Salivary gland glucagon is a fictitious substance due to tracer-degrading activity resistant to protease inhibitors

A high level of glucagon immunoreactivity was apparently detected in acid-saline extract from rat submandibular glands, but tracer glucagon added to the assay mixture was mostly damaged in spite of the presence of protease inhibitors commonly used in radioimmunoassay. Gel-filtration of the extract on a Bio-Gel P-10 column revealed strong tracer-degrading activity at the void fraction where the apparent immunoreactivity was eluted. Serial changes in apparent immunoreactivity of the extract fit well on the theoretical curve of an exponential tracer degradation. These findings indicate that the salivary gland glucagon is a fictitious substance due to tracer degradation during radioimmunoassay. Further study revealed that the glucagon molecule was hydrolyzed at the arginyl bonds and split into two fragments during incubation with the acid-saline extract from rat submandibular glands.     

X chromosome expression during oogenesis in the mouse.

The activities of glucose-6-phosphate dehydrogenase (G6PD) and lactate dehydrogenase (LDH) have been assayed in mouse oocytes at several stages of follicle development isolated from XX and XO female mice. Throughout the entire growth period the activity of G6PD was proportional to the number of X chromosomes present in the oocyte, whereas no difference in LDH activity was detected between XX and XO oocytes. It is concluded, therefore, that both X chromosomes are functional throughout oogenesis.     

Cell subpopulations in the late morula and early blastocyst of the mouse

Late morulae and early blastocysts consist of two main cell subpopulations which occupy different positions within the embryos. The cells of the outer layer have a polar surface phenotype. The outward-facing surface of this cell type has a discrete dense pole of short microvilli, whilst the inward-facing surface has a relatively sparse distribution of longer, thick microvilli. The inner cells lack short, dense microvilli but exhibit thick microvilli of variable density. After short-term isolation in medium low in Ca²⁺, the individual polar and apolar cells remain distinguishable. The expanded blastocyst also has two major cell subpopulations, but within each of these, heterogeneity is observed. The mural trophectodermal cells have a larger, more regular outward-facing area of sparse, short microvilli than do polar trophectodermal cells. The ICM consists of some cells that show extensive blebbing in medium low in Ca²⁺ and others that do not.     

An experimental approach to the study of factors affecting algal colonization in a sublittoral kelp forest

Factors influencing the early colonization by benthic macroalgae and the early successional sequence in a subtidal Ecklonia radiata (C. Agardh) J. Agardh forest in Port Jackson (Sydney) were investigated using controlled experiments involving the microscopic sampling of settlement plates. The preliminary results indicate that the Tolerance model of succession may be applicable. The effect of grazers, and competition between different algal types were important and could, later in the succession, lead to the operation of the mechanisms suggested by the Facilitation or Inhibition models. Controls for the use of settlement plates showed that thick settlement plates led to an over-estimation of initial algal growth compared with the growth on natural patches of substratum. Other results suggested that thick plates excluded grazers. Borders of plates were investigated, but the establishment of algae was random over the plates′ surfaces and border effects were the result of physical or biotic factors acting on algae after settlement. An experiment was developed for use in subtidal areas to test for artifacts due to cages. It was found that the presence of the cages influences the community by attracting small snails. Fish were excluded from some areas to monitor their effects on initial algal growth. Exclusion of fish was found to cause a reduction in algal growth as well as increases in numbers of small invertebrates such as amphipods and copepods. The results suggested that the amount of algae initially present was affected by invertebrate grazers, the abundances of which were, in turn, affected by predation. The species diversity of the early algal growth did not show any consistent trends between high and low grazing pressure. Grazing was important in determining the temporal variability of early growth of individual species. Siltation was positively correlated with early algal growth; a mechanism to account for this effect is suggested. The application of the methods used in this study to future experimental studies of sublittoral community dynamics is considered.     

