Subunit structure and higher order assembly of the hemocyanins of five members of the Naticidae family of marine gastropods: Calinaticina oldroydii (Dall), Euspira heros (Say), Neverita duplicata (Say), Polinices draconis (Dall), and Polinices lewisii (Gould)

1. The hemocyanins of the Naticidae family, E. heros, N. duplicata, P. draconis, P. lewisii and C. oldroydii were investigated by sedimentation velocity and scanning transmission electron microscopy. 2. At pH 8.0, 0.05 M Mg2+ E. heros hemocyanin is found to be predominantly in the tri-decameric state with a sedimentation coefficient (So20,w) of 131.3 (+/- 0.6) S. While the hemocyanin of N. duplicata is also mainly in the 130 S form, the hemocyanin of C. oldroydii is largely in the di-decameric form with a sedimentation coefficient close to 100 S. Other Naticidae hemocyanins, those of P. lewisii and P. draconis, have mixtures of the 100 S and 130 S di- and tri-decamers, and minor amounts of 150 S and faster sedimenting components. 3. The average particle masses based on STEM measurements are 8.85 x 10(6), 1303 x 10(6), and 17.1 x 10(6) da for the di-, tri-, and tetra-decameric assemblies of hemocyanin. 4. The subunit mol. wts of C. oldroydii hemocyanin and the published values for E. heros hemocyanin at alkaline pHs and in the presence of 8.0 M urea range from 4.2 x 10(5) to 4.8 x 10(5), suggesting the same decameric organization of the sub-assemblies of the Naticidae hemocyanins as for other molluscan hemocyanins. 5. The appearance of the larger hemocyanin particles in the electron micrographs support the hypothesis for their assembly that was based on similar studies of the hemocyanins of the Melongenidae family. According to this scheme the formation of higher aggregates is accomplished by the tail-to-head addition of each decameric unit to a central di-decamer which itself has the tail-to-tail Mellema and Klug arrangement of decamers. In this model all the higher aggregates terminate from either end with the same "collar" ends.     

Yeast myosin heavy chain mutant: maintenance of the cell type specific budding pattern and the normal deposition of chitin and cell wall components requires an intact myosin heavy chain gene

Recent studies with myosin heavy chain mutants in the slime mold Dictyostelium discoideum and the yeast Saccharomyces cerevisiae indicate that the myosin heavy chain gene is not essential for cell survival under laboratory growth conditions. However, cells lacking a normal myosin heavy chain gene demonstrate substantial alterations in growth and cell division. In this study, we report that a disruption mutant in the rod portion of the yeast myosin heavy chain gene, MYO1, produces abnormal chitin distribution and cell wall organization at the mother-bud neck in a high proportion of dividing cells. It is suggested that this phenotype is the cause of the cell division defect and the osmotic sensitivity of yeast MYO1 mutants. In the absence of a normal MYO1 polypeptide, yeast cells alter their cell type specific budding pattern. It is concluded that an intact myosin heavy chain gene is required to maintain the cell type specific budding pattern and the correct localization and deposition of chitin and cell wall components during cell growth and division.     

Purification and characterization of the restriction endonuclease RsrI, an isoschizomer of EcoRI

Rhodobacter sphaeroides strain 630 produces restriction enzyme RsrI which is an isoschizomer of EcoRI. We have purified this enzyme and initiated a comparison with the EcoRI endonuclease. The properties of RsrI are consistent with a reaction mechanism similar to that of EcoRI: the position of cleavage within the -GAATTC-site is identical, the MgCl2 optimum for the cleavage is identical, and the pH profile is similar. Methylation of the substrate sequence by the EcoRI methylase protects the site from cleavage by the RsrI endonuclease. RsrI cross-reacts strongly with anti-EcoRI serum indicating three-dimensional structural similarities. We have determined the sequence of 34 N terminal amino acids for RsrI and this sequence possesses significant similarity to the EcoRI N terminus.     

