bluuebunny/crossref_metadata_2025 huggingface dataset:
Long‐term outcome of surgical revascularization of supraaortic vessels
Authors: Uurto Ilkka T. , Lautamatti Visa , Zeitlin Rainer , Salenius Juha P.
Published: December 2002

The aim of this study was to evaluate long‐term results of different surgical reconstructions of supraaortic vessels, particularly the subclavian and innominate arteries. It is a retrospective review of 33 years experience in a teaching hospital; 80 surgical revascularizations were performed in 76 patients who suffered occlusive disease of subclavian or innominate artery from 1965 to 1998. These included 38 bypass (BP) Operations (28 carotid‐subclavian, 4 aorto‐subclavian, 3 aorto‐innominate, and 3 subclavian transpositions) and 42 endarterectomies (EA). All available patients (34) were reassessed clinically, and by triplex scanning. The mean clinical follow‐up was 9 months (range 1 to 116 months) for all patients and for control‐examined patients 158 months (range 6 to 346 months). Four patients were lost to follow‐up. The perioperative mortality was 2.5% (BP, 0%; EA, 5%). The overall patency rate for both the BP and the EA procedures was 95% at 1 and 5 years; 91% at 10 years (BP, 89%; EA, 93%) and 89% (BP, 87%; EA, 90%) at 15 years. Most of the patients (84%) were satisfied with the clinical result in the long term. We conclude that surgical revascularization of supraaortic vessels is an infrequent procedure, and all surgical techniques give good and durable long‐term outcome.
Functional and morphologic properties of a modified mesh for inguinal hernia repair
Authors: Junge Karsten , Klinge Uwe , Rosch Raphael , Klosterhalfen Bernd , Schumpelick Volker
Published: December 2002

Inguinal hernia repair is one of the most frequently performed operations. Next to conventional techniques, open and laparoscopic tension‐free methods using mesh implants to reinforce the abdominal wall are increasingly carried out, even becoming the standard procedure in many countries. Because of the benefits of material‐reduced meshes for incisional hernia repair, a new mesh modification for tension‐free inguinal hernia repair has been developed. In the present study this new low‐weight mesh (Vypro II) made of polypropylene and polyglactin multifilaments was compared to a common heavy‐weight polypropylene mesh (Prolene) regarding their functional consequences and the morphologic tissue response. After implantation in rats as an inlay, abdominal wall mobility was recorded by three‐dimensional photogrammetry and the tensile strength of the suture zone and the mesh itself was measured at 3, 21, and 90 days. Explanted tissue samples have been investigated for their histologic reaction in regard to the inflammatory infiltrate, vascularization, and connective and fat tissue ingrowth. Numbers of granulocytes, macrophages, fibroblasts, lymphocytes, and foreign giant body cells have been evaluated to reflect the quality of the tissue response. The cellular response was assessed by measuring DNA strand breaks and apoptosis (TUNEL), proliferation (Ki67), and cell stress (HSP70). The results indicated that restriction of abdominal wall mobility was significantly reduced with Vypro II compared to that seen with heavy‐weight mesh modification, and the inflammatory reaction and connective tissue formation were markedly diminished. Apoptosis and cell proliferation showed considerably lowered levels, and expression of cytoprotective HSP70 was significantly increased. The present study thus confirms the benefits of material‐reduced mesh modifications. The new low‐weight mesh (Vypro II) could be advantageous in inguinal hernia repair.
Modulation of renal sympathetic nerve activity during pneumoperitoneum in rats
Authors: Tanaka Kunihiko , Tsuchiya Yo , Ree Ken , Morita Hironobu
Published: December 2002

To examine neural control of renal function during pneumoperitoneum, renal sympathetic nerve activity (RSNA) was measured in pentobarbital‐anesthetized rats that had their entire nervous system intact or that had undergone lower thoracic dorsal rhizotomy or abdominal vagotomy. During pneumoperitoneum with intraabdominal pressure (IAP) of 10 mmHg, the mean arterial pressure did not change, but central venous pressure increased by 10 mmHg in all groups. In intact rats, the RSNA increased to 285 ± 22% during pneumoperitoneum and gradually recovered after release of the insufflation. The RSNA responses decreased during pneumoperitoneum in rats with dorsal rhizotomy or vagotomy compared to responses in intact rats. In intact rats the urine volume and Na+ excretion decreased during pneumoperitoneum and increased just after insufflation release. Dorsal rhizotomy, vagotomy, or renal denervation did not alter the antidiuretic and antinatriuretic responses during pneumoperitoneum; however, diuretic and natriuretic responses were completely abolished by either of these denervations following insufflation release. These results suggest that oliguria during pneumoperitoneum was not due to neural control of renal function but probably to a mechanical influence induced by the elevated LAP. On the other hand, diuretic and natriuretic responses after insufflation release were thought to be a neurally mediated response.
Laparoscopic appendectomy for perforated appendicitis
Authors: So Jimmy B. Y. , Chiong Ee‐Cherk , Chiong Edmond , Cheah Wei‐Keat , Lomanto David , Goh Peter , Kum Cheng‐Kiong
Published: December 2002

