bluuebunny/crossref_metadata_2025 huggingface dataset:
Common patterns of facial ontogeny in the hominid lineage
Authors: Rogers Ackermann Rebecca , Krovitz Gail E.
Published: September 2002

Recent evaluation of Neanderthal and modern human ontogeny suggests that taxon‐specific features arose very early in development in both lineages, with early, possibly prenatal, morphological divergence followed by parallel postnatal developmental patterns. Here we use morphometric techniques to compare hominoid facial growth patterns, and show that this developmental phenomenon is, in fact, not unique to comparisons between Neanderthals and modern humans but extends to Australopithecus africanus and to the hominoid lineage more broadly. This finding suggests that a common pattern of juvenile facial development may be more widespread and that the roots of ontogenetically early developmental differentiation are deep—perhaps predating the ape/human split of 6+ million years ago. Anat Rec (New Anat) 269:142–147, 2002. © 2002 Wiley‐Liss, Inc.
A pulse‐chase study tracking the conversion of macrophage‐endocytosed serum amyloid A into extracellular amyloid
Authors: Kluve‐Beckerman Barbara , Manaloor John J. , Liepnieks Juris J.
Published: September 2002

Objective

To determine whether serum amyloid A (SAA) is internalized by and processed in macrophages en route to deposition as extracellular amyloid.

Methods

SAA was tracked in cultures of peritoneal macrophages, using a pulse‐chase protocol. Macrophages were pulsed with either fluorescently (with Texas Red) tagged SAA (TxR‐SAA) or iodinated SAA (125I‐SAA). Cells were then rinsed and shifted to chase medium containing unlabeled SAA and amyloid‐enhancing factor (AEF) to induce amyloid formation. At selected times, TxR‐SAA in living cells was observed by confocal scanning microscopy. 125I‐SAA was visualized and quantified in cell lysates and medium by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and phosphorimaging. The presence of amyloid was confirmed by Congo red staining.

Results

Confocal microscopy immediately after the pulse revealed TxR‐SAA in endosomal vesicles, with no extracellular or cell surface accumulation. After 24 hours and 72 hours of chase, virtually all TxR‐SAA remained intracellular. By 10 days, extracellular fluorescence was very strong, indicating that SAA had moved out of cells. Congo red staining revealed amyloid colocalized with areas of extracellular fluorescence. Experiments using 125I‐SAA showed that while 90–95% of internalized 125I‐SAA was degraded within 24 hours, 5–10% persisted as intact SAA or SAA peptides. Immediately after the pulse, SAA was full‐length, but within 24 hours, discrete 125I‐SAA peptides were seen. Each peptide had an intact SAA amino‐terminus, as expected for AA protein. Amyloid was detected in cultures as early as 24 hours after initiation of treatment with SAA and AEF and appeared to be intracellular.

Conclusion

The results of this study provide direct evidence that SAA internalized by and processed in macrophages forms extracellular amyloid. Based on the presence of 125I‐AA protein in macrophage lysates prior to the appearance of extracellular TxR‐labeled amyloid, it was concluded that cleavage of SAA to AA occurs intracellularly.
Differences in idiopathic inflammatory myopathy phenotypes and genotypes between Mesoamerican Mestizos and North American Caucasians: Ethnogeographic influences in the genetics and clinical expression of myositis
Authors: Shamim Ejaz A. , Rider Lisa G. , Pandey Janardan P. , O'Hanlon Terrance P. , Jara Luis J. , Samayoa Eduardo A. , Burgos‐Vargas Ruben , Vazquez‐Mellado Janitzia , Alcocer‐Varela Jorge , Salazar‐Paramo Mario , Kutzbach Abraham Garcia , Malley James D. , Targoff Ira N. , La Torre Ignacio Garcia‐De , Miller Frederick W.
Published: September 2002

Objective

As part of a larger, worldwide study of the ethnogeography of myositis, we evaluated the clinical, serologic, and immunogenetic features of Mestizo (Mexican and Guatemalan) and North American Caucasian patients with idiopathic inflammatory myopathy (IIM).

Methods

Clinical manifestations, autoantibodies, HLA‐DRB1 and DQA1 alleles, and immunoglobulin Gm/Km allotypes were compared between 138 Mestizos with IIM and 287 Caucasians with IIM, using the same classification criteria and standardized questionnaires.

Results

IIM in Mestizo patients was characterized by a higher proportion of dermatomyositis (69% of adult Mestizos versus 35% of adult Caucasians; P < 0.001) and anti‐Mi‐2 autoantibodies (30% versus 7% of adults, respectively, and 32% versus 4% of children, respectively; P < 0.01). Genetic risk factors also differed in these populations. Whereas Mestizos had no HLA risk factors for IIM, HLA‐DRB1*0301, the linked allele DQA1*0501, and DRB1 alleles sharing the first hypervariable region motif 9EYSTS13 were major risk factors in Caucasian patients with IIM. Furthermore, different HLA‐DRB1 and DQA1 alleles were associated with anti‐Mi‐2 autoantibodies (DRB1*04 and DQA1*03 in Mestizos and DRB1*07 and DQA1*02 in Caucasians). Immunoglobulin γ‐chain allotypes Gm(1), Gm(17) (odds ratio for both 11.3, P = 0.008), and Gm(21) (odds ratio 7.3, P = 0.005) and κ‐chain allotype Km(3) (odds ratio 7.3, P = 0.005) were risk factors for IIM in Mestizos; however, no Gm or Km allotypes were risk or protective factors in Caucasians. In addition, Gm and Km phenotypes were unique risk factors (Gm 1,3,17 5,13,21 and Gm 1,17 23 21 and Km 3,3) or protective factors (Km 1,1) for the development of myositis and anti‐Mi‐2 autoantibodies (Gm 1,2,3,17 23 5,13,21) in adult Mestizos.

