Bioengineering of coagulation factor VIII for efficient expression through elimination of a dispensable disulfide loop.

BACKGROUND: Heterologous expression of factor VIII (FVIII) is about two to three orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels. OBJECTIVE: Disulfide bonds are vital to the proper folding, secretion and stability of most secretory proteins. In an effort to explore additional targeted bioengineering approaches, the role of disulfide bonds in FVIII secretion and function was probed in this study. METHODS AND RESULTS: Single and paired cysteine mutants were generated by substituting with serine or glycine residues and analyzed by transient transfection into COS-1 and CHO cells. Seven of the eight disulfide bonds in FVIII were found to be indispensable for proper secretion and function. However, elimination of the disulfide bond formed by C1899 and C1903 within the conserved A3 domain improved the secretion of FVIII. The addition of the C1899G/C1903G mutations to a previously described FVIII variant, 226/N6, with high secretion efficiency increased its secretion by 2.2-fold. Finally, the addition of the A1-domain mutation, F309S, in conjunction with the disulfide mutation had an additive effect, resulting in a net improvement in secretion of between 35 and 45-fold higher than wild-type FVIII in CHO cells. CONCLUSION: Such combined targeted bioengineering strategies may facilitate more efficient production of recombinant FVIII and contribute toward low-cost factor replacement therapy for hemophilia A.

Magnetic resonance imaging of relative glycosaminoglycan distribution in patients with autologous chondrocyte transplants.

RATIONALE AND OBJECTIVES: Autologous chondrocyte transplantation (ACT) is a potential treatment for full-thickness chondral lesions in the knee. Delayed gadolinium-enhanced magnetic resonance imaging of cartilage (dGEMRIC) has recently been developed as a sensitive and specific measure of cartilage glycosaminoglycans (GAGs). Under the conditions of dGEMRIC, T1 is directly related to the GAG concentration. Our aim for this study was to demonstrate the potential of dGEMRIC to evaluate ACT implants. METHODS: Eleven ACT implants were studied 2 to 24 months postoperatively by dGEMRIC. T1 values from three regions of interest were obtained to examine GAG content (1) in the implant, (2) in native cartilage adjacent to the implant, and (3) in native cartilage further removed from the implant (as "control"). RESULTS: One implant failed and therefore was not included. Four of the implants were studied between 2 and 6 months postoperatively and showed low T1 (GAG), less than 80% of the control native cartilage. Five of the six implants studied between 12 and 24 months postoperativley showed T1 (GAG) comparable to (>80%) of control. One 18-month graft showed low T1 comparable to the surrounding native cartilage, with normal GAG seen in cartilage far from the graft site. The GAG index (T1 values of the graft normalized to control) from the group of implants 6 months or less was 59% +/- 5% of control, whereas those at 12 to 24 months were 91% +/- 18% of control. The two groups were statistically different with a P value of 0.005. CONCLUSIONS: The GAG level in grafts that were implanted for less than 12 months appeared to be lower than that in the remote cartilage. At 12 months or greater, the grafts in this study had GAG levels that were comparable to both the adjacent and remote cartilage. This preliminary study of ACT implants has shown that it is feasible to apply the dGEMRIC technique in patients with ACT as a way to obtain information related to the composition of grafts. These results provide motivation and the pilot data with which to design further clinical studies.

Evidence for an amphipathicity independent cellular uptake of amphipathic cell-penetrating peptides.

The cellular uptake of a peptide set derived from membrane-permeable alpha-helical amphipathic peptides by stepwise alterations of structure forming propensity and charge was studied by confocal laser scanning microscopy (CLSM) combined with HPLC. For CLSM monitoring, an online protocol was employed that avoided bias of the uptake results by washout. Using this protocol, extensive fluorescence, approaching the intensity of the external peptide, was observed in the cytosol and nucleus within minutes in all cases, irrespective of the degree of amphipathicity. HPLC analyses of the cell lysates revealed the unmetabolized peptides to be the predominant source of the intracellular fluorescence. Significant amphipathicity-dependent differences became apparent only after washing the peptide-loaded cells, reflecting the effects of amphipathicity on resistance to wash out. Exposure of the cells to the peptides at 37 and 0 degrees C led to similar results, indicating the nonendocytic character of the uptake. With a view to practical applications, the results of the present study open the possibility of exploiting nonamphipathic peptides as vectors for translocating polar compounds into the cell interior, which would circumvent substantial obstacles currently connected with the use of amphipathic vector peptides, such as membrane toxicity and low solubility. Moreover, differences in the uptake of several members of the investigated peptide series into different cell types present a promising basis for the design of cell-type specific vector peptides.

