Osteogenic Cells Derived From Embryonic Stem Cells Produced Bone Nodules in Three-Dimensional Scaffolds.

An approach for 3D bone tissue generation from embryonic stem (ES) cells was investigated. The ES cells were induced to differentiate into osteogenic precursors, capable of proliferating and subsequently differentiating into bone-forming cells. The differentiated cells and the seeded scaffolds were characterized using von Kossa and Alizarin Red staining, electron microscopy, and RT-PCR analysis. The results demonstrated that ES-derived bone-forming cells attached to and colonized the biocompatible and biodegradable scaffolds. Furthermore, these cells produced bone nodules when grown for 3-4 weeks in mineralization medium containing ascorbic acid and beta-glycerophosphate both in tissue culture plates and in scaffolds. The differentiated cells also expressed osteospecific markers when grown both in the culture plates and in 3D scaffolds. Osteogenic cells expressed alkaline phosphatase, osteocalcin, and osteopontin, but not an ES cell-specific marker, oct-4. These findings suggest that ES cell can be used for in vitro tissue engineering and cultivation of graftable skeletal structures.

pH-dependent modulation of alkaline phosphatase activity in Serratia marcescens.

Serratia marcescens is an opportunistic pathogen responsible for causing nosocomial infections, corneal ulcer, necrotizing fasciitis, cellulites, and brain abscess. Alkaline phosphatase (APase) is believed to play an important role in the survival of several intracellular pathogens and their adaptation. We have studied the effect of low phosphate concentration and acid pH on the APase activities of S. marcescens. In a low phosphate medium, some strains of S. marcescens synthesize two different types of APases, a constitutive (CAPase) and an inducible (IAPase). Both the CAPase and IAPase isoenzymes completely lost their enzyme activities at pH 2.3, within 10 min of incubation at 0 degrees C. Acid-treated IAPase isoenzymes I, II, III, and IV solutions when adjusted to pH 7.8 showed recovery of 70%, 52%, 72%, and 60% of the lost activities, respectively. When the pH of the CAPase reaction mixture was raised to pH 7.8, the enzyme activity regained only 5% of its initial activity. Variations in protein concentration also affected the pH-dependent reversible changes of the IAPase activity. The higher the protein concentration, the faster the inactivation of enzyme activity observed at acidic pH at 0 degrees C. Conversely, the lower the protein concentration, the higher the rate of reactivation of enzyme activity observed for IAPase at alkaline pH. Protein interaction studies revealed a lack of similarity between CAPase and IAPase, suggesting separate genetic origin of these potentially virulent genes of S. marcescens.

Evidence on the presence of two distinct alkaline phosphatases in Serratia marcescens.

Certain strains of Serratia marcescens synthesized two different types of alkaline phosphatase (APase), constitutive (CAPase) and inducible (IAPase) APases, in low phosphate medium. Synthesis of the IAPase was repressed in the presence of high phosphate. Purification and separation of these electrophoretically distinct APases was achieved by using fractional (NH(4))(2)SO(4) precipitation, adsorption on a DEAE-cellulose column and elution of enzymes by a linear sodium chloride gradient. Starch gel electrophoresis of certain fractions revealed the separation of not only IAPase from CAPase but its separation into four distinct isozymes. CAPase gave maximum enzyme activity around pH 9.5, whereas for IAPase a broad range of enzyme activity was found between pH 8.5 and 10.5. Reversible inactivation at low pH occurred for IAPase but very little with CAPase. CAPase was more thermolabile than IAPase at 95 degrees C. The two APases were found to be distinct in their kinetic as well as immunological properties, suggesting two distinct enzyme species.

High temperature induced antibiotic sensitivity changes in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, which was resistant to a wide variety of antibiotics, became sensitive to several of these antibiotics when grown and tested at 46 degrees C. Cell wall antibiotics such as penicillin G and ampicillin were only effective when added to cells growing at 46 degrees C prior to a temperature shift to 37 degrees C. Antibiotics which penetrate the cytoplasmic membrane to express their inhibiting action present a pattern different from those which are active against the outer cell wall. In order that these compounds be effective, the permeability of the cytoplasmic membrane must be further altered with agents such as EDTA which allow the penetration of actinomycin D. Inhibitors of protein synthesis, such as streptomycin and chloramphenicol, have increased access to their sites of action in cells grown at 46 degrees C. Cells grown at 46 degrees C have 40% less lipopolysaccharide (LPS) than cells grown at 37 degrees C and the LPS aggregates were of large molecular size in cells grown at 46 degrees C. Growth at 46 degrees C affects the permeability properties of the outer cell wall more than the permeability properties of the cytoplasmic membrane and this was due, in part, to the selective release of LPS of LPS-protein complexes at elevated growth temperatures.

