In Vivo Substrates of the Lens Molecular Chaperones αA-Crystallin and αB-Crystallin

αA-crystallin and αB-crystallin are members of the small heat shock protein family and function as molecular chaperones and major lens structural proteins. Although numerous studies have examined their chaperone-like activities in vitro, little is known about the proteins they protect in vivo. To elucidate the relationships between chaperone function, substrate binding, and human cataract formation, we used proteomic and mass spectrometric methods to analyze the effect of mutations associated with hereditary human cataract formation on protein abundance in αA-R49C and αB-R120G knock-in mutant lenses. Compared with age-matched wild type lenses, 2-day-old αA-R49C heterozygous lenses demonstrated the following: increased crosslinking (15-fold) and degradation (2.6-fold) of αA-crystallin; increased association between αA-crystallin and filensin, actin, or creatine kinase B; increased acidification of βB1-crystallin; increased levels of grifin; and an association between βA3/A1-crystallin and αA-crystallin. Homozygous αA-R49C mutant lenses exhibited increased associations between αA-crystallin and βB3-, βA4-, βA2-crystallins, and grifin, whereas levels of βB1-crystallin, gelsolin, and calpain 3 decreased. The amount of degraded glutamate dehydrogenase, α-enolase, and cytochrome c increased more than 50-fold in homozygous αA-R49C mutant lenses. In αB-R120G mouse lenses, our analyses identified decreased abundance of phosphoglycerate mutase, several β- and γ-crystallins, and degradation of αA- and αB-crystallin early in cataract development. Changes in the abundance of hemoglobin and histones with the loss of normal α-crystallin chaperone function suggest that these proteins also play important roles in the biochemical mechanisms of hereditary cataracts. Together, these studies offer a novel insight into the putative in vivo substrates of αA- and αB-crystallin.     

Genetic Studies of the Prp17 Gene of Saccharomyces Cerevisiae: A Domain Essential for Function Maps to a Nonconserved Region of the Protein

The PRP17 gene product is required for the second step of pre-mRNA splicing reactions. The C-terminal half of this protein bears four repeat units with homology to the β transducin repeat. Missense mutations in three temperature-sensitive prp17 mutants map to a region in the N-terminal half of the protein. We have generated, in vitro, 11 missense alleles at the β transducin repeat units and find that only one affects function in vivo. A phenotypically silent missense allele at the fourth repeat unit enhances the slow-growing phenotype conferred by an allele at the third repeat, suggesting an interaction between these domains. Although many missense mutations in highly conserved amino acids lack phenotypic effects, deletion analysis suggests an essential role for these units. Only mutations in the N-terminal nonconserved domain of PRP17 are synthetically lethal in combination with mutations in PRP16 and PRP18, two other gene products required for the second splicing reaction. A mutually allele-specific interaction between prp17 and snr7, with mutations in U5 snRNA, was observed. We therefore suggest that the functional region of Prp17p that interacts with Prp18p, Prp16p, and U5 snRNA is in the N terminal region of the protein.     

Regulation of dynamin family proteins by post-translational modifications

Dynamin superfamily proteins comprising classical dynamins and related proteins are membrane remodelling agents involved in several biological processes such as endocytosis, maintenance of organelle morphology and viral resistance. These large GTPases couple GTP hydrolysis with membrane alterations such as fission, fusion or tubulation by undergoing repeated cycles of self-assembly/disassembly. The functions of these proteins are regulated by various post-translational modifications that affect their GTPase activity, multimerization or membrane association. Recently, several reports have demonstrated variety of such modifications providing a better understanding of the mechanisms by which dynamin proteins influence cellular responses to physiological and environmental cues. In this review, we discuss major post-translational modifications along with their roles in the mechanism of dynamin functions and implications in various cellular processes.     

AFX1 and p54 nrb : fine mapping, genomic structure, and exclusion as candidate genes of X-linked dystonia parkinsonism

We have mapped AFX1 and p54 ^nrb to a yeast artificial chromosome (YAC) contig of Xq13.1 that harbors the X-linked dystonia parkinsonism (XDP) locus DYT3. AFX1 is flanked by loci DXS7116 and Il2Rγ, and p54 ^nrb by loci DXS6673E and DXS7120. The exon-intron structure of both genes was analyzed. AFX1 is composed of three exons with most of exon 3 being untranslated. p54 ^nrb is made up of 12 exons ranging in size from 40 bp to 1227 bp. The start codon is in exon 3 and the stop codon in exon 12. Both genes are expressed in the brain, among other tissues. AFX1 and p54 ^nrb were excluded as candidates of DYT3 by sequencing of the exons and the flanking intronic sequences in an XDP patient and a control, and by Northern blot analysis. Received: 27 June 1997 / Accepted: 3 July 1997     

In vitro micropropagation of the tropical fruit tree Syzygium cuminii L.

