Protein-containing Media Research Articles

Composition-Dependent Protein-Material Interaction of Poly(Methyl Methacrylate-co-styrene) Nanoparticle Series.

Polymer nanoparticles continue to be of high interest in life science applications. Still, adsorption processes occurring in protein-containing media and their implications for biological responses are not generally predictable. Here, the effect of nanoparticle composition on the adsorption of bovine serum albumin (BSA), fibronectin (FN) and immunoglobulin G (IgG) as structurally and functionally different model proteins was explored by systematically altering the composition of poly(methyl methacrylate-co-styrene) nanoparticles with sizes in a range of about 550 nm. As determined by protein depletion from the suspension medium via a colorimetric assay, BSA and IgG adsorbed at similar quantities, while FN reached larger masses of adsorbed protein (up to 0.4 ± 0.06 µg·cm-2 BSA, 0.42 ± 0.09 µg·cm-2 IgG, 0.72 ± 0.04 µg·cm-2 FN). A higher content of styrene as the more hydrophobic polymer component enhanced protein binding, which suggests a contribution of hydrophobic interactions despite the particles exhibiting strongly negatively charged surfaces with zeta potentials of -44 to -52 mV. The quantities of adsorbed proteins were estimated to correspond to a confluent surface coverage. Overall, this study illustrated how protein binding can be controlled by systematically varying the nanoparticle bulk composition and may serve as a basis for establishing interfaces with a targeted level of protein retention and/or presentation.

Investigating the Impact of Shear Flow on Nanoparticle-Protein Interactions

Most nanoparticle-based therapies are intended for intravenous administration, exposing them to associated hemodynamic parameters and the presence of cells and biomacromolecules post-administration. While most efforts in nanomedicine development focus on formulation stability, the range of biologically-relevant approaches probing nanoparticle stability in biological media remains limited in scope. In the present study, we examine the role of surface chemistry in nanoparticle-protein interactions using three polystyrene latex nanoparticle chemistries. These nanoparticles were treated in media mimicking cell culture conditions, and the impact of static co-incubations versus flow on nanoparticle parameters was compared. Following treatment with protein-containing media, we performed an analysis of nanoparticle parameters using either the centrifugation-wash step or in-situ analyses to compare the effects of isolation protocols on nanoparticle physicochemical parameters. Overall, our findings show that flow and sample recovery methods significantly impacted the concentration and composition of surface-adsorbed proteins. Amine-modified latex nanoparticles showed the most pronounced susceptibility to flow and nanoparticle isolation techniques. The implications of this work lie in the development of more biologically-relevant and harmonized approaches in measuring the nanoparticle protein corona, since sample preparation techniques and analytical approaches used, may impact the translational scope and relevance of assays used to measure nanoparticle interactions with biological media.

Photoisomerization Neutralizes Vasoconstrictive Activity of a Heme Degradation Product.

Delayed cerebral ischemia (DCI) caused by cerebral vasospasm is the leading determinant of poor outcome and mortality in subarachnoid hemorrhage (SAH) patients, but current treatment options lack effective prevention and therapy. Two substance families of heme degradation products (HDPs), bilirubin oxidation end products (BOXes) and propentdyopents (PDPs), are elicitors of pathologic cerebral hypoperfusion after SAH. Z-configured HDPs can be photoconverted into the corresponding E-isomers. We hypothesize that photoconversion is a detoxification mechanism to prevent and treat DCI. We irradiated purified Z-BOXes and Z-PDPs with UV/Vis light and documented the Z–E photoconversion. E-BOX A slowly reisomerizes to the thermodynamically favored Z-configuration in protein-containing media. In contrast to vasoconstrictive Z-BOX A, E-BOX A does not cause vasoconstriction in cerebral arterioles in vitro and in vivo in wild-type mice. Our results enable a critical assessment of light-induced intrathecal photoconversion and suggest the use of phototherapy to prevent and cure HDP-mediated cerebral vasospasms.