Synthesis of tubulin and actin during the preimplantation development of the mouse

The relative rates of synthesis of actin and tubulin during mouse preimplantation development have been investigated utilizing O'Farrell's two-dimensional polyacrylamide gel system and internal protein markers. During mouse preimplantation development, rates of protein synthesis remain low and are little changed until the 8-cell stage when a rapid increase is evident. From the 8-cell stage on, a much higher rate of synthesis is maintained. The rate of synthesis of actin remains also at a steady low level in the unfertilized and fertilized ovum. However, by the 8-cell stage actin synthesis has increased 10-fold. Our measurements include the blastocyst, at that point in development actin synthetic rates are almost 90-fold higher than in the unfertilized ovum. While this precipitous increase is proceeding, incorporation of [³H]leucine into total protein increases only 7-fold. Synthesis of actin in the blastocyst represents 5.7% of total protein synthesis. The rate of tubulin synthesis, unlike actin, more closely parallels the increments in total protein synthetic rates. At the blastocyst stage it has increased 14-fold and its synthesis represents almost 2% of total protein synthesis. These results are discussed with reference to some of the physiological changes taking place during preimplantation development.     

Control mechanism of lymphocyte traffic. A study of the action of two sulfated polysaccharides on the distribution of 51Cr- and [3H]adenosine-labeled mouse lymph node cells

The effects of dextrans of varying molecular weights and of pentosan sulfate on the distribution of ⁵¹Cr-labeled mouse lymph node cells were studied in vivo, i.e., in recipients treated with the sulfated polysaccharides, and in vitro, i.e., by following the fate of cells treated in vitro, in intact syngeneic recipients. Both types of experiments demonstrate that dextrans, especially dextran sulfate (DXS) and pentosan sulfate (PS), considerably reduce lymph node entry of lymphocytes, with concomitant increases in the blood and, in the case of DXS, in both the blood and lungs. A parallel quantitative autoradiographic analysis of the distribution of [³H]adenosine-labeled cells confirmed the data with the ⁵¹Cr-labeled cells and, in adidtion, indicated that DXS and PS slow down circulation of lymphocytes through the marginal zone and red pulp of the spleen and, in the case of DXS, in the pulmonary capillary bed. Unusually large numbers of unlabeled lymphocytes were found in the endothelial wall of the post-capillary venules in lymph nodes of PS-treated mice.     

The roles of phenotype and position in guiding the fate of 16-cell mouse blastomeres

Newly formed 16-cell blastomeres were typed as larger or smaller, labelled with the short-term lineage marker FITC, and aggregated in various spatial arrays with 15 other age-matched unlabelled 16-cell blastomeres. The aggregates were cultured for 8 or 24 hr and the fluorescently labelled progeny identified. In all but 6 of 185 cases, the progeny developed as a physically coherent patch. Labelled larger cells placed on the outside of the aggregate generated mainly trophectoderm; when placed on the inside or randomly they always generated at least one trophectodermal offspring and in some cases also contributed cells to the inner cell mass (ICM). Labelled smaller cells placed on the inside of the aggregate generated mainly ICM; when placed on the outside or randomly they generated cells in the ICM alone, in trophectoderm alone, or in both tissues. From these results we conclude that phenotype is of major importance in determining the fate of larger cells whereas position strongly influences the fate of smaller cells.     

Production of human/mouse hybridomas secreting a human lymphokine,osteoclast activating factor

A human/mouse hybridoma was developed which has the property of secreting a human bone resorbing factor similar or identical to the osteoclast activating factor (OAF) isolated from human tonsil lymphocytes. Mouse plasmacytoma cells negative for OAF production were fused with an enriched subpopulation of human tonsil lymphocytes that had been activated with phytohemagglutinin (PHA) to produce OAF (G. E. Nedwin, M. A. Mohler, and R. A. Luben, submitted for publication). Culture supernatants from mixed hybridomas contained a bone resorbing protein shown to cause the release of ⁴⁵Ca from previously labeled mouse calvaria. The bone resorbing activity from these hybridomas was inhibited by the presence of OAF-specific monoclonal antibodies. Several hybridomas retained OAF production following limited dilution cloning. One clone, CD6.20, showed a biphasic dose-response curve for bone resorption similar to that of purified OAF from PHA-activated human tonsil lymphocytes. OAF production in the CD6.20 cell line has been retained for over 100 passages. Karyotype analysis of this cell shows the presence of human chromosomes 10 and 18 and the X chromosome.     

Amphetamine inhibition of alpha-bungarotoxin binding at the mouse neuromuscular junction.

Amphetamine inhibited neuromuscular transmission and prevented the irreversible blockade produced by α-bungarotoxin (α-BGT) in the isolated mouse phrenic nerve-diaphragm preparation. Similarly, amphetamine (1.35 × 10^?4 to 3 × 10^?3M) inhibited the binding of ¹²⁵I-α-BGT to mouse hemidiaphragms in a concentration-dependent manner; (+)-amphetamine was found to be twice as potent as its (-)-isomer with respect to inhibition of ¹²⁵I-α-BGT binding. It is suggested that amphetamine binds to the nicotinic, cholinergic receptor of skeletal muscle and may produce weakness and paralysis in amphetamine overdosage.     

