DNA protection with the DNA methylase M . BbvI from Bacillus brevis var. GB against cleavage by the restriction endonucleases PstI and PvuII

BamHI fragments of the Bacillus brevis var. GB plasmid pAD1 have been cloned in Escherichia coli HB101 using pBR322 plasmid as a vector. The analysis of the recombinant plasmids showed that additional PstI sites had appeared in cloned fragments of pAD1. Methylation of the recombinant plasmids in vitro by enzymes from B. brevis GB cells blocks cleavage at these additional PstI sites of cloned pAD1 fragments and at the PstI site of pBR322. Among DNA methylases of B. brevis GB, the cytosine DNA methylase M . BbvI is the most likely agent modifying the recognition sequences of PstI. The methylase can modify cytosine residues in PstI or PvuII sites if these recognition sequences are linked to G at 5'- or to C at 3'-termini. In particular, in vitro methylation of the SV40 DNA by B. brevis GB methylases protects one of the two PstI sites and two of the three PvuII sites. The described effect of the protection of the specific PstI and PvuII sites may be used for physical mapping of genomes and DNA cloning.     

In vitro activation and behavior of the ameboid sperm of Ascaris suum (Nematoda)

Summary A system is described for the study of activation and motility of Ascaris spermatozoa in vitro. Activation was accomplished by addition of the sperm-activating substances (SAS), extracted from the male accessory gland, to cells incubated in phosphate-buffered saline (pH 7.4) at 37–39° C under anaerobic conditions (95% N₂, 5% CO₂). Activation is characterized by a change from spherical to ameboid shape with coalescence of the refringent granules. The normal ameboid spermatozoa bear several stubby and needle-like filopodia at the lamellipodial margin. Within the lamellipodium are bundles of microfilament-like structures extending toward the pseudopodial membrane and concentrating within the needle-like filopodia. These filopodia exhibit a pendulous, sweeping motion with subsequent retraction and disappearence within the main lamellipodium. Membranes of the ameboid cells interact at the pseudopodial regions with partial fusion, as suggested by apparent membrane breakdown between interdigitating portions of the pseudopodia. Activation is complete in 5–15 min, is totally inhibited at 4° C and/or by an atmospheric environment, but can be reinitiated by transfer to anaerobic conditions at 22–9° C. Activation also requires favorable pH (6.8–8.7) and continual exposure to sufficiently high sodium concentrations (134–154mM), i.e., lowering of sodium concentration to 10 mM causes irreversible inactivation. Sodium may be replaced by potassium or lithium but not by Tris or sucrose. Proteinases (10 g/ml) can act as activators even though SAS lack detectable proteolytic activity against azoalbumin, azocasein, TAME and BTEE and SAS activation was not inhibited by TLCK or soybean trypsin inhibitor.Adult Ascaris suum were provided through the generosity of Wilson and Company, Cedar Rapids, Iowa, U.SA. This study was supported by grant number 5T01 HD00152 and postdoctoral fellowship 1F 32AI05646 from the National Institute of Health, U.S.A.     

In vitro development of theDrosophila chorion in a chemically defined organ culture medium

Summary TheDrosophila chorion is produced normally in isolated follicles in Robb's chemically defined culture medium. The complex architecture of the shell developed in vitro from follicles as young as early stage 10 is completely normal morphologically. In addition, the time required for in vitro development closely approximates that observed for in vivo development. Comparisons of insect culture media developed by Robb, Grace, Schneider, and Echalier show large variations in their ability to supportDrosophila chorion development.     

Time and process of sperm penetration into cumulus-free mouse eggs fertilized in vitro

Sperm penetration through the zona pellucida and fusion of the sperm head with the vitellus were observed continuously and filmed under phase optics in cumulus-free living mouse eggs inseminated in vitro with capacitated epididymal sperm. Most spermatozoa penetrated the zona pellucida, traversed the perivitelline space, and fused with the vitellus at an angle nearly perpendicular to the surface. The mean duration required for sperm to penetrate the zona pellucida was 20 minutes with a range of 15–26 minutes. Sperm traversed the perivitelline space in less than one second. The initial contact of sperm with the vitellus generally took place at the tip of the sperm head. When the tip of the sperm head contacted the vitellus there was an immediate reduction in the rate of flagellation, followed by the gradual sinking of the sperm head into the vitellus.     

