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.     

Involvement of the acyl chain of ceramide in carbohydrate recognition by an anti-glycolipid monoclonal antibody: the case of an anti-melanoma antibody,M2590, to GM3-ganglioside

The effect of the chain length of the fatty acid residue of the ceramide moiety of ganglioside G_M3 on the binding ability of monoclonal antibody M2590, which is specific for the carbohydrate structure of G_M3-ganglioside, was examined by means of a direct binding assay on thin layer chromatography plates (TLC immunostaining) and a quantitative enzyme-linked immunosorbent assay (ELISA). Derivatives of G_M3 with a long fatty acid chain reacted with the M2590 antibody, but those with a short fatty acid chain showed no reaction in either assay system. These results suggested that the acyl fatty acid moiety of the ganglioside played an important role in the formation or maintenance of the antigenic structure of the carbohydrate moiety of the ganglioside.     

Structure of the horseradish peroxidase isozyme C genes

We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.     

A novel IgA protease from Clostridium sp. capable of cleaving IgA1 and IgA2 A2m(1) but not IgA2 A2m(2) allotype paraproteins

Three bacterial strains of Bifidobacterium and Clostridium sp. from patients with inflammatory bowel disease (I.B.D.) and Streptococcus pneumoniae from a patient with pneumonia were identified to produce extracellular proteases cleaving IgA into Fab and Fc fragments. Although the proteases from the Bifidobacterium and the Streptococcus pneumoniae showed the characteristics of typical IgA1 proteases, cleaving the IgA of only the IgA1 subclass, the protease from Clostridium sp. revealed a dual substrate specificity, in that it cleaved both IgA1 and IgA2 of the A2m(1) allotype. The latter protease, however, did not show any activity with respect to the IgA2 of the A2m(2) allotype. Fc fragments isolated from the IgA1 and the IgA2 A2m(1) by digestion with the Clostridium sp. protease were identified to have an identical amino terminal residue of valine. The site of cleavage in both the alpha 1 and the alpha 2 of A2m(1) by the protease was assumed to be an identical peptide bond at Pro(221)-Val(222), which is a common one present just before the hinge of both the alpha 1 and the alpha 2 of the A2m(1) but not of the alpha 2 of the A2m(2). The protease was sensitive to ethylene-diamino tetraacetic acid, a chelating agent, similar to other already reported IgA1 proteases.     

Metabolism of n-3 polyunsaturated fatty acids and modification of phospholipids in cultured rabbit aortic smooth muscle cells

The metabolism of the linolenic acid family (n-3) of fatty acids, e.g., linolenic, eicosapentaenoic, and docosahexaenoic acids, in cultured smooth muscle cells from rabbit aorta was compared to the metabolism of linoleic and arachidonic acids. There was a time-dependent uptake of these fatty acids into cells for 16 hr (arachidonic greater than docosahexaenoic, linoleic, eicosapentaenoic greater than linolenic), and the acids were incorporated mainly into phospholipids and triglycerides. Eicosapentaenoic and arachidonic acids were incorporated more into phosphatidylethanolamine and phosphatidylinositol plus phosphatidylserine and less into phosphatidylcholine than linolenic and linoleic acids. Docosahexaenoic acid was incorporated into phosphatidylethanolamine more than linolenic and linoleic acids and into phosphatidylinositol plus phosphatidylserine less than eicosapentaenoic and arachidonic acids. Added linolenic acid accumulated mainly in phosphatidylcholine and did not decrease the arachidonic acid content of any phospholipid subfraction. Elongation-desaturation metabolites of linoleic acid did not accumulate. Cells treated with eicosapentaenoic acid accumulated both eicosapentaenoic and docosapentaenoic acids mainly in phosphatidylethanolamine and the arachidonic acid content was decreased. Added docosahexaenoic acid accumulated mainly in phosphatidylethanolamine and decreased the content of both arachidonic and oleic acids. The following conclusions are drawn from these results. The three n-3 fatty acids are utilized differently in phospholipids. The arachidonic acid content of phospholipids is reduced by eicosapentaenoic and docosahexaenoic acids, but not by linolenic acid. Smooth muscle cells have little or no desaturase activity, but have significant elongation activity for polyunsaturated fatty acids.     

Clostridium ramosum, an IgA protease-producing species and its ecology in the human intestinal tract

A bacterial strain isolated from feces of a patient with ulcerative colitis, which had been shown to produce a novel immunoglobulin A (IgA) protease (cleaving both the human IgA1 subclass and IgA2 subclass of A2m(1) allotype) extracellularly, was identified as Clostridium ramosum. By using a selective medium (propionate-rifampicin-gentamicin-colimycin-polymyxin medium) devised for C. ramosum, analysis of the population level of this organism was performed to determine its ecology in the human intestinal tract. C. ramosum was isolated in 20 of 25 fecal samples (80%) from patients with inflammatory bowel disease (I.B.D.) and in 112 of 135 samples (83%) from patients without I.B.D. (control group). C. ramosum was also isolated from 6 of 11 biopsy samples (55%) of the inflamed rectal mucosa from patients with ulcerative colitis and from five of 15 samples (33%) from the intact mucosa of the control group. The population levels of C. ramosum in most of the biopsy samples ranged from 2.3 to 5.0 log10 per gram. The IgA protease-positive C. ramosum was found in only four of 135 fecal samples (3%) and one of 15 biopsy samples (6.7%) from the control group. These results indicate that IgA protease-positive C. ramosum is not likely to play a role in the induction of I.B.D., unless the organism is first isolated from the patient with I.B.D.     