Albumin-gated zwitterion-stabilized mesoporous silica nanorod as a pH-responsive drug delivery system

Silica nanoparticles as drug delivery systems (DDS) have received huge attention in nanomedicine research. However, their drug release processes were usually discussed in the absence of proteins, which are abundant in real delivery media and likely to affect the release behaviors. In this work, novel pH-sensitive silica-based DDSs were constructed using the endogenous protein, human serum albumin (HSA), as the gatekeeper, and their release properties in protein-containing media were measured for the first time. As-synthesized mesoporous silica nanorod (MSNR) was modified with zwitterions to suppress the nonspecific adsorption of protein and to improve the dispersity. HSA was fixed onto MSNR through drug-protein interactions and covalent bonds, respectively. Benzoic-imine bonds were introduced into the designs to realize pH responsiveness. The fluorescence quenching effect of drugs on HSA was used to monitor the drug release in simulated body fluids containing proteins. The results indicated that protein gating could effectively reduce premature release in normal blood environment. The endogenous protein capping, high dispersity and stability, considerable loading content, low cytotoxicity and favorable responsiveness to acidic microenvironment enable the present structures to be promising carriers for chemotherapy drugs.

Protein corona formed on silver nanoparticles in blood plasma is highly selective and resistant to physicochemical changes of the solution.

Nanoparticles (NPs) in contact with protein-containing media such as biological fluids rapidly acquire a surface layer of proteins, known as the protein corona. The protein composition and structural properties of the protein corona are crucial for NP interactions with living cells. Although much has been learned about the protein corona phenomenon, further elucidation could benefit from extensive quantitative proteomics analysis. Herein we report a comprehensive quantitative characterization (>350 proteins) of the corona that formed on 60 nm silver NPs via interaction with human blood plasma, as a function of pH and temperature. By varying the pH and temperature one can access different conformational spaces and charge localizations of the plasma proteins, which in turn provide knowledge pertinent to how the proteome corresponds to binding affinity. Thirty-eight percent of the quantified proteins bind at all temperatures, 47% at all pH values, and of these most persistent proteins, approximately 60% do not significantly change in abundance within the protein corona. Evaluation of 544 protein properties (present in the Kyoto databank) suggests that binding of these proteins to NPs is determined by the extent of hydrophobicity, β-sheet propensity, α-helical structure (and turns), and amino acid composition. Protein binding is promoted by a larger amount of β-sheets, higher hydrophobicity, and a smaller amount of α-helices. Our work enhances researchers' knowledge of a long-standing, vexing aspect of the nano-bio interface.

Hyaluronan-decorated polymer nanoparticles targeting the CD44 receptor for the combined photo/chemo-therapy of cancer.

In the attempt to develop novel concepts in designing targeted nanoparticles for combination therapy of cancer, we propose here CD44-targeted hyaluronan-decorated double-coated nanoparticles (dcNPs) delivering the lipophilic chemotherapeutic docetaxel (DTX) and an anionic porphyrin (TPPS₄). dcNPs are based on electrostatic interactions between a negative DTX-loaded nanoscaffold of poly(lactide-co-glycolide), a polycationic shell of polyethyleneimine entangling negatively-charged TPPS₄ and finally decorated with hyaluronan (HA) to promote internalization through CD44 receptor-mediated endocytosis. DTX/TPPS₄-dcNPs, prepared through layer-by-layer deposition, showed a hydrodynamic diameter of around 180 nm, negative zeta potential and efficient loading of both DTX and TPPS₄. DTX/TPPS₄-dcNPs were freeze-dried with trehalose giving a powder that could be easily dispersed in different media. Excellent stability of dcNPs in specific salt- and protein-containing media was found. Spectroscopic behavior of DTX/TPPS₄-dcNPs demonstrated a face-to-face arrangement of the TPPS₄ units in non-photoresponsive H-type aggregates accounting for an extensive aggregation of the porphyrin embedded in the shell. Experiments in MDA-MB-231 cells overexpressing the CD44 receptor demonstrated a 9.4-fold increase in the intracellular level of TPPS₄ delivered from dcNPs as compared to free TPPS₄. Light-induced death increased tremendously in cells that had been treated with a combination of TPPS₄ and DTX delivered through dcNPs as compared with free drugs, presumably due to efficient uptake and co-localization inside the cells. In perspective, the strategy proposed here to target synergistic drug combinations through HA-decorated nanoparticles seems very attractive to improve the specificity and efficacy of cancer treatment.

