A nonradioactive method for isolating complementary DNAs endocing calmodulin-binding proteins

Calmodulin labeled with¹²⁵I or³⁴S has been used to screen expression libraries to isolate cDNAs encoding calmodulin-binding proteins (CBPs) from several eukaryotic systems. The use of radiolabeled calmodulin has, however, several disadvantages. We have developed a nonradiactive method to isolate cDNAs for CBPs using biotinylated calmodulin. Screening of a cDNA library in an expression vector with biotinylated calmodulin resulted in the isolation of cDNAs encoding CBPs. Avidin and biotin blocking steps, prior to incubation of the filters with biotinylated calmodulin, are found to be essential to eliminate the cDNAs that code for biotin-containing polypeptides. The cDNA clones isolated using this nonradioactive method bound calmodulin in a calcium-dependent manner. The binding of biotinylated calmodulin to these clones was completely abolished by ethylene glycolbis(\-aminoethylether)-N,N′-tetraacetic acid (EGTA), a calcium chelator. Furthermore, the isolated cDNAs were confirmed by probing the clones with³⁵S-labeled calmodulin. All the isolated clones bound to radiolabeled calmodulin in the presence of calcium but not in the presence of EGTA. The method described here is simple, fast, and does not involve preparation and handing of radiolabeled calmodulin. All the materials used in this method are commercially available; hence, this procedure should be widely applicable to isolate cDNAs encoding CBPs from any eukaryotic organism.     

Somatic embryogenesis and organogenesis inGuizotia Abyssinica

Summary Induction of somatic embryogenesis, shoot organogenesis, and subsequent plant regeneration in niger seem to be dependent on genotype, choice of explant, and composition of media growth regulators. Two distinct regeneration protocols have been developed for somatic embryogenesis and shoot organogenesis. Somatic embryogenesis was induced from epicotyls and cotyledonary explants (9 to 35%) (but not from hypocotyls and roots) in presence of 2,4-dichlorophenoxyacetic acid, 2,4,5-trichlorophenoxyacetic acid, and 2,4,5-trichlorophenoxypropionic acid. These embryos matured in MS medium containing Kinetin plus naphthalene acetic acid (NAA), Kinetin plus Zeatin, and Kinetin plus abscisic acid (ABA). Matured embryos could be germinated on LS and MS basal media without hormones. Non-embryogenic callus initiated on Linsmaier and Skoog’s (LS) medium from cotyledons of six different genotypes produced shoots (9 to 32%) on Murashige and Skoog’s (MS) medium fortified with 6-benzylaminopurine (BAP, 0.5 mg · liter^−1), and BAP (1 mg · liter^−1) plus NAA (0.1 mg · liter^−1). These shoots were rooted with 100% frequency by using indole-3-acetic acid or NAA and transferred successfully to the soil.     

v-myb blocks granulocyte colony-stimulating factor-induced myeloid cell differentiation but not proliferation.

To understand the effects of v-myb expression on mammalian hematopoietic cell differentiation, we have constructed a retroviral vector which can efficiently express v-myb gene product in mammalian cells. Infection of interleukin-3-dependent murine progenitor cell line 32D Cl3, which undergoes terminal differentiation to mature granulocytes in the presence of granulocyte colony-stimulating factor (GCSF), with this recombinant retrovirus does not abrogate its requirement of interleukin-3 for growth. However, expression of v-myb in these cells blocks their ability to differentiate in response to GCSF. Instead, the v-myb-infected cells proliferate indefinitely in the presence of GCSF. 32D Cl3 cells infected with empty vector carrying only the neomycin resistance gene responded to the addition of GCSF in a manner identical to that of the uninfected cells and underwent terminal differentiation into granulocytes. These results suggest that oncogenic forms of myb gene bring about transformation by blocking the differentiation signal derived by cytokines while promoting the proliferative signal transduction pathways.     

Synthesis of methyl 2,4-di-O-methyl-3-O-(2-O-methyl-alpha-L-rhamnopyranosyl)-alpha-L- rhamnopyranoside and methyl 2,4-di-O-methyl-3-O-[2-O-methyl-3-O-(2-O-methyl-alpha-L-fucopyranosyl)- alpha-L-rhamnopyranosyl]-alpha-L-rhamnopyranoside: di- and tri-saccharide segments of a phenolic glycolipid of Mycobacterium kansasii.

Syntheses of the title glycosides are described. The critical O-glycosylations were carried out in the presence of boron trifluoride etherate with a high degree of alpha-selectivity.     

Comparative analyses of human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vif mutants.

