Biosynthesis and structure of membrane and secretory immunoglobulins

Summary Almost all of the body's extracellular immunoglobulin (Ig) is derived from Ig-secreting plasma cells of lymphoid tissues. The secreted material is a heterogeneous mixture of different classes and specificities. Lymphoid tissues also contain a large number of essentially non-secretory cells — B lymphocytes — which bear Ig firmly associated with their plasma membranes. Ig molecules thus exist in two functionally different forms, as membrane-bound antigen receptors on the surface of B lymphocytes on the one hand, and as humoral secreted Ig antibodies on the other. On B cells, membrane-bound heavy chains have an apparent mol. wt. slightly larger than that of secreted heavy chains from plasma cells. Membrane-bound but not secreted heavy chains bind detergents, thus suggesting the presence of a hydrophobic region in membrane-bound heavy chains, which is absent in secreted heavy chains. Most investigations have dealt with immunoglobulin M. The two types of IgM heavy chains differ at their carboxy termini. Recent investigations at the nucleic acid level demonstrate that membrane-associated µ chains contain a 41-residue hydrophobic tail adjacent to the last constant domain, whereas secretory µ chains contain a 20-residue hydrophilic tail. At the present time, evidence is accumulating that all membrane-bound Ig heavy chain classes may contain similar hydrophobic structures necessary for anchorage of the molecules into the lipid bilayer.     

Correspondent reaction of mitotic recombination in yeast and sister chromatid exchange in human lymphocytes

Summary In the lower eukaryote Saccharomyces cerevisiae, 4,5,6-trichloro-2-(dichlorophenoxy)phenol and acridine orange cause different specific genetic alterations, either gene mutations or recombinations. These specific effects were used to characterize the mechanism of sister chromatid exchange (SCE) formation in human lymphocytes. Assuming that genetically active substances have comparable effects in lower and higher eukaryotes, the observations provide indirect evidence for a connection between induced mitotic recombination in yeast and SCEs in human lymphocytes and suggest that SCEs may be the consequence of a repair process.     

Immunological analysis of hemoglobin transition during metamorphosis of normal and isogenicXenopus

Summary Antisera against larval and adultXenopus hemoglobins as well as adult human hemoglobin showed no cross-reaction when tested by immunodiffusion against each heterologous antigen. In this test hemoglobin of a single animal produced two precipitation lines for larvae, but only one for adult stages. Immunoelectrophoresis also revealed more complex precipitation patterns for larval than for adult hemoglobins. Hemoglobin of the isogenic hybrid cloneXenopus laevis/X. gilli also reacted with antisera against normalXenopus hemoglobin.Quantitation of hemoglobins, analyzed by radial immunodiffusion showed fewer than 1% of adult hemoglobin in red cells of larvae, but 30% at completion of metamorphosis. Two weeks later adult hemoglobin attained over 90%, and in red cells of adultXenopus an average of 1% larval hemoglobin were detected.The relatively short transition period suggests that the loss of larval hemoglobin may be due to the elimination of larval red cells, and that the increase in adult hemoglobin may be indicative of a new cell line.     

Selective high-performance liquid chromatographic assays for hydralazine and its metabolites in plasma of man

Selective high-performance liquid chromatographic assays for hydralazine (I), hydralazine pyruvic acid hydrazone (II) and the acetylation metabolites, namely s-triazolo[3,4-a]-phthalazine (V) and 3-hydroxymethyl (VI) and 3-methyl-s-triazolo[3,4-a]phthalazine (VII) in human plasma were developed. Utilizing the fluorescence of these compounds or their derivatives the limits of detection could be extended down to 5 nmole/l (1 ng/ml) for I, 1 nmole/l (0.2 ng/ml) for II and 0.5 nmole/l (0.1 ng/ml) for V–VII. The intra-assay coefficients of variation for the assays ranged from 2 to 7% over the concentration range 5.0 to 0.05 μmole/l and the inter-assay variability in the slope of the standard curves ranged from 4 to 8%. An improved method for measuring the sum of I plus all its hydrazones (apparent I) was also developed. On addition of I to fresh plasma at 37°, half the added I was converted to II within 15 min and there was no detectable level of I, 2 h after the addition. The plasma level—time course of I, and its metabolites in a healthy volunteer (slow acetylator) following separate oral and intravenous administrations of I indicated that I contributed only a small fraction (4.3 and 4.7% respectively) to the area under the plasma level—time curve of apparent hydralazine.     

Regulation of phenotype in somatic cell hybrids derived by fusion of teratocarcinoma cell lines with normal or tumor-derived mouse cells

The phenotypes of somatic cell hybrids between murine embryonal carcinoma cell lines, F9 BrdU 7C12 and PCC4 aza 1, and normal murine splenic lymphocytes or thymoma-derived cell lines were compared. Analysis of morphology in vivo and in vitro of cell surface markers and of the karyotype of these cloned hybrid cells did not reveal any simple mechanism for the regulation of the phenotype of such hybrids. Hybrids of either the embryonal carcinoma cell phenotype or of a differentiated morphology (resembling neither parental cell) but not of lymphoid morphology can be derived from fusions of this type. Moreover, transition from one phenotype to the other (ECC → differentiated cell and differentiated cell → ECC) can be found with passage of clonally derived hybrid cell lines. Coordinate control of the phenotypic markers of the state of differentiation in these hybrid cells was found.     

