Primary structure of murine red blood cell-type pyruvate kinase (PK) and molecular characterization of PK deficiency identified in the CBA strain.

To clarify the molecular abnormality of pyruvate kinase (PK) deficiency identified in the mutant mice of CBA-Pk-1slc/Pk-1slc, we cloned murine red blood cell-type PK (R-PK) cDNA of those animals. The cDNA sequence spans 1827 bp, including an open reading frame that can encode 574 amino acids. Homology in the coding sequences between murine and human R-PK was 86.1% at nucleotide and 91.5% at amino acid levels. A homozygous missense mutation at nucleotide 1013 GGT-->GAT was identified in the cDNA sequence of the mutant, causing a single amino acid substitution at no. 338Gly-->Asp of the murine R-PK. Six amino acid residues, 335Val-336Ala-337Arg-338Gly-339Asp-340L eu, were encoded in exon 8 of both human and rat L (liver-type)/R-PK genes and were evolutionarily conserved in PK from bacteria through humans. 337Arg was reported to be important for substrate binding, suggesting that the amino acid change would impair substrate affinity of the PK subunit. A homozygous missense mutation at the catalytic domain has been identified in a human PK variant, PK Hong Kong (941ATT-->ACT, 314 Ile-->Thr). Although both 1013A and 941C gave rise to an amino acid change adjacent to the active site and may interfere with substrate binding to the subunit, the degree of anemia was much more severe in the human case. The erythroid-progenitor cell number increased in the spleen of Pk-1slc/Pk-1slc mice to a level approximately 66 times higher than that in normal CBA mice, suggesting that compensatory extramedullary erythropoiesis in the spleen of the mutant mice, but not in the human variant, might account for the observed difference in the phenotype.

[The normalizing effect of dalargin on the glucocorticoid and opioid levels of the blood in CBA and C57Bl/6 mice undergoing footshock stress]. / Normalizuiushchee vliianie dalargina na uroven' gliukokortikoidov i opioidov v krovi myshei linii CBA i C57Bl/6 v usloviiakh footshock-stressa.

Radioreceptor investigations showed that it is impossible to interpret changes in the activity of opioid receptor ligands of mu and delta types in plasma of mice under footshock stress (FSS) as activation or depletion of opioid system (OS). There occurred qualitative changes characterized by interstrain differences (ID). These are also typical for dynamics of development of FSS effects on blood corticosterone levels. Administration of dalargin diminishes qualitative and quantitative changes in OS function at different periods after FSS preventing early rise of corticosterone levels after the exposure. In spite of common features in dalargin action on opioid and steroid metabolism in mice of both strains, ID are also present.

Flow-cytometric enumeration of micronucleated polychromatic erythrocytes in mouse peripheral blood.

A flow-cytometric assay is described that can be used to determine the frequency and the DNA content of micronucleated polychromatic (PCE) and normochromatic (NCE) erythrocytes in mouse peripheral blood. Thiazole orange was used for discrimination between PCEs and NCEs, while Hoechst 33342 was used to detect micronucleated PCEs and NCEs. Up to 70,000 polychromatic erythrocytes can be analyzed in less than 10 min. This corresponds to 150-3,000 micronucleated polychromatic erythrocytes, 90-95% of which are true events as determined with a fluorescence microscope after sorting. Using X-rays as the inducing agent in dose-response experiments, a significant increase can be registered at doses of 0.02 Gy. It seems possible that the method will also allow the detection of clastogenic effects of other inducing agents at lower doses than previously possible.

The so-called interleukin-2 inhibitory activity of human serum is largely cytotoxic to mouse cells.