Although laparoscopic appendectomy for uncomplicated appendicitis is feasible and safe, its application to perforated appendicitis is uncertain. A retrospective study of all patients with perforated appendicitis from 1992 to 1999 in a university hospital was performed. A series of 231 patients were diagnosed as having perforated appendicitis. Of these patients, 85 underwent laparoscopy (LA), among whom 40 (47%) required conversion to an open procedure. An open appendectomy (OA) was performed in 146 patients. The operating time was similar for the two groups. Return of fluid and solid diet intake were faster in LA than OA patients (p<0.01). Postoperative infections including wound infections and abdominal abscesses occurred in 14% of patients in the laparoscopy group and in 26% of those with OA (p<0.05). The surgeon’s experience correlated with the conversion rate. Laparoscopic appendectomy is associated with a high conversion rate for perforated appendicitis. If successful, it offers patients faster recovery and less risk of infectious complications.
Pharmacotherapy for chronic hemorrhagic radiation proctitis
Authors: Gul Yunus A. , Prasannan Subhita , Jabar Faisal M. , Shaker Abdul R. H. , Moissinac Kevin
Published: December 2002

Endoscopic thermal therapy and formalin are being increasingly recommended for the treatment of chronic hemorrhagic radiation proctitis. It may be too early, however, to discard pharmacologic agents from the management process, especially in medical institutions where specialized equipment is unavailable. We prospectively assessed the effectiveness of medical therapy in 14 consecutive patients with chronic hemorrhagic radiation proctitis from July 1999 to June 2001. All 14 subjects were women (mean age 56 years), 13 of whom had had radio‐therapy for Cancer of the cervix. The median time to onset of Symptoms following irradiation was 16 months. Six patients had a hemoglobin level of<8 g/dl, and blood transfusion was required in 11 patients. In five patients (36%) initially treated with hydrocortisone enemas prior to referral, this treatment continued; and the remaining nine patients were commenced on sucralfate enemas. Two patients given rectal hydrocortisone continued to bleed and were treated with sucralfate enemas and topical formalin, respectively. Rectal sucralfate Suspension effectively procured symptomatic alleviation in all 11 patients. Rectal bleeding recurred in two patients who had been managed exclusively with hydrocortisone and sucralfate enemas, respectively, over a mean follow‐up of 6 months. Both patients were managed with topical formalin, which con‐trolled their Symptoms. Even though the number of subjects in this study is small, sucralfate enema can be recommended as an effective first‐line agent for managing patients with chronic hemorrhagic radiation proctitis. The use of more specialized therapy can therefore be reserved for cases where primary treatment failure occurs with sucralfate therapy.
Clinicoanatomical study on the infraportal bile ducts of segment 3
Authors: Ozden Ilgin , Kamiya Junichi , Nagino Masato , Uesaka Katsuhiko , Sano Tsuyoshi , Nimura Yuji
Published: December 2002

In some patients, bile ducts of segments 2 and 3 (B2 and B3) run caudally to the umbilical portion of the left portal vein (UP)—an infra‐portal course. We aimed to evaluate the frequency and clinical implications of this variation. Between January 1992 and October 2000, 108 patients underwent resection for hilar cholangiocarcinoma. The records of the 6 patients with infraportal left hepatic ducts were evaluated. An infraportal B3 was diagnosed in 6 patients (6%). No patient had an infraportal B2. An infraportal B3 could be demonstrated by computed tomography (CT) before biliary drainage, percutaneous transhepatic or endoscopic cholangiography, portography after percutaneous transhepatic biliary drainage (PTBD) via B3 and CT after PTBD via B3. Four patients (4/6) had a liver bridge covering Rex’s recess (B3 not in the bridge). The incidence of the bridge in 75 comparable patients was 9/75. In conclusion, common radiologic methods are sufficient for diagnosis of abnormal biliary anatomy. The presence of a liver bridge over Rex’s recess is suggestive of this variation. Separate biliary reconstruction for an infraportal branch is mandatory in an extended right hepatectomy for biliary tract cancer and may be necessary in liver transplantation with segments 2+3 grafting.
Molecular Embryogenesis of the Heart
Authors: Kirby Margaret L. , Waldo Karen L.
Published: December 2002

