Purification and characterization of actin from maize pollen

Pollen is an excellent source of actin for biochemical and physiological studies of the actomyosin system in higher plants. We have developed an efficient method to prepare relatively high levels of actin from the pollen of maize (Zea mays L.). The procedures of purification include acetone powder preparation, saturated ammonium sulfate fractionation, diethylaminoethyl-cellulose chromatography, a cycle of polymerization-depolymerization, and Sephacryl S-200 gel filtration. The average yield of actin is 19 milligrams per 100 grams of pollen grains extracted. This is comparable with those of Acanthamoeba castellanii and human platelets. The purified pollen actin is electrophoretically homogeneous and its molecular mass is 42 kilodaltons. The amino acid composition and circular dichroism spectrum of pollen actin are identical to those of muscle actin. The actin purified from pollen is able to polymerize to F-actin. The pollen F-actin activated the activity of the muscle myosin ATPase sevenfold.     

Infection of Anopheles darlingi fed on patients infected with Plasmodium vivax before and during treatment with chloroquine plus primaquine in Costa Marques, Rond?nia, Brazil.

Five patients with asexual and sexual parasites of Plasmodium vivax were treated orally with 600 mg chloroquine diphosphate (hour 0) followed with 300 mg at 8, 24 and 48 h later. Primaquine phosphate, 15 mg, was administered concurrently at h 0 and at 24 h intervals for 14 days. Anopheles darlingi were fed before the first dose (h -0.5) and 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 36, 48, 60 and 72 h later. Mosquitoes were examined for oocysts on day 8 and for sporozoites on day 15 after infection. Four of the five patients studied were still infective to mosquitoes from 1-5 h after the first dose of chloroquine plus primaquine. One of these and one other patient, who vomited 15 min after the first dose, became infective again at hours 10 and 12, respectively. Once produced, oocysts in mosquitoes fed on patients before, during and after chloroquine plus primaquine treatment appeared normal and produced sporozoite infected salivary glands. In view of these data, it is concluded that primaquine demonstrated rapid gametocytocidal activity and should be administered concurrently with chloroquine to reduce vivax malaria transmission.     

CD4-like molecules in human sperm

The expression of a molecule recognized by CD4 monoclonal antibodies was investigated on human sperm using immunolabelling, biochemical and immunochemical methods. Flow cytometry detected a significant fluorescence signal. SDS-PAGE analysis and Western blotting identified a molecule of 60 kDa, consistent with a CD4-like structure as confirmed after selective immunoseparation. Additional bands reacting with anti-CD4 were found in sperm extracts (73 kDa) and seminal fluid (90 kDa). These data indicate that sperm express a molecule similar to the receptor for HIV described on mononuclear cells.     

Evidence for allosteric coupling between the ribosome and repressor binding sites of a translationally regulated mRNA

Escherichia coli ribosomal protein S4 is a translational repressor regulating the expression of four ribosomal genes in the alpha operon. In vitro studies have shown that the protein specifically recognizes an unusual mRNA pseudoknot secondary structure which links sequences upstream and downstream of the ribosome binding site for rpsM (S13) [Tang, C. K., & Draper, D. E. (1989) Cell 57, 531]. We have prepared fusions of the rpsM translational initiation site and lacZ that allows us to detect repression in cells in which overproduction of S4 repressor can be induced. Twenty-five mRNA sequence variants have been introduced into the S13-lacZ fusions and the levels of translational repression measured. Sets of compensating base changes confirm the importance of the pseudoknot secondary structure for translational repression. An A residue in a looped, single-stranded sequence is also required for S4 recognition and may contact S4 directly. Comparison of translational repression levels and S4 binding constants for the set of mRNA mutations show that nine mutants are repressed much more weakly than predicted from their affinity for S4; in extreme cases no repression can be detected for variants with unchanged S4 binding. We suggest that the mRNA contains functionally distinct ribosome and repressor binding sites that are allosterically coupled. Mutations can relieve translational repression by disrupting the linkage between the two sites without altering S4 binding. This proposal assigns to the mRNA a more active role in mediating translational repression than found in other translational repression systems.     

Bulge loops used to measure the helical twist of RNA in solution.