Cervical versus intrauterine insemination of ewes using fresh or frozen semen diluted with aloe vera gel

This study was conducted at Belen de Escobar, Argentina, in March and April 1987. Experimental work on synchronization of estrus, deep-freeze conservation of ram semen and small fertility trials involving cervical and intrauterine (i.u.) insemination methods was undertaken. A total of 80 Corriedale ewes were used in seven insemination trials. Insemination trials were grouped into two experimental groups for comparison of 1) frozen semen diluted with an experimental extender and a control diluent inseminated cervically or i.u. in synchronized/superovulated ewes and 2) cervical insemination of fresh diluted or frozen semen in ewes inseminated at natural estrus or in ewes that were synchronized/superovulated. An overall ovulation rate of 8.7 +/- 0.5 was obtained by using a superovulatory regimen consisting of 3 mg Norgestomet implants and a total dose of 18 mg follicle stimulating hormone-pituitary (FSH-P). Numbers of ova recovered per ewe following superovulation ranged from 4.3 to 5.4. In experimental Group I, fertilization rates improved when laparoscopic intrauterine AI was used compared with cervical insemination (P<0.05). Fertility rates of i.u. and cervical insemination of frozen semen diluted with the experimental extender showed satisfactory fertilizing capacity. In experimental Group II, a lower number of fertilized ova were recovered from ewes inseminated with frozen semen (P<0.02), irrespective of their estrus manipulation.     

Sequence polymorphism of human complement factor H

Factor H is a major regulatory protein of the complement system. The complete cDNA coding sequence has been derived from overlapping clones, and a polymorphism at base 1277 has been characterized. In four clones there is a T at nucleotide 1277 and in two others there is a C. This T/C change represents a tyrosine/histidine polymorphism at position 384 in the derived amino acid sequence. Protein sequence studies on peptides generated by trypsin digestion of factor H, purified from pooled plasma from 12 donors, confirmed the presence of both tyrosine and histidine at this position. Tyrosine and histidine were observed in a ratio of 2 : 1, respectively, and therefore this polymorphism is likely to represent a sequence difference between the two most abundant charge variants, FH1 and FH2, of factor H.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Studies on the Tissue Distribution of the Puromycin-Sensitive Enkephalin-Degrading Aminopeptidases

An antiserum generated to the soluble form of the rat brain puromycin-sensitive enkephalin-degrading aminopeptidase was used to determine the tissue distribution of the soluble and membrane-associated forms of this enzyme. All tissues examined contained significant levels of the soluble enzyme form, with this enzyme accounting for greater than 90% of the arylamidase activity in brain, heart, and skeletal muscle. Native gel electrophoresis coupled with activity staining as well as inhibition studies were used to confirm the presence of this enzyme in various tissues. Serum was found not to contain this particular aminopeptidase. In contrast to the results obtained with the soluble enzyme form, brain was the only tissue found to contain the membrane-associated enzyme form. Although all tissues contained membrane-associated aminopeptidase activity only the brain enzyme could be maintained in solution in the absence of detergent. In addition, the brain membrane-associated enzyme could be distinguished from the membrane-associated aminopeptidase activity in other tissues on the basis of its sensitivity to inhibition by puromycin.     

Models of growth with density regulation in more than one life stage

Discrete-time models of growth of populations with nonoverlapping generations and density regulation in two life stages are studied. It is assumed that there is no delay in the effects of density. Assigning exponential, linear, or hyperbolic functions to describe the dependence of preadult survival and fecundity on density, nine models are obtained. The dynamics of the model resulting from using the exponential function to describe the density dependence of both preadult survival and fecundity is analyzed: for large values of the intrinsic rate of increase there may exist up to three equilibrium population sizes, two stable. This indicates that a life history with two episodes of density regulation can give origin to alternative stable states. The models are fitted to recruitment data from growth experiments of Drosophila laboratory populations obtained with the Serial Transfer System Type 2 (Ayala et al., 1973. Theor. Pop. Biol. 4, 331-356) and collected by other authors. The results of the fittings suggest that this recruitment data can be adequately described with the models.