Lysosomal acid lipase deficiency in rats: lipid analyses and lipase activities in liver and spleen

We report the biological characterization of an animal model of a genetic lipid storage disease analogous to human Wolman's disease. Affected rats accumulated cholesteryl esters (13.3-fold), free cholesterol (2.8-fold), and triglycerides (5.4-fold) in the liver, as well as cholesteryl esters (2.5-fold) and free cholesterol (1.33-fold) in the spleen. Triglycerides did not accumulate, and the levels actually decreased in the spleen. Analysis of the fatty acid composition of the cholesteryl esters and triglycerides showed high percentages of linoleic acid (18:2) and arachidonic acid (20:4) in both organs, especially in the liver. No accumulation of phospholipids, neutral glycosphingolipids, or gangliosides was found in the affected rats. Acid lipase activity for [14C]triolein, [14C]cholesteryl oleate, and 4-methyl-umbelliferyl oleate was deficient in both the liver and spleen of affected rats. Lipase activity at neutral pH was normal in both liver and spleen. Heterozygous rats showed intermediate utilization of these substrates in both organs at levels between those for affected rats and those for normal controls, although they did not accumulate any lipids. These data suggest that these rats represent an animal counterpart of Wolman's disease in humans.     

Biochemical systematics of Japanese fireflies of the subfamily Luciolinae and their flash communication systems

Japanese fireflies of the subfamily Luciolinae are biochemically analyzed using 13 allozymes, and the phylogenetic relationships obtained from this analysis are compared with their flash communication systems. As a result, the Japanese Luciolinae can be divided into three groups.Hotaria parvula andH. tsushimana together withLuciola yayeyamana andL. kuroiwae from the first group, and they use the same communication system.L. lateralis, Curtos okinawana, andC. costipennis make up the second group, and their communication systems are also the same.L. cruciata makes up the last one, and its communication system is different from the other fireflies of Luciolinae. Therefore, their taxonomical arrangement and communication systems are not congruent. However, the genetic similarity deduced by allozymic analysis of the members of the Japanese Luciolinae is highly consistent with their flash communication systems.     

Isolation of the Photoactive Reaction Center Complex that Contains Three Types of Fe-S Centers and a Cytochrome c Subunit from the Green Sulfur Bacterium Chlorobium limicola f. thiosulfatophilum, Strain Larsen

The photoactive reaction center (RC) complex from the greensulfur bacterium Chlorobium limicola f. thiosulfatophilum, strainLarsen, was isolated after solubilization and ammonium sulfatefractionation followed by ion-exchange chromatography. The spectrumof the complex was almost identical with that of the similarRC complex isolated by Feiler et al. [(1992) Biochemistry 31:2608–2614] except for the presence of cytochrome c₅₅₁instead of c₅₅₃ in the latter study. A molecular ratio of BChla to P840 of the isolated RC complex was assayed to be 25–35.SDSPAGE analysis revealed that the isolated complex containedthree major polypeptides with apparent molecular masses of 68,41 and 21 kDa, respectively. The 21-kDa polypeptide was identifiedto be a heme-binding protein by staining the gel for peroxidaseactivity. The cytochrome c₅₅₁ was oxidized by flash light ina biphasic manner with half times of 90 and 390 µs, respectively,that coincided with the reduction half times of P840⁺. Threedistinct iron-sulfur centers assigned to F_A, F_B and F_x, respectively,from their g-values were detected by EPR spectroscopy at cryogenictemperature. These results suggest that the present preparationcontains a minimal functional unit of the RC of this bacterium,and that this complex appears to lie on a evolutionary linebetween RC's of purple bacteria and photosystem I. (Received August 18, 1992; Accepted October 28, 1992)     

On-line monitoring of cell concentration of Perilla frutescens in a bioreactor

This article demonstrates the successful in situ real-time monitoring of the cell concentration of Perilla frutescens in a bioreactor by using a laser turbidimeter. It was found that turbidity measurements at 780 nm with the laser sensor were hardly affected by the red color of the anthocyanin produced by P. frutescens cells, nor by the aeration rate or agitation speed within the ranges investigated. There was an excellent linear relationship, with a correlation coefficient (r(2)) higher than 0.99, between the sensor's response and the cell concentration. The whole growth stage of the cells, i.e., lag, logarithmic, and stationary phases, in bioreactor cultivations, could be satisfactorily estimated on-line by means of the in situ turbidimeter. However, during the declining phase of the cells, an apparent deviation was observed between the on-line estimations and off-line measurements of cell concentrations by dry cell weight, while the wet cell weight could be estimated by the same turbidimeter system. We found that this deviation was caused by a decrease in the cell density due to an increase of the individual cell volume and a decrease of the cell dry weight during the declining phase. (c) 1993 John Wiley & Sons, Inc.