Development of the heart is a complex process involving primary and secondary heart fields that are set aside to generate myocardial and endocardial cell lineages. The molecular inductions that occur in the primary heart field appear to be recapitulated in induction and myocardial differentiation of the secondary heart field, which adds the conotruncal segments to the primary heart tube. While much is now known about the initial steps and factors involved in induction of myocardial differentiation, little is known about induction of endocardial development. Many of the genes expressed by nascent myocardial cells, which then become committed to a specific heart segment, have been identified and studied. In addition to the heart fields, several other “extracardiac” cell populations contribute to the fully functional mature heart. Less is known about the genetic programs of extracardiac cells as they enter the heart and take part in cardiogenesis. The molecular/genetic basis of many congenital cardiac defects has been elucidated in recent years as a result of new insights into the molecular control of developmental events.
Testicular Microlithiasis: Histologic and Immunohistochemical Findings in 11 Pediatric Cases
Authors: Drut Ricardo , Drut Rosa Mónica
Published: December 2002

Testicular microlithiasis (TM) is being recognized with increasing frequency because of the extensive use of ultrasound. TM has been linked to several pathological conditions of the testis, mainly with an increased risk for developing germ cell tumors. The pathogenesis of the microcalcospherites is unknown. We report a detailed morphologic and immunohistochemical analysis of 11 patients (age: 3 to 15 years) with TM. The microliths were related neither to the age of the children nor to the developmental stage of the testis. The microcalcospherites were PAS positive or collagen IV positive or surrounded by a collagen IV–positive band, extratubular structures consistently associated with double-layered annular tubules. Immature, smaller Sertoli cells commonly lined the inner layer of the annular tubules. Some microcalcospherites showed an interposed thin band of connective tissue cells between the concretion and the tubular basement membrane. The annular tubules seemed to result from progressive wrapping of the growing tubules around the concretions. Our findings favor the interpretation that the microliths are located outside the tubules and have been present there since very early stages of testicular development. The association of the calcospherites with Sertoli cells and annular tubules formation, like that of gonadal stromal tumor with annular tubules of the ovary and large cell–calcifying Sertoli cell tumor of the testis, favors the hypothesis that microliths may result from multifocal Sertoli cell dysfunction. Since both tumors are related to the Peutz-Jeghers syndrome, it is proposed that TM may result from the same genetic abnormalities. It is unclear how this may be related to the development of germ cell tumors. However, the presence of calcospherites in gonadoblastoma may indicate a combined Sertoli cell and germ cell derangement in the genesis of TM.
Aberrant Immunohistochemical Expression in Nonrhabdomyosarcoma Soft Tissue Sarcomas of Infancy: Retrospective Review of Clinical Material
Authors: Sebire Neil James , Ramsay Alan Drummond , Levitt Gillian , Malone Marian , Risdon Rupert Anthony
Published: December 2002

Malignant soft tissue tumors other than rhabdomyosarcoma (RMS) are uncommon in infancy, representing approximately 5% of pediatric sarcomas. The pathological categorization of non-RMS soft tissue malignancies from these young patients is complicated by variation in both morphologic and immunohistochemical features. A search covering an 11-year period identified 19 patients presenting at birth or in infancy with a clinical or referral diagnosis of soft tissue sarcoma. After histologic and immunohistochemical review, nine of these tumors were classified as primitive neuroectodermal tumor (PNET), three as infantile hemangiopericytoma (HPC), two as infantile fibrosarcoma (FS), and five as undifferentiated sarcoma. Those identified as undifferentiated sarcomas showed an atypical spindle and ovoid cell morphology, with cellular pleomorphism and high mitotic rate, but lacking the fascicular growth pattern of classic infantile fibrosarcoma. Immunohistochemical staining in this group showed variable weak positivity for a range of markers (desmin, smooth muscle actin, Myo-D1, PGP, NSE, S100, CO56, cytokeratin, and CD99), and did not fit readily into any distinct diagnostic category. In this series, tumors classified as soft tissue PNETs had a poor prognosis despite aggressive treatment. However, once RMS, PNET, and other rare specific lesions are excluded, the remaining undifferentiated sarcomas, despite their unusual morphology and immunohistochemistry, appear to behave in a similar favorable manner to infantile fibrosarcoma.
Limited Reliability of Lipid-laden Macrophage Index Restricts Its Use as a Test for Pulmonary Aspiration: Comparison with a Simple Semiquantitative Assay
Authors: Ding Yiling , Simpson Pippa M. , Schellhase Dennis E. , Tryka A. Francine , Ding Lieming , Parham David M.
Published: December 2002

