Normal Littermate Controls Research Articles

Molecular cloning and primary structure of myelin-associated glycoprotein.

Myelin-associated glycoprotein (MAG) may play a role in the cellular interactions leading to myelination. Using monoclonal antibodies and conventional antisera against MAG, we have isolated a cDNA clone from an expression library prepared from rat brain mRNA. The identity of the clone was confirmed by the exact match between its nucleotide sequence and two peptide sequences of 13 and 9 amino acids that we obtained by Edman degradation of two CNBr fragments of MAG. The cDNA clone hybridized to two size species of mRNA in rat approximately 3.5 kilobases in length. These mRNAs were present in brain but not liver and were expressed most abundantly at the time of active myelination (day 14). The mRNA for MAG was present at barely detectable levels in hypomyelinating jimpy mice compared to normal littermate controls. Therefore the MAG cDNA clone is both brain and myelin specific. DNA sequence analysis revealed that our MAG cDNA was derived from the same mRNA as clone p1B236, a randomly selected, brain-specific, partial cDNA isolated by Sutcliffe et al. [Sutcliffe, J. G., Milner, R. J., Shinnick, T. M. & Bloom, F. E. (1983) Cell 33, 671-682]. Analysis of the predicted protein sequence suggests that MAG has a long extracellular domain (499 amino acids), followed by a short transmembrane segment (20 amino acids) and an intracellular carboxyl-terminal domain (90 amino acids). The molecule has several glycosylation sites, three internal repeats homologous to a repeat in the neural cell adhesion molecule (N-CAM), and sites for phosphorylation near the carboxyl terminus. The primary structure reported here provides a molecular framework for further investigations into the function of the MAG molecule.

Vascular atrophy in the retinal degenerative rd mouse

We have observed that the lectin Ricinus communis agglutinin I (RCA), which binds to terminal galactose moieties, serves as a marker for vasculature in the mouse retina. The binding of fluorescein-isothiocyanate-conjugated RCA was used to study the development of retinal vasculature in normal mice and in rd (retinal degeneration) mutant mice, which exhibit a massive loss of photoreceptor cells during the first month of life. In the normal mouse, retinal capillaries develop in an ordered manner and are concentrated in three major zones between the inner limiting membrane and the outer plexiform layer. In the rd mouse, the vessels appear to form normally but begin to degenerate by the end of the second postnatal week. By the end of the fourth postnatal week there is approximately a 35% reduction in the total number of vascular profiles in the rd retina compared to normal littermate controls. This reduction in vascularity is temporally associated with the photoreceptor degeneration.

Acquired immobility response in weaver mutant mice

Weaver mutant mice, contrary to normal littermate controls, did not acquire the immobility response in a forced swimming task. In regard to swimming performance, the weaver mutants swam with less ability but with more vigor than normal mice. Results are discussed in terms of the role of the cerebellum in swimming behavior.

Pulmonary hypoplasia in chondrodystrophic mice.

Lungs of day-18 fetal mice with hereditary chondrodysplasia (cho) were examined histologically and biochemically for pulmonary hypoplasia. Compared with normal littermate controls, the mutant's lungs were smaller by 37 (wet weight) and 22% (dry weight). Total DNA and protein per whole lung were decreased by 13 and 19%, respectively. The significantly smaller-than-normal terminal sacs observed in histological sections of the mutant's lungs corresponded with the greater difference (37%) in lung wet weight. The developmental mechanism for this disorder was further explored by examining the volumes of thoracic cavity and amniotic fluid. The volume of the thoracic cavity of newborn mutants was less than half that of controls, suggesting that the pathogenetic mechanism for the hypoplastic lungs in chondrodysplastic mice includes thoracic dystrophy. Measurement of the amniotic fluid volume revealed polyhydramnios in the mutant, thereby ruling out oligohydramnios as a mechanism. The relevance of this study to human pulmonary hypoplasia in short-limb chondrodystrophy is discussed.

