Purification and characterization of poly(L-lactic acid)-degrading enzymes from Amycolatopsis orientalis ssp. orientalis

Polylactide or poly(l-lactic acid) (PLA) is a commercially promising material for use as a renewable and biodegradable plastic. Three novel PLA-degrading enzymes, named PLAase I, II and III, were purified to homogeneity from the culture supernatant of an effective PLA-degrading bacterium, Amycolatopsis orientalis ssp. orientalis. The molecular masses of these three PLAases as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 24.0, 19.5 and 18.0 kDa, with the pH optima being 9.5, 10.5 and 9.5, respectively. The optimal temperature for the enzyme activities was 50-60 degrees C. All the purified enzymes could degrade high-molecular-weight PLA film as well as casein, and the PLA-degrading activities were strongly inhibited by serine protease inhibitors such as phenylmethylsulfonyl fluoride and aprotinin, but were not susceptive to chymostatin and pepstatin. Taken together, these data demonstrated that A. orientalis ssp. orientalis produces multiple serine-like proteases to utilize extracellular polylactide as a sole carbon source.     

Data linkage using probabilistic decision rules: a primer

Electronic data linkage is increasingly being used by researchers and health professionals in the birth defects field as a tool for enhancing both research and service/care. However, in many cases, a common pre-existing ID number does not exist across different datasets, and common identifiers, such as names or dates of birth, which could be used to match records, may be known to contain errors or even legitimate differences over time. In such situations, probabilistic matching, which does not require that all identifying fields exactly agree in order for one to conclude that two records belong to the same individual, can be a valuable tool for improving data linkage. However, probabilistic matching is computationally complex and demanding, and not well understood by many who may wish to apply it in their work. Therefore, the purpose of this article is to provide an overview of one approach to probabilistic matching, including the step-by-step procedures involved in the calculation of indices corresponding to the likelihood that two records are a correct match. In addition, the use of multiple iterative protocols, in which several different matching strategies are used in order to maximize the number of linked records, is discussed. Finally, issues related to deduplication and verification of internal-consistency in the linked data set are also reviewed.     

Construction of sperm-specific lactate dehydrogenase DNA vaccine and experimental study of its immunocontraceptive effect on mice

Lactate dehydrogenase C4 (LDHC4) is a key enzyme for sperm metabolism. It is distributed specifically in testis and is highly immunogenic. In this study, two DNA vaccines pVAX1-hLDHC and pVAX1-mLDHC were constructed by inserting coding sequences of human and mice LDHC4 into the eukaryotic ex-pression vector pVAX1. The production of LDHC4 specific antibodies was induced in the sera of vac-cinated mice and the reproductive tract secretions of vaccinated female mice through immunization by mucosal surface instillation. Furthermore, the antibody titer increased with the times of immunization. In the mating experiment, the number of newborns of the vaccinated mice reduced significantly and some immunized female mice even lost the ability to bear any offsprings, suggesting that the difference between the immunized and control mice was statistically significant. Sperm agglutination analysis indicated that both the antisera from immunized mice and the reproductive tract secretions of vacci-nated female mice could agglutinate normal sperms. Results of immunohistochemistry showed that the antibodies present in the sera of immunized mice and the reproductive tract secretions of vaccinated female mice could specifically react with LDHC4 antigen, which mainly locates in the cytoplasm, acrosome membrane externa and acrosome capsule of the sperm. Taken together, our results indicated that the constructed contraceptive DNA vaccines did yield immunocontraceptive effects on mice and this would enable clinical trials in near future.     

Ising Model of Cardiac Thin Filament Activation with Nearest-Neighbor Cooperative Interactions

We have developed a model of cardiac thin filament activation using an Ising model approach from equilibrium statistical physics. This model explicitly represents nearest-neighbor interactions between 26 troponin/tropomyosin units along a one-dimensional array that represents the cardiac thin filament. With transition rates chosen to match experimental data, the results show that the resulting force-pCa (F-pCa) relations are similar to Hill functions with asymmetries, as seen in experimental data. Specifically, Hill plots showing (log(F/(1-F)) vs. log [Ca]) reveal a steeper slope below the half activation point (Ca₅₀) compared with above. Parameter variation studies show interplay of parameters that affect the apparent cooperativity and asymmetry in the F-pCa relations. The model also predicts that Ca binding is uncooperative for low [Ca], becomes steeper near Ca₅₀, and becomes uncooperative again at higher [Ca]. The steepness near Ca₅₀ mirrors the steep F-pCa as a result of thermodynamic considerations. The model also predicts that the correlation between troponin/tropomyosin units along the one-dimensional array quickly decays at high and low [Ca], but near Ca₅₀, high correlation occurs across the whole array. This work provides a simple model that can account for the steepness and shape of F-pCa relations that other models fail to reproduce.     

