Loss of the PGE1 requirement for MDCK cell growth associated with a defect in cyclic AMP phosphodiesterase

Prostaglandin E₁(PGE₁), one of the components in the hormone-supplemented, serum-free medium for Madin Darby Canine Kidney (MDCK) cells (Medium K-1), is required for both long-term growth and for dome formation. Variant cells have been isolated from MDCK populations, which lack the PGE₁, requirement for long-term growth in Medium K-1. These variants will be useful in identifying the molecular events initiated by PGE₁ which are necessary for the growth response to be observed. The growth and functional properties of five independently isolated PGE₁ independent clones have been examined. Normal MDCK cells grew at an equivalent rate in Medium K-1 and in serum-supplemented medium; the growth rate was lower in Medium K-1 lacking PGE₁. In contrast, PGE₁ independent clone 1 grew at an equivalent rate in Medium K-1 minus PGE₁, and in serum-supplemented medium. When PGE₁ was added to K-1 minus PGE₁, less growth of PGE₁ independent clone 1 was observed. A similar observation was made with one other PGE₁ independent clone which was studied. A hormone deletion study indicated that PGE₁ independent clone 1 still retained growth responses to the other four supplements in Medium K-1 (insulin, transferrin, T₃, and hydrocortisone). The molecular alterations associated with loss of the PGE₁ requirement for long-term growth were examined. At confluency, all of the PGE₁ independent clones studied had higher intracellular cyclic AMP levels following PGE₁ treatment, as compared with normal MDCK cells. The increased cyclic AMP levels in the variant cells could result from a number of different types of defects, including reduced cyclic adenylic acid (cyclic AMP) efflux, an increased affinity of PGE₂ for the PGE₁ receptor, or a defect in cyclic AMP metabolism. However, in all of the variant clones studied a decreased rate of cyclic AMP degradation by cyclic AMP phosphodiesterase was observed. Thus, the increased cyclic AMP levels in the PGE₁ independent variants may result from alterations which affect cyclic AMP metabolism. The effect of PGE₁ on dome formation by the variant cells was also examined. The frequency of dome formation by PGE₁ independent clone 1 was enhanced in a dosage-dependent manner, like normal MDCK cells. This observation suggests that PGE₁ affects MDCK cell growth and dome formation by different mechanisms.     

Ultrastructure and distribution of microbodies in leaves of grasses with and without CO2-photorespiration

Summary A comparative study was made of the ultrastructure, distribution and abundance of leaf microbodies in four species of temperate grasses with high and four tropical grasses with low CO₂-photorespiration. The temperate grasses were all festucoid; the tropical grasses included two panicoid species and two chloridoid. Comparisons of relative abundance were made by computing the average numbers of microbody profiles per cell section.Although microbodies were present in the green parenchymatous leaf cells in all grasses examined, their average number per cell was in general severalfold greater in the grasses with high CO₂-photorespiration than in those with low. Furthermore, whereas in the grasses with high CO₂-photorespiration the microbodies were distributed through the mesophyll, in those with low CO₂-photorespiration they were concentrated in the vascular-bundle-sheath cells and were smaller and relatively scarce in the mesophyll cells. The leaf microbodies of the eight grass species resembled one another in general morphology, but differed to some extent in regard to size and type of inclusion. Microbodies of all four festucoid species contained numerous fibrils with a discernible substructure. Those of the two panicoid species contained clusters of round bodies with transparent cores. The equivalence of the microbodies to peroxisomes as biochemically defined was shown cytochemically by employing 3,3-diaminobenzidine for the localization of catalase, a marker enzyme for the peroxisome. This reaction was blocked by the catalase inhibitor, aminotriazole.The observations on the relative abundance and distribution of peroxisomes in leaves of grasses with high CO₂-photorespiration versus those with low are consistent with the published biochemical data on the levels and distribution of peroxisomal enzymes in representatives of plants with high and low CO₂-photorespiration, and may help explain the differences in apparent photorespiratory levels between these two groups of plants.     

