Arginine utilization by Mycoplasma fermentans is not regulated by glucose metabolism: A 13C-NMR study

Abstract Electrofusion of protoplasts of two mutant strains of Hansenula polymorpha resulted in high fusion and hybrid yields when the calcium ions present in the conventional fusion medium replaced by zinc ions. The optimal fusion conditions were an alignment field of 0.4 kV cm⁻¹ strength and 2 MHz frequency for 30 s, followed by two consecutive pulses of 12 kV cm⁻¹ strength and 15 μs duration. With 0.05–0.1 mM zinc ions in the fusion medium an average clone number of 10⁴–10⁵ clones per 10⁸ input cells was reached. The presence of about 0.6 mM magnesium ions in the zinc fusion medium was essential.     

Solid-Supported Oligonucleotide Systems for Special Biomedical Applications

Abstract

The use of composite beads consisting of a 6 μm polystyrene core with 30 nm surface-bound silica particles to routine automatic oligodeoxynucleotide (ODN) synthesis is described.     

RNA11 protein is associated with the yeast spliceosome and is localized in the periphery of the cell nucleus.

The yeast rna mutations (rna2 through rna10/11) are a set of temperature-sensitive mutations that result in the accumulation of pre-mRNAs at the nonpermissive temperature. Most of the yeast RNA gene products are involved in and essential for mRNA splicing in vitro, suggesting that they code for components of the splicing machinery. We tested this proposal by using an in vitro-synthesized RNA11 protein to complement the temperature-sensitive defect of the rna11 extract. During the in vitro complementation, the input RNA11 protein was associated with the 40S spliceosome and a 30S complex, suggesting that the RNA11 protein is indeed a component of the spliceosome. The formation of the RNA11-associated 30S complex did not require any exogenous RNA substrate, suggesting that this 30S particle is likely to be a preassembled complex involved in splicing. The RNA11-specific antibody inhibited the mRNA splicing in vitro, confirming the essential role of the RNA11 protein in mRNA splicing. Finally, using the anti-RNA11 antibody, we localized the RNA11 protein to the periphery of the yeast nucleus.     

Cross-Homologies and Structural Differences Between Human Cholinesterases Revealed by Antibodies Against cDNA-Produced Human Butyrylcholinesterase Peptides

To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BuChE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BuChE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BuChE polypeptides interacted in immunoblots with denatured serum BuChE as well as with purified and denatured erythrocyte AChE. In contrast, native BuChE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BuChE dimers from muscle extracts, but not BuChE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BuChE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BuChE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BuChE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.     

Synthesis of the diastereomers of thymidine glycol, determination of concentrations and rates of interconversion of their cis-trans epimers at equilibrium and demonstration of differential alkali lability within DNA.

5,6-dihydroxy-5,6-dihydrothymidine (thymidine glycol) is a major product of the reaction of thymidine with reactive oxygen species, including those generated by ionizing radiation. Thymidine glycol exists as 2 diastereomeric pairs by virtue of the chirality of the C(5) and C(6) atoms. A simple procedure is described for synthesizing and purifying each of the diastereomeric pairs separately. After brominating thymidine, the two trans 5-bromo-6-hydroxy-5,6-dihydrothymidine (thymidine bromohydrin) C(5) diastereomers were easily separated by High Performance Liquid Chromatography. Each thymidine bromohydrin was quantitatively converted to the corresponding diastereomeric thymidine glycol pair by reflux in aqueous solution. The concentrations at equilibrium of the cis (5S,6R),(5R,6S) and trans (5S,6S),(5R,6R) forms of the thymidine glycol diastereomers were determined and were 80% cis and 20% trans for the 5S pair and 87% cis and 13% trans for the 5R pair. At equilibrium, the rate of cis-trans epimerization of the two sets of diastereomers was essentially identical. The 5S diastereomeric pair was significantly more alkali labile than the 5R pair due to the higher concentration of the 5S trans epimer at equilibrium. This differential alkali lability was also manifest when the thymine glycol moiety was present in chemically oxidized poly(dA-dT).poly(dA-dT) indicating that the chemical differences between the diastereomeric pairs are preserved in DNA. These chemical differences may affect the biological properties of this important oxidative derivative of thymine in DNA.     

Bulb mite,Rhizoglyphus robini (Astigmata: Acaridae) as an experimental animal

Rhizoglyphus robini Claparède (Acari: Astigmata: Acaridae) is proposed as a model laboratory animal for biological, ecological, physiological and toxicological studies. The mite is easy and inexpensive to rear, quite fecund, convenient to manipulate, and may rapidly be raised to gram quantities. Examples are presented of its use in soil pest ecology and control studies, and in physiological, biochemical and toxicological investigations. Efforts to explore the induction of detoxification systems by various chemicals, and a demonstration of its control by solarization, are also described.     

Characterization of dimer subunits of intermediate filament proteins

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.     

Epidemiological trends of dermatophytoses and dermatophytes in Jerusalem between 1954 and 1981

Data for dermatophyte infections analysed for five 3-year periods between 1954 to 1981 led to the following conclusions: (1) Tinea pedis, tinea cruris and tinea manuum showed an increase in the 50's and 60's and declined in the 70's; (2) Tinea unguium and tinea corporis showed an increase during the whole period; (3) At all these sites, the percentage of Trichophyton rubrum, the main etiologic agent, increased steadily over the periods while the percentage of Trichophyton mentagrophytes, the secondary etiological agent, decreased. Epidermophyton floccosum, the third etiological agent in these sites, showed no sharp fluctuations; (4) These three dermatophytes which show similar microclimatic requirements and favour the same microecological niches, were called glabrohydrophilic. In tinea corporis they form a definite subset, their percentage being similar to that at other glabrous sites; (5) Tinea capitis was at its peak in the 50's, decreased sharply until the second half of the 70's, its main etiological agent being Trichophyton violaceum. Since 1979, an increase of tinea capitis occurred due to the newly introduced Microsporum canis; (6) Dermatophytes favouring scalp hair were called trichophilic. In tinea corporis they form a definite subset, their percentage being similar to that of tinea capitis; (7) A comparison with other studies from this country shows that macroclimate (i.e. humid warm coastal climate compared with dry cooler inland-mountain climate) is not an important factor in the etiology of tinea.     

Effect of melphalan in vitro on induction of murine suppressor T cells by ConA

Summary The effect of treatment with melphalan in vitro on the activity of spleen cells from BALB/c mice was investigated. Incubation of spleen cells with 1.5–5 g melphalan/1×10⁷ inhibited subsequent mitogenic stimulation by ConA or PHA and the allogeneic response of BALB/c spleen cells against C57B1 target spleen cells. Incubation of spleen cells with ConA led to induction of suppressor T cells which when added to fresh cultures inhibited the allogeneic response. Preincubation of spleen cells with melphalan even at low concentrations (0.15–0.5 g 1×10⁷ cells) which do not directly affect mitogenic stimulation or allogeneic response partially inhibited the generation of suppressor T cells by ConA. Treatment with melphalan had no effect on already induced suppressor T cells as shown by incubation of spleen cells with melphalan (0.15–5 g/1×10⁷ cells) after incubation with ConA. Addition of cells treated with melphalan alone (without ConA) to fresh cultures led to an increase in the allogeneic response.