Characterization and compatibility of hydroxyethyl starch–polyethylenimine copolymers for DNA delivery

Hydroxyethyl starch (HES) has been proposed as a biodegradable polymer for shielding of DNA polyplexes, where the feasibility of this approach was shown both in vitro and in vivo. In this study, we report on the physicochemical characterization, the in vitro cytocompatibility and hemotoxicity of HES-decorated polyplexes. For this purpose, various HES molecules were coupled to a 22 kDa linear polyethylenimine (LPEI22) to produce a library of nine different HES–PEI conjugates. Particle analysis using dynamic light scattering showed that, neither the molar mass of HES nor the amount of HES in the polyplexes affected the particle diameter, as it was consistently around 70–80 nm. Imaging using atomic force microscopy and transmission electron microscopy showed that, both naked and HESylated polyplexes were in the same size range and had a spherical morphology. Meanwhile, the HES-mediated particle-shielding effect, manifested as reduction in the surface charge, strongly correlated with the molar mass of HES, where the charge decreased linearly with the increase in molar mass. Ethidium bromide binding assay showed that HES–PEI did not negatively affect DNA condensation at N/P ratios higher than 4. HES conjugation also showed a stabilizing effect against salt-induced particle disassembly, and particle aggregation in protein-containing media. Compatibility tests included cellular viability, as well as erythrocyte aggregation and hemolysis assays. HES–PEI conjugates showed lower cytotoxicity, no aggregation, and much lower hemolysis compared to unmodified PEI. In conclusion, these results show that the HES–PEI conjugates are promising gene delivery polymers with favorable physicochemical properties and compatibility profile.

The structural basis for peptide selection by the transport receptor OppA

Oligopeptide-binding protein A (OppA) from Lactococcus lactis binds peptides of an exceptionally wide range of lengths (4-35 residues), with no apparent sequence preference. Here, we present the crystal structures of OppA in the open- and closed-liganded conformations. The structures directly explain the protein's phenomenal promiscuity. A huge cavity allows binding of very long peptides, and a lack of constraints for the position of the N and C termini of the ligand is compatible with binding of peptides with varying lengths. Unexpectedly, the peptide's amino-acid composition (but not the exact sequence) appears to have a function in selection, with a preference for proline-rich peptides containing at least one isoleucine. These properties can be related to the physiology of the organism: L. lactis is auxotrophic for branched chain amino acids and favours proline-rich caseins as a source of amino acids. We propose a new mechanism for peptide selection based on amino-acid composition rather than sequence.

Daptomycin elimination by CVVH in vitro: evaluation of factors influencing sieving and membrane adsorption

Knowledge on the elimination of antibiotics by extracorporeal hemofiltration is a prerequisite for appropriate antimicrobial dosing in patients with renal failure. The present study set out to determine the clearance of the novel lipopetide antibiotic daptomycin from human whole blood by continuous venovenous hemofiltration (CVVH) in vitro. In addition, factors influencing daptomycin sieving and membrane adsorption were investigated. A recirculation model using different solvent media was established and daptomycin was added to the simulated blood circuit at varying concentrations. The concentration of daptomycin over time in the modelled blood compartment and the ultrafiltrate was measured by high performance liquid chromatography (HPLC). Mean Sieving coefficients (SCs) of daptomycin over time were calculated to 0.98 +/- 0.05, 0.33 +/- 0.02 and 0.40 +/- 0.03 at a baseline concentration of 60 microg/ml in Ringer lactate, Ringer lactate containing human albumin and in human whole blood, respectively. SCs of daptomycin in protein-containing media were higher than the free fraction in plasma of approximately 10%. Neither concentration of daptomycin nor addition of a second antibiotic showed significant impact on the SC. Adsorption of daptomycin to synthetic surfaces proved moderate and saturable, resulting in loss of around 20% of the amount initially added to the artificial blood circuit. In our in vitro setting the calculated clearance of daptomycin from whole blood exceeded the physiological clearance described for individuals with normal renal function. Investigation of clearance by CVVH in vivo seems necessary. Until sufficient clinical data are available for patients undergoing CVVH, monitoring of daptomycin concentrations in this population might be recommended in order to avoid sub-therapeutic exposure to daptomycin.

Daptomycin elimination by continuous venovenous hemofiltration: in vitro evaluation of factors influencing sieving and membrane adsorption

