Cholesterol Synthesis and Nerve Regeneration

Abstract: In this report, we examine the requirement of cholesterol biosynthesis and its axonal transport for goldfish optic nerve regeneration. Cholesterol, labeled by intraocular injection of [³H]mevalonolactone. exhibited a delayed appearance in the optic tectum. Squalene and other minor components were labeled but not transported. Following optic nerve crush, the amount of labeled cholesterol transport was elevated, while retinal labeling was not altered relative to control fish. A requirement for cholesterol biosynthesis is inferred from the inhibition of neurite outgrowth in retinal explants caused by the cholesterol synthesis inhibitor, 20, 25-diazacholes-terol. The inhibition of growth could be overcome by addition of mevalonolactone, but not cholesterol, to the medium. Intraperitoneal administration of 200 nmol of dia-zacholesterol resulted in 92-98% inhibition of retinal cholesterol synthesis and accumulation of labeled des-mosterol and other lipids in fish retina and brain which persisted for 2 weeks. Diazacholesterol-treated fish showed no reduction in the amount of lipid-soluble radioactivity transported following intraocular injection of [³H]mevalonolactone, but there were alterations in the chromatographic pattern of the transported labeled lipids. In contrast to its effects on neurite outgrowth in vitro , diazacholesterol did not inhibit optic nerve regeneration in vivo , as measured both by arrival of labeled rapidly transported protein at the tectum and by time required for the return of visual function.     

An RP4-prime containing a 285 kb fragment of rhizobium meliloti pSym megaplasmid: Structural characterization and utilization for genetic studies of symbiotic functions controlled by pSym

Summary We integrated the RP4 plasmid into a selected region of the pSym megaplasmid of Rhizobium meliloti 2011 by homologous recombination between pSym and a cloned fragment of pSym present in the RP4. This cointegrate was used to mobilize into Escherichia coli a Tn5 transposon located on pSym in the vicinity of the site of integration of the RP4. By this technique we obtained a series of RP4-primes that contained large fragments of the pSym megaplasmid and that were most probably generated by IS8 promoted deletions in the RP4-pSym cointegrate. One of them, pGMI42, which carries nitrogenase genes nifD and H as well as nodulation genes, was used for mutagenesis of the corresponding region of pSym after insertion of the Mu prophage into the tet gene. When various (pGMI-42:: Mu)::Tn7 were introduced into R. meliloti 2011 by conjugation, homologous recombination allowed insertion of Tn7 into pSym whereas the pGMI42::Mu was lost due to the suicide effect of Mu. In this way we obtained several symbiotic mutants deficient in either nodulation (Nod^-) or nitrogen fixation (Fix^-) in association with the host plant Medicago sativa.This paper is affectionately dedicated to the memory of Jean-Simon Julliot who initiated and inspired this work and who was killed by an avalanche on February 21, 1982     

The effect of ampicillin and tylosin on the faecal enterococci of healthy young chickens

Enterococcal isolates from young chickens were differentiated into one of three species, namely Enterococcus faecalis, Ent. faecium and Ent. gallinarum . The proportion of each species among the enterococcal population changed with time in birds not dosed with antibiotics. This pattern of change was modified in birds dosed with either tylosin or ampicillin even though ampicillin did not select for ampicillin resistance among the enterococcal population. A gradual increase in tylosin resistance was recorded with time among the enterococci of the 'undosed' control birds. This was associated with an increase in the proportion of Ent. faecium , a species commonly resistant to tylosin, among the enterococci of the birds as they grew older.     

Synthesis, biodistribution, and autoradiography of radiolabeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689)

35S- and 3H-labeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689) have been synthesized in our laboratory and used to study organ and cellular level distribution in C3H/Km mice bearing RIF-1 tumors. Tissue biodistributions obtained with 35S-WR-3689 showed that blood levels peak at 15 min postinjection and decline gradually over 60 min. At 30 min after drug injection the highest uptake is in kidney and submandibular salivary gland, with lowest levels in brain and moderate to low levels in the RIF-1 tumor, comparable to levels in skin and muscle. High resolution diffusible substance autoradiography with 3H-WR-3689 reveals a homogenous distribution of label over cells in liver and lung and nonuniform distribution of silver grains over the cytoplasm of cells in the kidney cortex, parotid and submandibular salivary glands, and small intestine. There are no indications of preferential nuclear location of label from protective drug in any tissue. Correlations of biodistribution and autoradiography data with measures of radioprotection in different tissues will be useful in interpreting mechanisms of radioprotection with this phosphorothioate.     