The cultivation of the exoerythrocytic stages ofPlasmodium berghei from sporozoites

Summary Cultures of embryonic rat brain and liver, and embryonic turkey brain were inoculated with sporozoites ofPlasmodium berghei. Sporozoites succeeded in establishing exoerythrocytic infections in approximately 10% of the cultures. The exoerythrocytic parasites developed to a late schizont stage with some showing early segmentation although free merozoites were not observed. The morphology and rate of development of the exoerythrocytic parasites in culture appear similar to that seen in vivo. This work was supported by ONR Contract No. N00014-76-C-1132 and Naval Medical Research and Development Command, Research Work Unit No. M0095PN.002.5058. the opinions and assertions contained herein are the private ones of the authors and are not to be construed as official or reflecting the views of the Navy Department or the naval service at large. The experiments reported herein were conducted according to the principles set forth in theGuide for the Care and Use of Laboratory Animals, Institute of Laboratory Resources, National Research Council, DHEW, Pub. No. (NIH) 74-23.     

Defined nutrient medium for the in vitro maintenance ofXenopus laevis oocytes

Summary A procedure is described for the isolation and culture of large numbers of follicle cell-freeXenopus laevis oocytes in all stages of development. The isolation procedure involves the incubation of pieces of ovary in a calcium-free solution OR2 containing 0.2% collagenase. A defined nutrient medium for the maintenance of the oocytes in vitro is presented. It is shown that this medium, referred to as DNOM, can maintain certain morphological and functional characteristics of oocytes for periods up to 3 weeks. Research supported by grant BMS 74-18790 from the National Science Foundation to JJE. Operated by Union Carbide Corporation for the U.S.E.R.D.A.     

The surface morphology of normal and malignant rat liver epithelial cells in culture

Summary A direct comparison has been made between normal parenchymal cells cultured from rat liver and malignant cells from both a rat liver tumor and a spontaneously transformed line derived from the parent rat culture. In all nondividing cells there was a 3- to 5-fold increase in the population of surface microvilli on the malignant cells compared to the normal cells. Notable variations in overall morphology were also observed when cells were incubated in arginine-deficient medium. During the course of this work T. D. Allen and P. T. Iype were supported by grants from the Medical Research Council and Cancer Research Campaign, and M. J. Murphy, Jr. was supported by a Special Fellowship of the Leukemia Society of America, Inc. and the Jean Shaland Fund.     

Serum Proteins Promoting [3H]Thymidine Uptake by Trypanosoma (Schizotrypanum) cruzi (Chagas) in vitro

SYNOPSIS. Five proteins capable of stimulating [³H]thymidine uptake by Trypanosoma cruzi in vitro were isolated from fetal calf serum by (NH₄)₂SO₄ precipitation and ion exchange column chromatography. The proteins were partially characterized by immunodiffusion, immunoelectrophoresis, polyacrylamide gel disc electrophoresis, and SDS electrophoresis. As estimated by SDS electrophoresis, using 4 standards, the molecular weight of protein 1 was 100,000, that of protein 2 was 76,000. and that of proteins 3–5 was 68,000 daltons.     

Effect of bioadctive glass templates on osteoblast proliferation and in vitro synthesis of bone-like tissue

Using in vitro synthesifzed bone tissue with cells aspirated fpom the patient's marrow is an appealing idea to avoid the profound limitations of biological of biologiaal and synthetic grafts. Procedures to synthesize bone tiqsue on vitro primapily relied on seeding various subqtpates with cellq that have osteogenia capacity in culture. It should be noted that in an in vitro system, msteoppogenitor cells, as well as bone themselves an papidiy change their phenotype, hence the substrate needs to promote the expression or the bone cell Phenotype. Furthermore, it needs to provide a template for bone deposition while gradually resorbing once bone tissue has been laid down. This paper presents initial evidence that optimally combines the requirements of the ideal template for in vitro synthesis of bone tissue. When made in popous dorm, and conditioned to detelop a bone-like surface prior to being seeded with pluripoteltial cells capable of expressing the osteoblastic phenotype, these templates lead to expeditious and a undalt in vitro synthesis of extracellular matrix with most important characteristics of bone tissue.     

In silico and pharmacological screenings identify novel serine racemase inhibitors

d-Serine is a coagonist of the N-methyl-d-aspartate (NMDA)-type glutamate receptor and its biosynthesis is catalyzed by serine racemase (SR). The overactivation of the NMDA receptor has been implicated in the development of neurodegenerative diseases, strokes, and epileptic seizures, thus, the inhibitors of SR have potential against these pathological states. Here, we have developed novel inhibitors of SR by in silico screening and in vitro enzyme assay. The newly developed inhibitors have lower IC₅₀ value comparing with that of malonate, one of the standard SR inhibitor. The structural features of novel inhibitors suggest the importance of central amide structure having a phenoxy substituent in their structure for the SR inhibitory activity. The present findings suggest the importance and rational development of new drugs for diseases of NMDAR overactivation.