The lipid-laden macrophage index (LLMI) is a semi-quantitative evaluation of alveolar macrophage lipid content used in diagnosis of pulmonary aspiration. To date, there are no published reports regarding the reliability of LLMI. We sought to evaluate the interobserver and intraobserver variability and validity of LLMI and to compare it to a simpler macrophage lipid content index (LCI). To evaluate reliability we compared both the LLMI and LCI of 26 bronchoalveolar lavage (BAL) specimens from 14 aspirators and 12 non-aspirators on 10 separate occasions by two observers. The ranges of means and standard deviations (SD) of LLMI for observer 1 (Obs 1) were 19–160 (5–31) for aspirators, and 0–48 (0–15) for non-aspirators; and those of observer 2 (Obs 2) were 77–249 (13–33) for aspirators and 47–170 (8–37) for non-aspirators. The ranges of means and SD of LCI for Obs 1 were 2–8 (0–2) for aspirators and 0–4 (0–1) for non-aspirators, compared with 2–9 (0–2) for aspirators and 1–6 (0–2) for non-aspirators for Obs 2. No statistical significance was found between LLMI and LCI by comparing coefficients of variation (CV) in either groups or observers. Poor agreement between the two observers was found using a Bland Altman analysis, with the difference of the two observations mostly exceeding zero and becoming larger as the average of the two observations became bigger. The combined sensitivity, specificity, and positive and negative predictive value (PPV and NPV) for both observers of the LLMI were 57%, 75%, 84%, and 69% and those of LCI were 58%, 92%, 93%, and 69%. We conclude that there is poor reliability for both methods. The LCI is simpler and appears to be at least as good as the LLMI.
Organ Weights in Human Fetuses after Formalin Fixation: Standards by Gestational Age and Body Weight
Authors: Guihard-Costa Anne-Marie , Ménez Françoise , Delezoide Anne-Lise
Published: December 2002

This study provides new standards of fetal organ weights (brain, heart, liver, pancreas, spleen, lungs, kidneys, adrenals, thymus, and thyroid) and body dimensions (crown–heel and crown–rump lengths, head circumference, and foot length). Subjects came from a large data-set including more than 4000 fetuses autopsied in fetopathology units of pediatric hospitals in Paris between 1986 and 2001. From this dataset, 673 subjects were carefully selected by exclusion of multiple pregnancies, macerated and malformed fetuses, subjects with abnormal karyotypes, and those with severe infections. Fetal age ranged from 9 to 42 gestational weeks, with a very large sample of fetuses in the first half of gestation. Each organ was weighed after fixation in formalin. The standards were computed in relation to age and body weight. The mathematical models used to fit the percentile growth curves were carefully selected for each organ or dimension. This study, based on reliable methodology, affords a whole set of accurate growth standards useful for pathologists.
Heterotopic Nephrogenic Rests in the Colon and Multiple Congenital Anomalies: Possibly Related Association
Authors: Jain Dhanpat , Martel Maritza , Reyes-Múgica Miguel , Parkash Vinita
Published: December 2002

Heterotopic renal tissue (HRT) in the wall of the colon is a very rare occurrence, with only five cases published. Our patient is only the second patient reported to have this abnormality in the absence of sirenomelia. We describe colonic HRT in a child, associated with multiple congenital anomalies. The congenital abnormalities were of the VACTERL type, accompanied by valvular cardiac anomalies that were clinically diagnosed as Shone syndrome. The HRT was not apparent clinically or grossly. Microscopically, multifocal islands of renal tissue consisting of glomeruli, cystically dilated tubules, and blastema were seen in all layers of the bowel, and simulated “cystic partially differentiated nephroblastoma.” Our case provides further support to the belief that VACTERL association and sirenomelia represent related entities.
Progressive Liver Fibrosis in Late-onset Argininosuccinate Lyase Deficiency
Authors: Mori Toshihiko , Nagai Kazushige , Mori Michio , Nagao Masayoshi , Imamura Masakatsu , Iijima Mikio , Kobayashi Keiko
Published: December 2002

A 4-month-old boy, with late-onset argininosuccinate lyase (ASL) deficiency with hepatomegaly, was treated by protein restricted diet and arginine supplementation; he was followed for 3 years. Hepatomegaly and mild liver dysfunction persisted without significant hyperammonemia. He maintained normal psychomotor development to the age of 12 months, but, at 3 years of age, his developmental status is in the borderline normal range. Liver biopsy performed at 12 months of age demonstrated swollen and pale hepatocytes with abnormal glycogen deposition and mild periportal fibrosis. A subsequent liver biopsy at 3 years of age showed progressive liver fibrosis in the periportal and central areas, which extended into the liver lobule. These findings suggest that liver impairment in ASL deficiency may advance without significant hyperammonemia and underline the importance of repeated liver biopsy in this disorder, even when the plasma ammonia level is well controlled.
Newborn with Anophthalmia and Features of Fryns Syndrome
Authors: Pierson Diane M. , Subtil Antonio , Taboada Eugenio , Butler Merlin G.
Published: December 2002

We report a newborn female with craniofacial malformations, bilateral anophthalmia, large abnormally shaped ears, short neck, small distal phalanges and nails, left diaphragmatic hernia, hypoplastic optic nerves, severe pulmonary hypoplasia, and an accessory spleen, and describe the autopsy findings. The infant expired at 18 h of life. The features were most consistent with Fryns syndrome although other conditions were considered including Matthew Wood syndrome. Anophthalmia, to our knowledge, has not been reported previously in Fryns syndrome; however, eye findings are common, particularly microphthalmia and cloudy cornea.
Danazol and plasma lipoprotein metabolism
Authors: Shepherd J.
Published: December 2002