Isolation of a constitutively expressed enzyme for hydrolysis of carbaryl in Pseudomonas aeruginosa.

A hydrolase constitutively expressed in Pseudomonas aeruginosa which converts carbaryl to 1-naphthol was purified 1,767-fold by using a combination of anion-exchange, hydroxylapatite, gel filtration, and hydrophobic interaction chromatography techniques. The presence of Triton X-100 in buffers was necessary for deaggregation and purification of the hydrolase. This is the first membrane-bound hydrolase involved in the hydrolysis of any methylcarbamate pesticide purified from a bacterial source to date. The enzyme exhibited a unique specificity of hydrolyzing only carbaryl (1-naphthyl N-methylcarbamate) but not any other methylcarbamates. The purified enzyme was a monomer with a molecular mass of 65,000 Da. The pH and temperature optima for the enzyme activity were 8.5 and 45 degrees C, respectively. No cofactor requirement for the hydrolase activity could be demonstrated, and none of the divalent cations studied affected the activity of the enzyme. Also, the enzyme activity was not affected by the thiols: dithioerythritol, dithiothreitol, and 2-mercaptoethanol. The Km and Vmax values for carbaryl were 9 microM and 7.9 mumol/min/mg of protein, respectively.

Presence of human immunodeficiency virus nucleic acids in wastewater and their detection by polymerase chain reaction.

The human immunodeficiency virus type 1 (HIV-1) released by infected individuals or present in human and hospital wastes can potentially cause contamination problems. The presence of HIV-1 was investigated in 16 environmental samples, including raw wastewater, sludge, final effluent, soil, and pond water, collected from different locations. A method was developed to extract total nucleic acids in intact form directly from the raw samples or from the viral concentrates of the raw samples. The isolated nucleic acids were analyzed for the presence of HIV-1 by using in vitro amplification of the target sequences by the polymerase chain reaction (PCR) method. HIV-1-specific proviral DNA and viral RNA were detected in the extracted nucleic acids obtained from three wastewater samples by this method. The specificity of the PCR-amplified products was determined by Southern blot hybridization with an HIV-1-specific oligonucleotide probe, SK19. The isolated nucleic acids from wastewater samples were also screened for the presence of poliovirus type 1, representing a commonly found enteric virus, and simian immunodeficiency virus, representing, presumably, rare viruses. While poliovirus type 1 viral RNA was found in all of the wastewater samples, none of the samples yielded a simian immunodeficiency virus-specific product. No PCR-amplified product was yielded when wastewater samples were directly used for the detection of HIV-1 and poliovirus type 1. The wastewater constituents appeared to be inhibitory to the enzymes reverse transcriptase and DNA polymerase.(ABSTRACT TRUNCATED AT 250 WORDS)

Microbiological and biotechnological aspects of metabolism of carbamates and organophosphates.

Several carbamate and organophosphate compounds are used to control a wide variety of insect pests, weeds, and disease-transmitting vectors. These chemicals were introduced to replace the recalcitrant and hazardous chlorinated pesticides. Although newly introduced pesticides were considered to be biodegradable, some of them are highly toxic and their residues are found in certain environments. In addition, degradation of some of the carbamates generates metabolites that are also toxic. In general, hydrolysis of the carbamate and organophosphates yields less toxic metabolites compared with the metabolites produced from oxidation. Although microorganisms capable of degrading many of these pesticides have been isolated, knowledge about the biochemical pathways and respective genes involved in the degradation is sparse. Recently, a great deal of interest in the mechanisms of biodegradation of carbamate and organophosphate compounds has been shown because (1) an efficient mineralization of the pesticides used for insect control could eliminate the problems of environmental pollution, (2) a balance between degradation and efficacy of pesticides could result in safer application and effective insect control, and (3) knowledge about the mechanisms of biodegradation could help to deal with situations leading to the generation of toxic metabolites and bioremediation of polluted environments. In addition, advances in genetic engineering and biotechnology offer great potential to exploit the degradative properties of microorganisms in order to develop bioremediation strategies and novel applications such as development of economic plants tolerant to herbicides. In this review, recent advances in the biochemical and genetic aspects of microbial degradation of carbamate and organophosphates are discussed and areas in need of further investigation identified.