Conclusion

IIM in Mesoamerican Mestizos differs from IIM in North American Caucasians in the frequency of phenotypic features and in the immune‐response genes predisposing to and protecting from myositis and anti‐Mi‐2 autoantibodies at 4 chromosomal loci. These and other data suggest the likelihood that the expression of IIM is modulated by different genes and environmental exposures around the world.
Effects of prasterone on corticosteroid requirements of women with systemic lupus erythematosus: A double‐blind, randomized, placebo‐controlled trial
Authors: Petri Michelle A. , Lahita Robert G. , Van Vollenhoven Ronald F. , Merrill Joan T. , Schiff Michael , Ginzler Ellen M. , Strand Vibeke , Kunz Arlene , Gorelick Kenneth J. , Schwartz Kenneth E.
Published: September 2002

Objective

To evaluate whether treatment with prasterone (dehydroepiandrosterone [DHEA]) would allow the dosage of prednisone (or an equivalent corticosteroid) to be reduced to ≤7.5 mg/day for 2 months or longer while maintaining stable or reduced disease activity in steroid‐dependent women with systemic lupus erythematosus (SLE).

Methods

In a double‐blind, randomized trial, 191 female SLE patients receiving prednisone (10–30 mg/day) were treated daily with either placebo, 100 mg of oral prasterone (an adrenal androgen), or 200 mg of oral prasterone for 7–9‐months. At monthly intervals, corticosteroid dosages were reduced by algorithm in patients whose SLE Disease Activity Index (SLEDAI) score was stable or improved. Patients for whom a sustained reduction in the dosage of prednisone (≤7.5 mg/day) was achieved for at least the last 2 months of the 7–9‐month treatment period were classified as responders.

Results

Response rates were 41% in the placebo group, 44% in the 100‐mg prasterone group, and 55% in the 200‐mg group (P = 0.110, 200 mg versus placebo). Among the 137 subjects (45 in the placebo group, 47 in the 100‐mg group, and 45 in the 200‐mg group) who had active disease at baseline (defined as SLEDAI score >2), 29%, 38%, and 51%, respectively, were responders (P = 0.031 for 200 mg prasterone versus placebo). Acne was the most common adverse event but was generally mild. Clinical and laboratory changes primarily reflected androgenic effects of prasterone.

Conclusion

Among women with lupus disease activity, reducing the dosage of prednisone to ≤7.5 mg/day for a sustained period of time while maintaining stabilization or a reduction of disease activity was possible in a significantly greater proportion of patients treated with oral prasterone, 200 mg once daily, compared with patients treated with placebo.
Inhibition of adjuvant‐induced arthritis by interleukin‐10‐driven regulatory cells induced via nasal administration of a peptide analog of an arthritis‐related heat‐shock protein 60 T cell epitope
Authors: Prakken Berent J. , Roord Sarah , Van Kooten Peter J. S. , Wagenaar Josée P. A. , Van Eden Willem , Albani Salvatore , Wauben Marca H. M.
Published: September 2002

Objective

To prevent and treat experimental arthritis via nasal administration of an altered peptide ligand (APL) from the major arthritogenic epitope in adjuvant‐induced arthritis (AIA) and to explore the mechanisms involved.

Methods

Peptides were administered nasally before and after induction of arthritis. Splenocytes and lymph node cells draining both the site of inflammation and the site of tolerance induction were used for cell transfer and were studied for antigen‐specific T cell characteristics. In addition, attempts were made to stop T cell tolerance in vitro, using anticytokine antibodies.

Results

Nasal administration of a modulatory APL of the heat‐shock protein 60 (Hsp60) 180–188 T cell epitope, alanine 183, had a suppressive effect in AIA that far exceeded that of the wild‐type epitope. In addition to its effectiveness in preventing AIA, alanine 183 may be effective in the treatment of ongoing AIA. The protective effect of alanine 183 can be passively transferred using activated splenocytes. Nasal administration of alanine 183 did not lead to detectable T cell proliferation or interleukin‐2 (IL‐2) production in mandibular lymph node cells, while transforming growth factor β (TGFβ), IL‐10, and IL‐4 were readily produced. Likewise, after nasally induced tolerance, followed by induction of arthritis, inguinal lymph node cells produced IL‐4, TGFβ, and IL‐10. After neutralizing in vitro the individual cytokines with anticytokine antibodies, only blocking of IL‐10 production led to reversal of tolerance, at the site of tolerance induction and the site of inflammation.

Conclusion

Nasal administration of an APL of Hsp60 180–188 induces highly effective protection against AIA through generation of regulatory cells that produce IL‐4, TGFβ, and IL‐10, whereas the induced tolerance is driven mainly by production of IL‐10.
Use of joint fluid analysis for determining cartilage damage in osteonecrosis of the knee
Authors: Saito Tomoyuki , Takeuchi Ryouhei , Mitsuhashi Shigeyuki , Uesugi Masaaki , Yoshida Takuji , Koshino Tomihisa
Published: September 2002

Objective

To assess the value of joint fluid analysis for determining cartilage degradation and prognosis in spontaneous osteonecrosis (ON) of the knee.

