A group of type I keratin genes on human chromosome 17: characterization and expression.

The human type I keratins K16 and K14 are coexpressed in a number of epithelial tissues, including esophagus, tongue, and hair follicles. We determined that two genes encoding K16 and three genes encoding K14 were clustered in two distinct segments of chromosome 17. The genes within each cluster were tightly linked, and large parts of the genome containing these genes have been recently duplicated. The sequences of the two K16 genes showed striking homology not only within the coding sequences, but also within the intron positions and sequences and extending at least 400 base pairs 5' upstream and 850 base pairs 3' downstream from these genes. Despite the strong homologies between these two genes, only one of the genes encoded a protein which assembled into keratin filaments when introduced into simple epithelial cells. While there were no obvious abnormalities in the sequence of the other gene, its promoter seemed to be significantly weaker, and even a hybrid gene with the other gene's promoter gave rise to a much reduced mRNA level after gene transfection. To demonstrate that the functional K16 gene that we identified was in fact responsible for the K16 expressed in human tissues, we made a polyclonal antiserum which recognized our functional K16 gene product in both denatured and filamentous form and which was specific for bona fide human K16.     

Molecular cloning and sequencing of a cDNA coding for mature human kidney cathepsin H

A cDNA library was established from human kidney RNA and screened with an extended oligonucleotide probe derived from the amino-acid sequence of human cathepsin H. A recombinant clone, pRF15, was isolated and characterized. DNA sequence analysis of its 1106-nucleotide-long insert revealed that pRF15 encodes the complete protein sequence of mature cathepsin H plus 28 amino acids of a propeptide, thus confirming that cathepsin H is synthesized as a larger precursor molecule and posttranslationally processed. Northern blot analysis indicated that cathepsin H is predominantly synthesized in kidney. A high degree of sequence homology was observed with rat cathepsin H, especially within the propeptide. The part of the prosequence coding for the "minichain" is conserved in the prosequence of aleurain, a plant thiol protease.     

Plasticin, a novel type III neurofilament protein from goldfish retina: increased expression during optic nerve regeneration.

The goldfish visual pathway displays a remarkable capacity for continued development and plasticity. The intermediate filament proteins in this pathway are unexpected and atypical, suggesting these proteins provide a structure that supports growth and plasticity. Using a goldfish retina lambda gt10 library, we have isolated a full-length cDNA clone that encodes a novel type III intermediate filament protein. The mRNA for this protein is located in retinal ganglion cells, and its level dramatically increases during optic nerve regeneration. The protein is transported into the optic nerve within the slow phase of axonal transport. We have named this protein plasticin because it was isolated from a neuronal pathway well known for its plasticity.     

Xylanase and xylosidase activities in avocado fruit

The activities of xylanase and xylosidase were demonstrated in mature avocado (Persea americana Mill.) fruits from different cultivars. When monitored on the day of harvest during the season at 1-month intervals, xylanase activity decreased and xylosidase activity increased between January and February and then remained stable until May. When monitored during the ripening process (January harvest), xylanase activity was constant, and xylosidase activity reached a peak at the climax of ethylene evolution and cellulase activity. Xylanase, which originated from Trichoderma viride and was added to the medium in which avocado discs were incubated, induced ethylene evolution.     

A novel cholinergic receptor mediates inhibition of chick cochlear hair cells.

The central nervous system provides feedback regulation at several points within the peripheral auditory apparatus. One component of that feedback is inhibition of cochlear hair cells by release of acetylcholine (ACh) from efferent brainstem neurons. The mechanism of hair cell inhibition, and the character of the presumed cholinergic receptor, however, have eluded understanding. Both nicotinic and muscarinic, as well as some non-cholinergic ligands can affect the efferent action. We have made whole-cell, tight-seal recordings from short (outer) hair cells isolated from the chick's cochlea. These are the principal targets of cochlear efferents in birds. ACh hyperpolarizes short hair cells by opening a cation channel through which Ca2+ enters the cell and subsequently activates Ca(2+)-dependent K+ current (Fuchs & Murrow 1991, 1992). Both curare and atropine are effective-antagonists of cholinergic inhibition at 3 microM, whereas trimethaphan camsylate and strychnine block at 1 microM. The normally irreversible nicotinic antagonist, alpha-bungarotoxin, reversibly blocked the hair cell response, as did kappa-bungarotoxin. The half-blocking concentration for alpha-bungarotoxin was 26 nM. It is proposed that the hair cell AChR is a ligand-gated cation channel related to the nicotinic receptor of nerve and muscle.     

Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Segmental release of amino acid neurotransmitters from transcranial stimulation

The present study used microdialysis techniques in an intact rabbit model to measure the release of amino acids within the lumbar spinal cord in response to transcranial electrical stimulation. Dialysis samples from the extracellular space were obtained over a stimulation period of 90 minutes and were examined using high pressure liquid chromatography. Neuronal excitation was verified by recerding corticomotor evoked potentials (CMEPs) from the spinal cord. A significant increase in the release of glycine and taurine compared to sham animals was measured after 90 minutes of transcranial stimulation. Glutamate and aspartate release was not significantly elevated. GABA concentrations were consistently low. CMEP components repeatedly showed adequate activation of descending fiber pathways and segmental interneuron pools during dialysis sampling. Since glycine, and to a lesser extent taurine, have been shown to inhibit motor neuron activity and are closely associated with segmental interneuron pools, suprasegmental modulation of motor activity may be, in part, through these inhibitory amino acid neurotransmitters in the rabbit lumbar spinal cord.     

Reversible dissociation of active octamer of cyanase to inactive dimer promoted by alteration of the sulfhydryl group

Cyanase is an inducible enzyme in Escherichia coli that catalyzes the reaction of cyanate with bicarbonate resulting in the decomposition of cyanate to ammonia and bicarbonate. In this study, the role of the single sulfhydryl group in each of the eight identical subunits of cyanase was investigated. Tetranitromethane, methyl methanethiosulfonate, N-ethylmaleimide, and Hg2+ all reacted with the sulfhydryl group to give derivatives which had reduced activities and which dissociated reversibly to inactive dimer. Association of inactive dimer to active octamer was facilitated by the presence of azide (cyanate analog) and bicarbonate, increased temperature and enzyme concentration, and presence of phosphate. Nitration of tyrosine residues by tetranitromethane occurred only in the absence of azide and bicarbonate, suggesting that at least some of the tyrosine residues become exposed when octamer dissociates to dimer. Site-directed mutagenesis was used to prepare a mutant enzyme in which serine was substituted for cysteine. The mutant enzyme was catalytically active and had properties very similar to native enzyme, except that it was less stable to treatment with urea and to high temperatures. These results establish that in native cyanase the sulfhydryl group per se is not required for catalytic activity, but it may play a role in stabilizing octameric structure, and that octameric structure is required for catalytic activity.     

Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., & Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by greater than 1.2 M guanidine hydrochloride; upon reconstitution, approximately 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, far-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structured intermediates (less than 1 min), formation of native enzyme is governed by two parallel slow and very slow first-order folding reactions (k1 = 1.3 X 10(-3) S-1 and k2 = 7 X 10(-5) S-1 at 20 degrees C). The rate constant of the association step following the slow folding reaction (determined by k1) must be greater than 10(6) M-1 S-1. The energy of activation of the slow folding step is of the order of 9 +/- 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), approximately 35% of the native helicity is preserved, as determined by circular dichroism.(ABSTRACT TRUNCATED AT 250 WORDS)