The polyadenylated RNA of zoospores and growth phase cells of the aquatic fungus,Blastocladiella

The stored poly(A) + RNA from zoospores of the aquatic fungus Blastocladiella emersonii represents 2.5% of the total RNA and has a model MW of 425,000 daltons and an average poly(A) isostich of 32 bases. The poly(A) + RNA also represents 2.5% of the total RNA from early growth phase cells and has a modal MW of 360,000 daltons and an average poly(A) isostich of 38 bases. The poly(A) + RNA from spores and 2-hr plants contains a structure resistant to RNases T₁, T₂, and A, which can be labeled with ³²PO₄ and which will bind to DBAE-cellulose. These characteristics strongly suggest that both the zoospore poly(A) + RNA and the 2-hr cell poly(A) + RNA are capped at the 5′ end; and, hence, it is unlikely that capping is involved in the control of protein synthesis during germination.Approximately 80% of the poly(A) + RNA of the spore is located in the membrane-enclosed ribosomal nuclear cap, and more than 90% of the poly(A) + RNA within the cap is found in the 80S monoribosome and heavier fractions.Synthesis of new poly(A) + RNA occurs very early during zoospore germination, and the labeled poly(A) + RNA rapidly enters the newly organized polysomes. The labeling data for early germination also suggest that cytoplasmic polyadenylation occurs.     

Maltosylamine, a specific inhibitor of beta-amylase.

β-Maltosylamine has been synthesized for the first time. It is an effective specific inhibitor of sweet potato β-amylase. This result extends the observation that 1-aminoglycosides are specific inhibitors of exoglycosidases which hydrolyze the corresponding glycose and also demonstrates that an enzyme acting with inversion, as well as those acting with retention of anomeric configuration, can be inhibited by glycosylamines. Maltosylamine, which acts as a reversible inhibitor of β-amylase, appears to be directed to the active site since it protects the essential sulfhydryl group of the enzyme from inactivation by N-ethylmaleimide.     

Analysis of migratory behavior of neural crest and fibroblastic cells in embryonic tissues

The precise migration of neural crest cells is apparently controlled by their environment. We have examined whether the embryonic tissue spaces in which crest cells normally migrate are sufficient to account for the pattern of crest cell distribution and whether other migratory cells could also distribute themselves along these pathways. To this end, we grafted a variety of cell types into the initial crest cell migratory pathway in chicken embryos. These cell types included (a) undifferentiated neural crest cells isolated from cultured neural tubes, intact crest from cranial neural folds, and crest derivatives (pigment cells and spinal ganglia); (b) normal embryonic fibroblastic cells from somite, limb bud, lateral plate, and heart ventricle; and (c) a transformed fibroblastic cell line (Sarcoma 180). Crest cells or their derivatives grafted into the crest migratory pathway all distributed normally, although in contrast to the result when neural tubes were graftedin situ, fewer cells were observed in the epithelium and few or none were localized in the nascent spinal ganglia. Grafted quail somite cells contributed to normal somitic structures and did not migrate extensively in the chicken host. Other fibroblasts did not migrate along cranial or trunk crest pathways, or invade adjacent tissues, but remained intact at the graft site. Sarcoma 180 cells, however, distributed themselves along the normal trunk crest pathway. Cranial and trunk crest cells and crest derivatives grafted ectopically in the limb bud or somite also dispersed, and were found along the ventral migratory pathway. Fibroblastic cells grafted into ectopic sites again remained intact and did not invade host tissue. We conclude (1) that neural crest cells and their derivatives are highly motile and invasive in their normal pathway, as well as in unfamiliar embryonic environments; and (2) that the crest pathway does not act solely to direct neural crest cells, since at least one transformed cell can follow the crest migratory route.     

Sister chromatid exchanges in human cells and Chinese hamster cells: Evidence that the rate of sister chromatid exchanges is a function of ploidy

The frequency of sister chromatid exchanges (SCE) in the chromosomes of the diploidy and polyploidy of Chinese hamster cells and human cells has been studied using BUdR-DAPI (bromodeoxyuridine, 4′-6-diamidino-2-phenylindol) fluorescence. The rate of SCEs per cell under constant control conditions is in proportion to the ploidy levels. In addition, the frequency of SCEs observed in a given human chromosome (nos. 1) is also directly proportional to the number of such chromosomes presented in the cells. The mean of SCEs in human chromosome numbers 1 is very similar (0.46–0.48) for diploid, triploid, and tetraploid cells. The results suggest that the rate of SCEs is a function of cellular ploidy levels.     

Comparison of phospho- and dephospho-ATP citrate lyase

The dephospho- form of rat liver citrate lyase has been prepared by treating purified [³²P]-ATP citrate lyase with a partially purified phosphatase. A comparison of the properties of the phospho- and dephosphoenzyme has been performed. The pH optima were the same for both forms of the enzyme in four different buffer systems although the optimum values varied identically for both enzyme forms with the buffer. Both the phospho- and dephosphoenzymes show the same kinetic properties except for the K_m observed for ATP in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system where it was 54 μm for the phosphoenzyme and 292 μm for the dephosphoenzyme. The present study also indicates that both enzymes are cleaved by trypsin and lysosomal proteases in a similar manner. Both forms of the enzyme tend to associate with mitochondria to the same extent and both enzymes have identical temperature stability curves.     

