Validation of a high-performance liquid chromatographic assay for the quantification of adenovirus type 5 particles

An anion-exchange–high-performance liquid chromatography (AE–HPLC) method for the quantification of adenovirus type 5 (Ad5) total particles was validated according to performance criteria of precision, specificity, linearity of calibration and range, limit of detection, limit of quantification, accuracy and recovery. The viral particles were detected by absorbance at 260 nm using photodiode array detector (PDA). Cesium chloride (CsCl) purified Ad5 and lysate samples were used for the validation of the method. Relative standard deviations (RSDs) for the inter-day, intra-day precision and reproducibility for both the lysate and the Ad5 standard were less than 10 and 2% for the peak area and retention time, respectively. The method was specific for Ad5 which was eluted at 8.0 min. The presence of DNA does not affect the recovery of Ad5 particles for accurate quantification. Based on the error in prediction to be less than 10%, the working range was established between 2×10¹⁰ and 7×10¹¹ VP/ml with correlation coefficient of 0.99975, standard deviation of 6.14×10⁹ VP/ml and a slope of 3.04×10⁵ VP/ml. The recovery of the method varied between 88 and 106% in all of the lysate samples investigated which is statistically similar to 100% recovery at 95% confidence interval.     

Manufacturing of recombinant adeno‐associated viruses using mammalian expression platforms

Manufacturing practices for recombinant adeno‐associated viruses (AAV) have improved in the last decade through the development of new platforms in conjunction with better production and purification methods. In this review, we discuss the advantages and limitations of the most popular systems and methods employed with mammalian cell platforms. Methods and systems such as transient transfection, packaging and producer cells and adenovirus and herpes simplex virus are described. In terms of best production yields, they are comparable with about 10⁴–10⁵ vector genomes produced per cell but transient transfection of HEK293 cells is by far the most commonly used. For small‐scale productions, AAV can be directly purified from the producing cell lysate by ultracentrifugation on a CsCl or iodixanol‐step gradient whereas large‐scale purification requires a combination of multiple steps. Micro/macrofiltration (i.e. including tangential flow filtration and/or dead‐end filtration) and chromatography based‐methods are used for large‐scale purification. Purified AAV products must then be quantified and characterized to ensure quality. Recent purification methods and current analytical techniques are reviewed here. Finally, AAV technology is very promising, but manufacturing improvements are still required to meet the needs of affordable, safe and effective AAV vectors essential for licensing of gene therapy clinical protocols.     

Modeling of scale-down effects on the hydrodynamics of expanded bed adsorption columns

Expanded-bed adsorption (EBA) is a technique for primary recovery of proteins starting from unclarified broths. This process combines centrifugation, concentration, filtration, and initial capturing of the proteins in a single step. An expanded bed (EB) is comparable to a packed bed in terms of separation performance but its hydrodynamics are that of a fluidized bed. Downstream process development involving EBA is normally carried out in small columns to minimize time and costs. Our purpose here is to characterize the hydrodynamics of expanded beds of different diameters, to develop scaling parameters that can be reliably used to predict separation efficiency of larger EBA columns. A hydrodynamic model has been developed which takes into account the radial liquid velocity profile in the column. The scale-down effect can be characterized in terms of apparent axial dispersion, D(axl,app), and plate number, N(EB), adapted for expanded bed. The model is in good agreement with experimental results obtained from 1- and 5-cm column diameters with buffer solutions of different viscosities. The model and the experiments show an increase of apparent axial dispersion with an increase in column diameter. Furthermore, the apparent axial dispersion is affected by an increase in liquid velocity and viscosity. Supported by visual observations and predictions from the model, it was concluded that operating conditions (liquid viscosity and superficial velocity) resulting in a bed-void fraction between 0.7 and 0.75 would provide the optimal separation efficiency in terms of N(EB).     

Role of spinal NMDA and non-NMDA receptors in the pressor reflex response to abdominal ischemia

Abdominal ischemia induces a pressor reflex caused mainly by C-fiber afferent stimulation. Because excitatory amino acids, such as glutamate, bind to N-methyl-D-aspartate (NMDA) and non-NMDA [dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)] receptors and serve as important spinal neurotransmitters, we hypothesized that both receptors play a role in the abdominal ischemia pressor reflex. In chloralose-anesthetized cats, NMDA receptor blockade with 25.0 mM dl-2-amino-5-phosphonopentanoate did not alter the pressor reflex (33 +/- 9 to 33 +/- 7 mmHg, P > 0.05, n = 4), whereas AMPA receptor blockade with 4.0 mM 6-nitro-7-sulfamylbenzo(f)quinoxaline-2,3-dione significantly attenuated the reflex (29 +/- 5 to 16 +/- 4 mmHg, P < 0.05, n = 6). Because several studies suggest that anesthesia masks the effects of glutamatergic receptors, this experiment was repeated on decerebrate cats, and in this group, NMDA receptor blockade with 25.0 mM dl-2-amino-5-phosphonopentanoate significantly altered the pressor reflex (36 +/- 3 to 25 +/- 4 mmHg, P < 0.05, n = 5). Our combined data suggest that spinal NMDA and AMPA receptors play a role in the abdominal ischemia pressor reflex.     