Detection of nucleic acids homologous to human immunodeficiency virus in wastewater.

Raw wastewaters were obtained from the cities of Belle Glade, Ocala and Gainesville in the state of Florida and were concentrated using several established methods for the recovery of human enteroviruses. The nucleic acids were then extracted from the wastewater concentrates, suspended in 2 x SSC with and without 2 N NaOH (for the detection of DNA and both DNA and RNA, respectively), and dot blotted onto hybridization membranes. These membranes were then hybridized with three 32P-end-labeled 18-mer oligonucleotides directed against the LTR, gag, and env regions of the human immunodeficiency virus type 1 (HIV-1). Autoradiographic analyses of these blots indicate that sequences homologous to HIV-1 genomic RNA and proviral DNA were found in Belle Glade wastewater but not in wastewater from Ocala and Gainesville. These findings may have implications in the wastewater treatment system as well as for detection of HIV-1 in clinical samples.

Biodegradation of halogenated organic compounds.

In this review we discuss the degradation of chlorinated hydrocarbons by microorganisms, emphasizing the physiological, biochemical, and genetic basis of the biodegradation of aliphatic, aromatic, and polycyclic compounds. Many environmentally important xenobiotics are halogenated, especially chlorinated. These compounds are manufactured and used as pesticides, plasticizers, paint and printing-ink components, adhesives, flame retardants, hydraulic and heat transfer fluids, refrigerants, solvents, additives for cutting oils, and textile auxiliaries. The hazardous chemicals enter the environment through production, commercial application, and waste. As a result of bioaccumulation in the food chain and groundwater contamination, they pose public health problems because many of them are toxic, mutagenic, or carcinogenic. Although synthetic chemicals are usually recalcitrant to biodegradation, microorganisms have evolved an extensive range of enzymes, pathways, and control mechanisms that are responsible for catabolism of a wide variety of such compounds. Thus, such biological degradation can be exploited to alleviate environmental pollution problems. The pathways by which a given compound is degraded are determined by the physical, chemical, and microbiological aspects of a particular environment. By understanding the genetic basis of catabolism of xenobiotics, it is possible to improve the efficacy of naturally occurring microorganisms or construct new microorganisms capable of degrading pollutants in soil and aquatic environments more efficiently. Recently a number of genes whose enzyme products have a broader substrate specificity for the degradation of aromatic compounds have been cloned and attempts have been made to construct gene cassettes or synthetic operons comprising these degradative genes. Such gene cassettes or operons can be transferred into suitable microbial hosts for extending and custom designing the pathways for rapid degradation of recalcitrant compounds. Recent developments in designing recombinant microorganisms and hybrid metabolic pathways are discussed.

Hydrolysis of carbaryl by a Pseudomonas sp. and construction of a microbial consortium that completely metabolizes carbaryl.

Two Pseudomonas spp. (isolates 50552 and 50581) isolated from soil degraded 1-naphthol and carbaryl, an N-methylcarbamate pesticide, respectively. They utilized these compounds as a sole source of carbon. 1-Naphthol was completely metabolized to CO2 by the isolate 50552, while the carbaryl was first hydrolyzed to 1-naphthol and then converted into a brown-colored compound by the isolate 50581. The colored metabolite was not degraded, but 1-naphthol produced by the isolate 50581 during the exponential phase of growth was metabolized by the isolate 50552. The two isolates were used to construct a bacterial consortium which completely catabolized carbaryl to CO2. No metabolite was detected in the cell cultures of the consortium. The isolate 50581 harbored a 50-kb plasmid pCD1, while no plasmid was detected in the isolate 50552. The isolated bacteria individually or as a consortium may be used for detoxification of certain industrial and agricultural wastes.

Multiple forms of alkaline phosphatase isoenzymes of Serratia marcescens.

Alkaline phosphatase (APase) isoenzymes produced by different strains of Serratia marcescens were examined. Variation of isoenzyme patterns with respect to number and their mobilities in starch gels after electrophoresis were observed. Ten strains gave a 1-isoenzyme pattern with 5 different mobilities; 7 strains gave a 2-isoenzyme pattern with 3 different mobilities; 9 strains gave a 3-isoenzyme pattern with 5 different mobilities; and 3 strains gave a 4-isoenzyme pattern. Three strains synthesized two electrophoretically distinct APases in low phosphate medium. A high concentration of inorganic phosphate induced the synthesis of one of these APase isoenzymes.

