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.     

Mechanism of inhibition caused by long-chain fatty acids in anaerobic digestion process

The inhibitory effect of long-chain fatty acids on the anaerobic digestion process was examined in batch experiments using synthetic substrates. The addition of long-chain fatty acids caused the appearance of the appearance of the lag period in the methane production from acetate and in the degradation of both long-chain fatty acids and n-butyrate. Methane production from hydrogen proceeded without lag period although its rate was lowered. Fermentation of glucose was not inhibited. Neutral fat in the whole milk was easily hydrolyzed to long-chain fatty acids, which brought about the inhibition. The addition of calcium chloride reduced the inhibitory effect of long-chain fatty but it did not do so after the culture had been exposed to long-chain fatty acids for more than several hours. The addition of calcium carbonate could not reduce the inhibition because of its insolubility.     

Significance of exercise and bed rest in pregnancy--study on the lying postures of gravidas during sleep (2)

For the purpose of determining the most reasonable lying posture for pregnant women, we investigated the lying positions of both 247 non-pregnant women and 302 pregnant women during sleep. As for the rate of each position during the entire period of observation, 33.2% of the non-pregnant women were in the supine position, 41.2% in the lateral position, 18.4% in Sims' position and 7.1% in the prone position. In the pregnant group, the rate of supine position, simple lateral position and Sims' position was 34.2%, 52.2% and 12.7%, respectively, but the rate of the prone position was limited to 0.8%. All of the gravidas assuming the prone position were at less than 16 weeks of gestation. Non-pregnant women could sleep in a variety of positions, but pregnant women could assume the prone position during sleep only in the stage when the abdomen is not yet prominent or distended. Pregnant women were restricted significantly, either consciously or unconsciously, with progress in gestation.     

Mechanisms of modulation of neuronal nicotinic receptors by substance P and OAG

Substance P is known to modulate neuronal nicotinicacetylcholine receptors (nAChRs) in the sympathetic nervous system.There are two conflicting proposals for the mechanism of this effect, an indirect action mediated by protein kinase C (PKC) and a direct interaction with receptor subunits. We studied the mechanisms of thiseffect in PC-12 cells. Substance P enhanced the decay of thenicotine-induced whole cell current. This effect was fast in its onsetand was not antagonized by guanosine5'-O-(2-thiodiphosphate), a G protein blocker, orstaurosporine, a nonselective PKC blocker. Staurosporine failed toreverse the inhibition by 1-oleoyl-2-acetyl-sn-glycerol (OAG), a synthetic diacylglycerol analog known to activate PKC. Theinhibitory effects of the peptide and OAG were preserved in excisedpatches, but substance P applied to the extra patch membrane wasineffective in the cell-attached patch configuration. We conclude thatsubstance P modulates neuronal nAChRs most likely by direct interactions with the receptors but independently from activation ofPKC or G proteins and that PKC does not participate in modulation by OAG.

     

Immunohistochemistry and immunocytochemistry of atrial natriuretic polypeptide in porcine heart

Summary Specific granules in porcine hearts were observed in atrial cardiocytes, Purkinje fibers, and transitional cells of the ventricle. These granule-containing cells were immunohistochemically stained by applying the avidin-biotin-peroxidase complex method using an antiserum against -human atrial natriuretic polypeptide (ANP). Immunoelectron microscopy of sections stained using the immunogold method indicated that these specific granules are storage sites of ANP. Furthermore, an impulse-conducting system consisting of immunoreactive cells was clearly distinguishable from nonimmunoreactive ventricular cardiocytes. We conclude that specific-granule-containing cells, i.e., ANP-producing cells, are located in both the atrial walls and the ventricular impulse-conducting system. The presence of ANP may be correlated with impulse conduction.     

Mutational analysis of structure--activity relationships in human tumor necrosis factor-alpha

To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.