High yield expression of leptospirosis vaccine candidates LigA and LipL32 in the methylotrophic yeast <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Background  

Leptospirosis, a zoonosis caused by Leptospira spp., is recognized as an emergent infectious disease. Due to the lack of adequate diagnostic tools, vaccines are an attractive intervention strategy. Recombinant proteins produced in Escherichia coli have demonstrated promising results, albeit with variable efficacy. Pichia pastoris is an alternative host with several advantages for the production of recombinant proteins.     

Active site substitution A82W improves the regioselectivity of steroid hydroxylation by cytochrome P450 BM3 mutants as rationalized by spin relaxation nuclear magnetic resonance studies

Cytochrome P450 BM3 from Bacillus megaterium is a monooxygenase with great potential for biotechnological applications. In this paper, we present engineered drug-metabolizing P450 BM3 mutants as a novel tool for regioselective hydroxylation of steroids at position 16β. In particular, we show that by replacing alanine at position 82 with a tryptophan in P450 BM3 mutants M01 and M11, the selectivity toward 16β-hydroxylation for both testosterone and norethisterone was strongly increased. The A82W mutation led to a ≤42-fold increase in V(max) for 16β-hydroxylation of these steroids. Moreover, this mutation improves the coupling efficiency of the enzyme, which might be explained by a more efficient exclusion of water from the active site. The substrate affinity for testosterone increased at least 9-fold in M11 with tryptophan at position 82. A change in the orientation of testosterone in the M11 A82W mutant as compared to the orientation in M11 was observed by T(1) paramagnetic relaxation nuclear magnetic resonance. Testosterone is oriented in M11 with both the A- and D-ring protons closest to the heme iron. Substituting alanine at position 82 with tryptophan results in increased A-ring proton-iron distances, consistent with the relative decrease in the level of A-ring hydroxylation at position 2β.     

Analysis of body measurements of newborn purebred Belgian Blue calves

At calving, purebred animals of the Belgian Blue (BB) breed are compromised by the incompatibility in size and shape of the dam and her calf, resulting in a very high incidence of dystocia problems. To clarify which body parts of the calf are of decisive importance to allow natural delivery and to investigate both the mean value as well as the variation among these body sizes within this breed (variation being an important condition for selection), measurements of nine body parts (body weight at birth (BW), body length (BL), length of the head (LH), shoulder width (SW), hip width (HW), heart girth (HG), withers height (WH) and the circumference of the fetlock of both the front (CFF) and the hind leg (CFH)) were assessed in 147 newborn purebred BB calves on 17 farms. Simple and partial correlations were assessed and we examined whether environmental factors (gender of the calf, parity of the cow, type of calving, season of birth and time of measurement after birth) were significantly associated with these specific calf measurements. The mean BW was 49.2 ± 7.1 kg. The average BL was 56.4 ± 4.5 cm and the mean LH was 24.4 ± 2.3 cm. Measurements obtained for SW and HW were 22.4 ± 2.2 and 22.9 ± 2.1 cm, respectively, whereas the mean WH was 71.1 ± 4.7 cm. Measurements of circumferences revealed a CFF of 17.9 ± 1.1 cm, a CFH of 18.0 ± 1.0 cm and a mean HG of 78.0 ± 5.4 cm. Partial correlations of the BW with eight body measurements were significant (P < 0.01) and ranged between 0.17 and 0.85; 0.42 and 0.88; and 0.24 and 0.88 when corrected for gender, parity and type of calving, respectively. BL (P < 0.01) and the CFF and CFH (P < 0.001) are larger in bull calves than in heifer calves. Calves born through caesarean section had broader SW (P < 0.01) and HW (P < 0.01) when compared with calves born after natural calving (defined as born per vaginam without assistance or with slight traction). Sizes of calves born out of multiparous cows were generally larger than of calves born out of heifers (SW: P < 0.001; HW: P < 0.05). As SW and HW are the broadest points of a BB calf, they are both candidates for being the limiting measures for calving ease, but the difference between HW and SW for the total data set was not different from zero (P > 0.05). In contrast to male calves in which no significant difference (between HW and SW) could be found, female calves show the difference between HW and SW that was significantly different from zero (P < 0.001); thus, in female calves, the HW is the most limiting factor of the calf's body. The significant variation in some body measures between the calves and the strong correlation within these sizes raises the possibility of selection towards smaller calves aiming to limit the dystocia problem in the BB breed. Furthermore, on the basis of our results, we were able to build equations for the farmer to use at the moment of calving containing the LH, the CF and the calf's gender to estimate SW and HW, the limiting body parts of the calf to be born naturally. Together with the knowledge of the pelvic size of the dam, this information gives the obstetrician or the farmer a more accurate prediction of the probability of natural calving at parturition.     