Mechanism of proton-pumping in the cytochrome b/f complex

Several models have been proposed to interpret the mechanism of proton-pumping associated with the electron transfer reactions in the cytochrome b/f complex. Energetics considerations suggest that the proton pump is coupled to the oxidation of cytochrome b by plastoquinone. Experiments performed in living cells under anaerobic conditions suggest that proton-pumping can occur through two independent mechanisms. When the two b cytochromes are reduced prior to a flash illumination i.e. after a long dark anaerobic incubation (>10 minutes), proton-pumping is very likely associated with the reduction of a semiquinone by cyt b which occurs at a site close to the inner face of the membrane. The electrogenic phase is associated with the tranfer of protons via a transmembrane channel. This process is not inhibited by 2-n-nonyl-4-hydroxyquinoline N-oxide (NQNO). Under repetitive-flash or under aerobic conditions, proton-pumping occurs according to a modified Q-cycle mechanism, which is inhibited by NQNO.Dedicated to Prof. L.N.M. Duysens on the occasion of his retirement     

A reinvestigation of the Gn-RH (gonadotrophin-releasing hormone) systems in the goldfish brain using antibodies to salmon Gn-RH

Summary The organization of Gn-RH systems in the brain of teleosts has been investigated previously by immunohistochemistry using antibodies against the mammalian decapeptide which differs from the teleostean factor. Here, we report the distribution of immunoreactive Gn-RH in the brain of goldfish using antibodies against synthetic teleost peptide.Immunoreactive structures are found along a column extending from the rostral olfactory bulbs to the pituitary stalk. Cell bodies are observed within the olfactory nerves and bulbs, along the ventromedial telencephalon, the ventrolateral preoptic area and the latero-basal hypothalamus. Large perikarya are detected in the dorsal midbrain tegmentum, immediately caudal to the posterior commissure. A prominent pathway was traced from the cells located in the olfactory nerves through the medial olfactory tract and along all the perikarya described above to the pituitary stalk. In the pituitary, projections are restricted to the proximal pars distalis. A second immunoreactive pathway ascends more dorsally in the telencephalon and arches to the periventricular regions of the diencephalon. Part of this pathway forms a periventricular network in the dorsal and posterior hypothalamus, whereas other projections continue caudally to the medulla oblongata and the spinal cord. Lesions of the ventral preoptic area demonstrate that most of the fibers detected in the pituitary originate from the preoptic region.     

Clastogen-induced chromosomal breakage as a marker for first trimester prenatal diagnosis of Fanconi anemia

Summary Using cultured trophoblast cells obtained by chorionic villus biopsy, we diagnosed Fanconi anemia (FA) in two pregnancies and excluded it in eight pregnancies at risk for the syndrome. Baseline chromosomal breakage and breakage induced by diepoxybutane (DEB) were analyzed. Increased breakage was used as a marker for the syndrome. Our results were unambiguous and provide a reliable method for prenatal detection of FA in the first trimester of pregnancy.     

Phase II study of vaccinia melanoma cell lysates (VMCL) as adjuvant to surgical treatment of stage II melanoma

Summary Patients with stage II melanoma were vaccinated with vaccinia virus-induced melanoma cell lysates (VMCL). The vaccine contained viable vaccinia virus, membranous fragments and no intact nuclei. A number of antigens defined by monoclonal antibodies were detected in the vaccine including the ganglioside GD3 and DR antigens. Administration of the vaccine was associated with depression of natural killer cell activity against melanoma and K562 target cells in the first 3–6 months of treatment. Leucocyte dependent antibody (LDA) activity against melanoma cells was induced or increased in titre in approximately half of the patients studied. Continued vaccination was associated in a number of patients with a decrease in LDA titres. Studies on a small sample of patients revealed that this was associated with the development of serum factors which inhibited LDA activity. LDA activity appeared directed to non-MHC antigens on melanoma cells which were of at least two specificities. One specificity which was shared with antigens on a number of non-melanoma carcinoma cells was removed by absorption on fetal brain and may be similar to oncofetal antigens described by other workers. Reactivity against melanocytes was induced in some patients and may underlie the development of vitiligo in several patients. These results suggest that vaccines prepared from VMCL may be a favourable method for increasing immune responses against melanoma.     

Enumeration of chlorine-stressed organisms with acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride (AOINT)

Direct microscopic enumeration ofEnterobacter cloacae with the acridine orange 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride technique (AOINT) was compared with spread plate counts on nonselective media to establish the usefulness of the former technique in the enumeration of chlorine-stressed cells. Results indicate that the techniques are comparable when the organisms are not stressed. However, AOINT is more sensitive than are plate counts in the detection of chlorine-stressed cells.