Detection and identification of poliovirus in environmental samples using nucleic acid hybridization.

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage phi X174. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples.

Microcosm for assessing survival of genetically engineered microorganisms in aquatic environments.

Laboratory-contained microcosms are important for studying the fate and survival of genetically engineered microorganisms. In this study, we describe a simple aquatic microcosm that utilizes survival chambers in a flowthrough or static renewal system. The model was used to study the survival of genetically engineered and wild-type strains of Escherichia coli and Pseudomonas putida in the lake water environment. Temperature-dependent studies indicated that the genetically engineered microorganisms survived better or at least as well as their wild-type counterparts at 15, 25, and 30 degrees C. The genetic determinants of the genetically engineered microorganisms also remained fairly stable within the host cell under the tested conditions. In the presence of organisms indigenous to lake water, E. coli was eliminated after 20 days, whereas P. putida showed an initial decline but was able to stabilize its population after 5 days. A herbicide, Hydrothol-191, caused a significant decline in numbers of P. putida, but no significant difference was observed between the genetically engineered microorganisms and the wild-type strain. The microcosm described is simple, can be easily adapted to study a variety of environmental variables, and has the advantage that the organisms tested are constantly exposed to test waters that are continuously renewed.

Novel method for monitoring genetically engineered microorganisms in the environment.

A method has been devised for directly detecting and monitoring genetically engineered microorganisms (GEMs) by using in vitro amplification of the target DNAs by a polymerase chain reaction and then hybridizing the DNAs with a specific oligonucleotide or DNA probe. A cloned 0.3-kilobase napier grass (Pennisetum purpureum) genomic DNA that did not hybridize to DNAs isolated from various microorganisms, soil sediments, and aquatic environments was inserted into a derivative of a 2,4-dichlorophenoxyacetic acid-degradative plasmid, pRC10, and transferred into Escherichia coli. This genetically altered microorganism, seeded into filter-sterilized lake and sewage water samples (10(4)/ml), was detected by a plate count method in decreasing numbers for 6 and 10 days of sample incubation, respectively. The new method detected the amplified unique marker (0.3-kilobase DNA) of the GEM even after 10 to 14 days of incubation. This method is highly sensitive (it requires only picogram amounts of DNA) and has an advantage over the plate count technique, which can detect only culturable microorganisms. The method may be useful for monitoring GEMs in complex environments, where discrimination between GEMs and indigenous microorganisms is either difficult or requires time-consuming tests.

Neisseria meningitidis: heterogenicity in the outer membrane proteins released into the growth medium.

Eight strains of Neisseria meningitidis belonging to different serogroups were analysed for their virulence in mice and their release of outer membrane proteins into the medium during growth. All strains released proteins. No detectable lipopolysaccharide was observed. However, SDS-PAGE showed a heterogenicity in the protein number and profile among the different strains of N. meningitidis tested.

Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate.

A Flavobacterium sp. (strain 50001), capable of degrading 2,4-dichlorophenoxyacetate (2,4-D), 2-methyl-4-chlorophenoxyacetate, and 2-chlorobenzoate and imparting resistance to mercury, harbored a degradative plasmid, pRC10. Cured strains of the Flavobacterium sp. lost the plasmid as well as the ability to degrade these chlorinated compounds. Comparison of this plasmid with the well-characterized 2,4-D-degradative plasmid pJP4 from Alcaligenes eutrophus showed regions of homology between the two plasmids. Restriction fragments of plasmid pRC10 which shared homology with the regions conferring 2,4-D-degradative genes (tfd) of plasmid pJP4 were cloned into a broad-host-range plasmid and studied in Pseudomonas putida. From the results obtained, the cloned DNA fragment expressed the genes for 2,4-D monooxygenase (tfdA) and 2,4-dichlorophenol hydroxylase (tfdB). In spite of the similarity in function, the size (45 kilobases) and restriction pattern of plasmid pRC10 were considerably different from those of pJP4 (80 kilobases). This may be due to the difference in the microbial background during evolution of the two plasmids.

Degradation of bromacil by a Pseudomonas sp.