Newborn rat retinal cells transplanted into a retinal lesion site in adult host eyes

We report the successful grafting of embryonic (newborn) rat retina into a lesion site (die-back zone) of an adult retina with a corresponding 90–100% survival rate. A penetrating lesion was made through the sclera, choroid and retina on the superior surface of the host eye and closed with microsutures. The lesion site was either allowed to stabilize for 5 weeks or immediately received a retinal graft. Retinas were removed from 1-day-old neonate donors, drawn through a small gauge needle and injected into the fresh or stabilized lesion site. Host animals were sacrificed and the eyes processed for light, scanning- and transmission-electron microscopic analysis at 1, 2, and 4 weeks after grafting. Analysis of sections through grafted tissue within the lesion site revealed that the neonatal cells not only survived at all times examined but also continued their development reminiscent of normal littermate controls. Examination of 4-week grafts revealed a laminar pattern similar to adult ganglion cell, inner plexiform, inner nuclear, and outer plexiform layers as well as developing photoreceptor neurons. The grafted tissue could easily be delivered into the retinal lesion site where it established a pattern of retinal layers within the die-back zone. In addition, the plexiform areas of the graft appeared to integrate with those of the host. The age of the adult retina lesion transplantation site had little effect on the graft/host integrative phenomenon. These studies show for the first time a method utilizing immature retinal grafts to fill and/or bridge the wound area of the lesioned adult mammalian retina. These observations also demonstrate the utility of using this model for the study of numerous retinal developmental phenomena.

The effect of hemopoietic microenvironment on splenic suppressor macrophages in congenitally anemic mice of genotype Sl/Sld.

Mechanisms underlying mononuclear phagocyte specialization are being probed by studying suppressor macrophages (M phi) as a reference population in mouse models with impaired blood monocyte formation. Splenic suppressor M phi, defined by PGE-mediated inhibition of Con A-induced T lymphocyte proliferation are induced by the i.p. administration of Corynebacterium parvum (CP). Mice severely depleted of bone marrow and blood monocytes by treatment with 89Sr fail to show this suppressor M phi response to CP, although M phi-forming stem cells, assessed as splenic M-CFC in vitro, are increased 20-fold. These observations suggest that radiosensitive bone marrow stem cells are necessary for the generation of both suppressor M phi and monocytes and that one such stem cell may be common to both types of mononuclear phagocytes. This notion was explored further by employing congenitally anemic mice of the genotype S1/S1d in which the hemopoietic microenvironment is genetically defective and thus unable to support the proliferation, differentiation, and function of stem cells. The congenital defect was found to be additionally expressed in the S1/S1d mouse by a monocytopenia of less than 10% of the values in normal congenic littermate controls and by the failure of splenic M-CFC to increase in response to CP. PGE-producing suppressor M phi expressing Fc gamma 2b receptors, however, were induced by CP in S1/S1d mice with no significant diminution of suppressor activity. These data establish the fact that significant impairment of the formation of monocytes is part of the overall hemopoietic defect in S1/S1d mice. PGE-producing suppressor M phi, however, were inducible at normal functional levels in the presence of a profound monocytopenia, and therefore appear to be independent of the mechanisms that regulate blood monocyte formation. Ablation of the bone marrow with 89Sr resulted in failure of CP to induce suppressor M phi in the spleens of the S1/S1d mice as in the littermate controls. Other observations in the present study, when taken with data from the 89Sr model, show the additional independence of these suppressor M phi from splenic M-CFC. In aggregate, these findings delineate three functionally definable populations of mononuclear phagocytes that appear to be independently regulated.

Exploration of a hole-board matrix in nervous mutant mice.

Nervous mutant mice and normal littermate controls explored a 4 X 4 hole matrix for 3 consecutive days. No difference was found between the groups for the following measures: total number of hole visits, visits to the center holes, and visits to contiguous holes or different holes. However, nervous mutants persevered more in visiting the same hole than controls. These results are discussed in terms of a possible role for the cerebellum in spatial working memory, similar to that suggested for the hippocampus.

Exploration and habituation in nervous mutant mice

Nervous mutant mice have selective damage to deep cerebellar nuclei and Purkinje cells. Compared to normal littermate controls, nervous mutant mice did not show habituation of a hole poke response to a large hole. The nervous mutants showed both signs of increased contact with a novel stimulus (number of rearing responses) and decreased exploration (number of hole pokes in small holes). Results are discussed in terms of a similarity between the behavioral effects of cerebellar damage and hippocampal damage.