Relative activities of linear and cyclic electron flows during chloroplast CO2-fixation

Evolution of oxygen and turnover of cytochromes b-563 and ? were measured upon illumination of isolated intact spinach chloroplasts with a series of flashes. The flash yield of cytochrome ? oxidation approximated the sum of the yields of cytochrome b-563 reduction and electron transfer through Photosystem II, regardless of whether HCO^?₃, 3-phosphoglycerate or O₂ served as the terminal electron acceptor. No absorbance contribution from cytochrome b-559 was discerned within the time range studied. Some pseudocyclic electron flow occurred when both HCO^?₃ and 3-phosphoglycerate were omitted, and possibly also during induction of photosynthesis; however, the flash yield data suggest that O₂ is not reduced at a significant rate during steady state photosynthesis. The maximum rate of cytochrome ? turnover (1000 μequiv./mg chlorophyll per h) was adequate to support the highest rates of photosynthesis observed in isolated chloroplasts.These results agree with the concept that cytochrome ? is a component both of the linear and cyclic pathways whereas cytochrome b-563 functions only in the cyclic pathway. NH₄Cl decreased the half time of cytochrome b-563 oxidation from 11.6 to 8.2 ms and decreased the half time of cytochrome ? reduction from 7.2 to 2.8 ms. The cyclic and linear pathways thus seem to be jointly regulated by a transthylakoid H⁺ gradient through a common control point on the reducing side of cytochrome ?. Cyclic turnover also increased during the induction phase of photosynthesis, when linear throughput is limited by the rate of utilization of NADPH. The slow rise in the P-518 transient correlated with increased cyclic activity under the above conditions.It is proposed that flexibility in the utilization of linear and cyclic pathways allows the chloroplast to generate ATP and NADPH in ratios appropriate to varying needs.     

The intracellular location of adenylyl cyclase in the cellular slime molds Dictyostelium discoideum and Polysphondylium pallidum

Adenylyl cyclase activity was low or not detectable on intact cells and in isolated plasma membranes, phagocytic vacuoles and nuclei of the two slime mold species examined. The entire activity of homogenates was sedimentable and concentrated in a light membrane fraction. When this fraction was centrifugated through sucrose density gradients the adenylyl cyclase activity sedimented differently from all other enzymes measured. The gradient fractions with the highest specific activity of adenylyl cyclase consisted mainly of small vesicles. No changes in adenylyl cyclase distribution were associated with development. The possibility that cellular slime mold adenylyl cyclase activity is associated with vesicles in vivo, as already suggested by Maeda & Gerisch [10], is discussed.     

Nickel,cobalt, and molybdenum requirement for growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum on H₂ and CO₂ as sole energy and carbon sources was found to be dependent on Ni, Co, and Mo. At low concentrations of Ni (<100 nM), Co (<10 nM) and Mo (<10 nM) the amount of cells formed was roughly proportional to the amount of transition metal added to the medium; for the formation of 1 g cells (dry weight) approximately 150 nmol NiCl₂, 20 nmol CoCl₂ and 20 nmol Na₂MoO₄ were required. A dependence of growth on Cu, Mn, Zn, Ca, Al, and B could not be demonstrated. Conditions are described under which the bacterium grew exponentially with a doubling time of 1.8 h up to a cell density of 2 g cells (dry weight)/1.     

Differentiation in cultures derived from embryonic chicken muscle: I. Muscle-specific enzyme changes before fusion in EGTA-synchronized cultures

It is known that myoblast fusion fails to occur in cultures containing EGTA (a calcium-specific chelator) but occurs very rapidly after EGTA medium is replaced with standard high-calcium medium. On the basis of a careful analysis of the time course of fusion in cultures switched from EGTA to standard medium, it is proposed that this method of synchronization be used routinely in studies of the timing of different processes during in vitro myogenesis. The kinetics of accumulation of total enzyme activity for creatine kinase and fructose diphosphate aldolase indicate that the increases characteristic of terminal muscle differentiation begin prior to the experimentally imposed onset of fusion in EGTA-synchronized cultures. Additionally, the accumulation of M-creatine kinase subunits, also typical for muscle differentiation, is shown by microcomplement fixation to begin before the switch from EGTA to standard medium. Creatine kinase isoenzyme patterns also show that the transition from B- to M-subunit-containing creatine kinases occurs in EGTA cultures not switched to standard medium. Like EGTA, 5-bromodeoxyuridine (BrdUrd) reversibly prevents myoblast fusion. By adding EGTA and BrdUrd in different sequences to muscle cell cultures, it is shown that they act at different stages in the course of in vitro myogenesis. Cells cultured in EGTA from 23 to 69 hr after plating fused very rapidly when switched to medium containing BrdUrd. In the reverse experiment, in which BrdUrd preceded EGTA, no fusion occurred. Parallel experiments with 5-fluorodeoxyuridine suggest that cell division is necessary to reverse the inhibitory effect of BrdUrd, but not that of EGTA; this is consistent with the observed kinetics of fusion after switching to standard medium. These data strongly support a model of myogenesis in vitro in which two processes (one BrdUrd-sensitive, the other EGTA-sensitive) occur sequentially. In the first process, myogenic cells give rise to cells capable of producing molecules necessary for (terminal) skeletal muscle differentiation, including both those required for cell fusion and specific isoenzymes. The second process, fusion itself, can occur in the presence of BrdUrd or in the absence of cell division.