Characterization of carbonic anhydrase from Neisseria gonorrhoeae.

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO(2) hydration and the similar behaviour of the kinetics of (18)O exchange between CO(2) and water at chemical equilibrium. The pH profile of the turnover number, k(cat), can be described as a titration curve with an exceptionally high maximal value of 1.7 x 10(6) s(-1) at alkaline pH and a pK(a) of 7.2. At pH 9, k(cat) is buffer dependent in a saturable manner, suggesting a ping-pong mechanism with buffer as the second substrate. The ratio k(cat)/K(m) is dependent on two ionizations with pK(a) values of 6.4 and 8.2. However, an (18)O-exchange assay identified only one ionizable group in the pH profile of k(cat)/K(m) with an apparent pK(a) of 6.5. The results of a kinetic analysis of a His66-->Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2-dimethylimidazole. The bacterial enzyme shows similar K(i) values for the inhibitors NCO(-), SCN(-) and N(3)(-) as HCA II, while CN(-) and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)-substituted NGCA with acetazolamide, NCO(-), SCN(-), CN(-) and N(3)(-) resemble the corresponding spectra obtained with human Co(II)-isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)-induced denaturation reveal a sensitivity of the CO(2) hydration activity to the reducing agent tris(2-carboxyethyl)phosphine (TCEP). However, the A(292)/A(260) ratio was not affected by the presence of TCEP, and a structural transition at 2.8--2.9 M GdnHCl was observed.     

Campylobacter fetus Uses Multiple Loci for DNA Inversion within the 5′ Conserved Regions of sap Homologs

Campylobacter fetus cells possess multiple promoterless sap homologs, each capable of expressing a surface layer protein (SLP) by utilizing a unique promoter present on a 6.2-kb invertible element. Each sap homolog includes a 626-bp 5' conserved region (FCR) with 74 bp upstream and 552 bp within the open reading frame. After DNA inversion, the splice is seamless because the FCRs are identical. In mutant strain 23D:ACA2K101, in which sapA and sapA2 flanking the invertible element in opposite orientations were disrupted by promoterless chloramphenicol resistance (Cm(r)) and kanamycin resistance (Km(r)) cassettes, respectively, the frequency of DNA inversion is 100-fold lower than that of wild-type strain 23D. To define the roles of a 15-bp inverted repeat (IR) and a Chi-like site (CLS) in the FCR, we mutagenized each upstream of sapA2 in 23D:ACA2K101 by introducing NotI and KpnI sites to create strains 23D:ACA2K101N and 23D:ACA2K101K, respectively. Alternatively selecting colonies for Cm(r) or Km(r) showed that mutagenizing the IR or CLS had no apparent effect on the frequency of the DNA inversion. However, mapping the unique NotI or KpnI site in relation to the Cm(r) or Km(r) cassette in the cells that changed phenotype showed that splices occurred both upstream and downstream of the mutated sites. PCR and sequence analyses also showed that the splice could occur in the 425-bp portion of the FCR downstream of the cassettes. In total, these data indicate that C. fetus can use multiple sites within the FCR for its sap-related DNA inversion.     