Methods

Synovial fluid was obtained from 30 knees with spontaneous ON (26 medial femoral condyles, 4 medial tibial plateaus) as well as from 50 knees with medial compartmental osteoarthritis (OA) as a control. Levels of chondroitin 6‐sulfate (C6S), C4S, and hyaluronic acid were measured with high‐performance liquid chromatography. The lesion size, appearance of the articular cartilage, and results of magnetic resonance imaging (MRI) were compared with the results of joint fluid analysis.

Results

The mean ± SD level of C6S was 82.2 ± 36.6 nmoles/ml in joint fluid from ON knees, which was significantly higher than the levels in knees with grade 2 (47.2 ± 20.0 nmoles/ml) and grade 3 (55.8 ± 29.2 nmoles/ml) OA. The C6S:C4S ratio was highest in lesions with mild articular changes and reflected the macroscopic alteration of cartilage overlying the ON lesion. The concentration of C6S in the 9 knees with lesions that covered ≥40% of the condyle (99.0 ± 32.9 nmoles/ml) was higher than that in the 17 knees with lesions that covered <40% of the condyle (67.2 ± 31.7 nmoles/ml). Knees with bone marrow edema on MRI had a higher level of C6S than did knees with a fibrous‐like appearance.

Conclusion

While radiologic staging was useful for indicating the size of the ON lesion, it was less valuable for determining articular cartilage damage. Joint fluid analysis may provide more precise information about articular cartilage degradation in ON, and the findings may also be of prognostic significance.
Discriminatory sonographic criteria for the diagnosis of carpal tunnel syndrome
Authors: Wong S. M. , Griffith J. F. , Hui A. C. F. , Tang A. , Wong K. S.
Published: September 2002

Objective

Sonographic examination of the median nerve has been suggested as a useful alternative to electrophysiologic study in the diagnosis of carpal tunnel syndrome. To determine its usefulness and the best diagnostic criterion, sonograms of patients with the disease were compared with sonograms of healthy subjects in a case–control study.

Methods

Patients with carpal tunnel syndrome and asymptomatic controls who were matched for age and sex were enrolled and underwent sonography of the wrists. Eight separate sonographic criteria were analyzed in each wrist. Data from the patient group and the control group were compared to establish optimal diagnostic criteria for carpal tunnel syndrome, using receiver operating characteristic analytic techniques.

Results

Thirty‐five patients with carpal tunnel syndrome and 35 asymptomatic controls were examined. Increased cross‐sectional area of the median nerve was found to be the most predictive measure of carpal tunnel syndrome, proximal to the tunnel inlet, at the tunnel inlet, and at the tunnel outlet, with significant differences between patients and controls. Using a receiver operating characteristic curve, a cut‐off value >0.098 cm2 at the tunnel inlet provided a diagnostic sensitivity of 89% and a specificity of 83%.

Conclusion

Sonographic measurement of the median nerve cross‐sectional area is both sensitive and specific for the diagnosis of carpal tunnel syndrome.
Effects of long‐term estrogen replacement therapy on osteoarthritis severity in cynomolgus monkeys
Authors: Ham Kimberley D. , Loeser Richard F. , Lindgren Bruce R. , Carlson Cathy S.
Published: September 2002

Objective

To determine the effects of long‐term estrogen replacement therapy (ERT) on the severity of osteoarthritis (OA) of the knee joint in surgically postmenopausal (bilaterally ovariectomized) female monkeys. A secondary aim was to evaluate the effect of soy phytoestrogen (SPE) treatment on the severity of OA.

Methods

Feral adult female cynomolgus macaques were ovariectomized bilaterally and then randomly divided into 3 age‐ and weight‐matched treatment groups. For 3 years, the first group received ERT with conjugated equine estrogens, the second group received SPE, and the third group received no treatment (controls). At necropsy, histologic lesions of OA were graded, and the area and thickness of cartilage and subchondral bone were measured. The data were summarized by principal components analysis, and the resulting factors and individual variables were compared using analysis of variance and analysis of covariance (age and weight as covariates).

Results

Cartilage lesions of OA were significantly less severe in the animals given ERT compared with those in the control group. This treatment effect remained significant when adjusted for age and weight. The factor representing subchondral bone was significantly higher, but the number of osteophytes was lower, in the ERT group compared with the control group. SPE treatment had no significant effect on cartilage or bone lesions of OA.

Conclusion

These results demonstrate that long‐term ERT significantly reduces the severity of OA lesions in this animal model.
Recognition of Granzyme B‐generated autoantigen fragments in scleroderma patients with ischemic digital loss
Authors: Schachna Lionel , Wigley Fredrick M. , Morris Steven , Gelber Allan C. , Rosen Antony , Casciola‐Rosen Livia
Published: September 2002

Objective

To examine whether autoantibodies recognizing granzyme B (GB)‐cleaved autoantigens are associated with ischemic digital loss (IDL) in limited systemic sclerosis (SSc).

Methods

Fifteen of 19 patients with limited SSc and IDL were matched by age, sex, race, and duration of disease to controls with limited SSc but without IDL. The sera were used to immunoblot HeLa cell lysates and chromosome preparations that had been incubated in vitro in the absence or presence of GB. Anticentromere antibodies (ACAs) were assayed by immunofluorescence and immunoprecipitation of in vitro‐translated centromere proteins (CENP‐B and CENP‐C). Immunoprecipitation of GB‐cleaved CENPs was also performed.

