Preparative Electrophoresis of Isotopically Labeled L-Cell Interferons

The preparative method of polyacrylamide gel electrophoresis was adapted for purification and characterization of isotopically labeled L-cell interferons. Re-covery of interferon activity was quantitative, and purification and resolution were comparable to those obtained by analytical polyacrylamide gel electrophoresis. Ultimate specific activities attainable ranged from 2 x 10(6) to 3 x 10(6) international units per mg of protein.     

Original Articles: Dynamic Redistribution of Isotopically Labeled Cohorts of Nitrogen Inputs in Two Temperate Forests

We compared simulated time series of nitrogen-15 (¹⁵N) redistribution following a large-scale labeling experiment against field recoveries of ¹⁵NH₄ ⁺ and ¹⁵NO₃ ⁻ in vegetation tissues. We sought to gain insight into the altered modes of N cycling under long-term, experimentally elevated N inputs. The study took place in two contrasting forests: a red pine stand and a mixed deciduous stand (predominantly oak) at the Harvard Forest, Massachusetts, USA. We used TRACE, a dynamic simulation model of ecosystem biogeochemistry that includes ¹⁵N/¹⁴N ratios in N pools and fluxes. We simulated input–output and internal fluxes of N, tracing the labeled cohorts of N inputs through ecosystem pools for one decade. TRACE simulated the peaks and timing of ¹⁵N recovery in foliage well, providing a key link between modeling and field studies. Recovery of tracers in fine roots was captured less well. The model was structured to provide rapid, initial sinks for ¹⁵NO₃ ⁻ and ¹⁵NH₄ ⁺ in both forests, as indicated by field data. In simulations, N in litter turned over rapidly, even as humus provided a long-term sink for rapidly cycling N. This sink was greater in the oak forest. Plant uptake fluxes of N in these fertilized plots were on the same order of magnitude as net assimilation fluxes in forest-floor humus. A striking result was the small rate of incorporation of N in humus resulting from the transfer of litter material to humus, compared with large fluxes of N into humus and its associated microorganisms through direct transfers from pools of inorganic N in soils. Received 19 May 1998; accepted 30 September 1998     

Bacteriochlorophyll and Heme Synthesis in Rhodopseudomonas spheroides: Possible Role of Heme in Regulation of the Branched Biosynthetic Pathway

Synthesis of heme, measured by incorporation of iron-59, and of bacteriochlorophyll was studied with wild-type and mutant strains of Rhodopseudomonas spheroides. The wild type formed heme from glycine and succinate at one-fortieth the rate of bacteriochlorophyll under anaerobic-light conditions. Added delta-aminolevulinate stimulated heme synthesis 10-fold without increasing bacteriochlorophyll production. Heme synthesis from glycine and succinate was increased when the magnesium branch of the biosynthetic path was curtailed by mutation or by p-fluorophenylalanine or 8-azaguanine. Synthesis of bacteriochlorophyll by the wild type from glycine and succinate stopped immediately after addition of puromycin, but heme production continued for a period. Porphyrins and other precursors did not appear upon addition of puromycin alone, but simultaneous addition of o-phenanthroline resulted in the accumulation of coproporphyrin. Production of this porphyrin by a mutant strain with impaired ability to form heme was unaffected by puromycin. Heme synthesis from glycine and succinate or from delta-aminolevulinate was decreased by limitation of methionine; it is suggested that coproporphyrin accumulation from glycine and succinate under conditions of methionine deficiency results from relief of feedback inhibition of delta-aminolevulinate synthase by heme. The development of delta-aminolevulinate synthase activity in response to low aeration is prevented by addition of delta-aminolevulinate. This repressive action of the latter is abolished when its conversion to heme is impeded by mutation or by methionine deficiency. It is suggested that heme, the quantitatively minor end product of the branched biosynthetic pathway, may regulate the flow of common intermediates when utilization of protoporphyrin by the magnesium branch is diminished. This regulation may be exerted by feedback inhibition of delta-aminolevulinate synthase and also by repression of enzyme formation.     