The potential long‐term impact of danazol on coronary risk hinges on its effect on lipoprotein metabolism rather than its influence on total plasma lipids. Danazol may exert a regulatory influence on three key processes in lipoprotein metabolism: hepatic lipase activity; low‐density lipoprotein receptor function; and lecithin:cholesterol acyl‐transferase activity. Danazol decreases plasma fibrinogen and lipoprotein (a) levels, promotes fibrinolysis and causes a rise in plasminogen. Such changes are beneficial as they inhibit the process of thrombosis. Androgenic properties of danazol produce effects on plasma lipids and lipoproteins which oppose estrogen‐induced changes. The usual recipients of danazol therapy are premenopausal females, in whom the absolute risk of ischemic heart disease is low. If the drug were shown to increase ischemic heart disease risk, detrimental factors must be weighed against its considerable and proven clinical benefits.
A new tool for teachers and researchers involved in e-mail tandem language learning
Authors: APPEL CHRISTINE , MULLEN TONY
Published: December 2002

This paper presents a new teacher interface for the Electronic Tandem Resources
(ETR) site, the student interface described in Appel & Mullen (2000), and a new version of the
site designed specifically for research purposes. The main features of the original site geared
towards the language learner were the creation of a virtual environment for tandem language
learning and the provision of tools and data intended to help foster the development of learner
autonomy. The new teacher interface supports the integration of tandem language learning activities
in the foreign language classroom and addresses the difficult issue of performance assessment and
task evaluation. Computer-mediated communication activities between students in different
countries are notoriously difficult for teachers to monitor. Nevertheless, there is evidence that
in certain situations it is beneficial for the teacher to be able to monitor these activities.
The teacher interface of the ETR site offers a user-friendly interface which requires only basic
computer skills, and gives teachers access to data such as the date of the most recently sent
messages, the number of words sent by students and the percentages of text written by each
student in their respective L1 and L2, without giving teachers access to the content of the
messages, thus preserving students’ privacy. Furthermore, a slightly different version has
also been designed for evaluation of the learning by the researcher investigating second
language learning in an electronic tandem environment. This interface has been designed for
setting up experiments and some of its features allow for control over variables related to
the experiment. The interface records time stamps for sent and received messages.
Feedback generation and linguistic knowledge in ‘SLIM’ automatic tutor
Authors: DELMONTE RODOLFO
Published: December 2002

SLIM is a prototype interactive multimedia self-learning linguistic software for
foreign language students at beginner-false beginner level. It allows students to work both in an
autonomous self-directed mode or in a way of programmed learning in which the process of
self-instruction is pre-programmed and monitored. In this latter mode it incorporates assessment
and evaluation tools in order to behave as an automatic tutor. It is organized into three basic
components: audiovisual materials; a linguistic database recording all language material in text
format; the supervisor. Audiovisual materials are partially taken from commercially available
courses; the linguistic database is a highly sophisticated classification of all words and
utterances of the course, both in written and spoken form, from all possible linguistic aspects.
The supervisor is both an attractive, enjoyable and strongly pedagogically based software that
allows the user to work on language materials. The most outstanding feature of SLIM is the use of
speech analysis and recognition which is a fundamental aspect of all second language learning
programmes. We also assume that a learning model can be represented by a finite state automaton
made up by a fixed number of possible states – corresponding to the macro and microlevels
at which the student's competence may be modelled – each one being internally constituted
by the actual linguistic objects of knowledge of the language that make it up.
Continuous Separation of Multicomponent Protein Mixtures by Annular Displacement Chromatography
Authors: Giovannini Roberto , Freitag Ruth
Published: December 2002

Displacement chromatography is a predominantly nonlinear mode of chromatography, which has certain advantages over the elution mode for preparative bioseparations. Whereas continuous production (and separation) processes have their theoretical benefits in this context, protein displacement chromatography has up to now only been performed in the batch mode. In this contribution, we demonstrate that the principle of continuous annular chromatography can be adapted to displacement chromatography. Separations of up to three standard proteins (two whey proteins, soybean trypsin inhibitor) were developed and optimized using a small (4 × 250 mm) batch column. These separations were subsequently transferred directly to the continuous system (500‐mL column). Separations of similar quality in terms of final product purity and recovery yield were obtained using the continuous system.
Genetic Programming Assisted Stochastic Optimization Strategies for Optimization of Glucose to Gluconic Acid Fermentation
Authors: Cheema Jitender Jit Singh , Sankpal Narendra V. , Tambe Sanjeev S. , Kulkarni Bhaskar D.
Published: December 2002