No specific interleukin-2 (IL-2) inhibitor has ever been demonstrated in human, mouse, or any other animal serum. Native mouse serum contains activities which completely inhibit IL-2-dependent and IL-2-independent in vitro proliferation of cells of different animal species by a non-cytotoxic mechanism. The decisive inhibitory component of mouse serum has a molecular weight of about 80,000, is heat-labile and has not been found in other animal sera. Also, native human serum completely suppresses the proliferation of various mouse cell types, predominantly by a cytotoxic effect caused by natural IgM antibodies and complement. Heat-inactivated human serum is no longer cytotoxic to mouse cells, and inhibits the proliferation of mouse cells much less than native serum. There is thus no evidence for a specific IL-2 inhibitor in mouse, human or other serum.

The activity of alkaline phosphatase in the sera of DBA/2NCrj and CBA/BrA mice with calcified tongue lesions.

Spontaneously occurring calcified lesions were found in the tongues of DBA/2NCrj and CBA/BrA mice. In the DBA/2NCrj strain, the frequency of the lesion was 80% (males) and 88% (females). The youngest age of a mouse with this lesion was 18 days after birth, and 3-4 lesions were found in the tongue of 6- to 8-week-old mice. In CBA/BrA mice, 20% of females and 48% of males had the lesions. No significant differences were found in the serum calcium concentrations in high and low lesion-developing strains, but the alkaline phosphatase activities in the high-developing DBA/2NCrj, DBA/LiA, and CBA/BrA strains were higher than in strains with no calcified lesions.

Experimental influenza infection: influence of stress.

The effect of immobilization stress on the course of various forms of influenza infection has been investigated. Influenza was produced in 10-14-week-old inbred mice by intranasal infection with pathogenic influenza virus strain A/PR/8/34 (H1N1) at different doses. Immobilization for 6 hr resulted in the appearance of virus-inhibiting activity in the serum of mice. This activity suppressed the reproduction of test-virus in tissue culture, it was resistant to acid pH 2.0 treatment and to heating at 56 degrees C. However, the high level of virus-inhibiting activity failed to protect the animals from subsequent development of lethal influenza infection. Immobilization stress caused a transient depression of virus induced interferon (IFN) production, as revealed by the use of virus inducer at early intervals after stress. Contemporarily, the stress could aggravate the course of virus infection promoting its transition from non-lethal form into a lethal one and virus penetration into brain.

[Damage to the hematopoietic system of BALB/c and CBA mice undergoing long-term repeated irradiation]. / Povrezhdenie krovetvornoi sistemy myshei linii BALB/c i CBA pri dlitel'nom povtornom obluchenii.

A higher death rate of BALB/c as compared to CBA mice was registered during long-term external irradiation. The changes in the content of the morphologically recognized bone marrow cells and blood formed elements in the exposed mice of the studied lines were different.

Large numbers of primitive stem cells are active simultaneously in aggregated embryo chimeric mice.

The possibility has been repeatedly raised that erythropoiesis results from clonal succession--the differentiation of one or a very small number of the most primitive stem cells that are sequentially activated to proliferate forming clones of differentiated cells and then eventually decline, to be replaced by new stem cell clones. We studied this possibility in chimeric mice made by combining embryos from two different strains so that they would have two distinct stem cell populations, each of which produces a different hemoglobin type (d and s). These were compared with F1 hybrids in which every stem cell produces both types. We measured the percentage of type d in seven to ten serial samples of circulating reticulocytes taken at three- to seven-day intervals and found that the variability in percent of this hemoglobin was only slightly higher in the chimeric mice than in F1 controls; SD ranged from 2.7% to 5.5% in the chimeric mice and from 3.4% to 3.9% in the controls. Using the binomial formula, the numbers of new clones formed during the reticulocyte life span, approximately three days, ranged from 33 to 118 in the individual chimeric mice. However, these numbers are underestimates because estimated numbers of clones depend inversely on variabilities, and the calculations did not exclude the contribution of experimental error to the overall variability. Total percentages of type d hemoglobin were also measured in seven to nine successive serial samples at 60- to 136-day intervals. These gave mean values similar to measures of newly synthesized hemoglobin in the same mice, but SD were larger, ranging from 5.3% to 8.4%. This reflects experimental error, both because of excess day-to-day variability found in this type of measurement and because there could not be fewer primitive stem cells activated to form clones of erythrocytes during the 45-day erythrocyte life span than during the three-day life span of reticulocytes. Since most and maybe all of the variation between successive samples in the same chimeric mouse appear to result from experimental error, many or even all of the primitive stem cells may simultaneously contribute to erythropoiesis.