Proteases are mitogenic to mesenchyme in vivo : A study using the chick embryo chorioallantoic membrane

The chorioallantoic membrane (CAM) of the 9 and 10 day chick embryo has been used as a system to investigate the control of growth of mesenchymal tissue in vivo. The mesenchyme (but not the chorionic epithelium or allantoic epithelium) responded to the mitogenic stimuli of trypsin, activated macrophages and the secreted products of activated macrophages. The response to trypsin mimics the response of chick embryo fibroblasts in vitro. Under the conditions used, the increased labeling index was observed in CAM mesenchyme by 6 h (the shortest time assayed) and remained at this level for at least 24 h. This system could be used to evaluate the mitogenicity in vivo of other substances known to be mitogenic to fibroblasts in vitro.     

Glucocorticoids down-regulate the number of 1, 25-dihydroxyvitamin D3 receptors in mouse intestine

This study examined the hypothesis that glucocorticoids might regulate intestinal receptors for 1,25(OH)₂vitamin D₃. Mice were treated with saline vehicle or dexamethasone in doses of 50–200 μg/100 gm body weight for 7 days. A dose-dependent fall in the number of receptors was found in dexamethasone treated animals (～30%); there was no change in the receptor affinity for 1,25(OH)₂D₃ (0.2 nM). The decline in receptor number required 5–7 days of dexamethasone treatment depending on dose. Adrenalectomy led to a 30% rise in receptor number which could be prevented by the 50 μg dose of dexamethasone. Receptor changes of equal magnitude were exhibited in vitamin D replete or deplete mice.     

Synthesis and secretion of IgG in synchronized mouse myeloma cells

A mutant of the MPC-11 mouse myeloma cell line which grows as a monolayer has been used to study the synthesis and secretion of IgG in relation to the cell cycle. The mitotic detachment method has been used to obtain a pure population of mitotic cells which were then allowed to progress through the G1, S, and G2 phases of the cell cycle. The synthesis and the rate of secretion of IgG have been studied in each phase of the cycle by incubation of cells with ¹⁴C-amino acids, followed by immunoprecipitation and quantitation of synthesized and secreted IgG_2b. The data are consistent with the idea that synthesis and secretion of Ig are not a cell cycle dependent event in myeloma cells.     

The determination of purine levels in human and mouse plasma

Variable levels of acetic anhydride have been recommended for addition to one of two reagents used in the glyoxylic acid method for the determination of tryptophan. For use of this reagent immediately after preparation it was shown that a minimum of 16% (v/v) of acetic anhydride should be included in the formulation to obtain near-maximum sensitivity. It was further demonstrated that reagent formulations with and without acetic anhydride changed with exposure to light. The observed changes are manifest as changes in the relative sensitivities of the assay. Several modifications are recommended to improve the sensitivity and stability of the acetic anhydride-containing reagent in this assay.     

Increases in microtubule assembly and in tubulin content in mitogenically stimulated mouse splenic T lymphocytes

We have examined the changes in the microtubule and tubulin contents in populations of mouse splenic T lymphocytes stimulated by the mitogen concanavalin A. Indirect immunofluorescence staining with antiserum to tubulin indicated that a more extensive microtubule network was assembled from the centrosome in those cells which had increased in size in response to the mitogen. Direct counts of microtubules from electron micrographs of the centrosome regions of cells showed approximately a 2-fold increase in microtubule number in 48 h stimulated populations and up to a 5-fold increase in the large, fully stimulated, blast cells. Determinations of tubulin and actin contents were made by the measurement of peptides specific to those proteins. As a percentage of total cell protein both of these cytoskeletal proteins increased during the first 24 h of stimulation. Tubulin increased 50% by 24 h and remained high in populations stimulated for 48 h. The tubulin content per cell increased 2.5-fold, from 0.20 to 0.51 μg/10⁶ cells, in the 48 h stimulated population. An increase in tubulin content was also seen following the stimulation of nude mouse B lymphocyte populations and of total splenic lymphocyte populations. Our results show that during lymphocyte stimulation there is a large increase in the numbers of microtubules assembled which is correlated with, and appears dependent on, a similar large increase in the cellular tubulin content.     

