Efficient biocatalyst for large-scale synthesis of cephalosporins, obtained by combining immobilization and site-directed mutagenesis of penicillin acylase

We describe the rational design of a new efficient biocatalyst and the development of a sustainable green process for the synthesis of cephalosporins bearing a NH? group on the acyl side chain. The new biocatalyst was developed starting from the WT penicillin acylase (PA) from Escherichia coli by combining enzyme mutagenesis, in position α146 and β24 (βF24A/αF146Y), and immobilization on an appropriate modified industrial support, glyoxyl Eupergit C250L. The obtained derivative was used in the kinetically controlled synthesis of cephalexin, cefprozil and cefaclor and compared to the WT-PA and an already described mutant, PA-βF24A, with improved properties. The new biocatalyst posses a very high ratio between the rates of the synthesis and two undesired hydrolyses (acylating ester and the amidic product). In particular, a very low amidase activity was observed with PA-βF24A/αF146Y and, consequently, the hydrolysis of the produced antibiotic was avoided during the process. Taking advantage of this property, higher conversions in the synthesis of cephalexin (99% versus 76%), cefaclor (99% versus 65%) and cefprozil (99% versus 60%) were obtained compared to the WT enzyme. Furthermore, the new mutant also show a higher synthetic activity compared to PA-βF24A immobilized on the same support, allowing the maximum yields to be achieved in very short reaction times. The production of cephalexin with the immobilized βF24A/αF146Y acylase has been developed on a pre-industrial scale (30 l). After 20 cycles, the average yield was 93%. The biocatalyst showed good stability properties and no significant decrease in performance.     

Microgeographic differentiation in historical Yemen inferred by morphometric distances

This study analyzed the variations in space of 8 body dimensions and 11 measures of the head of 1,244 adult Yemenite males, collected in 1933/34 by Coon in Yemen and in Hadhramawt. The aim was to evaluate the presence of geographic microdifferentiation of the populations settled in the different regions of Yemen at the time. Coon sub-divided the sample into six geographical areas according to birthplace and ethnicity of the individuals: Tihamah, the Western Mountains, the Central Plateau, the South Coast, the Eastern Mountains, and Hadhramawt. The results of ANCOVA (age as covariate) show that the observed differences of all variables among the six groups were highly statistically significant. Tukey's post-hoc test reveals higher statistically significant differences among four main groups: (1) Tihamah; (2) the Western Mountains and Central Plateau; (3) the Eastern Mountains; and (4) the Southern Coast and Hadhramawt. Multiple discriminant analysis carried out using only the data of the 11 measures of the head, the more "genetically" determined variables, confirmed these differences. Indeed, the first canonical variate well separates the groups with the Tihamah, Southern Coast and Hadhramawt on the one side and the Eastern Mountains, Western Mountains and Central Plateau on the other. The second canonical variate separates the Tihamah, Western Mountains and Central Plateau from the Eastern Mountains, Southern Coast and Hadhramawt. In conclusion, the Yemenite population seems to be composed of three morphologically distinct groups and an Eastern Mountains group which is positioned between the group formed by the Southern Coast and Hadhramawt and the Western Mountains and Central Plateau group. The Tihamah is the most distant from all the other groups. These differences are probably due to the presence/absence of geographical and cultural barriers that have favored/blocked the gene flow over the years. Indeed, the entire coastal bell, through the centuries, has constituted one of the principal commercial routes between the East, Africa, and the eastern Mediterranean, while the high inland mountains have isolated the remaining communities. This data is also confirmed by genetic studies. Finally, the average height (162.6 cm) of the global Yemenite population, compared to data from the other six middle-eastern Arab countries and Egypt, was found to be 3-6 cm less. This characteristic will be further studied, analyzing variations in average height according to the different age classes in order to evaluate any possible secular changes.     

Unbalanced 1q whole-arm translocation resulting in der(14)t(1;14)(q11-12;p11) in myelodysplastic syndrome

Unbalanced whole-arm translocations (WATs) of the long arm of chromosome 1, resulting in complete trisomy 1q, are chromosomal abnormalities detectable in both solid tumors and hematologic neoplasms. Among the WATs of 1q to acrocentric chromosomes, a few patients with der(1;15) described as a dicentric chromosome have been reported so far, whereas cases of der(1;14) are much rarer. We report on a case of der(1;14) detected as single anomaly in a patient with myelodysplastic syndrome. The aim of our work was to investigate the breakpoints of the (1;14) translocation leading to the der(1;14). Fluorescence in situ hybridization (FISH) experiments have been performed on chromosome preparations from bone marrow aspirate, using specific centromeric probes of both chromosomes, as well as a probe mapping to 1q11 band. FISH results showed that in our patient the derivative chromosome was monocentric with a unique centromere derived from chromosome 14. The breakpoints of the translocation were located in the short arm of chromosome 14 and in the long arm of chromosome 1, between the alphoid D1Z5 and the satellite II domains. The 1q breakpoint was within the pericentromeric region of chromosome 1, which is notoriously an unstable chromosomal region, involved in different chromosomal rearrangements.     