[Effect of fenazepam on the ACTH content of the blood plasma in inbred mice under stress exposure]. / Vliianie fenazepama na soderzhanie AKTG v plazme krovi inbredbykh myshei pri stressovykh vozdeistviiakh.

The influence of phenazepam on plasma ACTH level before and after exposure to stress in the "open field" test was investigated in C57BL/6, BALB/c and F1(C57BL/6 X BALB/c) mice. The differences in the dose-dependent drug effect on the hormone level between strains were studied. A correlation between ACTH level and a characteristic animal behaviour has been established for different strains.

Differences in some erythroid regulatory parameters between two inbred mouse strains.

Normal erythropoiesis in inbred CBA/Tr x CBA/Tr mice is shown to be associated with higher rates of red blood cell production and destruction than in age-matched, but significantly larger, C57Bl6/Tr x C57Bl6/Tr mice. As expected from these observations, serum Ep titers were significantly higher in the CBA/Tr mice than in the C57Bl6/Tr mice. These differences, which appear to result from variations in the operating point for erythropoiesis, can explain the different in vitro erythropoietin sensitivities of hematopoietic tissue from these two mouse strains.

Serum factor blocking the activity of virus-induced autoreactive T lymphocytes of mice.

Normal mouse serum was found to contain a factor blocking in vivo the effect of autoreactive cells accumulating in spleens of mice infected with tick-borne encephalitis (TBE), Langat, dengue type 2 (D2), and attenuated yellow fever (17D strain) viruses. The activity of the factor was manifested only, when the autoreactive lymphocytes had H-2 antigens identical with H-2 antigens of the serum donors. The hypothesis is discussed that serum factor protects the host against clones of autoreactive T lymphocytes generated in viral infections.

Calcium-dependent aggregation of human serum amyloid P component.

Normal human serum or isolated human amyloid P component was ultracentrifuged on density gradients containing either 10 mM EDTA or different concentrations of Ca2+ between 0.15 and 2.15 mM. In the presence of Ca2+ concentrations of 1 mM or more human P component sedimented more rapidly than it did in the presence of lower Ca2+ levels or of EDTA. This phenomenon was due to Ca2+-dependent aggregation of P component molecules and did not require the presence of any other serum constituents. It was completely inhibited by incorporating a physiological concentration (40 mg/ml) of serum albumin in the gradients, suggesting that free ionized Ca2+ is required to promote aggregation of the P component. P component from the mouse and the plaice (Pleuronectes platessa L.), a marine teleost, did not undergo the same Ca2+-dependent aggregation as human P component. These observations resolve a discrepancy existing in the literature concerning the sedimentation rate of human P component in density gradient ultracentrifugation and shed new light on its behaviour with respect to Ca2+ which may be relevant to the deposition of P component in amyloidosis.

[Characteristics of the blood system of inbred and mongrel mice]. / Kharakteristika sistemy krovi lineinykh i neinbrednykh myshei.

Peripheral blood and bone marrow indices were studied in inbred (101/H, CC57W, A2G, CBA/Lac, B6WV, C57BL/6, ACR) and mongrel mice. Normal values of the morphological composition of the blood and bone marrow were established. Comparative analysis demonstrated the existence of differences in the mentioned indices not only between the inbred and mongrel mice, but also within the individual animal strains.

The effect of serum inhibitor on the antigenic and mitogenic responses to E. coli bacteria.