This article presents two hybrid strategies for the modeling and optimization of the glucose to gluconic acid batch bioprocess. In the hybrid approaches, first a novel artificial intelligence formalism, namely, genetic programming (GP), is used to develop a process model solely from the historic process input‐output data. In the next step, the input space of the GP‐based model, representing process operating conditions, is optimized using two stochastic optimization (SO) formalisms, viz., genetic algorithms (GAs) and simultaneous perturbation stochastic approximation (SPSA). These SO formalisms possess certain unique advantages over the commonly used gradient‐based optimization techniques. The principal advantage of the GP‐GA and GP‐SPSA hybrid techniques is that process modeling and optimization can be performed exclusively from the process input‐output data without invoking the detailed knowledge of the process phenomenology. The GP‐GA and GP‐SPSA techniques have been employed for modeling and optimization of the glucose to gluconic acid bioprocess, and the optimized process operating conditions obtained thereby have been compared with those obtained using two other hybrid modeling‐optimization paradigms integrating artificial neural networks (ANNs) and GA/SPSA formalisms. Finally, the overall optimized operating conditions given by the GP‐GA method, when verified experimentally resulted in a significant improvement in the gluconic acid yield. The hybrid strategies presented here are generic in nature and can be employed for modeling and optimization of a wide variety of batch and continuous bioprocesses.
A Lab‐Built Respirometer for Plant and Animal Cell Culture
Authors: Lamboursain Laurence , St‐Onge Frédéric , Jolicoeur Mario
Published: December 2002

A very simple off‐line respirometer was developed to measure oxygen consumption rates of low respiring and shear‐sensitive cell suspensions. The respirometer is composed of a 10 mL glass syringe in which the plunger was substituted with a polarographic dissolved oxygen probe. Mechanical agitation is provided by means of a magnetic stirring bar inside the measuring chamber and a stir plate placed bellow the respirometer. Abiotic oxygen fluxes occurring in the measurement chamber such as oxygen diffusion and probe oxygen consumption were investigated. The apparent oxygen uptake rate was then corrected for abiotic oxygen fluxes, leading to accurate measurements of respiration rates ranging from 0.5 to 25.0 mM·h−1. Additionally, the effect of the stirring bar shape and of the test length on the integrity of plant ( Eschschzoltzia californica) and animal (NS0) cells was evaluated. Animal cells showed a higher resistance to mechanical stirring inside the respirometer compared to plant cells (0% of broken cells and 78.1% respectively for a polygonal stirring bar and a 15 min test). For plant cells, cell damage inside the measurement chamber was reduced by optimizing the stirring bar shape and reducing the test length to 5 min or less. This very simple design was shown to provide reliable and low‐cost quantification of the oxygen uptake rate of plant and animal cells and can be use even for more demanding measurements such as oxygen affinity studies.
Application of Fungi as Biocatalysts for the Reduction of Diethyl 1‐Oxoalkylphosphonates in Anhydrous Hexane
Authors: Brzezińska‐Rodak Małorzata , Żymańczyk‐Duda Ewa , Kafarski Paweał , Lejczak Barbara
Published: December 2002

Five different species of microorganisms, namely, Rhodotorula rubra, Rhodotorula glutinis, Cladosporium sp., Verticillium sp., and baker's yeast, turned out to be useful biocatalysts for enantioselective reduction of a variety of diethyl 1‐oxoalkylphosphonates. To suppress substrate decomposition, bioreductions were carried out under anhydrous conditions, using lyophilized cells immobilized on Celite R 630. The influence of reaction conditions such as biotransformation time and chemical additives on the yield of the reaction is discussed.
Continuous Desulfurization of Dibenzothiophene with Rhodococcus rhodochrous IGTS8 (ATCC 53968)
Authors: Schilling Bernhard M. , Alvarez Luis M. , Wang Daniel I. C. , Cooney Charles L.
Published: December 2002

Desulfurization of a model fuel system consisting of hexadecane and dibenzothiophene (DBT) by Rhodococcus rhodochrousIGTS8 was demonstrated in a 2‐L continuous stirred tank reactor (CSTR). The reactor was operated in a semicontinuous and continuous mode with and without recycling of the model fuel. A constant volumetric desulfurization activity A(t), (in mg HBP L−1 h−1) was maintained in the reactor with a feeding strategy of fresh cell suspension based on a first‐order decay of the biocatalyst. Maximum desulfurization rates, as measured by specific desulfurization activity, of 1.9 mg HBP/g DCW h were attained. Rates of biocatalyst decay were on the order of 0.072 h−1. Theoretical predictions of a respiratory quotient (RQ) associated with this biotransformation reaction agree well with experimental data from off‐gas analysis. In addition, the ratio of the specific desulfurization activity a(t), (in mg HBP/g DCW h) of recycled and fresh biocatalyst was determined and evaluated.
Carbon Mass Balance Evaluation of Cellulase Production on Soluble and Insoluble Substrates
Authors: Carlos Sáez Juan , Schell Daniel J. , Tholudur Arun , Farmer Jody , Hamilton Jenny , Colucci José A. , McMillan James D.
Published: December 2002

A methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near‐infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high‐performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (<72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.
True and Apparent Temperature Dependence of Protein Adsorption Equilibrium in Reversed‐Phase HPLC
Authors: Szabelski Paweł , Cavazzini Alberto , Kaczmarski Krzysztof , Van Horn Jennifer , Guiochon Georges
Published: December 2002

The adsorption behavior of bovine insulin on a C8‐bonded silica stationary phase was investigated at different column pressures and temperatures in isocratic reversed‐phase HPLC. Changes in the molar volume of insulin (Δ Vm) upon adsorption were derived from the pressure dependence of the isothermal retention factor ( k′). The values of Δ Vm were found to be practically independent of the temperature between 25 and 50 °C at –96 mL/mol and to increase with increasing temperature, up to –108 mL/mol reached at 50 °C. This trend was confirmed by two separate series of measurements of the thermal dependence of ln( k′). In the first series the average column pressure was kept constant. The second series involved measurements of ln( k′) under constant mobile‐phase flow rate, the average column pressure varying with the temperature. In both cases, a parabolic shape relationship was observed between ln( k′) and the temperature, but the values obtained for ln k′ were higher in the first than in the second case. The relative difference in ln( k′), caused by the change in pressure drop induced by the temperature, is equivalent to a systematic error in the estimate of the Gibbs free energy of 12%. Thus, a substantial error is made in the estimates of the enthalpy and entropy of adsorption when neglecting the pressure effects associated with the change in the molar volume of insulin. This work proves that the average column pressure must be kept constant during thermodynamic measurements of protein adsorption constants, especially in RPLC and HIC. Our results show also that there is a critical temperature, Tc ≈ 53 °C, at which ln( k′) is maximum and the insulin adsorption process changes from an exothermic to an endothermic one. This temperature determines also the transition point in the molecular mechanism of insulin adsorption that involves successive unfolding of the protein chain.
Mature Experimental Constructed Wetlands Treating Urban Water Receiving High Metal Loads
Authors: Scholz Miklas , Höhn Patrick , Minall Rowland
Published: December 2002

The aim was to assess over 2 years the treatment efficiencies of vertical‐flow wetland filters containing macrophytes and granular media of different adsorption capacities. Different concentrations of lead and copper sulfate (constant for 1 year each) were added to urban beck inflow water in order to simulate pretreated (pH adjustment assumed) mine wastewater. After 1 year of operation, the inflow concentrations for lead and copper were increased from 1.30 to 2.98 and from 0.98 to 1.93 mg/L, respectively. However, the metal mass load rates (mg/m2/d) were increased by a factor of approximately 4.9 for lead and 4.3 for copper. No breakthrough of metals was recorded. Lead and copper accumulated in the biomass of the litter zone and rhizomes of the macrophytes. Furthermore, microbiological activity decreased during the second year of operation. Bioindicators such as ciliated protozoa and zooplankton decreased sharply in numbers but diatoms increased. In conclusion, the use of macrophytes and adsorption media did not significantly enhance the filtration of lead and copper. Particulate lead is removed by filtration processes including straining. Furthermore, some expensive and time‐consuming water quality variables can be predicted with less expensive ones such as temperature in order to reduce sampling costs.
Adsorption of Cadmium Ion and Gallium Ion to Immobilized Metallothionein Fusion Protein
Authors: Terashima Masaaki , Oka Noriyuki , Sei Takamasa , Yoshida Hiroyuki
Published: December 2002

A fusion protein made from maltose binding protein (pmal) and human metallothionein (MT) was expressed using E. coli. The purified recombinant protein (pmal‐MT) was immobilized on Chitopearl resin, and characteristics of pmal‐MT for metal binding were evaluated. As expected from the tertiary structure of metallothionein, the pmal‐MT ligand adsorbed 12.1 cadmium molecules per one molecule of the ligand at pH 5.2. The pmal‐MT ligand also bound 26.6 gallium molecules per one molecule of the ligand at pH 6.5. Neither cadmium ion nor gallium ion bound to a control protein bovine serum albumin (BSA). Adsorption isotherms for both ions were correlated by Langmuir‐type equations. Two types of binding sites have been elucidated on the basis of HSAB (hard and soft acid and base) theory. It was suggested that gallium ion specifically binds to amino acid residues containing oxygen and nitrogen atoms, while cadmium ion binds to specific binding sites formed by multiple cysteine residues. The pmal‐MT ligand bound these metals in the concentration range of 0.2–1.0 mM, and the bound metal ions could be eluted under relatively mild conditions (pH 2.0). The pmal‐MT Chitopearl resin was stable and could be used repeatedly without loss of binding activity. Thus, this new ligand would be useful for recovery of toxic heavy metals and/or valuable metal ions from various aqueous solutions.
Enhanced Stress Tolerance in Escherichia coli and Nicotiana tabacum Expressing a Betaine Aldehyde Dehydrogenase/Choline Dehydrogenase Fusion Protein
Authors: Yilmaz Jenny Lindberg , Bülow Leif
Published: December 2002