Alterations in cerebellar glutamic acid decarboxylase (GAD) activity in a genetic model of torsion dystonia (rat)

Glutamic acid decarboxylase (GAD) activity was studied in specific brain regions of a newly identified genetic (rat) model of human torsion dystonia. GAD activity was found to be significantly increased in the deep cerebellar nuclei of dystonic rats at 16, 20, and 24 days of age. GAD activity in the other regions examined (vermis, cerebellar hemispheres, caudate nucleus, and globus pallidus) did not differ from that of age-matched normal littermate controls. Diazepam treatment significantly reduced the frequency of dystonic movements in the mutant.

Rescue of anti-influenza A virus cytotoxic T-lymphocyte responses in chemotherapy-suppressed mice.

The administration of cyclophosphamide (50 to 100 mg/kg) at 48 to 72 h before removal of murine lung or spleen mononuclear cells for culture rendered DBA/2 mice incapable of generating an effective cytotoxic T-lymphocyte response to influenza A virus-infected cells. The cytotoxic T-lymphocyte precursor frequency to influenza A virus in lung and spleen cells from cyclophosphamide-treated mice was significantly decreased when compared with that of normal littermate controls. The low cytotoxic T-lymphocyte activity in the lungs and spleens of cyclophosphamide-treated mice could be partially restored in vitro by human interleukin 2.

Effect of neonatal hypothyroidism on the serotonin system of the rat brain

The effects of neonatal thyroidectomy and thyroid hormone replacement therapy on the development of serotonin-contaning neurons in discrete rat brain nuclei were studied. Newborn male rats were rendered hypothyroid by the injection of 125 μCi 131I, and, after 45 days, were compared with normal littermate controls and 131I-injected animal subsequently maintainde by dialy T 4 injections. The serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) contents of discrete brain nuclei remove by punches of frozen brain slices were measured by HPLC with electrochemical detection. 5-HT and 5-HIAA contents were significantly increased in many nuclei of the hypothyroid rat brain. By blocking the biosynthesis ot 5-HT with p-chlorophynelalanine we found that the activity of tryptophan hydroxylase is an important step in the stimulatory effect of hypothyroidism on the 5HT and 5-HIAA contents. Futhermore, we demonstrated after blockage of monomine oxidase activity with pargyline, a less pronounced decline of 5-HIAA in neonatal hypothyroid animals, thus causing a relative accumulation of this metabolite. These results demonstrate theat there are important modifications of the 5-HT system in the brain of neonatal hypothyroid rats. This may have an important role in the development of hypothyroid-induced impairments of central nervous system function.

Altered brain copper and zinc content in quaking mice

Brain copper and zinc levels were determined in 21-day-old and “adult” C 3 HeB FeJ quaking mice and in normal littermate controls. Expressed per gram dry weight of brain, copper was increased 84% over normal mice at 21 days after birth, but was not significantly different from normal in the adults. Zinc was increased 23 to 24% at both ages. At both ages, brains from quaking mice had a significantly reduced content of solids, indicating increased water content in the mutant brain. Our study is the first to report copper and zinc content as a measure of both wet and dry brain weights. Our results indicate abnormal copper content in the quaking mutant. The relationship between copper content and other aspects of the quaking phenotype, including its seizure behavior and myelin deficit, remain to be established.

Immunoreactive neurotensin in the pancreas of genetically obese and diabetic mice. A longitudinal study.

Immunoreactive neurotensin (IR-NT) content in 2 N acetic acid extracts of pancreas was measured in genetically diabetic (C57BL/KsJ db/db and ob/ob) and obese (C57BL/6J ob/ob and db/db) mice and normal littermate controls from 5 to 24 wk of age to determine the relationship of any changes to the development of metabolic abnormalities. Pancreatic IR-NT in obese mice showed no consistent change compared with lean littermate controls. In contrast, diabetic mice demonstrated an increase in pancreatic IR-NT that occurred at 6-8 wk of age, and maximal about the time of islet B-cell failure (8-10 wk), and persisted over the study period. Pancreatic IR-NT eluted in two peaks on reverse phase high-pressure liquid chromatography, one of which exhibited a retention time similar to that of synthetic NT. These findings suggest that pancreatic IR-NT concentration is regulated by insulin, with elevated levels occurring in association with insulin deficiency and its metabolic consequences but not with insulin resistance. Taken together with the previous demonstration that NT influences pancreatic islet hormone secretion, the present findings support a possible role of endogenous NT in islet hormone regulation.

