Characterization of the central region containing the X-inactivation center and terminal region of the mouse X Chromosome using irradiation and fusion gene transfer hybrids

The irradiation and fusion gene transfer (IFGT) procedure provides a means of isolating subchromosomal fragments for use in the mapping of loci and for cloning probes from a particular area of a chromosome. Using this procedure, two large panels of somatic cell hybrids that contain mouse X Chromosome (Chr) fragments have been generated. These hybrid panels were generated by irradiating the monochromosomal mouse-hamster hybrid HYBX, which retains the mouse X Chr, with either 10 K or 50 K rads of X-irradiation followed by fusion with a recipient Chinese hamster cell line. IFGT hybrids retaining mouse material were generated at high frequency. These hybrids were used to orient loci in the X-inactivation center region that had not been resolvable in our interspecies backcross panel and also to map, within the terminal region of the X Chr, repeat elements detected by the probe p15-4. These hybrids not only complement existing interspecies meiotic mapping panels for the detailed analysis of specific regions of particular chromosomes, but also provide a potential source of material for chromosome-specific probe isolation.     

d-Hydantoinase from anaerobic microorganisms

Summary Of 373 anaerobic microbial isolates screened for the enzymatic conversion of dihydrouracil to N-carbamyl--alanine, several strains of Clostridium spp., C. glycolicum, C. subterminale and Peptococcus anaerobius were positive. These Clostridium and Peptococcus strains produced also N-carbamyl-d-amino acids from the respective 5-monosubstituted hydantoins. The d-hydantoinase activity from whole cell suspensions of P. anaerobius strain CRDA 303 was characterized with regard to pH and temperature stability and activity by using dihydrouracil (DHU) and isopropylhydantoin (IPH) as substrates. The d-hydantoinase from P. anaerobius was optimal at 60°C and at pH 6.5–9.5 for the substrate DHU. It was stable up to 55°C and at pH 5.0–9.5 and could be stored at 4°C under an aerobic atmosphere for at least 14 days. Offprint requests to: A. Morin     

Bacterial superantigens as anti-tumour agents: induction of tumour cytotoxicity in human lymphocytes by staphylococcal enterotoxin A

Summary Activation of lymphocytes by interleukin-2 (IL-2) induces lymphokine-activated killer (LAK) cells that show promising effects on tumour growth in clinical trials. We examined the effect of the superantigen staphylococcal enterotoxin A (SEA) on anti-tumour activity of freshly prepared human lymphocytes. Picomolar amounts of SEA rapidly induced cytotoxic activity against K562 and Raji cells as well as some natural-killer(NK)-resistant tumour cell lines. Cytotoxic activity was not dependent on target cell expression of either major histocompatibility complex (MHC) class I or II antigens as shown using mutated cell lines. Cell-sorting experiments showed that the activity was expressed by NK (CD5^–CD56⁺) as well as T (CD5⁺) cells, although the former contained the majority of cytotoxic activity. NK cells could not be directly activated by SEA. In contrast, SEA activated purified T cells to the same extent as in bulk cultures. It is suggested that SEA activation of NK cells is secondary to that brought about by lymphokines produced by T cells. Activation of LAK cells with SEA was comparable in magnitude as well as target cell spectrum to that of IL-2. In addition to the LAK-like cytotoxic activity induced by SEA, a superimposed cytotoxicity towards target cells expressing MHC class II antigens coated with SEA was observed. This staphylococcal-enterotoxin-dependent cell-mediated cytotoxicity (SDCC) was exclusively mediated by T cells. It is well established that MHC class II antigens function as receptors for staphylococcal enterotoxins on mammalian cells and that the complex between MHC class II antigen and — SEA apparently functions as a target structure for activated T cells with target cell lysis as a consequence. Activation of T lymphocytes with IL-2 also resulted in the capability to mediate SDCC. Staphylococcal enterotoxins represent a novel way of inducing anti-tumour activity in human lymphocytes, which could be of value in therapeutic applications.     