Bulge loops are commonly found in helical segments of cellular RNAs. When incorporated into long double-stranded RNAs, they may introduce points of flexibility or permanent bend that can be detected by the altered electrophoretic gel mobility of the RNA. We find that a single An or Un bulge loop near the middle of a long RNA helix significantly retards the RNA during polyacrylamide gel electrophoresis if n greater than or equal to 2. The mobility of an RNA containing two A2 bulges various periodically with the number of base pairs between the bulges. We interpret this to mean that A2 bulges varies periodically with the number of base pairs between the bulges. We interpret this to mean that Z2 bulges form torsionally stiff bends in the helix; the gel mobility reaches a minimum when the total helical twist between the bulges rotates the arms of the molecule into a cis conformation. The gel mobilities are proportional to the predicted end-to-end distance of the RNA if the average RNA helical repeat is 11.8 +/- 0.2 bp/turn and there is no helical twist (3 +/- 9 degrees) associated with the bulge (data obtained in 0.15 M Na+). Other sizes and sequences of bulges have very different effects on RNA helix conformation and flexibility. U2 bulges bend the helix to a much smaller degree than A2 bulges, while longer A or U bulge sequences probably allow bends of 90 degrees or more; all of these may be fairly flexible joints.(ABSTRACT TRUNCATED AT 250 WORDS)     

Limited Multiplication of Symbiotic Cyanobacteria of Azolla spp. on Artificial Media

We examined various media and conditions to isolate symbiotic cyanobacteria from the leaf cavities of Azolla spp. Cyanobacteria survived and multiplied to a limited extent on a medium with fructose, Casamino Acids, yeast extract, and NaNO₃ under 1% O₂. These cyanobacteria were antigenically identical to the endosymbionts.     

Peptide accumulation during growth of peptidase deficient mutants

Mutant strains of Salmonella typhimurium simultaneously lacking peptidases N, A, B and D accumulate a heterogeneous mixture of small, trichloroacetic acid-soluble peptides during growth in minimal medium. Approximately 20% of the labelled leucine supplied to a growing culture of the mutant strain is converted to peptides. These peptides accumulate inside the cells before being released into the growth medium. Although the origin of these peptides has not been established, there are several processes that might contribute peptides to this pool. These include (1) turnover of signal sequences, (2) turnover of attenuator peptides, and (3) degradation of prematurely terminated proteins. These results indicate that the same family of peptidases that catabolizes exogenously supplied peptides and functions in carbon-starvation-induced protein turnover also hydrolyzes peptides generated during normal exponential growth.     

Kinetics of bacteriophage lambda repressor synthesis directed by the PRE promoter: influence of temperature, multiplicity of infection, and mutation of PRM or the cro gene

Summary Expression of the P _RE (establishment) pathway for repressor synthesis is regulated both by phage-specific genetic elements and by physiological conditions. Here we describe the effects of temperature, multiplicity of infection, mutations in the cro gene, and a mutation in P _RM on P _RE-directed repressor synthesis. As Reichardt (1975a) has shown, repressor synthesis begins 5–15 min after infection by wildtype phage, and is shut off at 20–30 min after infection, depending on the temperature. At 43°, synthesis starts sooner, shuts off earlier, and leads to lower repressor levels than are attained at lower temperatures. Experiments with the temperature sensitive mutant crots20 demonstrate that, as had been shown previously in experiments at 30° and 37° C, cro protein is responsible for the shut-off of repressor synthesis at 43°. In addition to the effects of temperature, the kinetics of repressor synthesis are strongly affected by multiplicity of infection (moi). At mois greater than 10, repressor synthesis after infection by wildtype at 30° is dramatically inhibited. Unexpectedly, the P _RM mutation prm116, under certain conditions, can alleviate both cro-mediated shutoff and the inhibition of P _RE-directed repressor synthesis at high moi. These effects of prm116 are observed only at low temperature (30°–32° C) and at mois of about 6–10 or greater; they also appear to be cis-specific. Possible mechanisms for the effects of the prm116 mutation are discussed. Finally, these studies demonstrate that crots20, which was isolated as a temperature-sensitive lethal mutation in the cro gene (Herskowitz, unpublished), is temperature-sensitive with respect to the ability to shutoff P _RE-directed repressor synthesis; however, even at low temperature (30° C), the crots20 gene product is only partially active.     

Evidence for cytosolic benzo(a)pyrene carrier proteins which function in cytochrome P450 oxidation in rat liver

Solutions of cytosolic proteins from rat liver contain benzo(a)pyrene solubilizing activity capable of serving as a carrier between solid state benzo(a)pyrene and microsomal cytochrome P₄₅₀. Fractionation of benzo(a)pyrene-saturated cytosolic proteins on a Sephadex G-100 column or by sucrose density gradients produced benzo(a)pyrene peaks of about 46,000 daltons and a very high molecular weight material. The protein-bound benzo(a)pyrene obtained in both peaks was oxidized rapidly by microsomes in the presence of NADPH, indicating that the benzo(a)pyrene carrier activity is capable of presenting the substrate to the cytochrome P₄₅₀. Liver cytosolic proteins from rats receiving intraperitoneal injection of [¹⁴C] benzo(a)pyrene was chromatographed on a column of Sephadex G-75. Radioactivity eluted at the same positions of the chromatogram as did the carrier activities described above. These results indicate that these benzo(a)pyrene carrier proteins may have an $\text{in}\text{vivo}$ role in the metabolism of benzo(a)pyrene.