A gram-negative rod, identified as a Pseudomonas sp., was isolated from soil by using bromacil as the sole source of carbon and energy. During growth on bromacil or 5-bromouracil, almost stoichiometric amounts of bromide were released. The bacterium was shown to harbor two plasmids approximately 60 and 100 kilobases in size. They appeared to be associated with the ability to utilize bromacil as a sole source of carbon and also with resistance to ampicillin. This microorganism also showed the potential to decontaminate soil samples fortified with bromacil under laboratory conditions.

Bacterial metabolism of carbofuran.

Fifteen bacteria capable of degrading carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl methylcarbamate) were isolated from soil samples with a history of pesticide application. All isolates were gram negative and were oxidase- and catalase-positive rods; they occurred singly or as short chains. All of the identified isolates belonged to one of two genera, Pseudomonas and Flavobacterium. They were separated into three groups based on their mode of utilization of carbofuran. Six isolates were placed in group I; these isolates utilized carbofuran as a sole source of nitrogen. Seven isolates were placed in group II; these isolates utilized the pesticide as a sole source of carbon. Isolates of both groups I and II hydrolyzed carbofuran to carbofuran phenol. Two isolates, designated group III, also utilized carbofuran as a sole source of carbon. They degraded the pesticide more rapidly, however, so up to 40% of [14C]carbofuran was lost as 14CO2 in 1 h. The results suggest that these isolates degrade carbofuran by utilizing an oxidative pathway.

Isolation of a methyl parathion-degrading Pseudomonas sp. that possesses DNA homologous to the opd gene from a Flavobacterium sp.

Two mixed bacterial cultures isolated by soil enrichment were capable of utilizing methyl parathion (O,O-dimethyl O-p-nitrophenylphosphorothioate) and parathion (O,O-diethyl O-p-nitrophenylphosphorothioate) as a sole source of carbon. Four isolates from these mixed cultures lost their ability to utilize the pesticides independently in transfers subsequent to the initial isolation. One member of the mixed cultures, a Pseudomonas sp., however, hydrolyzed the pesticides to p-nitrophenol but required glucose or another carbon source for growth. The crude cell extracts prepared from this bacterium showed an optimum pH range from 7.5 to 9.5 for the enzymatic hydrolysis. Maximum enzymatic activity occurred between 35 and 40 degrees C. The enzyme activity was not inhibited by heavy metals, EDTA, or NaN3. Another isolate from the mixed cultures, a Flavobacterium sp., used p-nitrophenol for growth and degraded it to nitrite. Nitrite was assimilated into the cells under conditions during which the nitrogen source was excluded from the minimal growth medium. The hybridization data showed that the DNAs from a Pseudomonas sp. and from the mixed culture had homology with the opd (organophosphate degradation) gene from a previously reported parathion-hydrolyzing bacterium, Flavobacterium sp. The use of the opd gene as a probe may accelerate progress toward understanding the complex interactions of soil microorganisms with parathions.

Characterization of the developmentally regulated Bacillus subtilis glucose dehydrogenase gene.

The DNA sequence of the structural gene for glucose dehydrogenase (EC 1.1.1.47) of Bacillus subtilis was determined and comprises 780 base pairs. The subunit molecular weight of glucose dehydrogenase as deduced from the nucleotide sequence is 28,196, which agrees well with the subunit molecular weight of 31,500 as determined from sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The sequence of the 49 amino acids at the NH2 terminus of glucose dehydrogenase purified from sporulating B. subtilis cells matched the amino acid sequence derived from the DNA sequence. Glucose dehydrogenase was purified from an Escherichia coli strain harboring pEF1, a plasmid that contains the B. subtilis gene encoding glucose dehydrogenase. This enzyme has the identical amino acid sequence at the NH2 terminus as the B. subtilis enzyme. A putative ribosome-binding site, 5'-AGGAGG-3', which is complementary to the 3' end of the 16S rRNA of B. subtilis, was found 6 base pairs preceding the translational start codon of the structural gene of glucose dehydrogenase. No known promoterlike DNA sequences that are recognized by B. subtilis RNA polymerases were present immediately preceding the translational start site of the glucose dehydrogenase structural gene. The glucose dehydrogenase gene was found to be under sporulation control at the trancriptional level. A transcript of 1.6 kilobases hybridized to a DNA fragment within the structural gene of glucose dehydrogenase. This transcript was synthesized 3 h after the cessation of vegetative growth concomitant to the appearance of glucose dehydrogenase.