Results

GB‐cleaved autoantigens were immunoblotted by 16 of 19 IDL sera (84.2%) compared with 6 of 15 non‐IDL sera (40.0%) (odds ratio 8.0, 95% confidence interval 1.6–40.0). This association persisted after adjustment for ACA status. Furthermore, the presence of antibodies to centromere proteins as well as to GB‐cleaved antigens was highly specific for IDL, occurring in 12 of 19 IDL patients (63.2%) and in none of 15 controls (P < 0.0001). An identical 60‐kd GB‐generated fragment was recognized by 5 of 16 IDL sera (31.3%) and was demonstrated to arise through GB‐mediated cleavage of CENP‐C. GB‐cleaved CENP‐C fragments were recognized preferentially over the intact CENP‐C molecule by antibodies from patients with IDL.

Conclusion

The striking recognition of GB‐generated autoantigen fragments by sera from patients with limited SSc and IDL constitutes the first in vivo evidence that antibodies against GB‐generated centromeric peptide fragments identify a distinct clinical subset.
Minimizing a general loss function in off‐line quality control
Authors: Mak T. K. , Nebebe F.
Published: September 2002

We consider in the present paper the analysis of parameter designs in off‐line quality control. The main objective is to seek levels of the production factors that would minimize the expected loss. Unlike classical analyses which focus on the analysis of the mean and variance in minimizing a quadratic loss function, the proposed method is applicable to a general loss function. An appropriate transformation is first sought to eliminate the dependency of the variance on the mean (to achieve ‘separation’ in the terminology of Box). This is accomplished through a preliminary analysis using a recently proposed parametric heteroscedastic regression model. With the dependency of the variance on the mean eliminated, methods with established properties can be applied to estimate simultaneously the mean and the variance functions in the new metric. The expected loss function is then estimated and minimized based on a distributional free procedure using the empirical distribution of the standardized residuals. This alleviates the need for a full parametric model, which, if incorrectly specified, may lead to biased results. Although a transformation is employed as an intermediate step of analysis, the loss function is minimized in its original metric. Copyright © 2002 John Wiley & Sons, Ltd.
Behaviors of children with high‐functioning autism when paired with typically developing versus delayed peers: a preliminary study
Authors: Smith Tristram , Watthen Lovaas Nina , Ivar Lovaas O.
Published: September 2002

Children with high‐functioning autistic disorder (HAD) in a comprehensive behavioral treatment program (n = 9, age = 5–7 years) alternated between pairings with a typically developing peer and pairings with a peer who had both autism and developmental delay. All pairings took place in a free play setting. The children with HAD displayed much more interactive play and speech (as well as much less self‐stimulation) with typically developing peers than with delayed peers. They received frequent cues and consequences for interaction from typically developing peers, but not in sessions with delayed peers. These results suggest that placement with typically developing peers is critical for children with HAD in behavioral treatment. Copyright © 2002 John Wiley & Sons, Ltd.
Using habit reversal to treat chronic vocal tic disorder in children
Authors: Woods Douglas W. , Twohig Michael P.
Published: September 2002

In the present study, a nonconcurrent multiple baseline design was used to evaluate the effectiveness of simplified habit reversal in three children diagnosed with chronic vocal tic disorder. In two children, vocal tics were substantially reduced. In a third child, tics were not reduced, and the addition of a supplemental contingency management program was ineffective. Despite these mixed results, the treatment was viewed as acceptable to the children's parents in all cases as measured by a standard index of treatment acceptability. Implications of these findings are discussed. Copyright © 2002 John Wiley & Sons, Ltd.
Potential‐resolved electrochemiluminescence of Ru(bpy)32+/C2O42− system on gold electrode
Authors: Li Feng , Cui Hua , Lin Xiang‐Qin
Published: September 2002

The electrogenerated chemiluminescence of Ru(bpy)32+/C2O42− system on a pre‐polarized Au electrode was studied using a potential‐resolved electrochemiluminescence (PRECL) method. Two anodic ECL peaks were observed at 1.22 V (vs. SCE) (EP1), 1.41 V (vs. SCE) (EP2), respectively. The effects of the concentration of oxalate and Ru(bpy)32+, adsorbed sulphur, CO2, O2, pH of the solution and pretreatment of the Au electrode on the two PRECL peaks were examined. The surface state of the pre‐oxidized gold electrode was also studied using the X‐ray photoelectron spectroscopy (XPS) technique. Moreover, comparative studies on i–E and I–E curves were carried out and a possible mechanism involving both the catalytic and the direct electro‐oxidation pathways was proposed for the ECL of Ru(bpy)32+/C2O42− system. EP1 is attributed to the Ru(bpy)32/3+ reaction catalysed by C2O42− to generate Ru(bpy)32+*. EP2 is likely because C2O42− was oxidized at the electrode to form CO2−·, followed by reaction with Ru(bpy)33+ to generate Ru(bpy)32+*. Copyright © 2002 John Wiley & Sons, Ltd.
Measurement of free radicals and NO by chemiluminescence to identify the reperfusion injury in renal transplantation
Authors: Oehlschläger S. , Albrecht S. , Hakenberg O. W. , Manseck A. , Froehner M. , Zimmermann T. , Wirth M. P.
Published: September 2002

