Polyketide ß-branching: diversity, mechanism and selectivity.

Covering: 2008 to August 2020 Polyketides are a family of natural products constructed from simple building blocks to generate a diverse range of often complex chemical structures with biological activities of both pharmaceutical and agrochemical importance. Their biosynthesis is controlled by polyketide synthases (PKSs) which catalyse the condensation of thioesters to assemble a functionalised linear carbon chain. Alkyl-branches may be installed at the nucleophilic α- or electrophilic ß-carbon of the growing chain. Polyketide ß-branching is a fascinating biosynthetic modification that allows for the conversion of a ß-ketone into a ß-alkyl group or functionalised side-chain. The overall transformation is catalysed by a multi-protein 3-hydroxy-3-methylglutaryl synthase (HMGS) cassette and is reminiscent of the mevalonate pathway in terpene biosynthesis. The first step most commonly involves the aldol addition of acetate to the electrophilic carbon of the ß-ketothioester catalysed by a 3-hydroxy-3-methylglutaryl synthase (HMGS). Subsequent dehydration and decarboxylation selectively generates either α,ß- or ß,γ-unsaturated ß-alkyl branches which may be further modified. This review covers 2008 to August 2020 and summarises the diversity of ß-branch incorporation and the mechanistic details of each catalytic step. This is extended to discussion of polyketides containing multiple ß-branches and the selectivity exerted by the PKS to ensure ß-branching fidelity. Finally, the application of HMGS in data mining, additional ß-branching mechanisms and current knowledge of the role of ß-branches in this important class of biologically active natural products is discussed.

Partial recovery of the damaged rat blood-brain barrier is mediated by adherens junction complexes, extracellular matrix remodeling and macrophage infiltration following focal astrocyte loss.

Blood-brain barrier (BBB) dysfunction is a feature of many neurodegenerative disorders. The mechanisms and interactions between astrocytes, extracellular matrix and vascular endothelial cells in regulating the mature BBB are poorly understood. We have previously shown that transitory glial fibrillary acidic protein (GFAP)-astrocyte loss, induced by the systemic administration of 3-chloropropanediol, leads to reversible disruption of tight junction complexes and BBB integrity to a range of markers. However, early restoration of BBB integrity to dextran (10-70 kDa) and fibrinogen was seen in the absence of paracellular tight junction proteins claudin-5 and occludin. In the present study we show that in the GFAP-astrocyte-lesioned rat inferior colliculus, paracellular expression of adherens junction proteins (vascular endothelial (VE)-cadherin and ß-catenin) was maintained in vascular endothelial cells that lacked paracellular claudin-5 expression and which showed reversible post-translational occludin modification. Claudin-1 expression paralleled the loss and recovery of claudin-5, while claudin-3 or -12 immunoreactivity was not detected. In addition, the extracellular matrix, as visualized by laminin and fibronectin, underwent extensive reversible remodeling and perivascular CD169 macrophages become abundant throughout the lesioned inferior colliculus. At a time that GFAP-astrocytes repopulated the lesion area and tight junction proteins were returned to paracellular domains, the extracellular matrix and leukocyte profiles normalized and resembled profiles seen in control tissue. This study supports the hypothesis that a combination of paracellular adherens junctional proteins, remodeled basement membrane and the presence of perivascular leukocytes provide a temporary barrier to limit the extravasation of macromolecules and potentially neurotoxic substances into the brain parenchyma until tight junction proteins are restored to paracellular domains.

Chronic inflammatory pain and the neurovascular unit: a central role for glia in maintaining BBB integrity?

Pain is a complex phenomenon involving both a peripheral innate immune response and a CNS response as well as activation of the hypothalamic-pituitary-adrenal axis. The peripheral innate immune response to injury involves the rapid production and local release of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-/alpha), interleukin-1 (IL-1) and IL-6. Recent studies into the CNS response to peripheral chronic inflammatory pain strongly implicates a role for glia, and local synthesis of proinflammatory cytokines and growth factors. A characteristic feature of CNS inflammation is gliosis, in which inflammatory mediators activate glial cells (e.g. astrocytes and microglia, macrophages and leukocytes) which have been shown to induce and maintain hyperalgesia. In addition, inflammatory pain induces changes in blood-brain barrier (BBB) permeability and alters transport of clinically relevant drugs used to treat pain into the brain. Despite the increasing body of evidence for the involvement of glia in chronic pain and the role of glia in maintaining the BBB, few studies have addressed glial/endothelial interactions and the mechanisms by which glia may regulate the BBB during inflammatory pain. Further studies into the cellular mechanisms of glial/endothelial interactions may identify novel therapeutic targets for reversing chronic inflammatory induced BBB dysfunction and innovate therapies for modulating the severity of chronic inflammatory pain.