Exploitation of the semi-homothallic life cycle of Saccharomyces cerevisiae for the development of breeding strategies

A strain of Saccharomyces cerevisiae having desirable winemaking properties and high spore viability was bred from a semi-homothallic parent strain with similar winemaking properties but that produced sixfold fewer viable spores. Because the parent was homozygous for HO and for the MATa allele at both silent HMR and HML loci, it produced two MATa and two nonmating progeny per ascus. To obtain a segregant able to mate with the stable MATa progeny, a strain of the nonmating progeny, previously subjected to HO distruption with a KanMX4 cassette, was used. The resultant MATalphaho::KanMX4 transformant was mated to a MATa HO segregant and the diploid produced was sporulated to allow the isolation of a semi-homothallic diploid segregant designated 2D that lacked the KanMX4-disrupted HO allele as confirmed by sequence analysis. Genetic analysis indicated greater homozygosity in 2D than in the parent as assessed by PCR at five loci. The sugar consumption profiles of both 2D and the parent in grape juice fermentations were the same. Acetaldehyde levels and postfermentation biofilm formation were higher in 2D than in the parent. Because 2D has acceptable winemaking characteristics but produces significantly more viable spores than the parent strain, it will be useful in future breeding efforts.     

Intraspecific variability in the response of bloom-forming marine microalgae to changed climate conditions

Phytoplankton populations can display high levels of genetic diversity that, when reflected by phenotypic variability, may stabilize a species response to environmental changes. We studied the effects of increased temperature and CO(2) availability as predicted consequences of global change, on 16 genetically different isolates of the diatom Skeletonema marinoi from the Adriatic Sea and the Skagerrak (North Sea), and on eight strains of the PST (paralytic shellfish toxin)-producing dinoflagellate Alexandrium ostenfeldii from the Baltic Sea. Maximum growth rates were estimated in batch cultures of acclimated isolates grown for five to 10 generations in a factorial design at 20 and 24°C, and present day and next century applied atmospheric pCO(2), respectively. In both species, individual strains were affected in different ways by increased temperature and pCO(2). The strongest response variability, buffering overall effects, was detected among Adriatic S. marinoi strains. Skagerrak strains showed a more uniform response, particularly to increased temperature, with an overall positive effect on growth. Increased temperature also caused a general growth stimulation in A. ostenfeldii, despite notable variability in strain-specific response patterns. Our data revealed a significant relationship between strain-specific growth rates and the impact of pCO(2) on growth-slow growing cultures were generally positively affected, while fast growing cultures showed no or negative responses to increased pCO(2). Toxin composition of A. ostenfeldii was consistently altered by elevated temperature and increased CO(2) supply in the tested strains, resulting in overall promotion of saxitoxin production by both treatments. Our findings suggest that phenotypic variability within populations plays an important role in the adaptation of phytoplankton to changing environments, potentially attenuating short-term effects and forming the basis for selection. In particular, A. ostenfeldii blooms may expand and increase in toxicity under increased water temperature and atmospheric pCO(2) conditions, with potentially severe consequences for the coastal ecosystem.     

Low-temperature sensitivity and enhanced Bohr effect in red deer (Cervus elaphus) haemoglobin: a molecular adaptive strategy to life at high altitude and low temperature.

A study of the functional properties of haemoglobin from red deer (Cervus elaphus) whose habitat varies over a wide range of latitude, was performed. The oxygen-binding properties of the most common haemoglobin phenotype from the species living in Sardinia were examined with particular attention to the effect of pH, chloride, 2, 3-bisphosphoglycerate and temperature. Results indicate that red deer haemoglobin, like all haemoglobins from ruminants so far examined, is characterized by a low intrinsic oxygen affinity, with chloride being its main physiological modulator in vivo. The functional results and the low temperature sensitivity of the oxygen affinity are discussed in the light of the amino acid sequence of closely related ruminant haemoglobins.     

