Genetic engineering of doxorubicin production in Streptomyces peucetius: a review

The genetics and biochemistry of daunorubicin and doxorubicin production by Streptomyces peucetius is reviewed, with a focus on how such information can be used for the genetic engineering of strains having improved titers of these two antitumor antibiotics. Received 23 February 1999/ Accepted in revised form 22 March 1999     

A time since onset of injection model for hepatitis C spread amongst injecting drug users

Studies of hepatitis C virus (HCV) infection amongst injecting drug users (IDUs) have suggested that this population can be separated into two risk groups (naive and experienced) with different injecting risk behaviours. Understanding the differences between these two groups and how they interact could lead to a better allocation of prevention measures designed to reduce the burden of HCV in this population. In this paper we develop a deterministic, compartmental mathematical model for the spread of HCV in an IDU population that has been separated into two groups (naive and experienced) by time since onset of injection. We will first describe the model. After deriving the system of governing equations, we will examine the basic reproductive number $R_0$ , the existence and uniqueness of equilibrium solutions and the global stability of the disease free equilibrium (DFE) solution. The model behaviour is determined by the basic reproductive number, with $R_0=1$ a critical threshold for endemic HCV prevalence. We will show that when $R_0\le 1$ , and HCV is initially present in the population, the system will tend towards the globally asymptotically stable DFE where HCV has been eliminated from the population. We also show that when $R_0>1$ there exists a unique non-zero equilibrium solution. Then we estimate the value of $R_0$ from epidemiological data for Glasgow and verify our theoretical results using simulations with realistic parameter values. The numerical results suggest that if $R_0>1$ and the disease is initially present then the system will tend to the unique endemic equilibrium.     

Regulation of the Rev1–pol ζ complex during bypass of a DNA interstrand cross‐link

DNA interstrand cross‐links (ICLs) are repaired in S phase by a complex, multistep mechanism involving translesion DNA polymerases. After replication forks collide with an ICL, the leading strand approaches to within one nucleotide of the ICL (“approach”), a nucleotide is inserted across from the unhooked lesion (“insertion”), and the leading strand is extended beyond the lesion (“extension”). How DNA polymerases bypass the ICL is incompletely understood. Here, we use repair of a site‐specific ICL in Xenopus egg extracts to study the mechanism of lesion bypass. Deep sequencing of ICL repair products showed that the approach and extension steps are largely error‐free. However, a short mutagenic tract is introduced in the vicinity of the lesion, with a maximum mutation frequency of ~1%. Our data further suggest that approach is performed by a replicative polymerase, while extension involves a complex of Rev1 and DNA polymerase ζ. Rev1–pol ζ recruitment requires the Fanconi anemia core complex but not FancI–FancD2. Our results begin to illuminate how lesion bypass is integrated with chromosomal DNA replication to limit ICL repair‐associated mutagenesis.     