A size selective vascular barrier in the rat area postrema formed by perivascular macrophages and the extracellular matrix.

The fenestrated microvasculature of the area postrema shows a less restrictive blood-brain barrier than is found in other areas of the CNS. We have studied the expression and relationship of vascular endothelial tight junctional proteins, astrocytes, macrophages, and the extracellular matrix with the extravasation of fluorescently tagged dextrans and sodium fluorescein in the rat area postrema. Glial fibrillary acidic protein (GFAP) -positive astrocytes were present within the area postrema which was surrounded by a dense zone of highly GFAP-reactive astrocytes. Expression of the tight junction proteins claudin-5, -12 and occludin was absent, although diffuse cytoplasmic claudin-1 immunoreactivity was present. The extracellular matrix of the endothelium showed two non-fused thickened layers of laminin immunoreactivity. CD163 and CD169 immunoreactive perivascular macrophages were located within lacunae between these two laminin layers. Fluorescently tagged dextrans (10-70 kDa) passed from the vasculature but were retained between the inner and outer laminin walls and rapidly sequestered by the perivascular CD163 and CD169 macrophages. Three-kilodalton dextran diffused into the parenchyma, but was retained within the boundary of the area postrema at the interface with the highly reactive GFAP-astrocytes, while sodium fluorescein (0.3 kDa) passed from the area postrema into surrounding CNS areas. Our observations suggest that despite the absence of a tight blood-brain barrier, a size selective barrier restricting the movement of blood solutes into the parenchyma is present in the area postrema. We suggest that the rapid uptake by CD163 and CD169 macrophages together with the non-fused laminin immunoreactive layers of the extracellular matrix plays a size selective role in restricting movement of serum proteins and other blood borne macromolecules over 10 kDa in to the area postrema.

Screening cardiac ultrasonographic examination in patients with suspected cardiac disease in the emergency department.

BACKGROUND/OBJECTIVE: Our purpose was to evaluate the utility of a brief screening cardiac ultrasonographic (SCU) examination. We prospectively compared the SCU with conventional clinical evaluation in 124 emergency department (ED) patients with suspected cardiac disease. Furthermore, we assessed the impact and quality of SCU examinations as obtained by briefly trained ED personnel (EP). METHODS: Patients underwent clinical evaluation by an ED physician and SCU examination by a sonographer or cardiologist. Patient disposition, hospital stay length, and the number of full echo examinations were compared with the presence of significant findings on SCU. In patients who received a full echocardiogram during hospitalization, results of the initial clinical examination were compared with results of the SCU examination in the diagnosis of significant findings. A similar analysis, but with quality assessment, was performed on only those SCU examinations acquired by 4 EP. RESULTS: Of the 124 patients enrolled in the main study, 40 of 124 (32%) had significant findings on SCU. Of patients with abnormal SCUs versus normal SCUs, 16 of 40 (40%) versus 18 of 84 (21%) had hospital stay lengths >2 days (P < or =.05). Using the 36 inpatient full echo studies obtained for standard indications during hospitalization as a gold standard, initial clinical examination identified only 7 of 30 (23%) significant findings and had 16 false-positive diagnoses, whereas SCU identified 22 of 30 (73%) with 8 false positives. Although similar study results occurred with interpretation of 68 SCUs obtained by EP, quality was achieved in only 55% ED personnel versus 97% of sonographer-obtained SCUs (P <.05). CONCLUSIONS: An SCU examination detects significant findings misdiagnosed on initial clinical evaluation in the ED and provides prognostic data regarding length of hospital stay.

Synthesis of the marine natural product barbamide.

The first total synthesis of the trichlorinated natural product barbamide is described. The convergent approach involves coupling (S)-3-trichloromethylbutanoyl chloride with Meldrum's acid (2,2-dimethyl-1,3-dioxane-4,6-dione) to give 15 followed by addition of the novel secondary amine N-methyl-(S)-dolaphenine 2 (prepared in 6 steps and 24% overall yield from N-Cbz-L-phenylalanine) to give the beta-keto amide 16 which was converted directly to the required (E)-enol ether.

Negative correlation between oral malodour and numbers and activities of sulphate-reducing bacteria in the human mouth.