Further evidence for the presence of specific binding sites for prolactin in the rabbit brain. Preferential distribution in the hypothalamus and substantia nigra

In the present research we have extended our work on the presence of binding sites for prolactin in the rabbit brain focusing our attention on the brain areas with high dopamine cell bodies density. Among these areas the hypothalamus showed the highest specific binding of labeled ovine prolactin (oPRL). Clearly detectible specific binding was observed also in substantia nigra, whereas in other brain regions the specific binding was very small, except for the striatum where a low but not negligible binding was found in female rabbits. The binding of 125I-oPRL showed a hormonal specificity and Scatchard analysis of the binding showed no clear difference in dissociation constant (Kd) between hypothalamus, nigra and striatum.     

The anatomy of tissue cultured red raspberry prior to and after transfer to soil

The leaf, petiole, stem and root anatomy of an aseptically cultured red raspberry clone (Rubus idaeus L.) was studied before and 5 weeks after transfer to soil under controlled environmental conditions. Tissues persistent from culture showed little or no change with time in soil; they grew minimally and slight secondary wall deposition occurred. New organs formed in successive weeks after transplantation showed a graded increase in potential size and development. Some features, such as collenchyma formation, rapidly returned to control levels; this was seen in new leaves expanding in the first week after transplantation. Other features, such as sclerenchyma formation, did not occur in leaves expanding during the first 2 weeks after transplantation, even when these were a month or more in age. Some sclerenchyma was seen in leaves expanding in the third week after transplantation, increasing in later-formed leaves. Increasing the light intensity of transplant accelerated the return to control-type organ size and appearance. During acclimatization transitional forms of leaves, petioles, stems and roots develop that ranged anatomically from culture-to control-type. This trend is analagous to the normal developmental sequence of organ formation as it affects the potential for development of successily formed organs.     

Interspecies hydrogen transfer in anaerobic degradation of CMC to CH4 by a triculture of marine bacteria

Summary Study of CMC fermentation by a marine syntrophic association of an anaerobic cellulose-degrader, a carbohydrate-fermenter, and a methanogen. Altered fermentation pattern in general agreement with the concept of interspecies hydrogen transfer was obtained only with pregrowth of methanogen followed by inoculation of the two fermentative bacteria.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Calcium uptake by isolated rat intestinal cells

Intestinal cells were isolated by a combination of mechanical and enzymatic means, and their calcium uptake was assayed by a rapid filtration procedure. Calcium uptake was a time- and concentration-dependent process that was markedly elevated at 25 and 37°C, as compared to 0°C. Cells isolated from rat duodenum exhibited higher uptakes than cells from jejunum, which in turn took up more calcium than cells from the ileurn. Duodenal cells from vitamin D-deficient animals took up less calcium than cells from vitamin D-replete cells. In vivo vitamin D repletion with 1,25-dihydroxyvitamin D₃ raised calcium uptake by duodenal cells from treated animals toward that of cells from replete rats. Furthermore, calcium uptake by duodenal cells from vitamin D-deficient animals approximated that of ileal cells from replete rats. These findings with isolated cells parallel prior findings of tissue calcium transport and suggest that cellular calcium uptake may be related to the saturable component of intestinal calcium absorption. Isolated intestinal cells may therefore constitute one experimental model for the study of transcellular calcium transport.     

Entrainment and phase shifting of the circadian rhythm of cell division by light in cultures of the achlorophyllous ZC mutant ofEuglena gracilis

The photosynthesis-deficient ZC mutant ofEuglena gracilis Klebs (strain Z) was cultured at 16°C on an aerated, magnetically stirred, mineral medium containing 0.1% ethanol (pH 7.0). Cell division could be entrained by a 12: 12 light: dark cycle (LD: 12, 12) or even by a one-pulse skeleton photoperiod (LD: 1,23) The rhythm free-ran in DD for at least 8 days with a circadian period (=25.5 h) in populations that had been previously entrained by LD. The freerunning rhythm could be phase-shifted by a single 1-h light pulse (3000 lx). The strong (Type 0) phase-response curve derived from the resetting effects of such signals given at different circadian times was similar to that for the photosynthetic wild-type strain. These results demonstrate that the presence of a functional chloroplast compartment is not necessary for the circadian clock to function inEuglena and suggest that phase resetting of the circadian clock by light occurs via a similar pathway in both photosynthetic and non-photosynthetic cell types.