Lead dynamics in the forest floor and mineral soil in south-central Ontario

Recent studies have suggested that the residence time of Pb in the forest floor may not be as long as previously thought, and there is concern that the large pulse of atmospheric Pb deposited in the 1960s and early 1970s may move rapidly through mineral soils and eventually contaminate groundwater. In order to assess Pb mobility at a woodland (JMOEC) in south-central Ontario, a stable Pb isotope tracer ²⁰⁷Pb (8?mg?m^?2) was added to the forest floor in white pine (Pinus strobus) and sugar maple (Acer saccharum) stands, respectively, and monitored over a 2-year period. Excess ²⁰⁷Pb was rapidly lost from the forest floor. Applying first-order rate coefficients (k) of 0.57 (maple) and 0.32 (pine) obtained from the tracer study, and estimates of Pb deposition in the region, current predicted Pb concentrations in the forest floor are 1.5–3.1 and 2.1–5.8?mg?kg^?1 in the maple and pine plots, respectively. These values compare favorably with measured concentrations (corrected for mineral soil contamination) of 3.1–4.3?mg?kg^?1 in the maple stand and 2.6–3.6?mg?kg^?1 in the pine stand. The response time (1/k) of Pb in the forest floor at the sugar maple and white pine plots was estimated to be 1.8 and 3.1 years, respectively. The rapid loss of Pb from the forest floor at the JMOEC is much greater than previously reported, and is probably due to the rapid rate of litter turnover that is characteristic of forests with mull-type forest floors. In a survey of 23 forested sites that border the Precambrian Shield in south-central Ontario, Pb concentrations in the forest floor increased exponentially with decreasing soil pH. Lead concentrations in the forest floor at the most acidic survey sites, which exhibited mor-type forest floors, were approximately 10 times higher (～80?mg?kg^?1) than at the JMOEC, and pollution Pb burdens were up to 25 times greater. Despite the rapid loss of Pb from the forest floor at the JMOEC, the highest pollution Pb concentrations were found in the upper (0–1?cm) mineral soil horizon. Lead concentrations in the upper 30?cm of mineral soil were strongly correlated with organic matter content, indicating that pollution Pb does not move as a pulse down the soil profile, but instead is linked with organic matter distribution, indicating groundwater contamination is unlikely.     

Non-peptide alphavbeta3 antagonists. Part 6: design and synthesis of alphavbeta3 antagonists containing a pyridone or pyrazinone central scaffold

Two novel series of small-molecule RGD mimetics containing either a substituted pyridone or pyrazinone central constraint were prepared. Modification of the beta-alanine 3-substituent produced compounds that are potent and selective alpha(v)beta(3) antagonists and exhibit a range of physicochemical properties.     

The biometry of gills of 0-group European flounder

The gill surface area of 0-group, post-metamorphic Pleuronectes flesus L. was examined using digital image analysis software and expressed in relation to body mass according to the equation log Y=loga+c logW ( a =239·02; c =0·723). The components that constitute gill area, total filament length, interlamellar space and unilateral lamellar area were measured. The measurement of the length of every filament on all eight arches showed that commonly used methods of calculation can lead to an under-estimation of up to 24% of total filament length. Direct measurements of unilateral lamellar area with digital image analysis showed that previously reported gill area data for the same species was over-estimated by as much as 58%. In addition, in this species the neglect of gill pouch asymmetry after metamorphosis, can bring about a 14% over-estimation of total gill area.     

Diagnosis of Amyloidosis and Differentiation from Chronic,Idiopathic Enterocolitis in Rhesus (Macaca mulatta) and Pig-Tailed (M. nemestrina) Macaques