A study of the evolution of repeated DNA sequences in primates and the existence of a new class of repetitive sequences in primates.

A new class of lowly repetitive DNA sequences has been detected in the primate genome. The renaturation rate of this sequence class is practically indistinguishable from the renaturation rate of single-copy sequences. Consequently, this lowly repetitive sequence class has not been previously observed in DNA renaturation rate studies. This new sequence class is significant in that it might occupy a major fraction of the primate genome.Based on a study of the thermal stabilities of DNA heteroduplexes constructed from human DNA and either bonnet monkey or galago DNAs, we are able to compare the relative mutation rates of repetitive and single-copy sequences in the primate genome. We find that the mutation rate of short, interspersed repetitive sequences is either less than or approximately equal to the mutation rate of single-copy sequences. This implies that the base sequence of these repetitive sequences is important to their biological function.We also find that numerous mutations have accumulated in interspersed repeated sequences since the divergence of galago and human. These mutations are only recognizable because they occur at specific sites in the repeated sequence rather than at random sites in the sequence. Although interspersed repetitive sequences from human and galago can readily cross-hybridize, these site-specific mutations identify them as being two distinct classes. In contrast, far fewer site-specific mutations have occurred since the divergence of human and monkey.     

Alternative mating strategies in a desert grasshopper: a transitional analysis

We describe between- and within-individual variation in signalling behaviour for a population of male Ligurotettix coquilletti in a North American desert. For each observation date individual males were categorized as actively-signalling or inactive by the amount and regularity of their stridulation and their signalling location. Few males were active or inactive on all dates they were observed. By comparing the behaviour of individuals on consecutive observation dates, we identified the primary ways that individuals changed between active signalling and inactive behaviour. Behavioural transitions were frequently associated with movement between bushes. Inactive males became active upon moving to a vacant bush, and lone active males often became inactive after moving to a bush containing other males. Male-male aggression (primarily chases involving no contact between the participants, or only very brief contact) had a pronounced, short-term effect upon the signalling behaviour of males that received an attack. Approximately 33% of these recipients that had been stridulating became silent immediately following the chase. However, chases did not markedly alter the behaviour of recipients on the following observation date. Recipients were no more likely to change from inactive to active behaviour than vice versa between the observation dates immediately preceding and following the day of the chase. Although the transition analysis failed to detect marked behavioural changes among recipients, analyses based on longer periods of time revealed that males observed to be recipients were more likely to display inactive behaviour and less likely to mate than males observed to be initiators of aggression.     

Cell surface antigens of totipotent mouse teratocarcinoma cells grown in vivo: their relation to embryo, adult, and tumor antigens.

Cell surface antigens on mouse embryonal carcinoma (or teratocarcinoma) cells were investigated by means of a syngeneic antiserum prepared against small-size embryoid bodies from the ascites form of the OTT 6050 transplantable teratoma. These embryoid bodies consist of embryonal carcinoma cells which are usually covered by a yolk-sac-like epithelium. The choice of immunogen was based on the previous demonstration [Mintz, B., and Illmensee, K. (1975) Proc. Nat. Acad. Sci. USA72, 3585–3589] that embryonal carcinoma cells from this specific source are euploid, developmentally totipotent, and completely reversible to normalcy. In indirect immunofluorescence tests, anti-embryoid-body serum reacted with both cell types of the immunogen and with two in vitro lines of embryonal carcinoma cells. Absorption of antiserum with a pure yolk sac carcinoma derived from the epithelial component of the embryoid bodies enabled assessment of reactivity with the embryonal carcinoma component of the immunogen: The absorption revealed that some antigens recognized on the embryonal carcinoma cells were shared by the yolk sac epithelial cells but that some antigens were present only on the embryonal carcinoma cells. The antigens were not shared by sperm, which failed to fluoresce with unabsorbed antiserum and were ineffective when tested as absorbents of antiserum reactivity against embryoid body target cells. Unfertilized eggs also failed to fluoresce. Preimplantation embryos gave immunofluorescence evidence of some antigens shared with embryonal carcinoma cells (and some with yolk sac cells) during cleavage, and in the blastocyst on both inner cell mass and trophoblast. Postimplantation embryos were also antigen-positive (at least through Day 6) in immunofluorescence tests on endoderm as well as ectoderm cells. Absorption of the antiserum with various normal adult tissues showed substantial cross-reactivity, especially with ovary and testis. Other tumors were tested, but only hepatoma cells grown in vitro were reactive, thereby indicating lack of any general tumor recognition in the antiserum. The above results with syngeneic immunizations demonstrate that known totipotent teratocarcinoma cells possess surface molecules which, while not universal on normal cells or tumors, are shared with many other tissues, including developmentally plastic cells of early embryos, developmentally restricted cells of later embryos, and various adult tissues. Immunofluorescence tests of cleavage-stage (Day 2) embryos from matings of $\text{+}\text{t}{}^{12}\text{×}\text{+}\text{t}{}^{12}$ heterozygotes, yielding 40% mutant $\text{t}{}^{12}\text{t}{}^{12}$ homozygotes lethal on Day 3, were uniformly positive on all the embryos, including mutants and normals. Therefore, under these conditions, no evidence was adduced to support the hypothesis that surface components required for normal early development might be coded by the wild-type allele of t¹².     