Multiple shoots were obtained from nodal and shoot tip segments of 10 to 15-day-old seedlings of Syzygium cuminii L. on Murashige & Skoog (MS) revised medium supplemented with 6-benzyladenine (BA) (0.23–8.90 M) singly or in combination with -naphthaleneacetic acid (NAA), indole-3-acetic acid (IAA) or indole-3-butyric acid (IBA). Excised shoots were placed for root induction on MS medium containing NAA and/or IBA and then transferred to MS basal medium to form complete plantlets. The regenerated plantlets have been acclimatized and successfully transferred into the soil.     

Arginine residues involved in binding of tetrahydrofolate to sheep liver serine hydroxymethyltransferase.

The arginine residue(s) necessary for tetrahydrofolate binding to sheep liver serine hydroxymethyltransferase were located by phenylglyoxal modification. The incorporation of [7-14C]phenylglyoxal indicated that 2 arginine residues were modified per subunit of the enzyme and the modification of these residues was prevented by tetrahydrofolate. In order to locate the sites of phenylglyoxal modification, the enzyme was reacted in the presence and absence of tetrahydrofolate using unlabeled and radioactive phenylglyoxal, respectively. The labeled phenylglyoxal-treated enzyme was digested with trypsin, and the radiolabeled peptides were purified by high-performance liquid chromatography on reversed-phase columns. Sequencing the tryptic peptides indicated that Arg-269 and Arg-462 were the sites of phenylglyoxal modification. Neither a spectrally discernible 495-nm intermediate (characteristic of the native enzyme when substrates are added) nor its enhancement by the addition of tetrahydrofolate, was observed with the phenylglyoxal-modified enzyme. There was no enhancement of the rate of the exchange of the alpha-proton of glycine upon addition of tetrahydrofolate to the modified enzyme as was observed with the native enzyme. These results demonstrate the requirement of specific arginine residues for the interaction of tetrahydrofolate with sheep liver serine hydroxymethyltransferase.     

The multi-locus H-2D w16 region has an organization distinct from the D d region

Genomic DNA blot analyses using probes derived from the BALB/c 3 flanking region of the L ^d gene (L ^d 3 fl-C) and from near the BALB/c D3 ^d gene (50.2A) indicate that the B10.GAA37 mouse strain has a multi-locus D (D ^w16) region distinct from the five-gene organization observed in the D ^d and D ^q regions. To isolate the D ^w16 region class I genes, a genomic B10. GAA37-EMBL3 library was generated and screened with probes that preferentially hybridize to K and D region class I genes. Hybridization analyses of the isolated clones with L ^d derived oligonucleotide probes suggested that one of the clones contained the L ^w16 gene, whereas several other clones contained the L ^w16 gene. The sequence of the D ^w16 gene is most similar to that of the D ^p gene, particularly in the 3 half. Furthermore, the L ^w16 gene is quite similar in the 5 half and virtually identical in the 3 half to the L ^d gene, indicating that L ^w16, but not D ^w16, is a member of the L ^d gene family. Collectively, these data suggest that, through a D region recombination event, the novel D ^w16 region may have been assembled from primordial counterparts of the D ^p and L ^d genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60774-M60776, and M62759.     

Deuterium quadrupole coupling in N-acetylglycine and librational dynamics in solid poly(gamma-benzyl-L-glutamate).

To study the dynamics of peptide groups in solid proteins, we have accurately determined the principal components and molecular orientation of the electric field gradient tensor for the exchangeable deuterons in monoclinic N-acetylglycine by single-crystal deuterium nuclear magnetic resonance. These results are compared with the principal components of the amide deuterons in solid poly(gamma-benzyl-L-glutamate) measured in powder samples over a wide temperature range (140-400 K). The comparison indicates that in the solid polypeptide the N-D bonds undergo a small-amplitude torsional reorientation (libration) perpendicular to the peptide plane. To estimate dynamic rates, longitudinal relaxation times (T1 values) are reported for N-acetylglycine and poly(gamma-benzyl-L-glutamate). T1 values for the carboxyl and amide deuterons in N-acetylglycine are approximately 100 s, whereas for the amide deuterons in the polypeptide T1 approximately 1 s, also indicating that the N-D bonds are not stationary in the polypeptide. We determine from the reduced quadrupole coupling tensor the mean-square amplitude for the libration and show that it increases linearly with temperature. A simple qualitative theory for the relaxation times is presented on the basis of the assumption that the N-D reorientation is described either as a diffusion process in a square well or as a damped Langevin oscillator with a harmonic restoring force. The conclusion is that the short relaxation times of the polypeptide amide deuterons result from substantial frictional effects on reorientation that increase with temperature.     

Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells

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