Oxygen free radicals are generated during the reperfusion of ischaemic organs. Several experimental studies have demonstrated that the damage produced by reperfusion can be prevented by a scavenger of free radicals. Furthermore, a significantly improved 5 year graft survival rate after cadaveric renal transplantation has been reported in patients treated with scavengers of free radicals (Land et al., 1993). Therefore, a question remains to be answered: whether a routine monitoring of the radical‐mediated reperfusion injury with renal transplantation is useful, and whether there is a necessity for a generalized protective treatment in transplant patients. In a prospective trial, we evaluated a group of eight patients during and after renal cadaveric transplantation (three men, five women), using the chemiluminometric measurement of serum free radicals and NO. The serum quantities of free radicals and NO were significantly increased after reperfusion of the transplant kidney (p < 0.02). The mean time of noticeably increased levels of serum free radicals was 4.8 ± 1.2 h after reperfusion. The results thus showed an increased liberation of free radicals in the peripheral blood of transplant recipients as possible evidence of free radicals‐mediated reperfusion injury in renal transplantation. The generation of free radicals measured by chemiluminescence allow a controlled therapy to decrease the generation of free radicals with antioxidants during the early transplantation period e.g. in older recipients. Copyright © 2002 John Wiley & Sons, Ltd.
A mass balance study to assess the extent of contaminant removal achieved in the operations for the primary recovery of plasmid DNA from Escherichia coli cells
Authors: Ciccolini Laura A. S. , Shamlou Parviz Ayazi , Titchener–Hooker Nigel
Published: September 2002

Mass balances were performed on an alkaline lysis operation for the primary recovery of supercoiled plasmid DNA as part of a process for plasmid gene preparation. Escherichia coli DH5α/pSVβ was cultured in defined medium by fed‐batch fermentation and harvested at the end of the exponential phase. Alkaline lysis of the recombinant cells was performed at fixed shear rates ranging between 46 and 461 s−1, with neutralization 100 and 300 s after the initiation of the lysis. Mass balance calculations were used to optimize the operating conditions for carrying out the alkaline lysis operation. The results indicated that a plasmid yield of 75% and purity with respect to total DNA of 60% were achievable during the primary recovery operation. The influences of key contaminants, including the soluble proteins and the suspended solids, as they bear on the subsequent purification operations, were evaluated and discussed. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 796–805, 2002; DOI 10.1002/bit.10166
Direct recovery of glutathione S‐transferase by expanded bed adsorption: Anion exchange as an alternative to metal affinity fusions
Authors: Clemmitt R. H. , Chase H. A.
Published: September 2002

The use of expanded beds of ion‐exchange adsorbents for the direct recovery of a recombinant intracellular protein, glutathione S‐transferase (GST), from unclarified Escherichia coli homogenates is described. The results form the basis for a comparison between this approach for purifying GST and a chelating fusion strategy and highlight the need to consider the additional costs entailed by these more‐complicated approaches. The separation performance was investigated with respect to choice of anion or cation exchanger, adsorption pH, load volume, sample preparation, and stepwise elution protocol. Anion exchange was found to be more appropriate than cation exchange, as the low pHs involved in the latter caused a loss of activity. The optimal pH for adsorption was found to be 9 with a dynamic capacity from clarified homogenate in packed mode of 112 U mL−1 (11.2 mg GST mL−1). As increasing volumes of unclarified homogenate were applied to the expanded bed, the yield of GST in the eluate decreased, and the purification factor was found to increase and then decrease. This was due to the displacement of weakly bound proteins by GST and then its displacement by even more strongly binding proteins. The dynamic capacity of the anion exchanger, STREAMLINE DEAE, from unclarified homogenate in expanded mode decreased slightly to 85 U mL−1 (8.5 mg GST mL−1). The elution protocol for GST from the anion exchanger was then adjusted to try to maximize the degree of purification. Anion exchange expanded bed adsorption of GST from unclarified E. coli homogenate gave an eluted yield of 95.7% and 1.64‐fold purification. Interestingly, a decrease in the expression level of GST in the feedstream from 23 down to 13% caused a decrease in the dynamic capacity from 85 to 14.5 U mL−1 whereas the degree of purification remained similar. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 776–785, 2002; DOI 10.1002/bit.10167
Ethylene oxide and propylene oxide random copolymer/sodium chloride aqueous two‐phase systems: Wetting and adsorption on dodecyl–agarose and polystyrene
Authors: Huang Yingqing , Forciniti Daniel
Published: September 2002

Liquid/liquid partition chromatography is a mild yet powerful separation method for a variety of biological materials. This work demonstrates that it should be feasible to immobilize an ethylene oxide–propylene oxide (EO/PO) random copolymer solution and to use a solution of NaCl equilibrated against the polymer solution as the mobile‐phase (poly (EO–PO) [P(EO–PO)] and NaCl form two aqueous phases known as aqueous two‐phase systems). Three random copolymers with different molecular weights and EO/PO ratios were used. Dodecyl–agarose and polystyrene were tested as possible supports. The wetting energies of the aqueous two‐phase systems on these two kinds of surfaces were calculated as well as contact angles for each phase on the same surfaces. Finally, the thickness of P(EO–PO) adsorption layers on polystyrene lattices were measured by dynamic light scattering. Contact angle measurements indicate that indeed some EO/PO copolymers preferentially wet hydrophobic substrates, forming thin films. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 786–795, 2002; DOI 10.1002/bit.10169
Dependence of morphology on agitation intensity in fed‐batch cultures of Aspergillus oryzae and its implications for recombinant protein production
Authors: Amanullah A. , Christensen L. H. , Hansen K. , Nienow A. W. , Thomas C. R.
Published: September 2002