The present in vitro study set out to determine for the first time the continuous venovenous hemofiltration (CVVH) clearance of the novel lipopetide antibiotic, daptomycin, from human whole blood. Factors influencing daptomycin sieving and membrane adsorption were investigated in an in vitro setting. A recirculation model was established and daptomycin was added to the simulated blood circuit at different concentrations and in different solvent systems. The concentration of daptomycin over time in the modelled blood compartment and in ultrafiltrate was measured by HPLC. Mean sieving coefficients (SCs) of daptomycin over time were 1 ± 0.05, 0.3 ± 0.02 and 0.4 ± 0.03 in Ringer lactate, Ringer lactate containing human albumin and plasma, respectively. The CVVH clearance of daptomycin from whole blood exceeded the physiological clearance in an individual with normal renal function. Adsorption of daptomycin to synthetic surfaces proved moderate, resulting in loss of around 20% of the initial dose at 1 hour after the start of CVVH. Since mean SCs of daptomycin in protein-containing media were higher than the free fraction in plasma, our results suggest that besides protein binding, unknown factors such as intracellular drug partitioning influence sieving of daptomycin during hemofiltration. Due to the high in vitro CVVH clearance of daptomycin we determined from human whole blood, we recommend monitoring of daptomycin concentrations in patients undergoing hemofiltration. from 13th Scientific Symposium of the Austrian Pharmacological Society (APHAR). Joint Meeting with the Austrian Society of Toxicology (ASTOX) and the Hungarian Society for Experimental and Clinical Pharmacology (MFT) Vienna, Austria. 22–24 November 2007

Miniaturized Reference Electrodes. II. Use in Corrosive, Biological, and Organic Media

AbstractIn a recent paper [9] we reported the manufacturing and performance of miniaturized reference electrodes (MREs) with low sensitivity to chloride ions and pH. Here, we demonstrate the wide range applicability of a MRE based on an Ag/Ag2S internal reference element (IRE), imbedded in a photopolymerized hydrogel of improved composition, which contains the supporting electrolyte. Exchange current density, temperature coefficient, impedance value, and the voltammetric and potentiometric use of the Ag/Ag2S‐based MRE are discussed relative to the previously reported Ag/AgSCN, Ag/Ag3PO4, and Ag/AgCl‐based MREs. No special or extensive conditioning is required when moving these MREs from aqueous supporting electrolyte to an organic solution or from one organic medium to another, and the equilibration time in a new medium is very rapid (<6 min). The new Ag/Ag2S MRE has a highly stable potential in various media, including aqueous solutions (salt buffers and 20 wt.% H2SO4), biological samples (bovine serum albumin), mixed aqueous‐organic, and organic supporting electrolytes (methanol, ethanol, acetonitrile, propylene carbonate, methylene chloride, and DMSO). This is particularly advantageous when in the course of the electrochemical analysis an organic solution is being added to an aqueous supporting electrolyte. Such MREs are suitable for analyses of μL sample volumes and for use in protein‐containing media.

Assessment of cell concentration and viability of isolated hepatocytes using flow cytometry

The assessment of cell concentration and viability of freshly isolated hepatocyte preparations has been traditionally performed using manual counting with a Neubauer counting chamber and staining for trypan blue exclusion. Despite the simple and rapid nature of this assessment, concerns about the accuracy of these methods exist. Simple flow cytometry techniques which determine cell concentration and viability are available yet surprisingly have not been extensively used or validated with isolated hepatocyte preparations. We therefore investigated the use of flow cytometry using TRUCOUNT Tubes and propidium iodide staining to measure cell concentration and viability of isolated rat hepatocytes in suspension. Analysis using TRUCOUNT Tubes provided more accurate and reproducible measurement of cell concentration than manual cell counting. Hepatocyte viability, assessed using propidium iodide, correlated more closely than did trypan blue exclusion with all indicators of hepatocyte integrity and function measured (lactate dehydrogenase leakage, cytochrome P450 content, cellular ATP concentration, ammonia and lactate removal, urea and albumin synthesis). We conclude that flow cytometry techniques can be used to measure cell concentration and viability of isolated hepatocyte preparations. The techniques are simple, rapid, and more accurate than manual cell counting and trypan blue staining and the results are not affected by protein-containing media.

Inhibition of photosynthesis and bleaching of zooxanthellae by the coral pathogen Vibrio shiloi.

Vibrio shiloi is the causative agent of bleaching (loss of endosymbiotic zooxanthellae) of the coral Oculina patagonica in the Mediterranean Sea. To obtain information on the mechanism of bleaching, we examined the effect of secreted material (AK1-S) produced by V. shiloi on zooxanthellae isolated from corals. AK1-S caused a rapid inhibition of photosynthesis of the algae, as measured with a Mini-PAM fluorometer. The inhibition of photosynthesis was caused by (i) ammonia produced during the growth of V. shiloi on protein-containing media and (ii) a non-dialysable heat-resistant factor. This latter material did not inhibit photosynthesis of the algae by itself but, when added to different concentrations of NH4Cl, enhanced the inhibition approximately two- to threefold. Ammonia and the enhancer were effective to different degrees on zooxanthellae isolated from four species of coral examined. In addition to the rapid inhibition of photosynthesis, AK1-S caused bleaching (loss of pigmentation) and lysis of zooxanthellae. Bleaching was more rapid than lysis, reaching a peak (25% bleached algae) after 6 h. The factors in AK1-S responsible for bleaching and lysis were different from those responsible for the inhibition of photosynthesis, because they were heat sensitive, non-dialysable and active in the dark. Thus, the coral pathogen V. shiloi produces an array of extracellular materials that can inhibit photosynthesis, bleach and lyse zooxanthellae.