Structure of the Mitochondrial Aminolevulinic Acid Synthase,a Key Heme Biosynthetic Enzyme

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Preparation of Poliovirus Labeled with Phosphorus-33

Phosphorus-33 ((33)P), a weak (0.25 Mev) beta-emitting isotope of phosphorus with a half-life of 25 days, has been used to label poliovirus in cell culture. HeLa cell monolayers were depleted of phosphate and then labeled by incubating at 37 C in a medium (LM) containing about 10 muCi of (33)P as orthophosphate per ml. Labeled cells were infected at a high multiplicity with poliovirus type 1 and incubated for 8 hr in LM medium. Virus from infected cells was then concentrated and purified. Virus purity was confirmed by comparison of virus infectivity and radioactivity after CsCl density gradient centrifugation and by observing purified virus preparations with electron microscopy. With the method described, yields of about 10(10) to 5 x 10(10) plaque-forming units (PFU) of highly purified poliovirus with specific activities of about 3 x 10(-4) to 10(-3) disintegrations per min per PFU have been obtained from 1.5 x 10(8) to 3.0 x 10(8) HeLa cells.     

Preparation of Labeled Aflatoxins with High Specific Activities

Resting cells of Aspergillus parasiticus ATCC 15517 were used to prepare highly labeled aflatoxins from labeled acetate. High synthetic activity in growing cells was evidenced only during 40 to 70 hr of incubation. Glucose was required for high incorporation efficiency, whereas the concentration of the labeled acetate determined the specific activity of the product. When labeled acetate was continuously added to maintain a concentration near but not exceeding 10 mm, in a culture containing 30 g of glucose per liter, 2% of its labels could be recovered in the purified aflatoxins which have a specific activity more than three times that of the labeled acetate.     

Preparation of 15N Labeled Nucleosides and Large Scale Synthesis of Labeled Oligonucleotides with a New Type DNA-Synthesizer

Abstract

Microbiological and chemical methods for the preparation of ¹⁵N labeled nucleosides are described. Oligonucleotides are synthesized from the labeled nucleosides on a large scale by the phosphoramidite procedure using a self-developed DNA - Synthesizer. Preliminary ¹⁵N-NMR studies are reported.     

Preparation of Site-Specific Isotopically Labelled Zervamicins,the Antibiotic Peptaibols Produced by Emericellopsis Salmosynnemata

A simple procedure for the preparation of the specifically labelled peptide antibiotic zervamicins IC, IIA and IIB has been developed. The zervamicin molecules are labelled with stable isotopes by culturing the Emericellopsis salmosynnemata on a well-defined synthetic medium containing the highly isotopically enriched amino acid. To obtain the peptide with the specifically and highly enriched amino acid residue, precautions have been taken to prevent any de novo biosynthesis of the particular amino acid from unlabelled precursors. The enrichment of the labelled peptide is determined by mass spectrometric analysis. Following this method we have incorporated [2′,4′, 5′,6′,7′-²H₅]-L -Trp-1, [1′-¹⁵N]-L -Trp-1 and [2′, 3′,4′,5′,6′-²H₅]-L - Phl-16 into zervamicins IC, IIA and IIB on the preparative scale and without scrambling of the label. Thus, using the procedures described, isotopically labelled zervamicins can be prepared, allowing them to be studied by solid- state NMR.     

Reagents for the Preparation of Chromophorically Labeled Polyethylene Glycol-Protein Conjugates

We have developed a new class of reagents (2) for the covalent attachment of polyethylene glycol to proteins. These reagents (2) are the monomethoxypolyethylene glycol esters of 4-fluoro-3-nitrobenzoic acid. The reaction of 2 with lysine ε-amino groups produces a chromophore which can be used to quantitate the polyethylene glycol to protein molar ratio. Bovine (Zn, Cu) superoxide dismutase was used as a model protein for conjugation with 2. When monomethoxypolyethylene glycol of average molecular weight 2105 was used, a conjugate was obtained with a polyethylene glycol to protein molar ratio of 8.88 retaining 100% of native enzymatic activity; monomethoxypolyethylene glycol of average molecular weight 5210 yielded a conjugate with a polyethylene glycol to protein molar ratio of 9.96 retaining 73% of native enzymatic activity.     