We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed‐batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm−3. Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60–65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed‐batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below ≈40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle‐neck in this strain during fed‐batch fermentations. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 815–826, 2002; DOI 10.1002/bit.10181
Understanding viral partitioning in two‐phase aqueous nonionic micellar systems: 2. Effect of entrained micelle‐poor domains
Authors: Kamei Daniel T. , King Jonathan A. , Wang Daniel I. C. , Blankschtein Daniel
Published: September 2002

Unlike the partitioning behavior of hydrophilic, water‐soluble proteins, the partitioning behavior of viruses in the two‐phase aqueous nonionic n‐decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained using the excluded‐volume theory developed recently by our group. A central assumption underlying the excluded‐volume theory—that macroscopic phase separation equilibrium is attained—was therefore challenged experimentally and theoretically. Photographs of the two‐phase aqueous C10E4 micellar system were taken for different volume ratios to demonstrate that the entrainment of micelle‐poor (virus‐rich) domains in the macroscopic, top, micelle‐rich phase decreases with a decrease in the volume ratio. Partitioning experiments were then conducted with the model virus bacteriophage P22 and the model protein cytochrome c at different operating temperatures for different volume ratios. For bacteriophage P22, the measured viral partition coefficient at each temperature decreased by about an order of magnitude when the volume ratio was decreased from 10 to 0.1, which clearly indicated that entrainment is an important factor influencing viral partitioning. For cytochrome c, the measured protein partition coefficient did not change, which demonstrated that this entrainment effect negligibly influences protein partitioning. A new theoretical description of partitioning was also developed that combines the excluded‐volume theory with this entrainment effect. In this theory, one fitted parameter—the volume fraction of entrained micelle‐poor domains in the macroscopic, top, micelle‐rich phase—is used to account for the entrainment. To fit this parameter, only a single partitioning experiment is required for a given volume ratio, irrespectively of the partitioning solute. The new theoretical description of partitioning yielded very good quantitative predictions of the viral partition coefficients. Accordingly, it can be concluded that the primary mechanisms governing viral partitioning in the two‐phase aqueous C10E4 micellar system are the entrainment of micelle‐poor (virus‐rich) domains in the macroscopic, top, micelle‐rich phase and the excluded‐volume interactions that operate between the viruses and the micelles. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 203–216, 2002; DOI 10.1002/bit.10194
Induction of protein aggregation in an early secretory compartment by elevation of expression level
Authors: Schröder Martin , Schäfer Rolf , Friedl Peter
Published: September 2002

A variety of valuable therapeutic proteins are expressed in mammalian cells. Currently, rate‐limiting for secretion of recombinant glycoproteins are activities in the secretory pathway of eukaryotic cells, i.e., folding and glycosylation of the naked polypeptide chain. In this paper we provide evidence that elevation of expression level alone is sufficient to cause intracellular aggregation of a structurally relatively simple glycoprotein, antithrombin III (ATIII). Elevation of expression level by selection for increased drug resistance in Chinese hamster ovary cells stably expressing ATIII resulted in formation of disulfide‐bonded aggregates of ATIII. Aggregated ATIII displayed incomplete sialylation and Endo H‐sensitivity and located to the endoplasmic reticulum and the cis‐Golgi compartment in subcellular fractionations. To explore possible causes for aggregation of ATIII at elevated expression levels we investigated the influence of the two major energy sources of cultured mammalian cells, D‐glucose and L‐glutamine, on the ATIII‐yield. We found that utilization of D‐glucose was not limiting for synthesis of ATIII at elevated expression levels. However, the amount of ATIII‐synthesized per L‐glutamine consumed did not seem to increase steadily with expression level for ATIII, indicating that secretion of ATIII may be limited by the capacity of the cell to utilize L‐glutamine. © 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 78: 131–140, 2002; DOI 10.1002/bit.10206
High‐performance capillary electrophoretic analysis of hyaluronan and galactosaminoglycan‐disaccharides in gastrointestinal carcinomas. Differential disaccharide composition as a possible tool‐indicator for malignancies
Authors: Theocharis Achilleas D. , Theocharis Dimitrios A.
Published: September 2002

The glycosaminoglycans (GAGs) have documented implications for the growth and progression of malignant tumors. Gastrointestinal carcinomas (gastric, colon, rectum and pancreatic) are the most frequent malignancies occurring in human. GAGs, isolated from the tissues after digestion with papain, were analyzed by high‐performance capillary electrophoresis (HPCE) following treatment with chondroitinase ABC. The composition of GAGs in disaccharides derived from the various gastrointestinal carcinomas was compared with those of normal tissues. We report that human gastrointestinal carcinomas are characterized by increased concentrations of GAGs, which have quite different disaccharide composition which, in turn, is associated with marked increase of non‐sulfated (Δdi‐nonS) and 6‐sulfated (Δdi‐mono6S) Δ‐disaccharides. Particularly, a 12–51‐fold increase in Δdi‐nonS and a 3–42‐fold increase in Δdi‐mono6S content characterize these carcinomas, while the 4‐sulfated units (Δdi‐mono4S) showed a lower increase, about 0.5–1.5‐fold. Moreover, the quantitation of hyaluronan (HA)‐derived Δ‐disaccharides (Δdi‐nonSHA) also revealed a marked increase (1–12‐fold) in the malignant tissues. On the other hand, the content of the chondroitinase ABC‐resistant GAGs showed a low decrease, about 0.2–0.7‐fold. The high amounts of hyaluronan (HA) produced by these carcinomas and the ectopic production of chondroitin sulphate (CS) proteoglycans, in which (Δdi‐nonS) and (Δdi‐mono6S) predominated, suggest a close relation between the content of these GAGs and the malignant phenotype, the metastatic ability and the survival time. Copyright ­© 2002 John Wiley & Sons, Ltd.
Employment of detergent–tag/solute interactions in capillary electrophoresis of neutral polysaccharides
Authors: Tokarz Michal , Gustavsson Pär , Stefansson Morgan
Published: September 2002

