A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

Lack of association and linkage between HLA and familial polyposis coli

Summary We investigated possible association of and linkage between HLA and familial polyposis coli (FPC). In 182 individuals from 66 pedigrees of FPC and 108 individuals from a normal population, HLA-A,-B, and-C antigens were determined. When the frequencies of HLA antigens in 66 unrelated patients and in normal controls were compared, no association of FPC with HLA was observed. For the linkage analysis, HLA haplotypes of 17 affected sib pairs were investigated by the affected sib pair method. The number of pairs which shared two, one, and no haplotypes identical by descent was not significantly different from the number expected with random occurrence (P>0.95). Finally, seven families were analyzed using Morton's sequential test. A maximum lod score of-0.056 at a recombination fraction of 0.4, and a lod of-3.089 at a recombination fraction of 0.05 were obtained. Therefore, there is neither an association of nor linkage between FPC and HLA.     

Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was >70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)₂, 88; (Gly)₃, 82; (Gly)₄, 70; Gly-Gly-NH₂, 44, Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.     

Fate of sperm tail components after incorporation into the hamster egg

The changes in the cytoplasmic organelles of sperm tail in golden hamster eggs fertilized in vivo were observed by electron microscopy. Eggs were obtained from oviducts of hamsters that had been superovulated and inseminated by injection of cauda epididymal spermatozoa into the uteri. In the egg cytoplasm 10 hours after insemination, some of the mitochondria of the spermatozoon midpiece had begun to swell, and a number of multivesicular bodies were observed surrounding the midpiece. The fibrous sheath of the principal piece quickly disappeared prior to the first cleavage, whereas the axoneme and outer dense fibers were unaltered. During the two-cell stage, numerous multivesicular bodies gathered around the midpiece and fused with the mitochondria. The heterophagic vacuoles thus formed then gradually separated from the axial fibers. The outer dense fibers were disarranged and partially torn into small segments; then they seemed to dissociate into substructural granular components. The axonemal microtubules had begun to swell but were still present in the two blastomeres. It is indicated from these observations that at least the mitochondria of the tail constituents carried into the oocyte are digested into small molecular elements by the multivesicular bodies and are possibly distributed as nutrients for the blastomeres during the early stage of development.     

A kinetic description of antifreeze glycoprotein activity

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.     

Altered metabolism of bile acids in cholestasis: Determination of 1β- and 6α-hydroxylated metabolites

Trihydroxy and tetrahydroxy bile acid metabolites substituted at the C-1 or C-6 position were studied using the urine, serum and liver tissue from sixteen patients with cholestatic liver diseases. Following extraction, isolation and hydrolysis, bile acids were converted into the dimethylethylsilyl derivatives and assayed by capillary gas chromatography—mass spectrometry. Five 1β-hydroxylated bile acids, viz. 1β,3α,12α-trihydroxy-, 1β,3α,7β-trihydroxy-1, 1β,3α,7α,12α-tetrahydroxy-5β-cholanoic acids and an epimer of the first compound, and two 6α-hydroxylated bile acids, viz. 3α,6α,7α-trihydroxy-, 3α,6α,7α,12α-tetrahydroxy-5β-cholanoic acids, were completely or partially identified. Large amounts of 1β-hydroxylated and 6α-hydroxylated bile acids were found in the urine, whereas only trace amounts were detected in the serum and liver tissue. These findings indicate that altered metabolism, such as 1β- or 6α-hydroxylation of bile acids, is enhanced in cholestasis, and that the resulting hydroxylated metabolites are eliminated in the urine.     

Determination of tryptophan as the reduced derivative by acid hydrolysis and chromatography

A new procedure for the analyses of tryptophan and the total amino acid composition of proteins was based on the observations that pyridine borane reduces tryptophan in trifluoroacetic acid, while other amino acids remain intact [M. Kurata, Y. Kikugawa, T. Kuwae, I. Koyama, and T. Takagi (1980) Chem. Pharm. Bull. 28, 2274-2275; W.S.D. Wong, D.T. Osuga, and R.E. Feeney (1984) Anal. Biochem. 139, 58-67]. Concentrated HCl was used instead of trifluoroacetic acid for analytical purposes. The products were stable to hydrolysis in 6 N HCl, and the reduction did not interfere with hydrolysis and subsequent analyses. Quantitative recovery was achieved with most proteins when they were subjected to acid reduction in ice-cooled concentrated HCl with two incremental additions of pyridine borane. The reaction was terminated after 10 min by dilution with an equal volume of H2O, vacuum sealing, and hydrolyzing at 110 degrees C for 22 h. The yields of the expected values for cytochrome c, catalase, bovine serum albumin, subtilisin BPN', trypsin, chymotrypsin, beta-lactoglobulin, lysozyme, and pepsin were obtained. Ovotransferrin and ovalbumin, however, yielded values for tryptophan lower than literature values. With two different ion-exchange methods, the recoveries of all other amino acids were comparable to those obtained by acid hydrolysis with 6 N HCl. Since the same hydrolysate can be analyzed for both tryptophan and all the other amino acids, the procedure is a more convenient method than those requiring separate determinations. Initial results indicate that the method may be applied to high-performance liquid chromatographic procedures with adaptations of the protocols if necessary.