The Effects of Some Chlorinated Hydrocarbons on Certain Soil Bacteria

S ummary . Some common soil bacteria have been shown to tolerate the presence of chlorinated hydrocarbon fumigants. Several of these bacteria indicated an ability to utilize the chemicals as carbon or energy sources. The breakdown of these fumigants may yield compounds either beneficial or detrimental to plants grown in treated soil. Such information is critical to plant disease control since these chemicals are used for the control of plant pathogens, primarily nematodes. It is suggested that the side effects of soil treatment by which plants are stimulated in their growth is a direct consequence of enhanced beneficial microbial activity.     

Linkage of PGK1 to X-linked severe combined immunodeficiency (IMD4) allows predictive testing in families with no surviving male

Summary We present a linkage map of DNA probes around the X-linked severe combined immunodeficiency (IMD4) locus at Xq11-13. DXS159 and PGK1 show no cross-overs with the disease locus (Lod 3.01 at = 0.00). The order of loci is DXS1-DXS106-(DXS159-PGK1-IMD4)-DXS72-DXYS1. Members of families whose carrier status has been established by X-inactivation patterns were included in the analysis. As the probe (pSPT/PGK), which is used for investigation of X-inactivation patterns, has been shown to be linked to the disease itself, it is possible to assign phase in mothers of sporadic cases who have been shown to be carriers, even when they have no surviving male offspring.     

Perinatal Steroid Treatments Alter Alloparental and Affiliative Behavior in Prairie Voles

This experiment was designed to examine the hypothesis that perinatal manipulation of gonadal or adrenal steroids can alter the subsequent expression of juvenile parental (alloparenting) and affiliative behavior in prairie voles (Microtus ochrogaster). Corticosterone (PRECORT), testosterone (PRE-TP), or oil injections (PRESES) were given on Prenatal Days 12–20 or on Postnatal Days 1–6 (CORT6, TP6, or SES6, respectively). Alloparenting was reduced significantly in females in the CORT6 group and in males in the TP6 group. Sibling affiliative preferences were increased significantly in PRE-TP females and stranger preferences were increased in TP6 and CORT6 females. The results suggest timing is a critical factor determining whether hormones have a facilitative or inhibitory effect on alloparental and affiliative behavior in prairie voles. In this species, corticosterone and testosterone have similar organizational effects on affiliative behavior in females. Alloparental behavior is inhibited by postnatal corticosterone administration in females and by postnatal testosterone administration in males, whereas prenatal steroid administration had no significant effect on alloparenting in either gender.     

EagI andNotI linking clones from human chromosomes 11 and Xp

EagI and NotI linking libraries were prepared in the lambda vector, EMBL5, from the mouse-human somatic cell hybrid 1W1LA4.9, which contains human chromosomes 11 and Xp as the only human component. Individual clones containing human DNA were isolated by their ability to hybridise with total human DNA and digested with SalI and EcoRI to identify the human insert size and single-copy fragments. The mean (± SD) insert sizes of the EagI and NotI clones were 18.3 ± 3.2 kb and 16.6 ± 3.6 kb, respectively. Regional localisation of 66 clones (52 EagI, 14 NotI) was achieved using a panel of 20 somatic cell hybrids that contained different overlapping deletions of chromosomes 11 or Xp. Thirty-nine clones (36 EagI, 3 NotI) were localised to chromosome 11; 17 of these were clustered in 11q13 and another nine were clustered in 11q14–q23.1. Twenty-seven clones (16 EagI, 11 NotI) were localised to Xp and 10 of these were clustered in Xp11. The 66 clones were assessed for seven different microsatellite repetitive sequences; restriction fragment length polymorphisms for five clones from 11q13 were also identified. These EagI and NotI clones, which supplement those previously mapped to chromosome 11 and Xp, should facilitate the generation of more detailed maps and the identification of genes that are associated with CpG-rich islands. Received: 27 December 1995 / Revised: 30 January 1996