Discovery of a new source of rifamycin antibiotics in marine sponge actinobacteria by phylogenetic prediction

Phylogenetic analysis of the ketosynthase (KS) gene sequences of marine sponge-derived Salinispora strains of actinobacteria indicated that the polyketide synthase (PKS) gene sequence most closely related to that of Salinispora was the rifamycin B synthase of Amycolatopsis mediterranei. This result was not expected from taxonomic species tree phylogenetics using 16S rRNA sequences. From the PKS sequence data generated from our sponge-derived Salinispora strains, we predicted that such strains might synthesize rifamycin-like compounds. Liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis was applied to one sponge-derived Salinispora strain to test the hypothesis of rifamycin synthesis. The analysis reported here demonstrates that this Salinispora isolate does produce compounds of the rifamycin class, including rifamycin B and rifamycin SV. A rifamycin-specific KS primer set was designed, and that primer set increased the number of rifamycin-positive strains detected by PCR screening relative to the number detectable using a conserved KS-specific set. Thus, the Salinispora group of actinobacteria represents a potential new source of rifamycins outside the genus Amycolatopsis and the first recorded source of rifamycins from marine bacteria

Production of N -acyl homoserine lactones by the sponge-associated marine actinobacteria Salinispora arenicola and Salinispora pacifica

The structures of acyl homoserine lactone (AHL) compounds and their quantification were accomplished using an integrated liquid chromatography-mass spectrometry approach. The precursor and product ions, along with retention times of peaks, were searched against an in-house database of AHLs and structures confirmed by accurate mass and by comparison with authentic AHL standards. The two compounds, N-(3-oxodecanoyl)-L-homoserine lactone and N-(3-oxododecanoyl)-L-homoserine lactone, were characterised and quantified in Salinispora sp. cultures

Regulation of STIM1 and SOCE by the Ubiquitin-Proteasome System (UPS)

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca2+ entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3′s), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca2+ homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function

Subcellular optogenetic inhibition of G proteins generates signaling gradients and cell migration

Cells sense gradients of extracellular cues and generate polarized responses such as cell migration and neurite initiation. There is static information on the intracellular signaling molecules involved in these responses, but how they dynamically orchestrate polarized cell behaviors is not well understood. A limitation has been the lack of methods to exert spatial and temporal control over specific signaling molecules inside a living cell. Here we introduce optogenetic tools that act downstream of native G protein–coupled receptor (GPCRs) and provide direct control over the activity of endogenous heterotrimeric G protein subunits. Light-triggered recruitment of a truncated regulator of G protein signaling (RGS) protein or a Gβγ-sequestering domain to a selected region on the plasma membrane results in localized inhibition of G protein signaling. In immune cells exposed to spatially uniform chemoattractants, these optogenetic tools allow us to create reversible gradients of signaling activity. Migratory responses generated by this approach show that a gradient of active G protein αi and βγ subunits is sufficient to generate directed cell migration. They also provide the most direct evidence so for a global inhibition pathway triggered by Gi signaling in directional sensing and adaptation. These optogenetic tools can be applied to interrogate the mechanistic basis of other GPCR-modulated cellular functions

A Fast and Efficient Hplc Method for the Separation of Chromium Complexes in Chrome Tanning Solutions

An ion-pair reversed phase HPLC method is described to quantitatively determine individual chromium complexes in chrome tanning solutions. Separations are achieved within 12-40 minutes in contrast to 6-7 hours with the currently used ion-exchange method. Sample sizes of about one microlitre, as opposed to 10 millilitres with the ion-exchange method, are adequate for a separation, thus producing minimum waste. As described below in detail, precautions are taken in order td minimise any perturbation to complex equilibria that exist in chrome liquor so that the true composition of chrome liquor is obtained. Since the analysis can be performed within a very short time, monitoring the composition of tanning solution with time is possible without having to freeze-dry samples from each stage. The method described here is extremely flexible and simple requiring only a basic HPLC system and a reversed phase column which is temporarily modified to an 'ion-exchange' mode during separations but which is regenerated to the reversed phase mode at the end of the separations

An Ion-Exchange Atomic-Absorption Method for the Measurement of Ionized Calcium and Magnesium At Micromolar Concentrations

A quantitative ion-exchange/atomic absorption method is described for measuring the concentration of free (hydrated) calcium and magnesium in solution at micromolar levels. Sample solutions are pumped through a micro-column of strong acid-type cation-exchange resin until equilibrium has been achieved between resin and solution. After removal of interstitial solution by first air, then water, the sorbed metal ion is eluted from the resin with nitric acid directly into an atomic absorption spectrophotometer. In a 0.1 M 1:1 electrolyte, here KNO3, the amount of metal ion sorbed on the resin is directly proportional to the free metal ion concentration in solution over a concentration range of 1.25 to 5 x 10(-5) mol/L (12-50 mumol/L). Selectivities for free calcium and magnesium in the presence of complexing ligands such as citrate and phosphate compare well with calculated values

Lysinoalanine determination in sodium caseinate using the LKB Alpha Plus Amino Acid Analyser

Adaptation of the LKB Alpha Plus Amino Acid Analyser to separate and detect lysinoalanine (LAL) in caseinates is described. The separation of LAL was achieved by cation exchange followed by post-column derivatisation with ninhydrin. The effect on the separation of varying the pH and molarity of the Na+ ion in the mobile phase and the column temperatures was investigated, and the information gathered from these tests was used to compile the optimum procedure for use on the Alpha Plus. An isocratic system using 0.2 M Na+ concentration in a citrate buffer with a pH range from 4.9 to 5.5 and column temperatures ranging between 48 and 56 degrees C was found to be the most suitable for the separation of LAL. Many ninhydrin-positive substances which elute close to the LAL peak are likely to cause inaccurate estimations of LAL concentration, and an awareness of the consistent profile of these in a caseinate is essential in the analysis. The method was validated by achieving a recovery of 100% (+/- 3%), limit of detection of 20 ppm and a repeatability relative standard deviation of 7%

A rapid saponification procedure for the simultaneous HPLC determination of the natural levels of vitamins A, E and beta-carotene in milk

A rapid saponification procedure was developed for the simultaneous extraction of vitamins A, E and beta-carotene in milk, prior to HPLC analysis. A high accuracy, sensitivity and ruggedness were achieved by the use of an internal standard, beta-tocopherol, which enabled the determination of the low levels of vitamins occurring naturally. Vitamin losses during extraction and the subsequent HPLC analysis were minimized by the use of antioxidants and by preconditioning the silica column with alpha-tocopherol in order to prevent irreversible adsorption of vitamin E on the column. The results compared well with those obtained from a non-saponification approach using a modified Rose-Gottlieb extraction. Relative standard deviations for repeatability of 4.9, 3.5 and 3.3% were achieved for vitamins A, E and beta-carotene respectively. The rapid, single extraction and simultaneous HPLC analysis used for the 3 vitamins made the method well suited for routine analytical work

Cholecalciferol: Properties and Determination

Cholecalciferol (vitamin D) is the common form of vitamin D synthesized in animals, and the equivalent form in plants is ergocalciferol (vitamin D). Cholecalciferol is produced from provitamin D in human skin, on exposure to ultraviolet (UV) light. Due to avoidance of sunlight because of skin cancer concerns, vitamin D deficiency has become common in recent times, and dietary intake has become important. The best natural sources of vitamin D are fish liver oils, and fortification of food with vitamin D is a common practice. Vitamin D is commonly determined using immunoassays or liquid chromatography with UV or mass spectrometric detection