Isolation of a high molecular weight glycoconjugate derived from the surface of S purpuratus eggs that is implicated in sperm adhesion

Sea urchin sperm–egg adhesion is mediated by bindin, a sperm surface protein that has lectin-like activity. Bindin agglutinates eggs, and this interaction has been shown to be inhibited by glycopeptides released from the egg surface by protease treatment. In this study, we report the purification and properties of such an egg surface glycoconjugate that may be involved in sperm adhesion. The glycoconjugate was partially purified by gel filtration and affinity chromatography on bindin particles. Upon gel filtration on Sepharose CL 4-B, the glycoconjugate elutes near the void volume, suggesting that it has a molecular weight in excess of one million. In addition, we have found that the egg surface glycoconjugate agglutinates bindin particles, indicating that it is multivalent. Carbohydrate analysis indicates that the glycoconjugate is composed primarily of fucosc, xylose, galactose, and glucose. This purified egg surface component is the most potent inhibitor of bindin-mediated egg agglutination yet described.     

Computer simulation of engulfment and other movements of embryonic tissues

Using a model proposed earlier by Goel et al. and a set of plausible motility rules, a computer simulation of engulfment of two or more intact embryonic tissues is successfully carried out. The same motility rules are used to simulate the rounding up of a tissue, centralization of one tissue within another tissue (a phenomenon not yet observed), and phase inversion, a process which may have relevance to differentiation. The finnal structures bear a good resemblance to those observed experimentally. The software, in conjunction with an appropriate hardware configuration, allows a visual display of the dynamics of cellular movement. These simulations indicate that the range of inter-cellular interactions controlling these tissue rearrangements extends only one or two cell diameters.     

Primary in vitro antibody response of rabbit lymphoid cells and T-B cell collaboration in the absence of detectable mitogens

To investigate whether the antibody response and T-B-cell collaboration in vitro can be obtained in the absence of mitogens, a method of obtaining an in vitro primary anti-sheep red blood cell antibody response by rabbit spleen and lymph node cells was developed. We used Marbrook culture vessels and a specially prepared medium containing 10% autologous serum and maintained at pH 7.4–7.6. The system was shown to be devoid of any polyclonal mitogens as assessed by [³H]thymidine incorporation and by direct examination for blast cells in stained smears. The primary response increased continuously over the 5-day cultivation period and only IgM but not IgG plaque-forming cells (PFC) were detected. In over 20 experiments, the response ranged from 357 ± 17 to 4425 ± 110 PFC/10⁷ cultured cells with a median stimulation index of 52. The spleen cells required less antigen than the lymph node cells and 2-mercaptoethanol inhibited the response of the spleen cells but not that of the lymph node cells. Lymphocytes were separated into highly pure T- and B-cell populations by negative selection using antibody-coated human erythrocytes to rosette either T or B cells and Ficoll-Hypaque centrifugation to remove rosetted cells. Upon cultivation, B cells alone gave a low IgM response, whereas B cells reconstituted with T cells gave a response similar to that obtained with unseparated lymphoid cells. We concluded that: (a) optimal conditions for obtaining primary in vitro antibody responses using rabbit spleen and lymph node cells were established, (b) T-B-cell collaboration was demonstrated in the rabbit primary antibody response to sheep erythrocytes, and (c) the primary antibody response in vitro and T-B-cell collaboration may occur in the absence of detectable polyclonal mitogens.     

Simultaneous detection of 35S- and 32P-labeled proteins on electrophoretic gels

A new method for the determination of oxalic acid in urine, which does not require isolation of oxalic acid, was developed by derivatizing oxalic acid and separating and quantitating the product by automated liquid chromatography. Oxalic acid in urine was reacted with o-phenylenediamine to form the strongly uv-absorbing compound 2,3-dihydroxyquinoxaline. Isolation and quantitation of this derivative were accomplished using a reverse-phase C₈ column, 5% methanol in 0.1 m ammonium acetate buffer (pH 6.6) as eluant, and absorption at 314 nm. The method was linear from 1 to 151 μg oxalic acid/ml of sample and the conversion of oxalic acid to the dihydroxyquinoxaline over this concentration range was 94.9%. The precision of duplicates averaged ±1.1%. Analyses of urine before and after treatment with oxalate decarboxylase were employed to differentiate actual urinary oxalic acid from oxalogenic compounds. Under the conditions employed, no urine was found to contain inhibitors of oxalate decarboxylase. No significant contribution to the method was found in a study of 19 potentially interfering urinary constituents. Levels of oxalic acid found in 27 urine samples from patients by this method averaged 71% of levels found using an earlier colorimetric method.