Evidence for T cell-dependent immunity to Bacteroides fragilis in an intraabdominal abscess model.

It has been shown that active immunization of rats with the capsular polysaccharide of Bacteroides fragilis protects these animals against abscess development following intraperitoneal challenge with this species. Passive transfer of hyperimmune globulin from immunized animals to nonimmune recipients provided protection against B. fragilis bacteremia in challenged animals, but did not confer protection against abscess development. On the other hand, adoptive transfer of spleen cells from immunized animals to nonimmunized recipients resulted in protection against abscesses following challenge with B. fragilis. These data suggested that a T cell-dependent immune response was involved in protection against abscess development after immunization with B. fragilis capsular antigen. To determine the possible role of cell-mediated immunity prompted by the capsular antigen, inbred congenitally athymic OLA/Rnu rats and their phenotypically normal littermates were actively immunized. Despite the development of high titers of anti-B. fragilis capsular antibody, 100% of actively immunized athymic rats developed abscesses, as did 100% of unimmunized athymic control rats. However, no phenotypically normal littermate control rats that were actively immunized developed abscesses, while 100% of phenotypically normal unimmunized rats developed abscesses. Additional studies showed that adoptive transfer of T cell-enriched spleen cell preparations from Wistar/Lewis rats immunized with the capsular polysaccharide to nonimmune recipients also resulted in protection against B. fragilis-induced abscesses. We conclude that the protection afforded by immunization with B. fragilis capsule against intraabdominal abscesses caused by that organism is T cell-mediated and does not require the presence of serum antibody.

Brain-specific ceramide synthesis activity: change during brain maturation and in jimpy mouse brain

The synthesis of ceramide from lignoceric acid was studied in normally developing rat brain and jimpy mouse brain. The developmental pattern of the synthesis of both non-hydroxy and hydroxyceramide from free lignoceric acid in a brain-specific pathway requiring NADPH and cytosolic factors was similar to the synthesis from lignoceroyl CoA and both showed increasing activity during myelination. There were no differences in non-hydroxyceramide synthesis between jimpy and normal littermate control mouse brains by both pathways. HPLC analysis showed the same non-hydroxyceramide level in both types of brains.

Rejection of the intestinal parasite Nippostrongylus brasiliensis by mast cell-deficient W/Wv anemic mice.

The ability of W/Wv anemic mice to accumulate mucosal mast cells and to reject the intestinal parasite Nippostrongylus brasiliensis was examined. W/Wv mice did not accumulate mucosal mast cells in response to infections with N. brasiliensis. They eliminated a primary infection more slowly than did their normal littermate controls but were as refractory as controls to second and third infections. W/Wv mice had higher serum titers of worm-specific immunoglobulin E than did controls. These results indicate that mucosal mast cells are not an absolute requirement for N. brasiliensis rejection.

Effect of neonatal thyroid deficiency on the catecholamine, substance P, and thyrotropin-releasing hormone contents of discrete rat brain nuclei.

The effects of neonatal thyroidectomy and thyroid hormone replacement therapy on the development of catecholamine-, TRH-, and substance P-containing neurons in discrete rat brain nuclei were studied. Newborn male rats were rendered hypothyroid by the injection of 125 muCi 131I and, after 45 days, were compared with normal littermate controls and 131I-injected animals subsequently maintained on T4 injections. The peptide or catecholamine content of discrete brain nuclei removed by punches of frozen brain slices was measured by RIA or radioenzymatic assay, respectively. The success of the thyroidectomy was verified by criteria of weight, length, plasma T4, and pituitary GH content. Animals receiving T4 replacement therapy were indistinguishable from normal littermates. Substance P was measured in 32 different brain nuclei and was significantly increased in 19 of these areas in hypothyroid animals. No changes in norepinephrine were detected, and the dopamine content of all but 3 brain nuclei was increased by thyroidectomy. The TRH concentration was drastically reduced in the median eminence of hypothyroid animals and also changed in 3 other extrahypothalamic areas. All of the changes seen in catecholamine, TRH, and substance P distribution in hypothyroid animals were completely reversed by T4 replacement therapy. These results demonstrate changes in brain peptide neurotransmitters during the hypothyroid state and open new vistas for comprehension of biochemical mechanisms underlying central nervous system malfunction.