Amyloidosis is a progressive and ultimately fatal disease in which amyloid, an insoluble fibrillar protein, is deposited inappropriately in multiple organs, eventually leading to organ dysfunction. Although this condition commonly affects macaques, there is currently no reliable method of early diagnosis. Changes in clinical pathology parameters have been associated with amyloidosis but occur in late stages of disease, are nonspecific, and resemble those seen in chronic, idiopathic enterocolitis. A review of animal records revealed that amyloidosis was almost always diagnosed postmortem, with prevalences of 15% and 25% in our rhesus and pig-tailed macaque colonies, respectively. As a noninvasive, high-throughput diagnostic approach to improve antemortem diagnosis of amyloidosis in macaques, we evaluated serum amyloid A (SAA), an acute-phase protein and the precursor to amyloid. Using necropsy records and ELISA analysis of banked serum, we found that SAA is significantly elevated in both rhesus and pig-tailed macaques with amyloid compared with those with chronic enterocolitis and healthy controls. At necropsy, 92% of rhesus and 83% of pig-tailed had amyloid deposition in either the intestines or liver. Minimally invasive biopsy techniques including endoscopy of the small intestine, mucosal biopsy of the colon, and ultrasound-guided trucut biopsy of the liver were used to differentiate macaques in our colonies with similar clinical presentations as either having amyloidosis or chronic, idiopathic enterocolitis. Our data suggest that SAA can serve as an effective noninvasive screening tool for amyloidosis and that minimally invasive biopsies can be used to confirm this diagnosis.Abbreviations: SAA, serum amyloid AAmyloidosis is a pathologic condition that occurs spontaneously in humans, mammals, birds, and reptiles.⁴⁷ Secondary systemic amyloidosis, also referred to as reactive amyloidosis, is the most common form described in domestic animals.⁴⁶ It is a progressive disease in which an insoluble fibrillar protein consisting of β pleated sheets, amyloid, is deposited inappropriately in multiple organs, eventually leading to dysfunction.^40,46 Secondary amyloidosis is most often the result of chronic infections or inflammatory disease. In humans, it occurs with a wide variety of conditions including inflammatory bowel disease,³ osteoarthritis including rheumatoid and juvenile forms,^20,25 chronic infections such as tuberculosis, and hereditary disease such as familial Mediterranean fever.⁴³ Similarly, in nonhuman primates, the disease has been described with several conditions of chronic infection or inflammation including bacterial enterocolitis,^4,19,30,37 chronic indwelling catheters,⁹ parasitism,^2,4 respiratory disease,^30,37 trauma,³⁷ and rheumatoid arthritis.⁶Despite reported prevalences as high as 30% in rhesus (Macaca mulatta)⁴ and 47% in pig-tailed macaques (Macaca nemestrina),¹⁹ amyloidosis remains a challenge to diagnose. The current diagnostic ‘gold standard’ in macaques is histopathology of the affected organ;¹⁹ however, amyloid can be deposited in tissues for as long as 3 y before the development of clinical signs.¹⁶ Histologic diagnoses of amyloidosis typically are confirmed with Congo red staining, in which amyloid proteins appear apple-green and birefringent under polarized light. In addition, electron microscopy can detect the fibrillar amyloid proteins in tissues, and other histologic stains including methyl violet, sulphonated Alcian blue, and thioflavin S and T can be used but are less specific than is Congo red.³³ Although changes in clinical pathology parameters such as decreases in serum albumin and total protein have been associated with amyloidosis,^19,29 they are often nonspecific and resemble those seen in the frequently comorbid conditions chronic anorexia and chronic, idiopathic enterocolitis. Furthermore, imaging techniques such as abdominal X-ray and ultrasonography have been shown to be nondiagnostic in macaques with amyloidosis.¹⁹ Consequently, at our institution and in other macaque colonies, diagnosis of amyloidosis is often made at necropsy.The current standard of diagnosis in humans is biopsy with histopathology of affected organs, but unlike in nonhuman primates, minimally invasive tissue sampling has been extensively explored.¹⁷ Aspiration or biopsy of the subcutaneous abdominal fat pad has currently replaced many biopsy techniques as the preliminary diagnostic, with reported sensitivities ranging from 66% to 92%.^{5,24,28,39,44} Rectal biopsy was previously the preferred minimally invasive approach and is now often used adjunctively when subcutaneous abdominal fat is negative for amyloid but the clinical suspicion for amyloidosis remains high.^5,17 Additional tissue biopsy sites with limited morbidity such as skin, gingiva, and stomach have been reported with lesser sensitivities.^5,34,39,44 In contrast, limited information is published on the usefulness of minimally invasive biopsy techniques for diagnosing amyloidosis in macaques. One report found endoscopic biopsy of the stomach and colon to be of limited utility in diagnosing amyloidosis in a colony of pig-tailed macaques.¹⁹ Similarly, a single publication reported colonoscopy to be noninformative and labor-intensive in a colony of rhesus macaques.