Characterization and determination of titratable groups of proteins by linearization of titration curves. II. Application to lysozyme

The potentiometric acid-base titration curve of fully protonated lysozyme at ionic strengths of 0.10 and 1.0 m has been performed. The stoichiometry and the pK_a values of each titratable group have been determined through the linearization of titration curves. Two types of carboxylic groups with pK_a values of 3.76 and 5.02, the imidazole group with pK_a 7.37 and the amine group with pK_a 9.63, have been identified at an ionic strength of 0.10 m at 25.0°C. The number of titratable groups found per mole of protein has been 5.12 and 5.60 for the two types of carboxylic groups, 1.13 for the imidazole group, and 3.19 for the amino groups. The endpoint of the titration of the protein obtained by this method accords quite well with the endpoint obtained by the use of Gran function applied to the excess of strong base.     

Mesenchyme cells degrade epithelial basal lamina glycosaminoglycan

We investigated whether turnover of basal lamina glycosaminoglycan (GAG), an active process during epithelial morphogenesis, involves the mesenchyme. Fixed, prelabeled, isolated mouse embryo submandibular epithelia were prepared retaining radioactive surface components, as determined by autoradiographic and enzymatic studies, and a basal lamina, as assessed by electron microscopy. Recombination of mouse embryo submandibular mesenchyme with these epithelia stimulates the release of epithelial radioactivity when the labeled precursor is glucosamine or glucose but not when it is amino acid. The release is linear with time during 150 min incubation. Augmented release of epithelial label requires living mesenchyme which must be close proximity with the epithelia. Although heterologous mesenchymes, including lung, trachea, and jaw, stimulate the release of submandibular epithelial label, epithelial tissues do not. The label released by intact submandibular mesenchyme from prelabeled epithelia is in GAG and in two unique fractions: heterogeneous materials of tetrasaccharide or smaller size and N-acetylglucosamine. Enzymatic treatment of the heterogeneous materials revealed the presence of glycosaminoglycan-derived oligosaccharides. These unique products were not obtained by incubating prelabeled epithelia with a mesenchymal cell extract, suggesting that intact mesenchymal cells are required. N-Acetylglucosamine was also released when mesenchyme was recombined with living prelabeled epithelia which contained labeled basal laminar GAG. Our results establish that submandibular epithelial basal lamina GAGs are degraded by submandibular mesenchyme. We propose that one mechanism of epithelial-mesenchymal interaction is the degradation of epithelial basal laminar GAG by mesenchyme.     

Ethanolamine kinase from Culex pipiens fatigans

Ethanolamine kinase was partially purified from the larvae of Culex pipiens fatigans and its properties were studied. The enzyme was separated from choline kinase by acetic acid precipitation at pH 5.0 of a 13,000g supernatant of the larval homogenate. Alkaline phosphatase activity was removed from the enzyme preparation by the acid treatment followed by ammonium sulfate fractionation. The enzyme was localized in the cytosolic fraction and had a requirement for Mg²⁺ as a cofactor. The K_m values for ethanolamine and ATP were 4 × 10^?4 and 1.54 × 10^?4m, respectively. The affinity of the enzyme for nucleotide triphosphates was in the order, ATP > ITP > GTP while UTP and CTP were poorly utilized. p-Chloromercuribenzoate and N-ethylmaleimide inhibited the enzyme activity and reduced glutathione protected the enzyme from their inhibition. Choline and serine had no effect on the enzyme activity. The enzyme had a molecular weight of 44, 000 daltons as determined by gel filtration chromatography. Eggs contained the highest specific activity of the enzyme while adult insects had the highest total enzyme activity.     

Pollenanalytical research in the Grisons (Switzerland)

In the first part, an overview of the history of palynological researeh in Switzerland and particularly in the Grisons is given, with a map showing all investigated areas in the Grisons. The second part deals with the significance of the pollenanalytical research in the Grisons, namely, the history of vegetation during the Late and Post Glacial, climatic fluctuations and applications in geomorphology, archaeology and forestry research. Finally, some results concerning the history of vegetation during the Late and Post Glacial are discussed.I would like to thank my colleague H. Holzhauser for his help with the drawing of the figures.     

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus "in vivo", "in vitro" and in organ culture

The translocation of progesterone receptor from the cytosol into the nucleus was studied under "in vivo" and "in vitro" conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before 1h) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (less than 3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the "in vitro" incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same "in vitro" conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated "in vivo" by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.     