Smoking does not alter the dose-requirements and the pharmacodynamics of rocuronium.

PURPOSE: Controversial data about the effect of smoking on the dose-requirements and the pharamcodynamics of rocuronium have been reported recently. This study was conducted to evaluate the dose-requirements and the pharmacodynamics of rocuronium in smokers using target controlled infusion. METHODS: The dose-requirements of rocuronium for 60 min relaxation, using target controlled infusion, given under intravenous anaesthesia with propofol, fentanyl and nitrous oxide was studied in 37 smokers and 37 non-smokers. Initially 450 microg x kg(-1) rocuronium were administered, neuromuscular effects were quantified by recording the single twitch response of the adductor pollicis muscle after ulnar nerve stimulation using a force transducer, and the neuromuscular block was kept at 80% by target controlled infusion throughout the procedure. RESULTS: The dose-requirements for one hour relaxation were 867 +/- 116 microg x kg(-1) x hr(-1) for smokers (S) and 839 +/- 149 microg x kg(-1) x hr(-1) for non-smokers (NS). The duration to 10% and the spontaneous recovery from 25% to 75% of the control twitch response also showed no differences between S (17.2 +/- 3.4 min, 10.6 +/- 0.9 min) and NS (18.9 +/- 4.3 min, 10.9 +/- 3.2 min), as well as maximum block, onset time and infusion rate. CONCLUSION: Smoking does not alter the dose-requirements for rocuronium and no effects on the onset time, degree of block, time to maximum block, duration 10% and spontaneous recovery index were observed.

[The effect of different priming doses on the pharmacodynamics of cisatracurium]. / Die Wirkung unterschiedlicher Primingdosierungen auf die Pharmakodynamik von Cisatracurium.

OBJECTIVE: The aim of the study was to evaluate the effect of two different priming regimen on the onset time of 100 micrograms/kg cisatracurium, when compared to bolus administration. METHODS: 51 patients were randomly assigned and received either a bolus of 100 micrograms/kg cisatracurium, or a priming dose of 10 micrograms/kg cisatracurium followed after 4 min by 90 micrograms/kg cisatracurium, or a priming dose of 15 micrograms/kg cisatracurium followed after 4 min by 85 micrograms/kg cisatracurium. The neuromuscular monitoring was performed using a mechanomyograph (Groningen II Monitor). Anaesthesia was induced with propofol and fentanyl and maintained by continuous infusion of propofol. RESULTS: The priming combination of 15 micrograms/kg cisatracurium followed after 4 min by 85 micrograms/kg cisatracurium produced a statistically significant reduction in the onset time (95% block) (180 +/- 60 s) and time to complete block (210 +/- 48 s), when compared to the bolus group (240 +/- 60 s and 288 +/- 66 s) (p < 0.05). CONCLUSION: Our data indicate that the "priming principle" is an appropriate technique to shorten the onset time of cisatracurium. To achieve a maximum effect the priming combination of 15 micrograms/kg cisatracurium followed after 4 min by 85 micrograms/kg cisatracurium is recommended.

Structural requirements for cellular uptake of alpha-helical amphipathic peptides.

The structure of the cell-permeable alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) was modified stepwise with respect to its helix parameters hydrophobicity, hydrophobic moment and hydrophilic face as well as molecular size and charge. Cellular uptake and membrane destabilizing activity of the resulting peptides were studied using aortic endothelial cells and HPLC combined with CLSM. With the exceptions that a reduction of molecule size below 16 amino acid residues and the introduction of a negative net charge abolished uptake, none of the investigated structural parameters proved to be essential for the passage of these peptides across the plasma membrane. Membrane toxicity also showed no correlation to any of the parameters investigated and could be detected only at concentrations higher than 2 microM. These results implicate helical amphipathicity as the only essential structural requirement for the entry of such peptides into the cell interior, in accord with earlier studies. The pivotal role of helical amphipathicity was confirmed by uptake results obtained with two further pairs of amphipathic/non-amphipathic 18-mer peptides with different primary structure, net charge and helix parameters from I. The amphipathic counterparts were internalized into the cells to a comparable extent as I, whereas no cellular uptake could be detected for the non-amphipathic analogues. The mode of uptake remains unclear and involves both temperature-sensitive and -insensitive processes, indicating non-endocytic contributions.

