Chemoenzymatic formation of novel aminocoumarin antibiotics by the enzymes CouN1 and CouN7

The aminocoumarin antibiotics novobiocin, clorobiocin, and coumermycin A1 are highly potent inhibitors of the bacterial type II topoisomerase DNA gyrase. The key pharmacophore of both clorobiocin and coumermycin A1, the 5-methyl-2-pyrrolylcarbonyl moiety, targets the ATP-binding site of GyrB. The 5-methyl-2-pyrrolylcarbonyl group is transferred by the acyltransferases Clo/CouN7 from the carrier proteins Clo/CouN1 to the 3'-hydroxyl of the l-noviosyl scaffold during the late steps of clorobiocin and coumermycin A1 biosynthesis. We first examined the substrate specificity of the purified thiolation domain protein CouN1 in becoming primed by the phosphopantetheinyltransferase Sfp using a variety of synthetic CoA analogues of the 5-methyl-2-pyrrolylcarbonyl moiety. The acyl-S-CouN1 thioesters were then assayed as donors to the 3'-OH group of descarbamoylnovobiocin by the acyltransferase CouN7, resulting in 21 novel variants with heterocyclic acyl groups installed on the noviosyl moiety of the aminocoumarin scaffold. Scaleup of a 5-methylthiophene derivative yielded a compound with activity against both Gram-negative and Gram-positive bacteria. The minimal inhibitory concentration found for the Gram-positive bacteria was comparable to that of novobiocin.     

Role of conserved active site residues in catalysis by phospholipase B1 from Cryptococcus neoformans

Phospholipase B1 (PLB1), secreted by the pathogenic yeast Cryptococcus neoformans, has an established role in virulence. Although the mechanism of its phospholipase B, lysophospholipase, and lysophospholipase transacylase activities is unknown, it possesses lipase, subtilisin protease aspartate, and phospholipase motifs containing putative catalytic residues S146, D392, and R108, respectively, conserved in fungal PLBs and essential for human cytosolic phospholipase A2 (cPLA2) catalysis. To determine the role of these residues in PLB1 catalysis, each was substituted with alanine, and the mutant cDNAs were expressed in Saccharomyces cerevisiae. The mutant PLB1s were deficient in all three enzymatic activities. As the active site structure of PLB1 is unknown, a homology model was developed, based on the X-ray structure of the cPLA2 catalytic domain. This shows that the two proteins share a closely related fold, with the three catalytic residues located in identical positions as part of a single active site, with S146 and D392 forming a catalytic dyad. The model suggests that PLB1 lacks the "lid" region which occludes the cPLA2 active site and provides a mechanism of interfacial activation. In silico substrate docking studies with cPLA2 reveal the binding mode of the lipid headgroup, confirming the catalytic dyad mechanism for the cleavage of the sn-2 ester bond within one of two separate binding tracts for the lipid acyl chains. Residues specific for binding arachidonic and palmitic acids, preferred substrates for cPLA2 and PLB1, respectively, are identified. These results provide an explanation for differences in substrate specificity between lipases sharing the cPLA2 catalytic domain fold and for the differential effect of inhibitors on PLB1 enzymatic activities.     

Poly(ADP-Ribose) Polymerase Inhibitor PJ34 Reduces Brain Damage after Stroke in the Neonatal Mouse Brain

The poly(ADP-ribose) polymerase inhibitor PJ34 has recently been reported to increase cerebral blood flow, via the endothelial NO synthase, in the naive mouse brain throughout life. We addressed here the benefits of PJ34 after neonatal ischemia on hemodynamics and components of the neurovascular unit including the blood-brain barrier (BBB), microglia, and astrocytes. Nine-day-old mice were subjected to permanent MCA occlusion (pMCAo), and treated with either PBS or PJ34 (10 mg/kg). Mean blood-flow velocities (mBFV) were measured in both internal carotid arteries (ICA) and basilar trunk (BT) using Doppler-ultrasonography. BBB opening was assessed through somatostatin-receptor type-2 internalization and immunohistochemistry at 24 and 48 h. Lesion areas were measured 8 days after ischemia. In PBS-treated mice, pMCAo involved a drop in mBFV in the left ICA (p < 0.001 vs. basal), whereas mBFV remained stable in both right ICA and BT. PJ34 prevented this drop in the left ICA (NS vs. basal) and increased mBFV in the right ICA (p = 0.0038 vs. basal). No modification was observed in the BT. In contrast to PBS, BBB disruption extent and astrocyte demise were reduced in PJ34 mice only in the rostral brain at 48 h and 8 days post-pMCAo, respectively. Accordingly, 8 days after pMCAo, affected areas were reduced in the rostral brain (Bregma +0.86 and +0.14 mm), whereas total tissue loss was not reduced after PJ34 (4.0 ± 3.1%) vs. PBS (5.8 ± 3.4%). These results show that PJ34 reduced BBB permeability, astrocyte demise, and tissue loss (particularly in the rostral territories), suggesting that collateral supply mainly proceeds from the anterior ICA’s branches in the ischemic neonatal mouse brain.     