Neutral and inherently immobile polysaccharides are induced to migrate in an electric field through interactions with a detergent added to the electrophoretic electrolyte buffer. Before analysis the polysaccharides are converted to fluorescent derivatives to enable detection, but choice of a tag can also be utilized for modulation of the electrophoretic mobility. Three cases are discussed and exemplified, namely detergent‐solute, detergent‐solute + tag, and detergent–tag interactions. Anionic as well as cationic surfactants were exploited along with different derivatization reagents. Depending on the approach chosen, different kinds of information about sample composition and distribution(s) can be obtained, including degree of substitution, distribution of molecular weight (obtained in free solution without sieving media) and polymer conformation. A shift in polymer conformation upon a change in solvent composition can be monitored. Copyright © 2002 John Wiley & Sons, Ltd.
Analysis of glycoproteins and the oligosaccharides thereof by high‐performance capillary electrophoresis—significance in regulatory studies on biopharmaceutical products
Authors: Kakehi Kazuaki , Kinoshita Mitsuhiro , Nakano Miyako
Published: September 2002

This review describes the recent development in the analysis of glycoproteins using capillary electrophoresis with various separation techniques, and focuses especially on the analysis of recombinant glycoprotein pharmaceuticals. We include the analysis of glycoprotein multiforms (ie glycoform) as well as glycan analysis. The relationship between glycoprotein multiforms and oligosaccharide distributions in a glycoprotein sample is also discussed. Further, recent development in capillary electrophoresis–mass spectrometry is described. Copyright © 2002 John Wiley & Sons, Ltd.
Capillary electrophoresis for the analysis of glycosaminoglycans and glycosaminoglycan‐derived oligosaccharides
Authors: Mao Wenjun , Thanawiroon Charuwan , Linhardt Robert J.
Published: September 2002

Glycosaminoglycans are a family of polydisperse, highly sulfated complex mixtures of linear polysaccharides that are involved in many life processes. Defining the structure of glycosaminoglycans is an important factor in elucidating their structure–activity relationship. Capillary electrophoresis has emerged as a highly promising technique consuming an extremely small amount of sample and capable of rapid, high‐resolution separation, characterization and quantitation of analytes. Numerous capillary electrophoresis methods for analysis of intact glycosaminoglycans and glycosaminoglycan‐derived oligosaccharides have been developed. These methods allow for both qualitative and quantitative analysis with a high level of sensitivity. This review is concerned with separation methods of capillary electrophoresis, detection methods and applications to several aspects of research into glycosaminoglycans and glycosaminoglycan‐derived oligosaccharides. The importance of capillary electrophoresis in biological and pharmaceutical samples in glycobiology and carbohydrate biochemistry and its possible applications in disease diagnosis and monitoring chemical synthesis are described. Copyright © 2002 John Wiley & Sons, Ltd.
Determination of rat hepatocellular glutathione by reversed‐phase liquid chromatography with fluorescence detection and cytotoxicity evaluation of environmental pollutants based on the concentration change
Authors: Toyo'oka Toshimasa , Tanabe Junko , Jinno Hideto
Published: September 2002

Three methods for the determination of rat hepatocellular thiols by high‐performance liquid chromatography (HPLC) with fluorescence (FL) detection have been developed. The thiols in the cells were tagged with three fluorogenic reagents, SBD‐F, ABD‐F and DBD‐F. These reagents could permeate into cells and effectively reacted with thiols to produce highly fluorescent derivatives. These derivatives fluoresced in the long wavelength region at around 530 nm (excitation at around 380 nm). The five biological thiols tagged were perfectly separated by reversed‐phase liquid chromatography and were sensitively and selectively detected without any interference from endogenous substanaces. The main thiol in the cells was reduced GSH and the concentration was at the mM level. The proposed procedures were applied to the determination of hepatocellular GSH after treatment of environmental pollutants such as volatile organic compounds (VOC) and endocrine disrupting chemicals (EDC). From the comparison of intracellular GSH concentration, the test compounds were classified into four groups: compounds of strong depletion (eg triphenyltin chloride, hexachlorocyclohexene, nonylphenol, bromoacetic acid, 4‐chlorobenzyl chloride and 1,3‐dichloropropene), slight decrease (eg bisphenol A, benzo[a]pylene, carbon tetrachloride and benzene), slight increase (eg bromoform and toluene), and no effect (eg 1,1,1‐trichloroethane, 1,1,2‐trichloroethane and 1,2‐dichloroethane). Although the decrease of GSH concentration does not reflect the cytotoxicity of chemicals, the proposed procedure utilizing isolated rat hepatpcytes seems to be useful for investigating the bioactivation of VOC, and EDC, etc. Copyright © 2001 John Wiley & Sons, Ltd.
Application of capillary electrophoresis in the analysis of novel synthetic dideoxynucleoside analogues with potential anti‐HIV activity
Authors: Balayiannis G. , Papaioannou D. , Karamanos N. K.
Published: September 2002

