Homozygous Control Mice Research Articles

Alterations in growth, haematopoiesis and serum chemistry profiles infitness 1 4226SB mutant mice

We investigated patterns of growth, haematopoiesis and alterations in serum chemistry profiles that were associated with anN-ethyl-N-nitrosourea-induced mutation in thefitness 1 locus in chromosome 7 in mice. Mice hemizygous for thefit 1 mutation [c fit 14226SB/Df(c Mod2 sh1)VT] had growth retardation characterised by decreased body weights, shorter body lengths and tail lengths at 40 and 60 days of age compared to normal homozygous control mice [cch+/cch+]. Haemograms revealed that mice hemizygous for thefit 1226SB mutation that were killed at 40 days had microcytic, hypochromic anaemia that was mildly regenerative in nature. No significant differences were detected in total leucocyte counts, differential leucocyte counts and platelet counts between hemizygous mutant mice and control mice. Haemograms from the surviving mice hemizygous for thefit 14226SB mutation that were killed at 60 days indicated that the haemoglobin concentrations, mean corpuscular volumes and mean corpuscular haemoglobin concentrations were significantly decreased compared to normal controls. The reticulocyte counts and the platelet counts from the mice hemizygous for thefit 14226SB mutation were mildly increased compared to normal controls killed at 60 days. Serum chemistry analyses at 40 days of age indicated that the hemizygousfit 1 mutant mice had hypoproteinaemia characterised by hypoalbuminaemia and hypoglobulinaemia. They also developed hypoglycaemia, mild hyperphosphataemia and moderate elevation in the activity of alkaline phosphatase in serum. Quantitative isozyme analysis indicated that the increase in total serum alkaline phosphatase activity in the hemizygous mutant mice was due to an increase in the bone isozyme. In addition to those alterations in the serum chemistry profile noted above, hemizygous mutant mice killed at 60 days had an extremely mild increase in urea nitrogen concentration and mildly increased activities of alanine aminotransferase and aspartate aminotransferase in serum. From these data, it was concluded that thefit 14226sb mutation m mice causes growth retardation, microcytic, hypochromic anaemia, and alterations in serum chemistry profiles that suggest lesions in one or more organ system(s). The exact mechanism(s) by which thefit 14226SB mutation mediates these lesions remains to be elucidated.

Constitutive (pro)insulin release from pancreas of transgenic mice expressing monomeric insulin.

To evaluate the role of protein aggregation and calcium in the sorting of insulin for regulated vs. constitutive release from the intact pancreas, we targeted the expression of a monomeric mutant form of human (pro)insulin (B9/B27) to the pancreatic beta-cells of transgenic mice. This mutant insulin does not form dimers or hexamers, but can aggregate at high concentration in the presence of calcium. A homozygous line (171) was produced that expressed 55% of the total (pro)insulin message in their beta-cells as the mutant form and had normal pancreatic total (pro)insulin content [measured as immunoreactive insulin (IRI)]. Fasting glucose levels in these transgenics and in homozygous control mice expressing native human (pro)insulin were normal, although levels were abnormally elevated during ip glucose tolerance testing. In the presence of extracellular calcium, regulated IRI release from the isolated perfused pancreas of the transgenic mice was undetectable in the absence of secretagogues and responded with normal phasic kinetics when stimulated with increasing steps of glucose, with glucose plus isobutylmethylxanthine, or with arginine. Without extracellular calcium (0 calcium plus EGTA), normal pancreas did not release IRI in either the presence or absence of secretagogues. In contrast, without calcium or secretagogues, transgenic pancreas spontaneously and constitutively released IRI at high levels equivalent to those elicited by glucose (22 mM) plus calcium from normal pancreas. This release was partially inhibited by glucose or arginine. Constitutive secretion was acutely sensitive to calcium; inhibition occurred within minutes after the addition of calcium and quickly returned to its characteristic level (with overshoot) when calcium was subsequently removed. Somatostatin, at a concentration that caused 50% inhibition of normal glucose-stimulated secretion, did not affect constitutive release. Control pancreas from the transgenic mice, expressing native human (pro)insulin, responded normally to secretagogues and did not constitutively release hormone in the absence of calcium. It is concluded that expression of monomeric human insulin in pancreatic beta-cells from transgenic mice did not interfere with normal phenotypic insulin secretion, indicating that the functional secretory apparatus was not impaired. Constitutive secretion of IRI from the intact pancreas requires both the expression of a monomeric form of insulin and the absence of extracellular calcium, two conditions that reduce aggregation. These results are consistent with the hypothesis that protein aggregation favors sorting to the regulated pathway, whereas suppressed aggregation causes sorting for constitutive release.

The purification and characterization of an inhibitor of protein synthesis from the muscle of dystrophic mice and homozygous control mice

Homogenates of hindlimb muscle were obtained from homozygous (+/+) control mice and dystrophic (dy/dy) mice of the ReJ 129 strain and subjected to purification by ion exchange chromatography, gel filtration, centrifugation on sucrose gradients, and sodium dodecyl sulphate gel electrophoresis. A proteinaceous material inhibitory to protein synthesis that was detected previously in fractions from dystrophic muscle (Petryshyn and Nicholls 1978) was present at reduced levels of activity in muscle fractions from homozygous controls. Although the specific activity after the sucrose gradient step was similar in the 2 groups of animals, the activity per animal was 3 times higher in the dystrophic mice. Evidence is presented to support the view that the inhibitory material is identical in the 2 groups and also that the active protein, of an approximate molecular weight of 120 000, can be denatured by heat and 2-mercaptoethanol to the less active form that was described previously, of an approximate molecular weight of 70 000.