Relationship of lipoprotein-associated phospholipase A2 and oxidized low density lipoprotein in carotid atherosclerosis

Plasma levels of lipoprotein-associated phospholipase A₂ (Lp-PLA₂) and oxidized low density lipoprotein (oxLDL) have been identified as risk factors for cardiovascular disease. Lp-PLA₂ is the sole enzyme responsible for the hydrolysis of oxidized phospholipids on LDL particles in atherosclerotic plaques. We have studied the relationship between Lp-PLA₂ and oxLDL in carotid endarterectomy (CEA) tissues and in matched plasmas. In extracts from CEA anatomical segments, the levels of oxLDL were significantly associated with the levels of Lp-PLA₂ protein (r = 0.497) and activity (r = 0.615). OxLDL and Lp-PLA₂ mass/activity were most abundant in the carotid bifurcation and internal segments where plaque was most abundant. In extracts from CEA atheroma, the levels of oxLDL and Lp-PLA₂ were significantly correlated (r = 0.634). In matched plasma and atheroma extracts, the levels of Lp-PLA₂ were negatively correlated (r = − 0.578). The ratio of Lp-PLA₂ to oxLDL was higher in atheromatous tissue (277:1) than in normal tissue (135:1) and plasma (13:1). Immunohistochemical experiments indicated that in plaques, oxLDL and Lp-PLA₂ existed in overlapping but distinctly different distribution. Fluorescence microscopy showed both oxLDL and Lp-PLA₂ epitopes on the same LDL particle in plasma but not in plaque. These results suggest that the relationship between Lp-PLA₂ and oxLDL in the atherosclerotic plaque is different from that in the plasma compartment.     

An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.     

A self-priming microfluidic diaphragm pump capable of recirculation fabricated by combining soft lithography and traditional machining

Fluid transport is crucial in the development of microanalytical devices. While there are many micropump designs available, most are incapable of sustaining recirculation of fluid at microL/min to mL/min levels. We have designed and fabricated a positive displacement micropump by combining soft lithography with traditional bulk machining. The micropump is actuated through pneumatic pressure. The pump is self-priming and is suitable for recirculating fluid through a microfluidic device containing mammalian cell culture. By custom designing the volume of the pumping chamber, tight control of the output flow rate can be obtained by changing the actuation frequency. It can also be fabricated easily on plastic substrates without access to expensive microfabrication equipment.     

Composition and diversity of microbial communities recovered from surrogate minerals incubated in an acidic uranium-contaminated aquifer

Our understanding of subsurface microbiology is hindered by the inaccessibility of this environment, particularly when the hydrogeologic medium is contaminated with toxic substances. In this study, surrogate geological media contained in a porous receptacle were incubated in a well within the saturated zone of a pristine region of an aquifer to capture populations from the extant communities. After an 8-week incubation, the media were recovered, and the microbial community that developed on each medium was compared to the community recovered from groundwater and native sediments from the same region of the aquifer, using 16S DNA coding for rRNA (rDNA)-based terminal restriction fragment length polymorphism (T-RFLP). The groundwater and sediment communities were highly distinct from one another, and the communities that developed on the various media were more similar to groundwater communities than to sediment communities. 16S rDNA clone libraries of communities that developed on particles of a specular hematite medium incubated in the same well as the media used for T-RFLP analysis were compared with those obtained from an acidic, uranium-contaminated region of the same aquifer. The hematite-associated community formed in the pristine area was highly diverse at the species level, with 25 distinct phylotypes identified, the majority of which (73%) were affiliated with the beta-Proteobacteria. Similarly, the hematite-associated community formed in the contaminated area was populated in large part by beta-Proteobacteria (62%); however, only 13 distinct phylotypes were apparent. The three numerically dominant clones from the hematite-associated community from the contaminated site were affiliated with metal- and radionuclide-tolerant or acidophilic taxa, consistent with the environmental conditions. Only two populations were common to both sites.     

Saturation mutagenesis of toluene ortho-monooxygenase of Burkholderia cepacia G4 for Enhanced 1-naphthol synthesis and chloroform degradation

Directed evolution of toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 previously created the hydroxylase alpha-subunit (TomA3) V106A variant (TOM-Green) with increased activity for both trichloroethylene degradation (twofold enhancement) and naphthalene oxidation (six-times-higher activity). In the present study, saturation mutagenesis was performed at position A106 with Escherichia coli TG1/pBS(Kan)TOMV106A to improve TOM activity for both chloroform degradation and naphthalene oxidation. Whole cells expressing the A106E variant had two times better naphthalene-to-1-naphthol activity than the wild-type cells (V(max) of 9.3 versus 4.5 nmol.min(-1).mg of protein(-1) and unchanged K(m)), and the regiospecificity of the A106E variant was unchanged, with 98% 1-naphthol formed, as was confirmed with high-pressure liquid chromatography. The A106E variant degrades its natural substrate toluene 63% faster than wild-type TOM does (2.12 +/- 0.07 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1) [mean +/- standard deviation]) at 91 microM and has a substantial decrease in regiospecificity, since o-cresol (50%), m-cresol (25%), and p-cresol (25%) are formed, in contrast to the 98% o-cresol formed by wild-type TOM. The A106E variant also has an elevated expression level compared to that of wild-type TOM, as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Another variant, the A106F variant, has 2.8-times-better chloroform degradation activity based on gas chromatography (V(max) of 2.61 versus 0.95 nmol.min(-1).mg of protein(-1) and unchanged K(m)) and chloride release (0.034 +/- 0.002 versus 0.012 +/- 0.001 nmol.min(-1).mg of protein(-1)). The A106F variant also was expressed at levels similar to those of wild-type TOM and 62%-better toluene oxidation activity than wild-type TOM (2.11 +/- 0.3 versus 1.30 +/- 0.06 nmol.min(-1).mg of protein(-1)). A shift in regiospecificity of toluene hydroxylation was also observed for the A106F variant, with o-cresol (28%), m-cresol (18%), and p-cresol (54%) being formed. Statistical analysis was used to estimate that 292 colonies must be screened for a 99% probability that all 64 codons were sampled during saturation mutagenesis.     