The effect of serum inhibitory factors on the induction of the in vitro immune response and mitogenic response to Escherichia coli antigens in murine spleen cell cultures was investigated. Both normal and congenitally athymic mice served as spleen cell and serum donors. Cultured CBA/J spleen cells produced anti-bacterial antibody in the presence of mouse serum, but the mitogenic response to the bacterial antigen was suppressed by the addition of mouse serum. The addition of mouse serum from either CBA/J or nude mice to nude spleen cell cultures led to suppression of both the antigenic and mitogenic responses to bacterial antigen. The polyclonal antibody response by CBA/J spleen cell cultures to SRBC induced by bacteria was significantly enhanced by the addition of either CBA/J or nude mouse serum. Conversely, the nude spleen cell polyclonal response to SRBC was significantly inhibited by high concentrations of nude or CBA/J mouse serum. These data indicate that inhibition of the specific E. coli and nonspecific polyclonal antibody responses by mouse serum occurs only in cultures of spleen cells taken from mice lacking a population of T lymphocytes.

The effects of cell density, incubation temperature, syngeneic serum, and syngeneic red blood cells on mouse lymphocyte responses in vitro.

Various parameters affecting the quantitative assessment of mouse mixed lymphocyte response (MLR) in microplates were investigated. In agreement with the findings of others cell density was found to be particularly critical, maximal proliferative efficiency occurring at 3 X 10(6) viable cells/cm2. Slight reduction in the degree of viable cell--cell interaction caused by either reduction in cell density or insertion of irradiated cells into the cultures greatly reduced the efficiency of the proliferation. An incubation temperature of 33-34 degrees C was found to have distinct advantages over 37 degrees C, there being improved cell survival and reproducibility at this lower temperature. Transformation of mouse lymphocytes was strongly inhibited by low concentrations of normal syngeneic serum, the degree of inhibition depending on the concentration of foetal calf serum (FCS) in culture and on the method of preparation of serum. Inhibition of mouse cell responses was also caused by syngeneic red blood cells (RBC), in contrast to the pronounced enhancement of guinea pig lymphocyte responses to phytohaemagglutinin (PHA) in the presence of syngeneic RBC.

Prolactin and growth hormone levels in different inbred strains of mice: patterns in association with estrous cycle, time of day, and perphenazine stimulation.

These experiments were designed to compare prolactin (PRL) and growth hormone (GH) secretion in strains of mice with varying incidences of mammary tumors. In addition to the basal levels, PRL concentrations were compared after stimulation with perphenazine. Although pituitary concentrations of PRL and pituitary serum concentrations of GH appeared to be generally higher in strains with high incidence of mammary tumors, basal PRL levels in single decapitate serum samples seemed to have little correlation with the incidence of mammary tumors in different strains. However, PRL concentrations in sera after perphenazine injection followed a pattern characteristic of the mammary tumor incidence rate of the strain: C3H/St and CBA/St--the two high-incidence strains--had lower levels of PRL; C57BL/St and BALB/cST--the two low-incidence strains--had higher levels; and DBA/2St--the medium-incidence strain--had an intermediate level. PRL contents of the pituitary glands were depleted virtually equally in all strains except the BALB/cSt. These results suggested that the rate of metabolism of PRL in strains with high incidence of mammary tumors may be faster than in those with low incidences. Perphenazine had no influence on GH secretion in most mice. The strain-specific differences in PRL and GH concentrations were usually present even during cyclical and diurnal fluctuations. Serum PRL and GH levels were generally higher during the follicular phase and lower during the luteal phase of the estrous cycle in both C3H/St and C57BL/St strains. There were signs of episodic secretion of PRL together with evidences of circadian periodicity in the secretion of PRL and GH in mice of both strains. The levels of GH were usually high during the morning hours of those of PRL high during the evening hours. The data show a high degree of specificity in the secretion of PRL and GH im mice of different inbred strains, and it is possible that these strain-specific differences may be an important factor in the development of mammary tumors.