The Design of a Pharmaceuttcally Acceptable Liposomal Formulation of Recombinant Interleukin-2 (Ril-2) for Locoregional Anticancer Immunotherapy

Liposomes composed of egg-phosphatidylcholine and egg-phosphati-dylglycerol containing recombinant interleukin-2 (rIL-2) were prepared and characterised for use in locoregional anticancer immunotherapy. During the encapsulation studies it was observed that the protein precipitated. Therefore, the impact of the precipitation phenomenon on the characteristics and in vivo performance of rIL-2 liposomes was studied. Recombinant IL-2 was diluted in various aqueous media and the amount of precipitated protein determined. Also, the in vitro bioactivity, chemical stability, and in vivo antitumor efficacy of liposomes prepared with precipitated rIL-2 or non-precipitated rIL-2 were assessed.Massive protein precipitation (60 up ± 95% of total protein) was observed upon dilution of rIL-2 in salt-containing media, but not upon dilution in 5% glucose or water. Liposomes prepared with precipitated rIL-2 were shown to release 50% of the entrapped rIL-2 over a three-day period at 37° in protein-containing media. Loss of rIL-2 bioactivity and chemical integrity was observed during storage at 4° over a 4-week period. Locoregional administration of precipitated rIL-2 in the guinea pig Line 10 tumor model resulted in significantly more tumor growth inhibition than administration of non-precipitated rIL-2. Liposomes containing non-precipitated rIL-2 were found to elicit similar antitumor effects as precipitated rIL-2. The results point to the importance of proper characterisation of new rIL-2 formulations as the physical properties of formulated rIL-2 may strongly influence its bioactivity.

Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA

Plasmid-free and plasmid-harbouring E. coli JM109 strains were investigated in shaken flasks, stirred tanks in batch and continuous operation. The shaken flask cultivations were performed in M9 minimal medium and in media with various protein supplements. The host hardly grows on M9 minimal medium as opposed to the plasmid-harbouring cells, which grow well on this medium. All of the investigated cells propagate well on protein-containing media. The influence of the combinations of repressor plasmid pRK248cI, the protection plasmid EcoR4 and the production plasmid pMTC48 were determined on the initial specific growth rate of the E. coli JM109 without gene expression, on the yield coefficient of cell growth, acetate concentration and acetate yield coefficient in the yeast extract-containing (HM) medium. The influence of various media on the induction of the gene expression were evaluated. In cultivation media with protein supplement, the growth rate and yield coefficient increased. The variation of the volumetric and specific β-lactamase activities with the cultivation time were determined in a stirred tank reactor in HM medium. With increasing dilution rate the process performance decreased. Simple relationships exist between the substrate uptake rate and the specific growth rate of the continuous cultivated cells in M9 and HM media. The influence of the dilution rate on the cell mass concentration, colony forming units, acetate formation, yield coefficients of growth and acetate formation, substrate uptake rate, CO 2 production rate, ammonium formation rate and β-lactamase activity in M9 and HM media were determined as well. Carbon balances of the batch and continuous cultivations indicated high carbon recoveries. On account of the higher growth rate of plasmid-harbouring cells than that of the plasmid-free cells, the behaviour of the investigated plasmid-free and plasmid-harbouring E. coli JM109 cells deviates from the published properties of other plasmid-free and plasmid-harbouring E. coli cells.

Sequence of theCandida albicansgene encoding the secretory aspartate proteinase

The gene encoding the Candida albicans aspartate proteinase that is secreted by cells grown in protein-containing media was cloned from a C. albicans genomic bank. The base sequence of the insert shows a 1173 bp open-reading frame and indicates an amino acid sequence typical of aspartate proteinases, with amino acid sequence homology to other enzymes of this class and a putative signal peptide consisting of 50 amino acids upstream of the active enzyme.