Differentiation of the uterus in preparation for gestation: a model for the action of progesterone

During the preimplantation stages of pregnancy, rising titers of progesterone alter the metabolism of the uterine endometrium to permit implantation of the blastocyst. In this model of progestational differentiation, it is proposed that endometrial pyridine nucleotide metabolism is a key target of progestogen action. The hormone may modulate NAD metabolism to promote NADP synthesis while inhibiting NAD breakdown to ADP ribose and nicotinamide. The result of such an action would impair uterine DNA synthesis and cell division, but provide increased NADP for coenzyme-limited synthetic processes and cytodifferentiation. As a result, the endometrium differentiates and becomes sensitive to decidual-inducing stimuli (the blastocyst). The decidual stimulus reverses the process by rapidly inhibiting NADP production, and by dramatically increasing poly ADP ribosylation of nuclear protein, thus facilitating DNA synthesis and the wave of cell division associated with the initiation of decidualization. The background information and evidence in support of this model are presented.     

X-chromosome activity in heterokaryons and hybrids between mouse fibroblasts and teratocarcinoma stem cells

To determine whether 2X-active cells contain factors capable of reactivating the inactive mammalian X chromosome, fibroblast lines, having a cytologically or genetically marked inactive X, were fused with 2X-active mouse embryos or ovarian teratocarcinoma stem cells. Fusions with 2–16 cell embryos were uninformative because no mitosis occurred in heterokaryons. Fusions with 2X-active teratocarcinoma cells, and screening for re-expression of alleles on the inactive X showed that reactivation did not occur with detectable frequency in heterokaryon. Hybridization of HPRT^?M. musculus × M. caroli cells with XO HPRT^? teratocarcinoma cells yielded hybrids with a frequency of >10^?6; these hybrids all expressed the Hpt allele on the inactive M. caroli X, but not the M. caroliGpd or Pgk. Late replication-banding studies of hybrids and 6-thioguanine-resistant revertants showed that the reactivated Hp⁺ allele was still located on the late replicating X. Similar results were obtained with hybridization of this line to 1X-active (male-derived) fibroblast lines, indicating that hybridization per se, rather than a specific factor contributed by the teratocarcinoma cell partner, was reponsible for the frequent localized derepression of the Hpt⁺ allele on the inactive X.     

Genetic control of cell-type-specific levels of mouse beta-galactosidase.

Tissue analyses of mouse β-galactosidase (BGS) activity indicate previously unobserved genetic complexities. The existing genotypic classification of Bgs^h (high-activity) vs Bgs^d (diminished-activity) alleles has been based exclusively on strain differences in brain [Felton, J., Meisler, M., and Paigen, K. (1974). J. Biol. Chem.249, 3267–3272]. However, it now appears that other tissues in a strain with “high BGS” in brain can be relatively low in activity, or that tissues in a “low-BGS” strain can be relatively high. In addition, different cell types within a tissue (e.g., exocrine vs endocrine pancreas) can vary in BGS activity independently of each other. A given tissue ranges, among strains tested here, between 1.8- and 3.6-fold over the activity of the lowest strain. These new results suggest that complex mechanisms may be involved in tissue- and cell type-specific control of BGS activity. The hypothesis is proposed that there may be “controlling genes” determining specific enzyme levels by causing a limited somatic amplification of the structural gene and by controlling the degree of amplification in a given cell type and strain.