Morphine addiction and withdrawal alters brain peptide concentrations

This study demonstrates that, during morphine addiction and withdrawal in rats profound alterations in the concentrations of a variety of brain peptides occur. Somatostatin, cholecystokinin, neurotensin and substance P concentrations increased during morphine addiction. Naloxone-induced withdrawal decreased brain concentrations of TRH, somatostatin, neurotensin and substance P. Naloxone alone decreased thalamic substance P and neurotensin concentrations. Vasoactive intestinal peptide concentrations were unaltered by any of the treatments. The fall in the tissue concentration of somatostatin during naloxone-induced withdrawal correlated well with the fall in the circulating growth hormone, suggesting that this could be secondary to somatostatin release. Our data support the hypothesis that brain peptides, acting locally in the brain as neuromodulators, play an important role in the genesis of the syndromes of morphine addiction and withdrawal.     

Macrophage activation for tumor cytotoxicity: Control of cytotoxic activity by the time interval effector and target cells are exposed to lymphokines

Macrophages continuously exposed to lymphokines (LK) and target cells throughout a 48-hr cytotoxicity assay exhibit 3-fold more tumoricidal activity than do cells optimally treated with LK before addition of tumor cells. Increased cytotoxic activity induced by continuous LK treatment was not due to direct toxic effects of LK on tumor target cells or to alterations in target cell susceptibility to cytopathic effects of LK-activated macrophages. Moreover, sensitivities of responsive macrophages to LK activation signals and time courses for onset and loss of tumoricidal activity during continuous exposure or LK pulse were identical. Analysis of macrophage or LK dose responses and time courses for development of cytotoxicity each suggest that differences in tumoricidal activity between macrophages continuously exposed or pulsed with LK were quantitative: the number of cytotoxic events was increased 2.7 ± 0.2-fold (mean ± SEM for 11 experiments) during continuous LK treatment. Optimal levels of macrophage tumoricidal activity then occur only if effector cells, target cells and activation stimuli are simultaneously present for a defined time interval: tumor cells need not be present during the initial 2 to 3 hr of culture; LK can be removed after 8 hr with little or no loss of cytotoxic activity. However, removal of LK or target cells during the critical 4- to 8-hr interval decreased levels of cytotoxicity 3-fold. Thus, nonspecific effector function by LK-activated macrophages in controlled by both the physicochemical nature of the LK mediator and the time interval effector and target cells are exposed to LK.     

BCG-induced granulomatous pulmonary inflammation and splenomegaly in mice: A T-cell-dependent response

When C57BL/6 mice were injected iv with BCG in an oil-in-saline emulsion, they developed intense pulmonary granulomatous inflammation (PGI) and splenomegaly as well as chemotactic activity for macrophages and angiotensin-converting enzyme (ACE) in their lung fluids. PGI, splenomegaly, and levels of chemotactic activity and ACE were markedly reduced in T-cell-deficient “B” mice. The capacity to develop PGI was fully restored and splenomegaly was partially restored in “B” mice by the provision of syngeneic thymocytes, spleen cells, or purified T cells. These results indicate that the full expression of BCG-induced PGI is dependent upon thymus-derived cells and is associated with high levels of chemotactic activity for macrophages and ACE in the lung lavage fluid. Although BCG-induced splenomegaly appears to be T cell dependent, it did not reach its full magnitude in reconstituted “B” mice.     

Studies on the effects of the antitumor agent camptothecin and derivatives on DNA. Camptothecin potentiated cleavage of DNA by bleomycin in vitro

Addition of sodium camptothecin (2a, Fig. 1) in comparable low concentrations to the glycopeptide antitumor antibiotic bleomycin (BLM) leads to enhanced rates of single-strand scission of PM2-covalently closed circular DNA, whereas sodium camptothecin alone has no effect. A similar enhancement of DNA scission by sodium camptothecin is produced with the 1 : 1 bleomycin-iron complex alone or in conjunction with NADPH as an additional reductant. The interpretation that camptothecin may substitute for the reducing requirement of the antibiotic is supported by its oxidation at 37°C by the 1 : 1 bleomycin iron complex, by iron salts or more efficiently by hydrogen peroxide to the known hemiacetal (3, Fig. 1).Electrochemical studies of 2a, its analogues and selected model compounds established that the α-pyridone ring D is most susceptible to a one-electron reduction at a reversible potential of ?0.95 ± 0.01 V. The reduced camptothecin is a transient species readily capable of donating an electron. This process may by compatible with a coupled reduction of the sequestered Fe(III) in the glycopeptide antibiotic necessary for the expression of antibiotic and antitumor properties. The results may provide a mechanistic rationale for the observed potentiation of the antitumor activity of bleomycin by camptothecin in vivo.