Influence of Human Food Consumption on the Time Budget of Vervets

In nonhuman primate groups that utilize crop and food-raiding strategies, the inclusion of human food in the diet can have a pervasive impact on the activity budget. Human food is usually of higher quality and energy per unit than wild food. Thus, metabolic demands may be reached sooner on a human food diet, which in turn leads to improved foraging efficiency. In particular, a reduction in time spent feeding is associated with an increase in time spent resting. We investigated changes in time budget in a group of vervets (Chlorocebus aethiops pygerthrus) living in a tourist and cultivated area of Entebbe, Uganda (Zoo group). Saj collected 123 hr of focal observations on 17 individuals. Human food constituted half of the diet of Zoo group. Access to human food resulted in a high proportion of time resting and a low proportion of time feeding. The group had a small average daily range and a small home range in relation to other reports on unprovisioned groups. The time budget differences suggest that the juveniles spent more time moving and feeding and less time resting than adults did, and that adult females were more social than adult males were. These patterns have been observed in both provisioned and unprovisioned vervet groups. In contrast to a report from an unprovisioned group (Harrison, 1983), lactating females in this provisioned situation were able to maintain feeding and activity levels similar to those of other adults. However, adult females spent significantly less time consuming human food than juveniles did. We suggest that the risk associated with human food consumption may explain the low consumption of human food by females with dependent offspring. The differences may also reflect the different nutritional requirements of lactating females or the tendency for juveniles to be more exploratory than other group members.     

Postnatal maturation of neuroepithelial bodies and carotid body innervation: A quantitative investigation in the rabbit

The intrapulmonary airways contain oxygen-sensitive chemoreceptors which may be analogous to the arterial chemoreceptors: the neuroepithelial bodies (NEB). While the NEB are prominent in the neonatal lung, physiological studies indicate that the carotid bodies are still relatively inactive at birth. This points to an unequal degree of development of both during the early neonatal period. As a reflexogenic chemoreceptor function depends on a well-developed innervation, we undertook a comparative investigation of the development of the NEB and the carotid body glomus cell innervation. Two morphological aspects of the innervation of NEB and carotid body glomus cells were quantified in rabbits of different age groups. The total sectional area of intracorpuscular and intraglomerular nerve endings per NEB or glomus cell group, respectively, was measured and the area percentage of mitochondria and synaptic vesicles was determined. In the NEB, no significant difference in total sectional area of the nerve endings between the age groups was observed, while in the carotid body there was a significant increase in the adult age group. In addition, the area percentage of mitochondria and synaptic vesicles of the nerve endings did not change significantly with age in the NEB, while in the carotid body these increased and decreased, respectively, with age. These observations point to a shift from morphologically efferent nerve endings, rich in synaptic vesicles, to morphologically afferent nerve endings, rich in mitochondria. Our interpretation of these findings is that, at birth, the NEB innervation is more mature than the carotid body glomus cell innervation and that the latter matures at a later time than the former. These findings support the theory that the NEB may act as complementary chemoreceptors to the carotid body during the early postnatal period.     

Allocation of carbon to growth and secondary metabolites in birch seedlings under UV-B radiation and CO2 exposure

In plants, the allocation of carbon to secondary metabolites has been shown to be determined by both the availability of resources (e.g., CO₂ concentration) and by specific stress factors (e.g., ultraviolet [UV]‐radiation). It has been suggested that, in combination, CO₂ and UV‐B radiation may differentially affect plant growth and morphogenic parameters, and elevated CO₂ may ameliorate the effects of UV‐B radiation. In the present work, the effects of increased atmospheric CO₂ concentration and UV‐B radiation on growth and the accumulation of different types of secondary metabolites were studied in silver birch (Betula pendula Roth). Seedlings were exposed to 350 and 700 μmol mol^?1 of CO₂ in a greenhouse. At both CO₂ levels, additional UV‐B was either present (8.16 kJ m^?2 day^?1 of biologically effective UV‐B irradiance) or absent. The time course of accumulation of individual secondary compounds and the shifts in allocation of carbon between biomass and the secondary metabolites (phenolic acids, flavonoids, condensed tannins) were studied during a 1‐month‐long exposure. Additionally, the activities of enzymes ( l ‐phenylalanine ammonia‐lyase [PAL], EC 4.3.1.5; peroxidase, EC 1.11.1.7; polyphenol oxidase, EC 1.10.3.1) were determined for leaves. UV‐B radiation significantly increased biomass, PAL activity, and the accumulation of phenolic acids and flavonoids in seedlings. Elevated CO₂ concentration increased the activities of all the enzymes studied and the accumulation of condensed tannins in leaves, especially with UV‐B radiation. Because the observed UV‐B induction of flavonoids was smaller under a high CO₂ concentration, it was suggested that the excess of carbon in the atmosphere may moderate the effect of UV‐B by increasing the metabolic activity of leaves (high enzyme activities) and by changing the allocation of internal carbon between different primary and secondary metabolites in the plant. Our results demonstrate the significant increase in the allocation of carbon to secondary metabolites without any large change in growth due to the elevation of CO₂ concentration and UV‐B radiation. There also was a stronger impact of CO₂ than UV‐B on the phenolic metabolism of birch seedlings.