The majority of cases of oral malodour are thought to be due to bacterial activities in the mouth, but many of the bacterial species responsible have not been identified. Volatile sulphide compounds have been proposed as constituents of oral malodour. Therefore, the relation between intensity of odour and numbers of bacteria in the mouth that are sulphide-producing from sulphate was investigated. Numbers of such dissimilatory sulphate-reducing bacteria (SRB) and sulphide reduction rates were evaluated in samples from different oral sites in relation to measures of oral malodour. Results showed that sulphate-reducing bacterial numbers and activities were negatively correlated with malodour, as determined by organoleptic assessment and measurement with a sulphide-monitoring instrument, the Halimeter. The data indicate that sulphide produced by oral SRB may not be an important contributor to oral malodour. A rather poor correlation was observed between Halimetric and organoleptic values, indicating that these methods may measure different aspects of oral malodour intensity.

Synthesis of probes for the active site of leucine dehydrogenase.

A series of novel 3-substituted 2-oxobutanoic acids were prepared and incubated with leucine dehydrogenase giving in one case both a kinetic resolution at C-3 and reductive amination of the ketone. This is the first example of an amino acid dehydrogenase catalysed kinetic resolution and reductive amination.

Witnessed embolization of a right atrial mass during transesophageal echocardiography: implications regarding the safety of esophageal intubation.

A patient with chronic lung disease and a right atrial density that was difficult to distinguish on a transthoracic echocardiogram underwent transesophageal echocardiography (TEE) that demonstrated two mobile masses attached to the anterior right atrial wall. During the TEE procedure, the patient experienced coughing and retching due to the esophageal intubation, and the embolization of one of the right atrial masses was observed. This case is the first to document this mechanism of pulmonary embolism (a mechanism that was suspected in two prior reports), and it questions the safety of procedures that induce retching and coughing in patients with mobile right atrial masses.

Methylation of the NMDA receptor agonist L-trans-2,3-pyrrolidine-dicarboxylate: enhanced excitotoxic potency and selectivity.

This study investigated the excitotoxic properties of a novel series of NMDA analogues in which a methyl group was introduced to the 5-position of the pyrrolidine ring of L-trans-2,3-PDC, a previously identified NMDA receptor agonist. While all of these compounds induced NMDA-receptor-mediated injury, methylation increased in vivo excitotoxic potency 1000-fold. Injections (1 mu 1) in rat dorsal hippocampus of cis- and trans-5-methyl-L-trans-2,3-PDC (0.1 nmol) induced 50-70% neuronal damage to areas CA1 and CA4, comparable to that induced by 100 nmol of L-trans-2,3-PDC. Further, cis- and trans-methylated analogues induced distinct patterns of hippocampal pathology consistent with differential excitotoxic vulnerability of neurons expressing NMDA receptors. Neuronal damage produced by the 5-methyl-L-trans-2,3-PDCs could be blocked by coadministration of MK-801 (3 mg/kg ip), but not NBQX (25 nmol). Biochemical and physiological assays confirmed the action of the analogues as NMDA agonists, but did not provide an explanation for differences in excitotoxic potency between the methylated and nonmethylated 2,3-PDCs. or example, the activity of the compounds as inhibitors of 3H-glutamate binding (IC50 values: 0.4, 1.4, and 1.2 microM for cis-5-methyl-,trans-5-methyl-, and L-trans-2,3-PDC, respectively), agonists at NR1A/NR2B receptors (EC50 values: 5, 49, and 16 microM for cis-5-methyl-,trans-5-methyl-, and L-trans-2,3-PDC, respectively), and in vitro excitotoxins in cortical cultures varied only two- to fivefold as a consequence of methylation. Potential roles of NMDA receptor subtypes and transport in these effects are discussed. As potent and selective NMDA excitotoxins, cis- and trans-5-methyl-L-trans-2,3-PDC will be of value studying excitotoxic mechanisms, MDA-receptor-mediated pathology, and NMDA receptor heterogeneity.

Irreversible inhibition of high-affinity [3H]kainate binding by a novel photoactivatable analogue: (2'S,3'S,4'R)-2'-carboxy-4'-(2-diazo-1-oxo-3, 3,3-trifluoropropyl)-3'-pyrrolidinyl acetate.