Releases of Norepinephrine and Dopamine in Ventriculocisternal Perfusions in Hepatectomized and Laparotomized Rats

Abstract: The completely hepatectomized rat has frequently been used as a model to study changes in the economy of norepinephrine (NE) and dopamine (DA) in hepatic coma. Hypothermia characteristically develops in hepatectomized rats and also occurs in patients in hepatic coma and is associated with improved survival in both. The aims of the present study were to measure both release and uptake of NE and release of DA in brain in warm (37°C) and cool (30–32°C) rats at 3–5 h after laparotomy or hepatectomy. Ventriculocisternal perfusions of the brain were performed on rats under basal conditions and during releases evoked by 40 m M K⁺. Basal releases of NE and DA and evoked release of DA were greater in the warm hepatectomized rats than in all other groups. In some studies, 10⁻⁵ M amitriptyline was added to the perfusates to assess whether neuronal uptake was changed after hepatectomy. Uptake of released NE was equally robust in cool hepatectomized as in cool laparotomized rats but could not be measured in warm hepatectomized rats because of amitriptyline toxicity in these rats. Decreases in NE and increases in DA content were found in most areas of the brain after perfusion. Increased releases of NE and DA may contribute to the pathogenesis of hepatic encephalopathy.     

Lignification related enzymes in Picea abies suspension cultures

Activity of a number of enzymes related to lignin formation was measured in a Picea abies (L) Karsten suspension culture that is able to produce native-like lignin into the nutrient medium. This cell culture is an attractive model for studying lignin formation, as the process takes place independently of the complex macromolecular matrix of the native apoplast. Suspension culture proteins were fractionated into soluble cellular proteins, ionically and covalently bound cell wall proteins and nutrient medium proteins. The nutrient medium contained up to 5.3% of total coniferyl alcohol peroxidase (EC 1.11.1.7) activity and a significant NADH oxidase activity that is suggested to be responsible for hydrogen peroxide (H2O2) production. There also existed some malate dehydrogenase (EC 1.1.1.37) activity in the apoplast of suspension culture cells (in ionically and covalently bound cell wall protein fractions), possibly for the regeneration of NADH that is needed for peroxidase-catalysed H2O2 production. However, there is no proof of the existence of NADH in the apoplast. Nutrient medium peroxidases could be classified into acidic, slightly basic and highly basic isoenzyme groups by isoelectric focusing. Only acidic peroxidases were found in the covalently bound cell wall protein fraction. Several peroxidase isoenzymes across the whole pI range were detected in the protein fraction ionically bound to cell walls and in the soluble cellular protein fraction. One laccase-like isoenzyme with pI of approximately 8.5 was found in the nutrient medium that was able to form dehydrogenation polymer from coniferyl alcohol in the absence of H2O2. The total activity of this oxidase towards coniferyl alcohol was, however, several orders of magnitude smaller than that of peroxidases in vitro. According to 2D 1H-13C correlation NMR spectra, most of the abundant structural units of native lignin and released suspension culture lignin are present in the oxidase produced dehydrogenation polymer but in somewhat different amounts compared to peroxidase derived synthetic lignin preparations. A coniferin beta-glucosidase (EC 3.2.1.21) was observed to be secreted into the culture medium.     

Signaling through CD14 attenuates the inflammatory response to Borrelia burgdorferi, the agent of Lyme disease

Lyme disease is a chronic inflammatory disorder caused by the spirochetal bacterium, Borrelia burgdorferi. In vitro evidence suggests that binding of spirochetal lipoproteins to CD14, a pattern recognition receptor expressed on monocytes/macrophages and polymorphonuclear cells, is a critical requirement for cellular activation and the subsequent release of proinflammatory cytokines that most likely contribute to symptomatology and clinical manifestations. To test the validity of this notion, we assessed the impact of CD14 deficiency on Lyme disease in C3H/HeN mice. Contrary to an anticipated diminution in pathology, CD14(-/-) mice exhibited more severe and persistent inflammation than did CD14(+/+) mice. This disparity reflects altered gene regulation within immune cells that may engender the higher bacterial burden and serum cytokine levels observed in CD14(-/-) mice. Comparing their in vitro stimulatory activity, live spirochetes, but not lysed organisms, were a potent CD14-independent stimulus of cytokine production, triggering an exaggerated response by CD14(-/-) macrophages. Collectively, our in vivo and in vitro findings support the provocative notion that: 1) pattern recognition by CD14 is entirely dispensable for elaboration of an inflammatory response to B. burgdorferi, and 2) CD14-independent signaling pathways are inherently more destructive than CD14-dependent pathways. Continued study of CD14-independent signaling pathways may provide mechanistic insight into the inflammatory processes that underlie development of chronic inflammation.