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.     

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.     

Immunotargeting of daunomycin to localized and metastatic human colon adenocarcinoma in athymic mice

Summary A monoclonal antibody (designated SF25), which recognizes a protein antigen expressed on a large number of human colon carcinomas, was used for drug targeting. Daunomycin-antibody conjugates were prepared by two previously described procedures. In one, the drug was bound to the antibody through a spacer of small molecular mass (cis-aconitic acid), while in the other a dextran bridge served as the link between drug and antibody. High substitution rates of drug to antibody were obtained using the latter binding procedure. Both conjugates were tested in vitro against two human colon carcinoma cell lines, LS180 and KM-12. The efficacy of a daunomycin-dextran-SF25 antibody conjugate was tested against colon carcinoma LS180 tumors transplanted at different sites into athymic mice. The specific conjugate was significantly more inhibitory to a subcutaneous tumor growth than its components or their mixture. SF25 antibody alone showed antitumoral effects against all three forms of transplanted tumor tested, namely, local, metastatic or intrahepatic, whereas daunomycin, on its own, was effective only against the subcutaneous tumor. Binding of daunomycin to dextran partially improved its inhibitory activity against the metastatic tumor. The conjugate, daunomycin-dextran-SF25 antibody reduced the number of metastatic foci, increased the survival rate and delayed death. Yet against lymph node metastases it was not significantly better than a mixture of both constituents. However, results obtained with an intrahepatic tumor, a model that mimics the natural progression of the disease, resembled those described with the subcutaneous tumor. Daunomycin-dextran-SF25 antibody was significantly more effective than all components separately and than a mixture of drug and antibody, provided a highly drug-substituted conjugate was used.     

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.     

Plant oligoadenylates: enzymatic synthesis, isolation, and biological activities

An enzyme that converts [3H, 32P]ATP, with a 3H:32P ratio of 1:1, to oligoadenylates with the same 3H:32P ratio was increased in plants following treatment with human leukocyte interferon or plant antiviral factor or inoculation with tobacco mosaic virus. The enzyme was extracted from tobacco leaves, callus tissue cultures, or cell suspension cultures. The enzyme, a putative plant oligoadenylate synthetase, was immobilized on poly(rI) . poly(rC)-agarose columns and converted ATP into plant oligoadenylates. These oligoadenylates were displaced from DEAE-cellulose columns with 350 mM KCl buffer, dialyzed, and further purified by high-performance liquid chromatography (HPLC) and DEAE-cellulose gradient chromatography. In all steps of purification, the ratio of 3H:32P in the oligoadenylates remained 1:1. The plant oligoadenylates isolated by displacement with 350 mM KCl had a molecular weight greater than 1000. The plant oligoadenylates had charges of 5- and 6-. HPLC resolved five peaks, three of which inhibited protein synthesis in reticulocyte and wheat germ systems. Partial structural elucidation of the plant oligoadenylates has been determined by enzymatic and chemical treatments. An adenylate with a 3',5'-phosphodiester and/or a pyrophosphoryl linkage with either 3'- or 5'-terminal phosphates is postulated on the basis of treatment of the oligoadenylates with T2 RNase, snake venom phosphodiesterase, and bacterial alkaline phosphatase and acid and alkaline hydrolyses. The plant oligoadenylates at 8 X 10(-7) M inhibit protein synthesis by 75% in lysates from rabbit reticulocytes and 45% in wheat germ cell-free systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

In vitro selective effect of melphalan on human T-cell populations

Summary In vitro treatment with 2 g/2×10⁶ cells melphalan (l-PAM: l-phenylalanine mustard) significantly decreased the total number of T lymphocytes from peripheral blood (PBL) of healthy human donors and of the OKT₄ population (precursor suppressor/helper/inducer) T cells as defined by monoclonal antibodies OKT₃ and OKT₄, respectively. No changes in the OKT ₈ ⁺ lymphocyte population (cytotoxic/mature suppressor cells) were observed following the same treatment. Preincubation of PBL with l-PAM at concentrations that do not affect the rate of DNA synthesis in PHA-stimulated lymphocytes inhibited the generation of T suppressor lymphocytes by ConA, as shown by their effect on PHA stimulation. Treatment of allogeneic PBL with l-PAM had no effect on mature suppressor T cells already induced by Con A, as shown by incubation of PBL with l-PAM after incubation with ConA.