Cellular uptake of an alpha-helical amphipathic model peptide with the potential to deliver polar compounds into the cell interior non-endocytically.

Evidence that multiple, probably non-endocytic mechanisms are involved in the uptake into mammalian cells of the alpha-helical amphipathic model peptide FLUOS-KLALKLALKALKAALKLA-NH2 (I) is presented. Extensive cellular uptake of N-terminally GC-elongated derivatives of I, conjugated by disufide bridges to differently charged peptides, indicated that I-like model peptides might serve as vectors for intracellular delivery of polar bioactive compounds. The mode of the cellular internalization of I comprising energy-, temperature-, pH- and ion-dependent as well as -independent processes suggests analogy to that displayed by small unstructured peptides reported previously (Oehlke et al., Biochim. Biophys. Acta 1330 (1997) 50-60). The uptake behavior of I also showed analogy to that of several protein-derived helical peptide sequences, recently found to be capable of efficiently carrying tagged oligonucleotides and peptides directly into the cytosol of mammalian cells (Derossi et al., J. Biol. Chem. 269 (1994) 10444-10450; Lin et al., J. Biol. Chem. 270 (1995) 14255-14258; Fawell et al., Proc. Natl. Acad. Sci. USA 91 (1994) 664-668; Chaloin et al., Biochemistry 36 (1997) 11179-11187; Vives et al., J. Biol. Chem., 272 (1997) 16010-16017).

[Recurrent abdominal colic, myalgia and mononeuritis multiplex]. / Rezidivierende abdominelle Koliken, Myalgien und Mononeuritis multiplex.

The case of a 30-year-old male, who presented with a three months history of fever, night sweats, weight loss and myalgia is reported. Subsequently abdominal cramps, bloody diarrhea and mononeuropathy multiplex developed. An abdominal and renal angiogram showed changes of vascular structures diagnostic for polyarteritis nodosa. An immunosuppressive treatment (Prednisolon 100 mg/day and Cyclophosphamid 200 mg/day) was started. However, diffuse peritonitis as the aftermath of bowel infarction, which comprised the total length of the jejunum and the proximal parts of the ileum, developed at the third week of this treatment. Despite immediate surgical resection of the ischemic bowel septic complications occurred and the patient died.

Multiple receptor domains interact to permit, or restrict, androgen-specific gene activation.

A critical problem within transcription factor families is how diverse regulatory programs are directed by highly related members. Androgen and glucocorticoid receptors (AR, GR) recognize a consensus DNA hormone response element (HRE), but they activate target genes with precise specificity, largely dependent on the promoter and cell context. We have assessed the role of different receptor domains in hormone-specific response by testing chimeras of AR and GR for their ability to activate the androgen-specific enhancer of the mouse sex-limited protein (Slp) gene. Although all of the mutant receptors activated simple HREs, only a few activated the androgen-specific element. One component shared by receptors functional on the AR-specific target was the AR DNA binding domain. Activation was not due to differential DNA affinity but rather to the AR DNA binding domain escaping suppression directed at the GR DNA binding domain in this enhancer context. A further mechanism increasing specific activation was cooperation of receptors at multiple and weak HREs, which was accentuated in the presence of both the AR N terminus and ligand binding domain. These domains together increased recognition of weak HREs, as demonstrated by in vitro DNase I footprinting and transactivation of mutant enhancers. Further, AR N-terminal subdomains reported to interact directly with the ligand binding domain relieved an inhibitory effect imposed by that domain. Therefore, functions intrinsic to AR augment steroid-specific gene activation, by evading negative regulation operating on the domains of other receptors and by enhancing cooperativity through intra- and inter-receptor domain interactions. These subtle distinctions in AR and GR behavior enforce transcriptional specificity established by the context of nonreceptor factors.

Medial subluxation of the patella without previous lateral retinacular release.

This case presented a 17-year-old patient with persistent complaints localized to the right patellofemoral joint. Clinical examination demonstrated increased medial translation of the patella on manual stress. In contrast to previous published reports on medial patellar subluxation, this patient had not undergone prior lateral retinacular release. Arthroscopic examination documented medial tracking of the patella as well as excess medial translation. Imbrication of the patient's lateral patellar retinaculum centralized patella tracking and diminished medial translation on stress testing as observed arthroscopically and clinically. This case illustrates that medial patellar subluxation is a subtle problem that may be overlooked in the patient presenting with patellofemoral complaints and should be included in the differential diagnosis.

