Transient gene expression in HEK293 cells: peptone addition posttransfection improves recombinant protein synthesis.

Gene expression by large-scale transfection of mammalian cells is becoming an established technology for the fast production of milligram and even gram amounts of recombinant proteins (r-proteins). However, efforts are still needed to optimize production parameters in order to maximize volumetric productivities while maintaining product quality. In this study, transfection efficiency and volumetric productivity following transient gene expression in HEK293 cells were evaluated using green fluorescent protein (GFP) and human placental secreted alkaline phosphatase (SEAP) as reporter genes. We show that a single pulse of peptones (protein hydrolysates) to the cultures performed in a low serum (1%, v/v) and in serum-free medium results in a significant increase in volumetric protein productivity. Sixteen peptones from different sources were tested and almost all of them showed a positive effect on r-protein production. This effect, however, is time- and concentration-dependent. By using Tryptone N1 (a casein peptone, TN1) to feed the cultures at 24 h posttransfection (hpt), a 2-fold increase in volumetric SEAP productivity was obtained 5 days posttransfection. This effect was shown to be equal to that obtained when the culture was fed with a supplementary 4% (v/v) of serum. The positive effect of TN1 on protein production was also demonstrated with Tie2 protein ectodomain produced in serum-free medium. HPLC analysis of amino acids consumption/production during control batch and TN1 pulse culture showed some major differences in amino acid metabolism when using TN1 pulse. Asparagine, glycine, histidine, threonine, leucine, and valine show accumulation in the medium over the cultivation period instead of being consumed as observed in unfed sample (except for asparagine, which remained unchanged). Isoleucine, tyrosine, methionine, and phenylalanine all remained unchanged or slightly fluctuated in TN1-fed culture after the feeding pulse, while they were all steadily consumed in the control run. The relative abundance of SEAP's mRNA suggests that the improvement in protein yield results both from an increase of the translational activity and transcription efficiency. Further understanding of mechanisms by which amino acids/peptides regulate transcriptional and translational machinery in mammalian cells should facilitate the design of new strategies for the improvement of r-protein production by large-scale transfection.

Production of tomato flavor volatiles from a crude enzyme preparation using a hollow-fiber reactor.

In recent years there has been an increase in the interest in the production of compounds by isolation from natural sources or through processes that can be deemed "natural". This is of particular interest in the food and beverage industry for flavors and aromas. Hexanal, organoleptically known to possess "green character", is of considerable commercial interest. The objective of this study was to determine if the enzyme template known to be responsible for the synthesis of hexanal from linoleic acid (18:2) in tomato fruits could be harnessed using a hollow-fiber reactor. A hollow-fiber reactor system was set up and consisted of a XAMPLER ultrafiltration module coupled to a reservoir. The enzyme template was extracted from ripe tomato fruits and processed through an ultrafiltration unit (NMWC of 100 kDa) to produce a retentate enriched in soluble and membrane-associated lipoxygenase (LOX) and hydroperoxide lyase (HPL). This extract was recirculated through the lumen of the hollow-fiber ultrafiltration unit with the addition of substrate in the form of linoleic acid, with buffer addition to the reaction flask to maintain a constant retentate volume. Product formation was measured in the permeate using solid phase microextraction (SPME) developed for this system. At exogenous substrate concentrations of 16 mM and a transmembrane pressure of 70 kPa, hexanal production rates are in the order of 5.1 microg/min. Addition of Triton X-100 resulted in membrane fouling and reduced flux. The reactor system has been run for periods of up to 1 week and has been shown to be stable over this period.

Immediate surgical management of severe renal injuries in multiple-injured patients.

Between 1969 and 1980, 1,132 renal injuries were recorded in 1,120 patients. Of the 129 patients with a severe renal injury (laceration, rupture, pedicle injury), 118 (91%) had associated injuries and 101 (86%) required an immediate laparotomy by general surgeons for intra-abdominal injury. Immediate surgical management was performed on 90 severe renal injuries and resulted in a nephrectomy rate of 5 of 55 lacerations (9%), 16 of 17 ruptures (94%), and 10 of 18 pedicle injuries (56%). Seven of the 18 pedicle injuries (39%) had immediate repair with salvage of the kidney. Conservative management was done on 32 severe renal injuries. Autopsy revealed 10 severe renal injuries in 9 patients who died in the operating room or on admission. A delayed renal operation was required in 9 (28%), and total renal loss (nephrectomy plus main renal artery injury) occurred in 7 of the 32 cases (22%). The severe degrees of renal injury did not all resolve spontaneously. Immediate surgical management of the patient with a severe renal injury resulted in a nephrectomy rate comparable with that of conservatively managed renal injuries.

Immediate exploration of the unilateral acute scrotum in young male subjects.

Physical exertion, sexual activity or trauma can precipitate cremasteric contraction and result in torsion of a testicle with an underlying congenital anomaly. A high index of suspicion is required by the primary care doctor if acute torsion is to be diagnosed early for immediate reduction. The duration of torsion and the degree of twisting determine the long-term salvage rate of testicular tissue. An aggressive policy of immediate exploration of the unilateral acute scrotum in young men resulted in exploration of twice as many cases of acute epididymitis as torsion of the testis or its appendages but also resulted in an immediate orchiopexy (salvage) rate of 90 per cent and a long-term salvage rate of 73 per cent of patients with acute torsion of the testis.