Inhibitory effects of amitriptyline on the stimulation-induced Ca 2+ increase in parotid acini

The present study demonstrates the effects of the antidepressant, amitriptyline, and the acetylcholine antagonist, atropine, on the stimulation-induced rise in cytosolic, free Ca 2+ (Ca 2+). The changes in Ca i 2+ of collagenase-isolated rat parotid acini were measured by means of the Ca 2+ -sensitive dye, fura-2. It was found that stimulation by carbachol resulted in a maximal increase of 582 ± 34 nM (mean ± S.E.) in Ca i 2+ with a k S of 5.8 ± 1.3 μM. Adrenaline caused a rise of 380 ± 22 nM in Ca i 2+ with a k S of 0.5 ± 0.2 μM. Amitriptyline and atropine were found to inhibit the carbachol-induced rise in Ca i 2+ with dissociation constants (k I of 105 and 1.25 nM, respectively, in the absence of agonist. The adrenergic-induced rise in Ca i 2+ was inhibited by amitriptyline with a k I of 45 nM. Amitriptyline was found to inhibit both receptor classes by a competitive or mixed type of inhibition. Similarly, atropine exerted the same type of inhibition on the acetylcholine receptor. Amitriptyline and atropine were found to be mutually exclusive for competing for substrate binding on the receptor. These findings are consistent with a common binding site for amitriptyline and atropine on the acetylcholine receptor, possibly in close proximity with, but different from the substrate binding site. The stimulation-induced cell shrinkage evoked by the loss of electrolytes and water from the acini was measured by a 90° light scattering signal. It was found that this method makes possible the detection of autonomic side-effects of antideprassants on acini suspended in protein-containing media.

Automated high-performance liquid chromatographic extraction and quantification procedure for lipoxygenase metabolites

A number of methods have been used to measure various lipoxygenase metabolites in aqueous samples. These methods, however, suffer from three major limitations: first, they require extensive extraction and isolation from protein-containing media; second, mainly due to the first limitation, they have poor recoveries; and third, these methods usually require a two-step procedure, one for the actual extraction and the other for the quantification of the lipoxygenase metabolites. We have developed a fully automated high-performance liquid chromatographic method which circumvents these limitations. As a result, we are able to obtain high recoveries of various lipoxygenase metabolites from protein-containing samples (i.e. biological samples) while simultaneously quantifying each metabolite. The method employs a column venting technique, whereby the fatty acids are extracted by a pre-column and the proteins are vented to waste. The pre-column eluate is then directed through the analytical column which separates the lipoxygenase metabolites. The described method is reproducible and minimizes both the time and the cost involved in assaying a sample.

IDENTIFICATION OF PROTEIN-CONTAINING PAINT MEDIA BY QUANTITATIVE AMINO ACID ANALYSIS

In the present study the amino acid analyses of five protein-containing media were first ascertained. Then several specimens, both antique and modern, whose identity was unknown to the analyst, were assayed. The application of quantitative amino acid assay was found to be a useful and accurate means of determining the nature of protein media when sufficient peptide (protein) material was present.

Streptomycin II, An Antibiotic Substance Produced by a New Species of Streptomyces*

The production of streptomycin by actinomycetes is limited to certain strains of a single species of the genus Streptomyces, known as S. griseus. In addition to the first 2 streptomycin-producing strains isolated in 1943, only 3 other streptomycin-producing strains have thus far been reported. Although over a hundred cultures of S. griseus, isolated from different substrates, have now been tested in our laboratory for their antibiotic properties, only one culture was found capable of producing streptomycin; several others were capable of producing antibiotic substances such as grisein, which differed, however, from streptomycin. Similar results were obtained by Carvajal, who isolated 2 streptomycin-producing strains of S. griseus. No other organisms culturally different from S. griseus have been found capable of producing streptomycin. In connection with a study of the bacterial population of the waters around Bikini and Rongelap Atolls during the recent atomic bomb experiments in the Marshall Islands, a culture of an actinomycete was isolated from one of the Bikini soils. This culture showed strong inhibition of various Gram-positive and Gram-negative bacteria, as well as of acid-fast bacteria. This culture belonged to the genus Streptomyces, but was distinct from streptomycin-producing S. griseus. This culture was very similar, both morphologically and culturally, to S. griseolus (Actinomyces 96) described by one of us in 1919. It differed, however, in some of its cultural properties and in its ability to produce an active antibiotic substance. Because of these differences and also because of the peculiar natural substrate from which this culture was isolated, it is proposed to designate it as Streptomyces bikiniensis. It produces on synthetic and on organic media containing glucose, a dark gray aerial mycelium, which consists of dichotomous branches with straight chains of conidia. A thick complete pellicle is formed on the surface of stationary cultures, with protein-containing media being pigmented brown to black.