Magnetic resonance imaging for the evaluation of acute posterolateral complex injuries of the knee.

Clinical evaluation of posterolateral complex injuries of the knee can be difficult. To determine if magnetic resonance imaging can assist in decision-making in the treatment of posterolateral complex injuries, six consecutive patients with acute posterolateral knee trauma were imaged preoperatively with standard magnetic resonance imaging sequences. Magnetic resonance imaging findings were then correlated with results of examination under anesthesia or open lateral reconstruction. There were five complete lateral complex injuries (grade III) and one partial tear. Magnetic resonance imaging was able to accurately depict the extent of injury preoperatively in each case. All patients had concomitant anterior cruciate ligament tears. There was one partial posterior cruciate ligament tear. Visualization of the arcuate complex, biceps femoris tendon, lateral capsule, iliotibial band, popliteal tendon, and lateral collateral ligament was possible. A characteristic bone contusion on the anteromedial femoral condyle was present in all patients with complete posterolateral disruptions. Lateral complex injuries of the knee can be very accurately demonstrated on magnetic resonance imaging, and this imaging technique can be used to clarify the exact nature of the injury to allow better surgical planning.

Involvement of an octamer-like sequence within a crucial region of the androgen-dependent Slp enhancer.

Androgen dependence of the mouse sex-limited protein (Slp) gene is conferred by an enhancer encompassing a consensus hormone response element (HRE) and sites for several nonreceptor factors. The footprint IV (FPIV) region of the enhancer plays a key role in hormone- and tissue-specific response, both in vitro and in vivo. We characterized FPIV-binding factors by methylation interference analysis and UV cross-linking of several complexes evident in gel mobility-shift assays. The footprinting analysis revealed that distinct base contacts within the multiple nuclear protein-DNA complexes occurred primarily within a sequence similar to an octamer transcription factor (Oct-1) binding site. With additional data on approximate molecular weights from UV cross-linking, several plausible candidates were tested for their DNA binding and functional activity at FPIV. Oct-like protein binding in gel-shift assays with several cell and tissue extracts was evident using specific competitors and antibodies, but was lower in affinity for FPIV than for an Oct-1 consensus site. Site-directed mutation of the FPIV sequence to a consensus Oct-1 element within the Slp enhancer context increased Oct-1 binding in vitro, but greatly reduced hormonal induction in vivo. This suggested that Oct-1 is not directly involved in response, or alternatively, that Oct-1 bound to the lower-affinity site interacts with neighboring factors significantly differently than Oct-1 bound to a consensus sequence. A sequence overlapping the Oct-like element that was similar to a hepatic nuclear factor-4 (HNF-4) site showed no ability to bind HNF-4 in vitro, nor the related orphan receptor, chicken ovalbumin upstream promoter factor (COUP-TF). Intriguingly, however, expression of COUP-TF in transfection had a dramatic inhibitory effect on response of the androgen-specific enhancer (C' delta9), but did not affect other enhancer configurations that can also be induced by glucocorticoid (C 'delta2). This underscores that, despite extensive sequence identity of C' delta9 and C' delta2, components of the androgen-specific transcription complex differ significantly from that of one that is more generally steroid responsive.

Repression of cAMP-induced expression of the mouse P450 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) by androgens.

In primary cultures of mouse Leydig cells, testosterone represses the cAMP-induced de novo synthesis of P450 17 alpha-hydroxylase/C17-20 lyase (P450c17) protein and the accumulation of P450c17 mRNA, via an androgen receptor (AR)-mediated mechanism. To examine the mechanism by which androgens repress the cAMP-induced expression of the mouse Cyp17 gene, constructs containing 5'-flanking sequences of the mouse Cyp17 linked to the chloramphenicol acetyltransferase (CAT) reporter gene were cotransfected into MA-10 tumor Leydig cells with a mouse AR expression plasmid. In the presence of dihydrotestosterone, the cAMP-induced expression of a reporter construct containing -1021 bp of Cyp17 promoter sequences was repressed. In contrast, no repression by dihydrotestosterone was observed when the -1021 bp Cyp17-CAT construct was cotransfected with a human AR expression plasmid missing the second zinc finger of the DNA-binding domain, indicating that DNA binding is involved in AR-mediated repression of Cyp17 expression. Analysis of deletions -346 bp of 5'-flanking region of the mouse Cyp17 promoter are sufficient to confer androgen repression of the cAMP-induced expression of Cyp17. Deoxyribonuclease I footprinting analysis indicated that the AR interacts with sequences between -330. and -278 bp of the Cyp17 promoter. This region overlaps with the previously identified cAMP-responsive region located between -346 and -245 bp of the Cyp17 promoter. These results suggest that AR-mediated repression involves binding of the AR to sequences in the cAMP-responsive region of the Cyp17 promoter, possibly interfering with the binding of the protein(s) that mediate cAMP induction of Cyp17.