Virion infectivity factor (vif), a gene found in all lentiviruses, plays an essential role in virus replication in certain target cells. We examined the replication competence of the human immunodeficiency virus type 2 (HIV-2) vif mutant in different T-cell lines and primary cells in comparison with that of the HIV-1 vif mutant. Both mutant viruses were unable to replicate in peripheral blood-derived mononuclear cells but replicated with wild-type efficiency in certain T-cell lines, such as SupT1 and MOLT-4/8. These results confirm the importance of vif in the infection of relevant target cells and imply that some cellular factor(s) could compensate for vif function. However, HIV-1 and HIV-2 vif mutant viruses also show differential replications in other cell lines, suggesting either different threshold requirements for the same cellular factor(s) or the involvement of different factors to compensate for vif-1 and vif-2 functions. By cross complementation experiments, we showed that vif-1 and vif-2 have similar functions. Our studies further indicate the existence of two kinds of nonpermissive cells: H9 is unable to complement HIV-1 delta vif but is susceptible to a one-round infection with HIV-1 delta vif produced from permissive cells. In contrast, U937 is nonpermissive for HIV-2 delta vif produced from permissive cells but, once infected, is able to complement the delta vif function. In both types of nonpermissive cells, a step prior to proviral DNA synthesis is affected.     

Cryosurgery of the monkey (macaque) prostate. II. Apparent immunopathologic alterations following cryostimulation.

Previous investigation of the development of humoral immunologic responsiveness following cryostimulation of the monkey prostate up to 30 days postoperatively disclosed low to modest titres of antibodies to prostatic tissue antigens. In the present study, the possible occurrence and predominance of a cellular response and its ensuing immunopathologic effects on the prostate and other accessory sexual glands of these animals, not initially examined, together with further serologic evaluation and correlation of this cellular response with the presence of antiprostatic antibodies, has been made 41 to 90 days following freezing. A reduction in the size of the prostate observed following freezing was accompanied by what were suggestive of immunopathologic alterations. Alterations occurred principally in the caudal lobe, and were characterized by what appeared to be specific periacinar foci of lymphocytic infiltrates. These lymphocytes were observed to infiltrate onto the acinar epithelial cells with subsequent separation of epithelial cells from the basal lamina and epithelial cell destruction. Other observed alterations in the prostate consisting of stromal fibrosis, periodic presence of inflammatory cells, proliferation of regenerating glands, and squamous metaplasia were in consonance with previous histologic studies of the prostate following cryosurgery by others. Antiprostatic antibodies were present in only one of the seven animals evaluated at the time which these observations were made. The present preliminary observations provide evidence suggestive of the development of a specific cellular immunologic response following cryosurgery of the prostate. Fending confirmation of the study of a larger series of animals, these observations may be of potential significance in providing an explanation of reported cases of eradication of human prostatic carcinomas following cryotherapy.     

An active center tryptophan residue in dihydrofolate reductase: chemical modification, sequence surrounding the critical residue, and structural homology considerations.

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei contains three tryptophan residues and the amino acid sequence surrounding each tryptophan has been determined. Oxidation of one of these residues by N-bromosuccinimide at pH 6.5 can be correlated with the complete loss of enzymatic activity. Following denaturation in urea, the oxidized enzyme was alkylated with dimethyl(2-hydroxy-5-nitrobenzyl) sulfonium bromide. Based on amino acid analyses and absorbance measurements at 410 nm, 2.2 mol of hydroxynitrobenzyl groups was incorporated per mol of protein. Presumably, hydroxynitrobenzyl adducts are formed with the two nonessential tryptophans. From the amino acid compositions of the two major thermolytic peptides containing the hydroxynitrobenzyl label and the partial sequences of two cyanogen bromide peptides containing the tryptophans, it was deduced that tryptophan-5 and tryptophan-129 were modified and, therefore, by difference, tryptophan-21 is the functional residue which becomes oxidized. The amino acid sequence surrounding tryptophan-21 is -Leu-

-Trp-His-Leu-Pro-. In reductases from four other species, this region of the sequence is highly homologous; such a conservation in this vicinity of the primary structure may indicate a functional involvement. The proline residues at positions 20 and 24 may serve to position tryptophan-21 into the appropriate configuration for optimum substrate-binding interactions.     

Interferon-gamma-induced astrocyte class II major histocompatibility complex gene expression is associated with both protein kinase C activation and Na+ entry

Astrocytes can be induced by interferon-gamma (IFN-gamma) to express class II major histocompatibility complex (MHC) antigens. This study was undertaken to elucidate the intracellular signaling pathways involved in IFN-gamma induction of class II MHC. We examined the effects of Na+/H+ antiporter and protein kinase C (PKC) inhibitors on class II expression and Na+ influx in astrocytes. We found that amiloride and ethyl isopropylamiloride, inhibitors of Na+/H+ exchange, blocked IFN-gamma-induced class II gene expression. IFN-gamma stimulated Na+ influx, and this increased influx was inhibited by amiloride. Treatment of astrocytes with the PKC inhibitor H7 also blocked the increase in Na+ uptake induced by IFN-gamma, indicating that IFN-gamma-induced PKC activation is required for subsequent Na+ influx. IFN-gamma treatment produced an increase of total PKC activity, which was associated with a rapid translocation of PKC activity from cytosolic to particulate fraction. H7 and another PKC inhibitor, staurosporine, inhibited IFN-gamma-induced class II gene expression. However, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate, a potent PKC activator, did not affect class II expression. Taken together, our data indicate that both IFN-gamma-induced PKC activation and Na+ influx are required for class II MHC expression in astrocytes but that activation of PKC alone is not sufficient for ultimate expression of this gene.