A Simple Biosynthetic Method for Preparation of High Specific Radioactivity Phosphatidyl-choline

Chemical changes in lysozyme during heating at 150~250°C for 20min were investigated by means of IR, ESR, and CD spectroscopies and gel permeation chromatography, and further a tryptic hydrolysate from the lysozyme heated at 200°C was analyzed by ion exchange chromatography. At 150°C, polymerization through disulfide linkages was observed, and at180°C, both polymerization and degradation occurred. When the temperature was raised to 200°C, remarkable changes in the structure of lysozyme, such as cleavage and recombination of peptide bonds, occurred. Over 200°C, polymerization and degradation occurred more violently.     

Preparation of Labeled Casein fron Goat Milk after Lysine-14C Injection

Hydrogen peroxide in methylotrophic yeasts can be metabolized in at least two distinct ways. Addition of exogenous hydrogen peroxide removes the dependance of catalase on endogenously-produced hydrogen peroxide resulting enhanced rates of alcohol oxidation. Exogenous hydrogen peroxide is also efficiently degraded by cytochrome c peroxidase (CCP), a competitive reaction which does not result in enhanced alcohol oxidation. To overcome the influence of cytochrome c peroxidase, artificial peroxisomes were prepared by coimmobilization of alcohol oxidase and catalase. These artificial peroxisomes mimic the peroxide-induced rate enhancement observed with whole cells.     

酶标HBV DNA探针的研制与应用

本实验采用一种非放射性物质——碱性磷酸酶标记乙肝病毒HBV DNA制备分子探针。碱性磷酸酶在苯醌作用下与单链DNA联结,形成DNA和酶的共价复合物,即酶标探针。此探针通过分子杂交与待测DNA结合,与酶的底物作用显色,几小时内可观察结果,其最低检测量约为10pg。用此探针检测乙肝病人血清中的HBV DNA,与~(32)P标记的探针比较,酶标探针可检测出~(32)P标记探针检出率的95.7％。结果表明,所合成的酶标探针具有准确、简便、快速、安全而经济的优点,具有应用前景。     

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina ¹. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes ^{2 3 4} and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.Download video file.^{(77M, mov)}     

Biosynthetic polypeptide libraries

Methodological advances and new applications have fueled significant growth in the practice of polypeptide library screening.     

纳他霉素的生物合成基因研究

纳他霉素是一种多烯大环内酯类抗真菌抗生素,能专一地抑制酵母和霉菌,作为天然的防腐剂用于食品和饲料行业。概述了纳他霉素的化学结构,作用机理以及基因调控方面的研究,包括合成基因,修饰基因和调节基因。并展望了在纳他霉素基因工程研究方面的前景。     

Biosynthetic studies of spiroleptosphol

Studies of the biosynthesis of spiroleptosphol (1) revealed that 1 comprised a heptaketide (C1, C5–C10, and C12–C18 moiety), two methyl carbons (C19 and C20) from methionine, and a C₃ unit (C3, C4, and C11 moiety) derived through the TCA cycle.     

Differential Activation of Staphylococcus aureus Heme Detoxification Machinery by Heme Analogues

The reactive nature of heme enables its use as an enzymatic cofactor while rendering excess heme toxic. The importance of heme detoxification machinery is highlighted by the presence of various types of these homeostatic systems in Gram-positive and Gram-negative microorganisms. A number of pathogens possess orthologs of the HssRS/HrtAB heme detoxification system, underscoring a potential role this system plays in the survival of bacteria in heme-rich environments such as the vertebrate host. In this work, we sought to determine the role of this system in protection against metalloporphyrin heme analogues identified by previous studies as antimicrobial agents. Our findings demonstrate that only toxic metalloporphyrins maximally activate expression of the Staphylococcus aureus heme detoxification system, suggesting that the sensing mechanism of HssRS might require a component of the associated toxicity rather than or in addition to the metalloporphyrin itself. We further establish that only a subset of toxic metalloporphyrins elicit the oxidative damage previously shown to be a significant component of heme toxicity whereas all toxic noniron metalloporphyrins inhibit bacterial respiration. Finally, we demonstrate that, despite the fact that toxic metalloporphyrin treatment induces expression of S. aureus heme detoxification machinery, the HrtAB heme export pump is unable to detoxify most of these molecules. The ineffectiveness of HrtAB against toxic heme analogues provides an explanation for their increased antimicrobial activity relative to heme. Additionally, these studies define the specificity of HssRS/HrtAB, which may provide future insight into the biochemical mechanisms of these systems.