Contextual dependence of steroid receptor function on an androgen-responsive enhancer.

The enhancer of the mouse sex-limited protein (Slp) gene includes a consensus hormone response element (HRE) that interacts with several auxiliary elements for steroid induction. The 160-bp fragment. C' delta 2, confers response to androgen or glucocorticoid in transfection, while a 120-bp subfragment, C' delta 9, is activated only by androgen in some cells. Site-directed mutants were tested to identify elements affecting differential response of androgen or glucocorticoid receptors (AR, GR). While most mutations of C' delta 2 affected induction by either steroid similarly, disruptions of the consensus HRE or an octamer-like sequence were more severe for GR than AR activity. An HRE half-site was critical to androgen-specific induction of C' delta 9 but had little impact in the nonspecific C' delta 2 context. In DNase I footprinting, full-length AR and GR bound similarly to the consensus HRE but dissimilarly to nonconsensus sites. Intriguingly, NF-kappa B bound the region of C' delta 2 absent from C' delta 9. Expression of I kappa B decreased response of C' delta 2, but not C' delta 9, confirming a permissive role of NF-kappa B in steroid activation. In this case, different factors may associate with receptors in the presence of NF-kappa B than those that confer androgen specificity in NF-kappa B's absence, suggesting that exclusion of some factors from a specific transcription complex is as crucial as inclusion of others. This dissection of C' delta 2 and C' delta 9 in vitro reveals subtle distinctions in AR and GR interactions that may underlie specific hormonal response in vivo.

Multiple sclerosis: prospective analysis of TNF-alpha and 55 kDa TNF receptor in CSF and serum in correlation with clinical and MRI activity.

The possibility of antagonizing tumor necrosis factor-alpha (TNF-alpha) in vivo with antibodies or soluble TNF receptor has focused much interest on the role of this cytokine in the natural course of MS. We studied nine patients prospectively and serially for one year (14 time points, 131 observations). TNF-alpha and the 55 kDa soluble TNF receptor were measured every 4 weeks in the serum and at defined time points in the CSF. Each value was correlated to clinical symptoms and to MRI measurements obtained on the same day. All patients with relapsing-remitting disease showed periodic increases of TNF concentrations. Overall, the association between serum TNF-alpha levels and bursts of Gd-DTPA enhancement on cranial MRI was not sufficiently tight to reach statistical significance. However, serum TNF levels > 50 pg/ml and measurable CSF levels were always associated with Gd-DTPA enhancing MRI lesions. Isolated high serum TNF peaks were noted during episodes of infection, hay fever or psychic stress. After treatment with glucocorticoids, TNF levels were suppressed for several months, whereas new Gd-DTPA enhancing lesions continued to appear. The concentrations of the soluble 55 kDa TNF receptor did not show marked fluctuations. These results are consistent with an active role of TNF-alpha in MS during periods of disease activity and provide further support for the clinical evaluation of anti-TNF therapies.

Specific steroid response from a nonspecific DNA element.

A fundamental dilemma of steroid hormone regulation is how specific transcription is attained in vivo when several receptors recognize the same DNA sequence in vitro. We have identified an enhancer of the mouse sex-limited protein (Slp) gene that is activated by androgens but not by glucocorticoids in transfection. Induction requires a consensus hormone response element (HRE) and multiple auxiliary elements within 120 base pairs. Androgen specificity relies on a dual function to augment androgen but prevent glucocorticoid action from a site that both receptors can bind. The nonreceptor factors are the dominant force in transcriptional specificity, although HRE sequence variations can affect the stringency and magnitude of hormonal response. The effect of HRE variations suggests that receptor position is altered relative to the other factors. Thus protein interactions that elicit specific gene regulation are established by the array of DNA elements in a complex enhancer and can be modulated by subtle sequence differences that may influence precise protein contacts.

Water states in living systems. I. Structural aspects.

Formation and special consequences of water-ordering are studied and discussed. Based on the knowledge of the water states, a special dynamic clustering (called dynamic frustration) is suggested. This can be an essential mechanism for life processes. A frustration mechanism and its consequences are discussed.