Isolation of hemopoietic pluripotent stem cells from spleens of thiamphenicol-pretreated mice

The present report describes the bulk isolation of pluripotent stem cells (PSC) (assayed as day-9 CFU-S, colony-forming-units-spleen). As starting material, spleens, highly enriched with PSC, were used from mice that were bled and treated with thiamphenicol (TAP). In subjecting the spleen cells to a two-stage centrifugal elutriation procedure and a subsequent Percoll gradient centrifugation stage a 30-fold enrichment in the CFU-S concentration was achieved. The splenic PSC seeded with a characteristic low efficiency in the spleens of irradiated mice (f = 2%). Correcting the colony number for this, we obtained a cell mixture consisting of 88% PSC, contaminated with 4% committed precursor cells and about 10% ganuloid cells.     

Phase behavior of isolated photoreceptor membrane lipids is modulated by bivalent cations

The phase behavior of isolated photoreceptor membrane lipids is further investigated by ³¹P-NMR, in view of earlier discrepant results [(1979) Biochim. Biophys. Acta 558, 330–337; (1982) FEBS Lett. 124, 93–99]. We present evidence that the discrepancy is due to bivalent cations. When resuspended in aqueous media at neutral pH in the absence of bivalent cations, the isolated photoreceptor membrane lipids largely adopt the bilayer configuration. However, upon addition of such cations (Ca²⁺ Mg²⁺) or when resuspended in their presence, the formation of other phases (hexagonal H_II, lipidic particles) results. The rate of this transition depends on cation concentration and temperature. The transition is not easily reversed by addition of EDTA. Implications with regard to photoreceptor membrane structure and function need further study.     

ACTH-induced inhibition of endogenous rat brain protein phosphorylation in vitro: Structure activity

ACTH_1–24 inhibits the endogenous phosphorylation in vitro of distinct SPM protein bands. Using N-terminal fragments of ACTH, the structure-activity requirements for this effect were studied. A rather complex interaction of the ACTH fragments with endogenous SPM phosphorylation was observed. The effects were not only dependent on the primary structure of the peptide used, but also on the protein band studied and the ATP/SPM ratio used in the incubation system. ACTH_1–24 did not interfere with the ATP-hydrolyzing activity of the SPM preparation, nor did it influence the endogenous phosphatase activity. Therefore, a direct interaction of ACTH with SPM protein kinase(s) is likely to be responsible for its effect on phosphorylation.     

Influence of serum albumin on fecundity and weight of the progeny of the tsetse fly Glossina palpalis palpalis

Females of a membrane-fed colony of G. p. palpalis (food source: fresh defibrinated bovine blood) were fed on derivatives of bovine blood, in which the red cell fraction remained unchanged while the serum fraction was replaced by an artificial solution. This solution consisted of a simple saline, isotonic to serum and enriched by bovine serum albumin with increasing concentrations. Physical parameters such as pH and osmolarity of all media were similar to controls. The absence of serum albumin in the food medium resulted in sterility of the females and caused high mortality. The presence of increased levels of serum albumin in the medium increased the fecundity until it was similar to that of flies fed on the standard diet of fresh defibrinated bovine blood. The offspring size was positively correlated with the concentration of the serum albumin in the mother's diet. However, a high concentration of albumin (>6%) resulted in increased adult mortality. Thus, the presence of optimum amounts of free serum albumin in bovine blood appears necessary for the development and production of viable larvae of the tsetse fly G. p. palpalis.

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Résumé Les femelles d'une population de G. p. palpalis élevées sur membrane (aliment: sang frais défibriné de bovin) ont été nourries de dérivés de sang bovin dont les globules rouges n'avaient pas été modifiés, mais dont le sérum avait été remplacé par une solution artificielle. Cette solution était simple, saline, isotonique au sérum et enrichie d'albumine de sérum bovin en concentrations croissantes. Les témoins ont montré l'identité des paramètres physiques, tels que le pH et l'osmolarité, pour toutes les solutions. L'absence d'albumine de sérum dans l'aliment donné aux femelles a provoqué leur stérilité et une mortalité élevée. L'augmentation des quantités d'albumine de sérum a accru la fécondité jusqu'à ce que celle-ci soit identique à celle des glossines avec un régime normal composé de sang frais défibriné de bovin. Il y avait une corrélation directe entre la taille des descendants et la concentration d'albumine de sérum dans l'alimentation de la mère. Toutefois, une forte concentration d'albumine (plus de 6%) a augmenté la mortalité imaginale. La présence de quantités optimales d'albumine de sérum libre dans le sang bovin semble donc nécessaire au développement et à la production de larves viables de la mouche tsé-tsé, G. p. palpalis.

     

Comparison of the in vivo clearance of des-AA and des-AABB rat fibrin monomers prepared by different methods

Solubilization of fibrin monomers (Fm's) is usually performed with dilute acetic acid, urea or sodium bromide. These solvents can affect the biological properties of Fm's. Therefore we describe a new method to keep Fm's in solution, under milder conditions i.e. by generating them in Dcate solutions and avoiding non-physiological conditions. The in vivo behaviour of iodinated rat Fm's injected in rats and prepared by this new method was compared with that of Fm's dissolved in acetic acid, urea or sodium bromide.Fm's prepared in Dcate solutions accumulate rapidly, within 10 minutes after injection, in all organs tested, predominantly in kidney, liver and lung, probably by interaction with endothelial cells. The blood radioactivity remains nearly constant during the first 90 minutes and decreases thereafter exponentially. Fm's dissolved in sodium bromide behave similarly. However, Fm's dissolved in acetic acid or urea behave differently and do not accumulate in organs. This suggests that Fm's loose their capability to accumulate in organs and probably to interact with endothelial cells when they have been dissolved in acetic acid or urea.The slow exponential clearance phase does not differ significantly between the various Fm's and their $\text{T}\text{1}\text{2'S}$ are estimated to lie between 5 and 7 hours.     

Enhancement of Endogenous Release of Glutamate and γ-Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long-Term Potentiation

The effect of long-term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and gamma-aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K(+)-induced Ca(2+)-independent release components of these amino acids. In contrast, K(+)-induced Ca(2+)-dependent release of both glutamate and GABA increased approximately 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage φR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10³ transconjugants per g of soil when 10⁷ donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx^r Rp^rP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Evidence for the deficiency of β-glucosidase-activating factor in fibroblasts of patients with I-cell disease

Summary Reduced activity of -glucosidase was shown in the cultured skin fibroblasts of four patients with I-cell disease when the enzyme was tested without the use of detergents. In the presence of taurocholate and triton X100 -glucosidase activity was normal. This suggested a deficiency of a -glucosidase-activating factor in I-cell fibroblasts rather than of the enzyme itself. The deficiency of -glucosidase activity was corrected to some extent by mixing cell lysates, and more effectively by cocultivation and fusion of I-cell disease and Gaucher fibroblasts. These results present evidence for the presence of a -glucosidase-activating factor in normal and Gaucher fibroblasts. In fibroblasts of patients with I-cell disease this activator is probably deficient, as is the case for most lysosomal enzymes.     

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5–6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2–3-fold). There were no α-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.