¹⁵ Retrospective studies of macaque colonies have shown a predilection for amyloid deposition in the intestines and liver,^4,30,38 suggesting that endoscopic or percutaneous biopsy of these tissues may reliably provide definitive antemortem diagnosis for amyloidosis.In addition to biopsy, identification of the relevant amyloid precursor protein within the blood is an integral part of the diagnosis of amyloidosis in human patients¹⁷ and holds promise as a screening tool in macaque colonies because of its high throughput potential in comparison to biopsy. Serum amyloid A (SAA), an acute-phase protein, can be found circulating in the blood and is the precursor for amyloid formation and deposition in secondary systemic amyloidosis. Specifically, when elevated SAA persists in the bloodstream, it ultimately progresses to amyloid deposition in tissues.^13,45 Profound elevations in SAA occur in the bloodstream as a result of acute inflammation, but these elevations are transient as SAA then is rapidly degraded and removed from the peripheral circulation.^7,45 Although the exact role of chronic inflammation and SAA in the pathogenesis of secondary, systemic amyloidosis is not well understood, SAA is pathologically persistently elevated in human patients with chronic inflammatory disease that develop secondary systemic amyloidosis. In contrast, serum SAA remains at normal lower levels in human patients without amyloidosis but ongoing chronic inflammatory disease.^13,14,26 Furthermore, quantification of SAA is more effective than are organ function tests as a prognostic measure of amyloid disease and is routinely used to monitor disease progression and response to treatment in humans.¹⁴ In rhesus and pig-tailed macaques, SAA is elevated in subjects with amyloidosis as compared with those that are clinically normal.^8,19 The ability to distinguish between healthy animals and those with subclinical amyloidosis would be clinically useful. Human studies indicate that establishing a diagnosis of secondary amyloidosis in its early stages followed by prompt treatment of the inciting chronic inflammatory process can arrest the progression of amyloidosis and can even result in disease remission in some cases.^{21,23,31,32,36} Of equal interest would be the ability to distinguish amyloidosis from chronic, idiopathic enterocolitis, a common disease among macaque colonies^12,35 that has considerable clinical overlap with the late stages of amyloidosis but different therapeutic options and prognosis than does systemic amyloidosis. Although there is no definitive treatment for amyloidosis in humans or macaques, recent human case reports suggest that antiinflammatory therapy with newer targeted monocolonal antibody medications, such as IL6 receptor antagonists, can successfully reverse the disease. This outcome has been demonstrated in several cases by both the reduction of circulating SAA to normal levels and by the histologic disappearance of amyloid proteins in biopsies of affected tissues.^{21,23,31,32,36} Accurate antemortem diagnosis of amyloidosis in macaques potentially would support further investigations into the novel application of these drugs for the treatment of amyloidosis in both human and macaque patients.We hypothesize that SAA, in addition to being a useful screening method for identifying animals with amyloidosis, can be used to distinguish between macaques with this disease and those with chronic, idiopathic enterocolitis. We further hypothesize that, in agreement with retrospective studies from macaques at other institutions, the intestines and liver will be commonly affected in amyloidotic macaques in our own colonies and that minimally invasive biopsy of these tissues can provide definitive, antemortem diagnosis of amyloidosis.     

Orthosteric Binding of ρ-Da1a,a Natural Peptide of Snake Venom Interacting Selectively with the α1A-Adrenoceptor

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α_1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of ³H-prazosin or ¹²⁵I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α_1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α_1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α_1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of ³H-prazosin or ¹²⁵I-HEAT, and the IC₅₀ of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca²⁺ release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α_1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106^3.32A and the S188^5.42A/S192^5.46A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86^2.64A, F288^6.51A and F312^7.39A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86^2.64 was identified as a key interaction point for ¹²⁵I-HEAT, as the variant F86^2.64A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α_1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86^2.64, F288^6.51 and F312^7.39) and agonist (F288^6.51 and F312^7.39) ligands. Its selectivity for the α_1A-adrenoceptor may result, at least partly, from its interaction with the residue F86^2.64, which appears to be important also for HEAT binding.     

Role of purine metabolism in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium × hortorum) hypocotyl cultures

Somatic embryogenesis in geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μ M thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6-diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μ M adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP-induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy-tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.