A photolabile trifluoromethyldiazoketone derivative of kainate (KA), (2'S,3'S,4'R)-2'-carboxy-4'-(2-diazo-1-oxo-3, 3,3-trifluoropropyl)-3'-pyrrolidinyl acetate (DZKA), was synthesized and evaluated as an irreversible inhibitor of the high-affinity KA site on rat forebrain synaptic plasma membranes (SPMs). In the absence of UV irradiation, DZKA preferentially blocked [3H]KA binding with an IC50 of 0.63 microM, a concentration that produced little or no inhibition at AMPA or NMDA sites. At 100 microM, however, DZKA inhibited [3H]AMPA and L-[3H]glutamate binding by approximately 50%. When examined electrophysiologically in HEK293 cells expressing human KA (GluR6) or AMPA (GluR1) subtypes, DZKA acted preferentially at KA receptors as a weak agonist. DZKA also exhibited little or no excitotoxic activity in mixed rat cortical cultures. Irreversible inhibition was assessed by pretreating SPMs with DZKA (50 microM) in the presence of UV irradiation, removing unbound DZKA, and then assaying the reisolated SPMs for radioligand binding. This protocol produced a selective and irreversible loss of approximately 50% of the [3H]KA sites. The binding was recoverable in SPMs pretreated with DZKA or UV alone. Coincubation with L-glutamate prevented the loss in [3H]KA binding, suggesting that the inactivation occurred at or near the ligand binding site. These results are consistent with the action of DZKA as a photoaffinity ligand for the KA site and identify the analogue as a valuable probe for future investigations of receptor structure and function.

Nutritional aspects of dissimilatory sulfate reduction in the human large intestine.

In contrast to other anaerobic ecosystems, such as marine and estuarine sediments, there is a lack of information on the nutritional requirements of human gut sulfate-reducing bacteria (SRB). Various substrates stimulated sulfate reduction in mixed culture, including short-chain fatty acids and other organic acids, alcohols, and amino acids (but not sugars or aromatic compounds). However, the use of sodium molybdate as a specific inhibitor of sulfate reduction caused an accumulation of ethanol and malonate only, and reduced the rate of utilization of lactate. This indicates the importance of these electron donors for sulfate reduction. Since ethanol and lactate are primarily utilized by members of the Desulfovibrio genus, the results suggest a physiologically important role for this group. Experiments with two strains of Desulfovibrio desulfuricans isolated from human feces demonstrated that both were able to reduce sulfite, thiosulfate or nitrate in the absence of sulfate. In addition, one strain (DsvUC1) was able to grow by fermentative metabolism, although the second strain (DsvFD1) showed more restricted fermentative growth. The data indicate that desulfovibrios are ecologically the most significant group of SRB in the human colon, and that colonic isolates belonging to this genus are versatile, in terms of both the electron acceptors and donors that they are able to utilize.

L-trans-2,3-pyrrolidine dicarboxylate: characterization of a novel excitotoxin.

This study investigated the in vitro and in vivo excitotoxic properties of a novel conformationally constrained analogue of L-glutamate, L-trans-2,3-pyrrolidine dicarboxylate (L-trans-2,3-PDC). When tested for excitotoxic activity in rat cortical cultures, L-trans-2,3-PDC mimicked the action of NMDA in both acute (30 min) and chronic (24 h) exposure paradigms. This neurotoxicity was attenuated by co-addition of MK-801 (10 microM). Microinjections of L-trans-2,3-PDC into the dorsal hippocampus of male rats also induced a selective pattern of pathology indicative of an NMDA receptor excitotoxin. In contrast to the equipotency observed in vitro, 100 nmol of L-trans-2,3-PDC were needed to produce cellular damage comparable to that induced by 25 nmol of NMDA. Consistent with an action at NMDA receptors, L-trans-2,3-PDC-induced damage could be significantly reduced by co-administration of MK-801 (3 mg/kg i.p.), but not by NBQX (25 nmol). In radioligand binding assays L-trans-2,3-PDC inhibited the binding of 3H-L-glutamate to NMDA receptors (IC50 1 microM), although it also exhibited some cross reactivity with KA and AMPA receptors. L-trans-2,3-PDC was also identified as a competitive inhibitor (Ki = 33 microM) of 3H-D-aspartate uptake into rat forebrain synaptosomes. In contrast to the action of a transported substrate, such as L-glutamate, L-trans-2,3-PDC did not exchange with 3H-D-aspartate that had been previously loaded into the synaptosomes.

Growth, incidence and activities of dissimilatory sulfate-reducing bacteria in the human oral cavity.

Viable counts and activities of sulfate-reducing bacteria were determined in the oral cavities of 12 healthy volunteers. Of these, 10 harboured viable sulfate-reducing bacteria populations. Six separate sites were sampled: the posterior tongue, anterior tongue, mid buccal mucosa, vestibular mucosa, supragingival plaque and subgingival plaque. Sulfate-reducing bacteria occurred in all areas, with the highest incidence in supragingival plaque. Viable counts and sulfate-reducing activities in each of the regions varied from 0 to 10(8) cfu (g wet weight)-1 and from 0 to 50 nmol (g wet weight)-1 h-1, respectively. As sulfate-reducing bacteria can be detected in the oral cavity, they may potentially be involved in terminal oxidative processes carried out by the microflora of the mouth.