Somatostatin inhibits the ventilatory response to hypoxia in humans

The effects of a 90-min infusion of somatostatin (1 mg/h) on ventilation and the ventilatory responses to hypoxia and hypercapnia were studied in six normal adult males. Minute ventilation (VE) was measured with inductance plethysmography, arterial 02 saturation (SaO2) was measured with ear oximetry, and arterial PCO2 (Paco2) was estimated with a transcutaneous CO2 electrode. The steady-state ventilatory response to hypoxia (delta VE/delta SaO2) was measured in subjects breathing 10.5% O2 in an open circuit while isocapnia was maintained by the addition of CO2. The hypercapnic response (delta VE/delta PaCO2) was measured in subjects breathing first 5% and then 7.5% CO2 (in 52-55% O2). Somatostatin greatly attenuated the hypoxic response (control mean -790 ml x min-1.%SaO2 -1, somatostatin mean -120 ml x min-1.%SaO2 -1; P less than 0.01), caused a small fall in resting ventilation (mean % fall - 11%), but did not affect the hypercapnic response. In three of the subjects progressive ventilatory responses (using rebreathing techniques, dry gas meter, and end-tidal Pco2 analysis) and overall metabolism were measured. Somatostatin caused similar changes (mean fall in hypoxic response -73%; no change in hypercapnic response) and did not alter overall O2 consumption nor CO2 production. These results show an hitherto-unsuspected inhibitory potential of this neuropeptide on the control of breathing; the sparing of the hypercapnic response is suggestive of an action on the carotid body but does not exclude a central effect.     

Biochemical variation in the Sunni Muslims of Pulwama district, Jammu and Kashmir

Baseline data are presented on phenotype and gene frequency distributions of nine red-cell enzyme systems (ADA, AK, EsD, GLO I, PGM1, AP, GPI, PGM2, SOD) in the Sunni Muslims of Kashmir valley in the northernmost Indian border state of Jammu and Kashmir.     

Dose-dependent response of Trichoderma harzianum in improving drought tolerance in rice genotypes

Planta - This study demonstrates a dose-dependent response of Trichoderma harzianum Th-56 in improving drought tolerance in rice by modulating proline, SOD, lipid peroxidation product and DHN / AQU...     

A fertile revertant from petaloid cytoplasmic male-sterile carrot has a rearranged mitochondrial genome

 A spontaneously derived fertile plant was recovered from a petaloid cytoplasmic male-sterile (CMS) carrot inbred line. Genetic analysis indicated a single nuclear gene was responsible for the restoration to fertility. Within a family segregating for the nuclear restorer in combination with the sterility-inducing cytoplasm, fertile plants were recovered that could not restore fertility when crossed to sterile genotypes. Genetic analysis indicated cytoplasmic reversion for fertility, and Southern analysis, comparing mtDNA organization of the fertile revertant and its CMS progenitor, identified mitochondrial genome rearrangements. Hybridization of cosmids representing a 108-kb subgenomic circle of the sterile line to DNA of a fertile maintainer and fertile revertant lines indicated a similar mtDNA organization for these genotypes that was distinct from that of the sterile line. Six restriction fragments totalling 43.2 kb were common to the fertile maintainer and revertant and absent in the sterile; other restriction fragments totalling 38.2 kb were present only for the sterile line. Unique fragments of low stoichiometry, two for the fertile maintainer and three for the revertant, distinguished these lines. The reversion to fertility in the sterile line could have resulted from the amplification of a mitochondrial submolar genome highly homologous to that found in the fertile maintainer line. Received: 4 October 1997/Accepted: 12 December 1997     

Autogeny and endocrine involvement in the reproduction of tropical flesh flies (Diptera: Sarcophagidae)

Autogeny is highly developed among the Sarcophagidae in tropical Africa. Unlike their temperate region relatives, the tropical species examined can mature the first batch of eggs without an adult protein meal, and they do so without the long period of delay usually seen in autogenous species. Protein deprivation, however, does reduce the number of eggs matured by about one-third. The tropical and temperate flies appear to share a common endocrine mechanism controlling egg maturation. Removal of the medial neurosecretory cells of the pars intercerebralis prevents egg maturation. Reimplantation of clusters of MNC restores the ability to mature eggs. Extirpation of the corpora allata reduces the percentage of females that mature eggs to about 70%, and the females that do mature eggs produce fewer eggs. The significant difference between the tropical and temperate species centers on the trigger that sets the neuroendocrine system into action. For the tropical species, the brain is activated immediately and does not require the trigger of a protein meal.     

Experimental studies of migration in bugs of the genus Dysdercus

Summary Using a tethered flight technique, migration was studied in the African cotton stainer bugs Dysdercus fasciatus Sign., D. nigrofasciatus Stål, and D. superstitiosus (F.) (Heteroptera: Pyrrhocoridae). The flight system of males of all three species matures in 4–6 days post-eclosion, and flight can continue throughout life. Fed females of D. fasciatus do not fly, but rather histolyse the flight muscles and develop oocytes. Fed females of D. nigrofasciatus and D. superstitiosus exhibit some flight at 4 days post-eclosion, but flight is essentially absent at 6 days when flight muscle histolysis and oögenesis are in progress. Starved females of all three species undertake considerable flight and do not histolyse the flight muscles or develop oöcytes. In starved pairs coupling frequency is lowered permitting increased migration. The three species are thus facultative migrants using starvation as a releasing cue. D. fasciatus feeds on baobab (Adansonia) fruits which are seasonally abundant and is the most opportunistic species with the highest reproductive potential and no flight in the presence of food. D. nigrofasciatus feeds on a variety of herbaceous annuals and perennials; it follows an intermediate migration and reproductive strategy. Finally D. superstitiosus is the most catholic in its food habits and has the lowest reproductive potential. Both of the latter species retain some flight capacity even when fed, presumably so that individuals can locate suitable host plants.