Membrane inlet for mass spectrometric measurement of catalysis by enzymatic decarboxylases

Membrane inlet mass spectrometry (MIMS) uses diffusion across a permeable membrane to detect in solution uncharged molecules of small molecular weight. We point out here the application of MIMS to determine catalytic properties of decarboxylases using as an example catalysis by oxalate decarboxylase (OxDC) from Bacillus subtilis. The decarboxylase activity generates carbon dioxide and formate from the nonoxidative reaction but is accompanied by a concomitant oxidase activity that consumes oxalate and oxygen and generates CO₂ and hydrogen peroxide. The application of MIMS in measuring catalysis by OxDC involves the real-time and continuous detection of oxygen and product CO₂ from the ion currents of their respective mass peaks. Steady-state catalytic constants for the decarboxylase activity obtained by measuring product CO₂ using MIMS are comparable to those acquired by the traditional endpoint assay based on the coupled reaction with formate dehydrogenase, and measuring consumption of O₂ using MIMS also estimates the oxidase activity. The use of isotope-labeled substrate (¹³C₂-enriched oxalate) in MIMS provides a method to characterize the catalytic reaction in cell suspensions by detecting the mass peak for product ¹³CO₂ (m/z 45), avoiding inaccuracies due to endogenous ¹²CO₂.     

New insights into the structure and dynamics of actinomycetal community during manure composting

Despite advancing knowledge about the functional role of actinomycetes in degrading lignocellulosic materials, definitive knowledge concerning the diversity and dynamics of the actinomycetal community in composting is still lacking. In this study, real-time polymerase chain reaction (PCR) coupled with denaturing gradient gel electrophoresis (DGGE) and clone library construction were applied to investigate actinomycetal diversity and dynamics in a pilot-scale composting. Quantitative real-time PCR data revealed that actinomycetes accounted for 18–86 % of bacteria and that the fraction peaked during the maturing phase, indicating that Actinobacteria were critical to the compost ecosystem. Qualitatively, actinomycetal communities displayed distinct temporal variations during composting. Fourteen distinct genera of actinomycetes and an unknown group were observed in manure composts. Redundancy analysis indicated that temperature exerted an influence over the actinomycetal communities. Specifically, pathogenic Corynebacterium species dominated in the initial phase, whereas the genera Saccharomonospora and Thermobifida were abundant in the thermophilic phase. In maturing composts, mesophilic Micrococcineae members were most prevalent. The dominant thermophiles along with Micrococcineae may jointly facilitate the degradation of lignocellulosic materials during composting. Together, our research revealed a more detailed ecological and potential functional role for actinomycetes in the compost ecology.     

Pyramiding and evaluation of the brown planthopper resistance genes Bph14 and Bph15 in hybrid rice

The brown planthopper (BPH) is the most devastating insect pest in rice-producing areas. Shanyou 63 has become a widely cultivated hybrid in China over the last two decades; however, this line has become increasingly susceptible to bacterial blight (BB), blast, and BPH, resulting in a rapid decline in its use in rice production. In this study, a molecular marker-assisted selection (MAS) introgression of Bph14 and Bph15 was performed to improve the BPH resistance of Minghui 63 and its derived hybrids such as Shanyou 63. The effect of pyramiding genes was then comprehensively evaluated using three tests that comprised seedbox screening, feeding rate, and antixenosis for settling in the field. The results showed that the improved hybrids containing a single BPH resistance gene showed enhanced resistance (lower resistance score, honeydew weight and number of BPH settling) compared to conventional hybrids, while pyramiding two genes provided even higher resistance. Moreover, both Bph14 and Bph15 are partial dominance genes, and have a strong dosage effect on the resistance to BPH in the hybrid background, which is useful for breeding BPH-resistant hybrids. Field trial data demonstrated that yields of improved hybrid rice were higher than or similar to the control (Shanyou 63) under natural field conditions. These improved versions could be used in breeding programs for “green super rice.”     

State of gonads of yearlings of triploid rainbow trout Oncorhynchus mykiss exposed to androgenic hormone under high-mountain conditions of South Vietnam

Changes in the morphology of gonads under the effect of methyltestosterone in juveniles of triploid rainbow trout Oncorhynchus mykiss incubated and cultivated in a trout farm under climatic conditions of South Vietnam have been studied. It was shown that the effect of the androgenic hormone intensifies the formation and development of testes in triploid rainbow trout at an age of 1+, decreasing the proportion of sterile individuals and the proportion of fish with anatomical and cytological anomalies in the structure of gonads. The relationship between the found anomalies in the structure of the genital system of trout and its triploidization, hormonal effect, and abiotic environmental conditions is discussed.