Effects of long-term storage on the stability of OpMNPV DNA contained in TM Biocontrol-1

Orgyia pseudotsugata multicapsid nucleopolyhedrovirus (OpMNPV) DNA was extracted from samples representing 10 lots of TM Biocontrol-1 stored at -10 degrees C for 5-15 years and digested with the restriction enzymes BglII, PstI, and SalI. DNA from the OpMNPV virus strain (MEM-75-STANDARD) used to produce the TM Biocontrol-1 lots was also extracted and digested. No restriction fragment length polymorphisms were observed in any of the samples and there was no evidence of DNA degradation. This indicates that long-term cold storage of TM Biocontrol-1 had no adverse effect on the quality of the OpMNPV DNA. In addition to the expected >23 kb OpMNPV DNA, extracts from lots 4a, 5b, and 6 contained 10 additional nucleic acid segments, ranging in size from 0.9 to 4.2 kb. The electrophoretic profile of these segments was characteristic of O. pseudotsugata cypovirus (OpCPV). RNase A/DNase I treatment showed that the nucleic acid contaminants were composed of RNA, suggesting that lots 4a, 5b, and 6 contained OpCPV as well as OpMNPV. Bioassay results have shown that there is a decrease in efficacy of stored TM-biocontrol-1, but this did not appear to be directly correlated with the length of time in storage.     

Large scale purification and refolding of HIV-1 protease fromEscherichia coli inclusion bodies

The protease encoded by the human immunodeficiency virus type 1 (HIV-1) was engineered inEscherichia coli as a construct in which the natural 99-residue polypeptide was preceded by an NH₂-terminal methionine initiator. Inclusion bodies harboring the recombinant HIV-I protease were dissolved in 50% acetic acid and the solution was subjected to gel filtration on a column of Sephadex G-75. The protein, eluted in the second of two peaks, migrated in SDS-PAGE as a single sharp band ofM _r ≈ 10,000. The purified HIV-1 protease was refolded into an active enzyme by diluting a solution of the protein in 50% acetic acid with 25 volumes of buffer atpH 5.5. This method of purification, which has also been applied to the purification of HIV-2 protease, provides a single-step procedure to produce 100 mg quantities of fully active enzyme.     

The effect of tosyl lysine chloromethyl ketone on the activity of uridine diphosphoglucose pyrophosphorylase of the cellular slime mold Dictyostelium discoideum

We report here that the specific activity of UDPG pyrophosphorylase in extracts of $\text{D. discoideum}$ amoebae and preculmination-stage cells increases as a function of the length of their exposure to tosyl lysine chloromethyl ketone, an irreversible inhibitor of a number of serine and sulfhydryl proteases. This compound also stabilizes the activity of the enzyme in crude extracts of amoebae. These results can be interpreted, with some assumptions, as evidence in support of the hypothesis that the levels of the enzyme are maintained in $\text{D. discoideum}$ by a balance of synthesis and degradation.     

Basis of unique red cell membrane properties in hereditary ovalocytosis.

Hereditary ovalocytes from a Mauritian subject are extremely rigid, with a shear elastic modulus about three times that of normal cells, and have increased resistance to invasion by the malaria parasite Plasmodium falciparum in vitro. The genetic anomaly resides in band 3; the protein gives rise to chymotryptic fragments with reduced mobility in SDS/polyacrylamide gel electrophoresis, but this is a result of anomalous binding of SDS and not a higher molecular weight. Analysis of the band 3 gene reveals (1) a point mutation (Lys56----Glu), which also occurs in a common asymptomatic band 3 (Memphis) variant and governs the electrophoretic properties, and (2) a deletion of nine amino acid residues, including a proline residue, encompassing the interface between the membrane-associated and the N-terminal cytoplasmic domains. The interaction of the mutant band 3 with ankyrin appears unperturbed. The fraction of band 3 capable of undergoing translation diffusion in the membrane is greatly reduced in the ovalocytes. Cells containing the asymptomatic band 3 variant were normal with respect to all the properties that we have studied. Possible mechanisms by which a structural change in band 3 at the membrane interface could regulate rigidity are examined.