The aim of this study was the development of a capillary electrophoretic method for the analysis of a series of novel synthetic dideoxynucleoside analogues with potential anti‐HIV activity. These analogues consist of a tetrahydrofuranyl or a tetrahydropyranyl ring as the pseudosugar part and bear a hydroxyethyl side‐chain and a nucleobase of the pyrimidine (eg thymine or uracil) or the purine (adenine) type with cis or trans configuration. Analysis of these derivatives was performed by capillary zone electrophoresis using 25 mM phosphate pH 3.00 and 4.00 as operating buffers for pyrimidine and purine analogues, respectively, and detection of separated species at 254 nm. Copyright © 2001 John Wiley & Sons, Ltd.

Abbreviations used:

ddN

2′,3′‐dideoxynucleosides

iso‐ddN

isodideoxynucleosides.
Determination of malondialdehyde by capillary electrophoresis, application to human plasma and relation of its levels with prematurity
Authors: Korizis K. N. , Exarchou A. , Michalopoulos E. , Georgakopoulos C. D. , Kolonitsiou F. , Mantagos S. , Gartaganis S. P. , Karamanos N. K.
Published: September 2002

Malondialdehyde (MDA) is considered as the most important marker for monitoring lipid peroxidation, which is strongly associated with the development of serious diseases in adults and premature neonates. In this paper we report a method for determination of free MDA in human plasma using capillary zone electrophoresis. MDA was separated and determined as conjugate with tetrabutylammonium hydrogen sulphate (TBAS). Analysis was performed using 20 mM borare, pH = 9.3, as operating buffer and detection of the MDA–TBAS adduct at 267 nm. The method has a linear range up to 80 µM with a detection limit of 0.2 µM. The method was applied to the analysis of MDA in plasma of healthy adults, normal‐gestation infants and of preterm neonates. Plasma proteins were successfully removed following centrifugation through a centricon‐3 membrane. Results showed that the method can be easily and accurate applied for the determination of MDA in human plasma and that the level of MDA in pretern neonates is significantly higher (p ≤ 0.001) as compared to the two other cases. This suggests that MDA analysis may be useful to monitor the development and process of diseases related to lipid peroxidation, oxidative stress and prematurity. Copyright © 2001 John Wiley & Sons, Ltd.

Abbreviations used:

BHT

2,6‐ditetrabutyl‐4‐methylphenol

MDA

molandialdehyde

ROP

retinopathy of prematurity

TBA

thiobarbituric acid

TBAS

tetrabutylammonium hydrogen sulphate.
Natural Capitalists' indictment of traditional capitalism: A reappraisal
Authors: Desrochers Pierre
Published: September 2002

In Natural Capitalism, the best seller by Paul Hawken and Amory and L. Hunter Lovins (1999), the authors summarize widespread beliefs on earlier economic development and indict past practices as inherently wasteful. In building their case, the authors allegedly identify a number of the failures of traditional capitalism. This article contends, however, that a number of these reflect more the mischaracterization by Hawken and the Lovinses of technology, economic and policy history than real shortcomings of past private‐sector behaviour.

The first section of this paper deals with traditional capitalism's alleged sins of omission and provides evidence showing how the characteristics that Hawken and the Lovinses desire did indeed hold in the market system: (i) substantial resource‐productivity gains; (ii) generalized success in ‘closing the loop’ on industrial waste and (iii) a long‐term productive outlook. The second section illustrates how politicians, far from being the benevolent actors that Hawken and the Lovinses envision, typically engaged in obstructionist policy dynamics that tended more to hamper rather than facilitate the adoption of more efficient and environmentally friendly business practices. While there is much to be said on behalf of some of the insights contained in Natural Capitalism, a better understanding of past successes and failures in the area of business–environment interactions might have led the authors to different policy prescriptions. Copyright © 2002 John Wiley & Sons, Ltd. and ERP Environment.
Companies' participation in EMAS: The influence of the public regulator
Authors: Glachant Matthieu , Schucht Simone , Bültmann Alexandra , Wätzold Frank
Published: September 2002

When adopted in 1993, the European Union's Eco‐Management and Audit Scheme (EMAS) was viewed as emblematic of a new policy approach involving more flexible and market‐based environmental instruments. A few years after coming into force, EMAS does not appear to be a tremendous success in terms of industrial participation. Apart from in Germany and Austria, participation is insignificant and comparatively very far behind that in ISO 14001, the environmental management standard of the International Organization for Standardization. The paper seeks to explain this modest result. It focuses on the influence of the European and national regulators on industrial participation. Using a comparative analysis of the implementation of EMAS in France, Germany, the Netherlands and the United Kingdom, it argues that the most powerful participation leverage has been the granting of regulatory relief for registered companies. This leads one to be pessimistic as to the future of EMAS. The possibility and scope for a lighter regulatory touch are primarily nationally specific since they are related to the national regulatory traditions. Consequently, the systematic and comprehensive use of this leverage is unlikely to generalize. Copyright © 2002 John Wiley & Sons, Ltd. and ERP Environment.