Neuroprotective effect of free radical scavengers on beta-N-oxalylamino-L-alanine (BOAA)-induced neuronal damage in rat hippocampus.

The neurotoxin beta-N-oxalylamino-L-alanine (BOAA), found in Lathyrus sativus seeds, is thought to be the causative agent of neurolathyrism. We have investigated the neuroprotective effects of free radical scavengers on BOAA-induced toxicity following focal injection (1 microliter) of BOAA and comparing the pathological outcome with the effects of injections of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), kainate (KA) or N-methyl-D-aspartate (NMDA) into the dorsal hippocampus of male Wistar rats. Cellular damage was assessed histologically. BOAA (50 nmol) induced a highly selective pattern of hippocampal damage identical with that seen with AMPA (1 nmol). BOAA-induced neurotoxicity, but not AMPA, KA (0.5 nmol) or NMDA (25 nmol)-induced neurotoxicity, was prevented in a dose-dependent manner by focal co-injection of four potential free radical scavengers; dimethyl sulphoxide (DMSO) (1750-7000 nmol), dimethylthiourea (DMTU) (8000 nmol), dimethylformamide (DMF) (7000 nmol) and mannitol (1000 nmol). These findings suggest that hippocampal damage induced by BOAA involves an interaction between AMPA receptors and free radicals.

Decapitation Reduces the Metabolism of Gibberellin A20 to A1 in Pisum sativum L., Decreasing the Le/le Difference.

When the metabolism of [13C,3H]gibberellin (GA)20 in Pisum sativum L. was investigated using decapitated plants and stem sections, no evidence was obtained for the recently postulated inhibitor of GA20 3[beta]-hydroxylase (V.A. Smith [1992] Plant Physiol 99: 372-377). Instead, the results are consistent with the hypothesis that the mutation le reduces GA1 production by altering the structure or level of the 3[beta]-hydroxylase.

Neuronal damage induced by beta-N-oxalylamino-L-alanine, in the rat hippocampus, can be prevented by a non-NMDA antagonist, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline.

The neurotoxin beta-N-oxalylamino-L-alanine (BOAA), found in Lathyrus sativus seeds, is thought to be the causative agent of neurolathyrism. We have investigated the in vivo mechanism of action of BOAA by focal injection (1 microliter) in the dorsal hippocampus of male Wistar rats and comparing the pathological outcome with the effects of injections (1 microliter) of alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA), kainate (KA) or N-methyl-D-aspartate (NMDA). Cellular damage induced by the excitatory amino acids in the pyramidal (CA1-CA4) and dentate granule neurones (DG) was assessed histologically 24 h after the injection. The study shows that BOAA (50 nmol) induces hippocampal toxicity with a highly selective pattern of regional cellular damage. The CA1, CA4 and DG subfields show 70-90% neuronal injury whereas CA2 and CA3 show only minimal damage. This pattern of cellular damage is similar to that induced by AMPA (1 nmol) and NMDA (25 nmol) but not KA (0.5 nmol). BOAA-induced neurotoxicity is prevented in a dose-dependent manner by focal co-injection of the non-NMDA receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) (1-25 nmol) but not by a dose of MK-801 (3 mg/kg i.p.) which is neuroprotective against an injection of NMDA. Delayed focal injections of NBQX (25 nmol) up to 2 h after the BOAA injection result in a significant protection of all pyramidal and granular cell regions. These results indicate that the in vivo hippocampal toxicity of BOAA is mediated by AMPA receptors rather than by KA or NMDA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Shoot elongation in Lathyrus odoratus L.: Gibberellin levels in light- and dark-grown tall and dwarf seedlings.

The levels of gibberellin A1 (GA1), GA20, GA19, GA8, GA29 and GA81 (2-epiGA29) were measured in tall (L-) and dwarf (ll) sweet-pea plants grown in darkness and in light. In both environments the apical portions of dwarf plants contained less GA1; GA8 and GA19, but more GA20, GA29, and GA81 than did those of tall plants. It is concluded that the partial block in 3ß-hydroxylation of GA20 to GA1 is imposed by allele l in darkness as well as in the light. Furthermore, darkness does not appear to enhance elongation in sweet pea by increasing GA1 levels. The reduction of the pool size of GA19 in dwarf plants supports recent theories on the regulation of GA biosynthesis, formulated on the basis of observations in monocotyledonous species. Darkness results in decreased GA20, GA29, and GA81 levels in the apical portions of tall and dwarf plants and possible reasons for this are discussed.