Interaction of ouabain with the Na(+) pump in intact epithelial cells

To determine the specificity and efficacy of [(3)H]ouabain binding as a quantitative measure of the Na(+) pump (Na(+), K(+)-ATPase) and as a marker for the localization of pumps involved in transepithelial Na(+)-transport, we analyzed the interaction of [(3)H]ouabain with its receptor in pig kidney epithelial (LLC-PK(1)) cells. When these epithelial cells are depleted of Na(+) and exposed to 2 muM [(3)H]ouabain in a Na(+)-free medium, binding is reduced by 90 percent. When depleted of K(+) and incubated in a K(+)- free medium, the ouabain binding rate is increase compared with that measured at 5 mM. This increase is only demonstable when Na(+) is present. The increased rate could be attributed to the predominance of the Na(+)-stimulated phosphorylated form of the pump, as K(+) is not readily available to stimulate dephosphorylation. However, some binding in the K(+)-free medium is attributable to pump turnover (and therefore, recycling of K(+)), because analysis of K(+)-washout kinetics demonstrated that addition of 2 muM ouabain to K(+)-depleted cells increased the rate of K(+) loss. These results indicate that in intact epithelial cells, unlike isolated membrane preparations, the most favorable condition for supporting ouabain binding occurs when the Na(+), K(+)-ATPase is operating in the Na(+)-pump mode or is phosphorylated in the presence of Na(+). When LLC-PK(1) cells were exposed to ouabain at 4 degrees C, binding was reduced by 97 percent. Upon rewarming, the rate of binding was greater than that obtained on cells kept at a constant 37 degrees C. However, even at this accelerated rate, the time to reach equilibrium was beyond what is required for cells, swollen by exposure to cold, to recover normal volume. Thus, results from studies that have attempted to use ouabain to eliminate the contribution of the conventional Na(+) pump to volume recovery must be reevaluated if the exposure to ouabain was done in the cold or under conditions in which the Na(+) pump is not operating.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Complex mixtures of antibodies generated from a single production qualitatively and quantitatively evaluated by native Orbitrap mass spectrometry

Composite antibody mixtures designed to combat diseases present a new, rapidly emerging technology in the field of biopharmaceuticals. The combination of multiple antibodies can lead to increased effector response and limit the effect of escape variants that can propagate the disease. However, parallel development of analytical technologies is required to provide fast, thorough, accurate, and robust characterization of these mixtures. Here, we evaluate the utility of native mass spectrometry on an Orbitrap platform with high mass resolving power to characterize composite mixtures of up to 15 separate antibodies. With this technique, unambiguous identification of each antibody in the mixtures was achieved. Mass measurements of the intact antibodies varied 7 ppm on average, allowing highly reproducible identification and quantitation of each compound in these complex mixtures. We show that with the high mass-resolving power and robustness of this technology, high-resolution native mass spectrometry can be used efficiently even for batch-to-batch characterization.     

Hexazonium pararosaniline as a fixative for animal tissues

Hexazonium pararosaniline is a valuable reagent that has been used in enzyme activity histochemistry for 50 years. It is an aqueous solution containing the tris-diazonium ion derived from pararosaniline, an aminotriarylmethane dye, and it contains an excess of nitrous acid that was not consumed in the diazotization reaction. Other investigators have found that immersion for 2 min in an acidic (pH 3.5) 0.0015 M hexazonium pararosaniline solution can protect cryostat sections of unfixed animal tissues from the deleterious effects of aqueous reagents such as buffered solutions used in immunohistochemistry, while preserving specific affinities for antibodies. In the present investigation hexazonium pararosaniline protected lymphoid tissue and striated muscle against the damaging effects of water or saline. The same protection was conferred on unfixed sections treated with dilute nitrous or hydrochloric acid in concentrations similar to those in hexazonium pararosaniline solutions. Model tissues (solutions, gels or films containing gelatin and/or bovine albumin) responded predictably to well known cross-linking (formaldehyde) or coagulant (mercuric chloride) fixatives. Hexazonium pararosaniline solutions prevented the dissolution of protein gels in water only after 9 or more days of contact, during which time considerable swelling occurred. It is concluded that there is no evidence for a “fixative” action of hexazonium pararosaniline. The protective effect on frozen sections of unfixed tissue is attributable probably to the low pH of the solution.     

A new method for surface staining large slices of fixed brain using a copper phthalocyanine dye

Here we describe a method for gross staining of gray matter in slices of formaldehyde-fixed human brain. After protection of white matter with 4% phenol at 60°C for 5 min followed by a cold water wash, the gray matter was stained for 10-15 min at 20-25°C with 1% aqueous copper(II) phthalocyanine tetrasulfonic acid tetrasodium salt (CPTS). The staining resisted all attempts to be washed from the gray matter. Stained slices can be stored indefinitely in slightly acidified water, or plastinated as permanent dry specimens.