devising a sensitive, nonradioactive method to detect minute quantities of protein, we have partially overcome this limitation and have extensively classified the proteins of the bundles. To isolate hair bundles, we glue the saccular macula of the bullfrog to a glass coverslip, expose the tissue to a molten agarose solution, and allow the agarose to solidify to a firm gel. By rotating the gel disk with respect to the fixed macula, we isolate the hair bundles by shearing them at their mechanically weak bases. The plasma membranes of at least 80% of the stereocilia reseal. To visualize the proteins of the hair bundle, we covalently label them with biotin, separate them by SDS-PAGE, and transfer them to a charged nylon membrane. We can detect less than 500 fg of protein by probing the membrane with streptavidin-alkaline phosphatase and detecting the chemiluminescent product from the hydrolysis of the substrate 3-(4-methoxyspiro-(1,2-dioxetane-3,2'-tricyclo-[3.3.1. 1(3.7)]decan)-4-yl) phenyl phosphate (AMPPD). These techniques reveal a distinct constellation of proteins in and associated with hair bundles. Several proteins, such as calmodulin, calbindin, actin, tubulin, and fimbrin, have previously been described. A second class of proteins in the preparation appears to be derived from extracellular sources. Finally, several heretofore undescribed bundle proteins are identified and characterized by their membrane topology, subcellular localization, and glycosidase and protease sensitivities.     

Making an effort to listen: mechanical amplification in the ear

The inner ear's performance is greatly enhanced by an active process defined by four features: amplification, frequency selectivity, compressive nonlinearity, and spontaneous otoacoustic emission. These characteristics emerge naturally if the mechanoelectrical transduction process operates near a dynamical instability, the Hopf bifurcation, whose mathematical properties account for specific aspects of our hearing. The active process of nonmammalian tetrapods depends upon active hair-bundle motility, which emerges from the interaction of negative hair-bundle stiffness and myosin-based adaptation motors. Taken together, these phenomena explain the four characteristics of the ear's active process. In the high-frequency region of the mammalian cochlea, the active process is dominated instead by the phenomenon of electromotility, in which the cell bodies of outer hair cells extend and contract as the protein prestin alters its membrane surface area in response to changes in membrane potential.     

Divalent counterions tether membrane-bound carbohydrates to promote the cohesion of auditory hair bundles

The cell membranes in the hair bundle of an auditory hair cell confront a difficult task as the bundle oscillates in response to sound: for efficient mechanotransduction, all the component stereocilia of the hair bundle must move essentially in unison, shearing at their tips yet maintaining contact without membrane fusion. One mechanism by which this cohesion might occur is counterion-mediated attachment between glycan components of apposed stereociliary membranes. Using capillary electrophoresis, we showed that the stereociliary glycocalyx acts as a negatively charged polymer brush. We found by force-sensing photomicrometry that the stereocilia formed elastic connections with one another to various degrees depending on the surrounding ionic environment and the presence of N-linked sugars. Mg²⁺ was a more potent mediator of attachment than was Ca²⁺. The forces between stereocilia produced chaotic stick-slip behavior. These results indicate that counterion-mediated interactions in the glycocalyx contribute to the stereociliary coherence that is essential for hearing.     

High-frequency stable transformation of cotton (Gossypium hirsutum L.) by particle bombardment of embryogenic cell suspension cultures

Stable transformation of cotton (Gossypium hirsutum L.) at a high frequency has been obtained by particle bombardment of embryogenic cell suspension cultures. Transient and stable expression of the β-glucuronidase (GUS) gene was monitored in cell suspension cultures. Transient expression, measured 48 h after bombardment, was abundant, and stable expression was observed in over 4% of the transiently expressing cells. The high efficiency of stable expression is due to the multiple bombardment of rapidly dividing cell suspension cultures and the selection for transformed cells by gradually increasing the concentrations of the antibiotic Geneticin (G418). Southern analysis indicated a minimum transgene copy number of one to four in randomly selected plants. Fertile plants were obtained from transformed cell cultures less than 3 months old. However, transgenic and control plants from cell cultures older than 6 months produced plants with abnormal morphology and a high degree of sterility. Received: 20 January 1999 / Revision received: 1 October 1999 / Accepted: 11 October 1999     

Menaquinone (vitamin K2) biosynthesis: evidence that the Escherichia coli menD gene encodes both 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid synthase and alpha-ketoglutarate decarboxylase activities.

The formation of 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC), the first identified intermediate in the menaquinone biosynthetic pathway, requires two reactions. They are the decarboxylation of alpha-ketoglutarate by an alpha-ketoglutarate decarboxylase, which results in the formation of succinic semialdehyde-thiamine PPi (TPP) anion, and the addition of the succinic semialdehyde-TPP anion to isochorismate carried out by the enzyme SHCHC synthase. Evidence is provided to support the conclusion that both enzymatic activities are encoded by an extended menD gene which is capable of generating a bifunctional 69-kDa protein. Consistent with the requirement for TPP in the decarboxylation of alpha-ketoglutarate, the translated amino acid sequence contains the characteristic TPP-binding motif present in all well-characterized TPP-requiring enzymes.     

spoVG sequence of Bacillus megaterium and Bacillus subtilis.

We have sequenced the stage V sporulation specific gene spoVG in both Bacillus megaterium and Bacillus subtilis. The open reading frames encode polypeptides of 96 and 97 residues, respectively, and have an 88.6% amino acid identity. Both genes have putative rho-independent terminators. No significant amino acid or nucleotide homology of either gene was found when compared with sequences contained in either the Genbank or EMBL data bases.