Sex-limited effects of the expression of the db gene in mice during puberty.

The autosomal recessive gene diabetes (db) produces a condition similar to human insulin-dependent diabetes mellitus in certain strains of inbred mice. In this investigation, the effects of expression of the db gene on the development of the submandibular glands, electrophoretic protein patterns in salivas, fasting blood glucose levels, and glycosylated hemoglobin levels were evaluated in mice undergoing puberty. Three sex-limited effects of the db gene were observed in diabetic male mice: (1) a compromise of the development of the specialized submandibular glands with the extensive tubular portion normally found in males. (2) failure to develop a salivary protein pattern unique to male mice, and (3) attainment of higher levels of fasting blood glucose than found in female diabetic mice. Since it has been documented that homozygous mice fail to develop functional gonads, apparently due to insufficient production of gonadotropin, it is likely that the compromised development of the specialized submandibular glands, and, consequently, the male salivary protein pattern, is a result of decreased testosterone production. Experiments in which diabetic mice were treated with testosterone support that conclusion, since testosterone caused transformation of the salivary protein pattern to one identical with that of normal male littermate controls and increased the tubular portion of the submandibular glands.

The responses of hemopoietic precursor cells in mice to bacterial cell-wall components.

The influence upon different cellular and humoral parameters of hemopoiesis of three structurally unrelated, highly purified bacterial cell-wall components (BCWC) was investigated. The spleens of C57BL/6 mice assayed 6 days after the injection of either lipid A or outer-membrane lipoportein, but not murein, showed a marked increase in granulocyte-macrophage, eosinophil, and megakaryocyte progenitor cell levels. The number of pluripotent hemopoietic stem cells (CFU-S) also increased in the spleens of mice treated with either lipid A or lipoprotein. Similar results were obtained following the injection of lipoprotein or lipid A into CBA or C57BL/6.nu mice. Genetically anemic Wf/Wf mice were found to have spontaneously elevated numbers of splenic progenitor cells, which increased further after the injection of lipid A. The proportions of the different splenic progenitor cell types were similar in both untreated and lipid A treated Wf/Wf mice, and in normal littermate controls. When tested in vitro, unfractionated or partially purified post-lipid A serum was found to stimulate the growth of granulocyte-macrophage progenitor cells (GM-CFC), but no detectable stimulation of eosinohphil, megakryocyte, or erythroid progenitor cells was observed. The data suggest that the rise in splenic levels of the different progenitor cells is not mediated by the corresponding types of CSF, but more likely by proliferation and differentiation of CFU-S.

Changes in polyploidization of exocrine pancreas in db/db diabetic and normal mice.

Polyploidy in tele-insular and peri-insular exocrine pancreas of C57BL/KsJ-db/db diabetic mice and normal littermate controls was quantitated, because the diseased state has been shown to produce an increase in the percentage of polyploid islet B-cells. Three diabetic and 3 normal animals were sacrificed at 4.5, 7, 9.5, 12, 14.5, and 17 weeks of age. Semi-automatic nuclear size analysis on Feulgen stained sections separated the nuclei into polyploid classes, with the following results: 1) In both normal and diabetic mice, percentages of polyploid cells in peri-insular and tele-insular regions differed significantly from one another, and from that of the islet B-cells. Per-cent polyploidy was highest in the peri-insular regions, and lowest in the islets. 2) In the tele-insular regions, per cent polyploidy did not differ between normals and diabetics at 4.5 weeks. By 17 weeks, polyploidy in the diabetics was essentially unchanged, while that of the normal animals had risen and become significantly elevated over that of both the 4.5 week normal and the 17 week diabetic animals. 3) In peri-insular regions, per cent polyploidy did not differ significantly between normals and diabetics throughout the period studied.