A common theme in the amino acid sequences of actin and many actin-binding proteins?

The amino acid sequences of several actin regulatory proteins have recently been determined. Do these proteins function by mimicking actin-actin interaction sites?     

Localization of a 24-kilodalton glycoprotein in adult Schistosoma mansoni using immunofluorescence and immunoelectron microscopy

Studies on schistosome protective immune responses have focused mainly on antigens of the parasite's syncytial surface. One of the characterized schistosome antigens, a 24-kDa glycoprotein, has been considered important in mechanisms of immune evasion by the parasites. In the present study, using affinity-purified antibodies to the 24-kDa protein for immunofluorescence and immunoelectron microscopy, we demonstrated an association of the 24-kDa antigen with the discoid bodies (the major syncytial inclusion bodies; DBs) and the surface membrane complex (most likely the apical plasma membrane) of adult Schistosoma mansoni. This is consistent with previous observations that the 24-kDa antigen appeared to be localized to the syncytial membrane and DB fractions. The present results also support the suggestion that the DBs are the precursor organelles of the apical plasma membrane.     

Effect of long-term hypophysectomy on ovarian follicle populations and gonadotrophin-induced adenosine cyclic 3',5'-monophosphate output by follicles from Booroola ewes with or without the F gene

Long-term (i.e. approximately 70 days) hypophysectomy led to a significant (P less than 0.05) reduction in ovarian weight but no reduction in the total number of antral follicles (greater than 0.1 mm in diameter). In hypophysectomized ++ Booroola ewes (N = 8) follicles were always less than or equal to 3 mm and in hypophysectomized FF Booroola ewes (N = 6) follicles were always less than or equal to 2 mm in diameter; in ewes of both genotypes follicles reached diameters which were approximately 40% of their predicted final size at ovulation. Under in-vitro conditions, follicles from the FF and ++ hypophysectomized ewes produced significant increases in cAMP within 1 h of exposure to gonadotrophins (P less than 0.05) although no genotypic differences in cAMP production were noted. We conclude that ovarian follicles in FF and ++ ewes have absolute requirements for pituitary hormone on reaching diameters of 2 mm and 3 mm respectively and that appreciable numbers of antral follicles in ewes of both genotypes remain responsive to pituitary gonadotrophins despite prolonged deprivation of these hormones.     

Platelet-activating factor and serum components from oestrous mice co-operate to mimic the activity of 'early pregnancy factor' in the rosette inhibition assay

When male mouse spleen cells were incubated with a combination of platelet activating factor (PAF, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and sera from female mice in oestrus, the cells displayed a markedly increased rosette inhibition titre (RIT) when subsequently tested in the rosette inhibition assay. Neither PAF nor oestrous mouse sera alone could induce this effect, the combined action was required. Lyso-PAF could not substitute for the PAF, nor could male mouse sera nor the sera from females in dioestrus or metoestrus substitute for the oestrous mouse serum requirement. Pro-oestrous mouse sera could replace oestrous mouse sera but were less effective in their dose-responses. Studies on the mechanism of action of the PAF and oestrous mouse serum components suggested that the PAF stimulated the production and release of soluble factors (termed S2 factors) which by themselves could induce increased RIT values when applied to fresh spleen cells. The PAF-stimulated cell populations were rendered refractory to the action of these S2 factors and did not display increased RIT values, unless oestrous mouse serum was added. This serum acted to reverse the refractory state, allowing the S2 factors to exert their effect, and so cells treated with PAF and oestrous mouse serum displayed increased RIT values.     

Avoidance of used endometrial sites by blastocysts of the bank vole, Clethrionomys glareolus

In bank voles blastocysts arising from mating immediately after parturition do not implant in used zones of the preceding pregnancy but in the regions between them. However, if mating is postponed until 12 or more days after parturition blastocysts do implant in used zones of the previous pregnancy. Implantations also occur at zones used two pregnancies previously. It seems likely that the absence of implantations at recently used zones of the uterus is determined by the uterus rather than blastocysts.     

Mapping of a putative surface-binding site of human coagulation factor XII

We have localized the binding epitope(s) of two murine monoclonal antibodies (B7C9 and P5-2-1) that were shown previously to inhibit the activation of human coagulation factor XII by negatively charged surfaces. A factor XII cDNA expression library in lambda gt11 was screened with antibody B7C9, and 16 immunoreactive bacteriophage were isolated. Fusion proteins from each of the recombinant phage were reactive with both monoclonal antibodies. Two of the phage cDNA inserts were found to code for amino acid residues -6-+31 and +1-+47 of factor XII, respectively, thereby defining the limits of the antigenic peptide to amino acids +1-+31. Each of the remaining 14 recombinant phage contained longer factor XII cDNA inserts that included sequences coding for the amino-terminal 31 amino acid residues. These results were confirmed by direct binding of antibody B7C9 to synthetic peptides containing amino acids 1-14 and 1-28 of factor XII. Further experiments with a set of nested peptides also indicated that amino acid residues 1-4 were essential but not sufficient for binding of B7C9 to the peptides. Hydrophobicity analysis of the amino-terminal region of plasma factor XII revealed a highly hydrophilic region between amino acid residues 5 and 15 that contained positively charged lysine residues at positions 8, 11, and 13. We conclude that a major epitope(s) recognized by monoclonal antibodies B7C9 and P5-2-1 is present in the amino-terminal 28 amino acids of factor XII. It is proposed that binding of these antibodies to factor XII blocks interaction of the positively charged region between residues 5 and 15 with negatively charged surfaces, thereby inhibiting activation.     

Functional consequences of alterations to amino acids located in the catalytic center (isoleucine 348 to threonine 357) and nucleotide-binding domain of the Ca2+-ATPase of sarcoplasmic reticulum

The sequence of 10 amino acids (ICSDKTGTLT357) at the site of phosphorylation of the rabbit fast twitch muscle Ca2+-ATPase is highly conserved in the family of cation-transporting ATPases. We changed each of the residues flanking Asp351, Lys352, and Thr353 to an amino acid differing in size or polarity and assayed the mutant for Ca2+ transport activity and autophosphorylation with ATP or P1. We found that conservative changes (Ile----Leu, Thr----Ser, Gly----Ala) or the alteration of Cys349 to alanine did not destroy Ca2+ transport activity or phosphoenzyme formation, whereas nonconservative changes (Ile----Thr, Leu----Ser) did disrupt function. These results indicate that very conservative changes in the amino acids flanking Asp351, Lys352, and Thr353 can be accommodated. A number of mutations were also introduced into amino acids predicted to be involved in nucleotide binding, in particular those in the conserved sequences KGAPE519, RDAGIRVIMITGDNK629, and KK713. Our results indicate that amino acids KGAPE519, Arg615, Gly618, Arg620, and Lys712-Lys713 are not essential for nucleotide binding, although changes to Lys515 diminished Ca2+ transport activity but not phosphoenzyme formation. Changes of Gly626 and Asp627 abolished phosphoenzyme formation with both ATP and Pi, indicating that these residues may contribute to the conformation of the catalytic center.