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5‐fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector‐transformed control plants.
Rhamnolipid Biosurfactant Production by Strains of Pseudomonas aeruginosa Using Low‐Cost Raw Materials
Authors: Rahman K. S. M. , Rahman Thahira J. , McClean Stephen , Marchant Roger , Banat Ibrahim M.
Published: December 2002

This study was aimed at the development of economical methods for higher yields of biosurfactant by suggesting the use of low‐cost raw materials. Two oil‐degrading strains, Pseudomonas aeruginosa GS9–119 and DS10–129, were used to optimize a substrate for maximum rhamnolipid production. Among the two strains, the latter produced maxima of 4.31, 2.98, and 1.77 g/L rhamnolipid biosurfactant using soybean oil, safflower oil, and glycerol, respectively. The yield of biosurfactant steadily increased even after the bacterial cultures reached the stationary phase of growth. Characterization of rhamnolipids using mass spectrometry revealed the presence of dirhamnolipids (Rha‐Rha‐C10–C10). Emulsification activity of the rhamnolipid biosurfactant produced by P. aeruginosa DS10–129 was greater than 70% using all the hydrocarbons tested, including xylene, benzene, hexane, crude oil, kerosene, gasoline, and diesel. P. aeruginosa GS9–119 emulsified only hexane and kerosene to that level.
Effect of Long‐Term Cobalt Deprivation on Methanol Degradation in a Methanogenic Granular Sludge Bioreactor
Authors: Zandvoort Marcel H. , Geerts Roy , Lettinga Gatze , Lens Piet N. L.
Published: December 2002

The effect of the trace metal cobalt on the conversion of methanol in an upflow anaerobic sludge bed (UASB) reactor was investigated by studying the effect of cobalt deprivation from the influent on the reactor efficiency and the sludge characteristics. A UASB reactor (30 °C; pH 7) was operated for 261 days at a 12‐h hydraulic retention time (HRT). The loading rate was increased stepwise from 2.6 g chemical oxygen demand (COD)·L reactor−1·d−1 to 7.8 g COD·L reactor−1·d−1. Cobalt deprivation had a strong impact on the methanogenic activity of the sludge. In batch tests, the methanogenic activity of the sludge with methanol as the substrate increased 5.3 (day 28) and 2.1 (day 257) times by addition of 840 nM of cobalt. The sludge had an apparent Km for cobalt of 948 nM after 28 days of operation and 442 nM at the end of the run. Cobalt deprivation during 54 days of operation led to a methanol conversion efficiency of only 55%. Continuous addition of cobalt (330 nM) for 33 days improved the methanol removal efficiency to 100%. In this period of cobalt dosing, the cobalt concentration in the sludge increased 2.7 times up to 32 μg·g TSS−1. Upon omission of the cobalt addition, cobalt washed‐out at a stable rate of 0.1 μg·g VSS−1·d−1. At the end of the run, the cobalt concentration of the sludge was similar to that of the seed sludge.
Continuous Production of High‐Content Fructooligosaccharides by a Complex Cell System
Authors: Sheu Dey‐Chyi , Duan Kow‐Jen , Cheng Chih‐Yu , Bi Jia‐Lin , Chen Jer‐Yi
Published: December 2002

A complex biocatalyst system with a bioreactor equipped with a microfiltration (MF) module was employed to produce high‐content fructooligosaccharides (FOS) in a continuous process initiated by a batch process. The system used mycelia of Aspergillus japonicus CCRC 93007 or Aureobasidium pullulans ATCC 9348 with β‐fructofuranosidase activity and Gluconobacter oxydans ATCC 23771 with glucose dehydrogenase activity. Calcium carbonate slurry was used to control pH to 5.5, and gluconic acid in the reaction mixture was precipitated as calcium gluconate. Sucrose solution with an optimum concentration of 30% (w/v) was employed as feed for the complex cell system, and high‐content FOS was discharged continuously from a MF module. The complex cell system was run at 30 °C with an aeration rate of 5 vvm and produced more than 80% FOS with the remainder being 5–7% glucose and 8–10% sucrose on a dry weight basis, plus a small amount of calcium gluconate. The system worked for a 7‐day continuous production process with a dilution rate of 0.04 h−1, and the volumetric productivity for total FOS was more than 160 g L−1 h−1.
Reversible Immobilization of Invertase on Sepabeads Coated with Polyethyleneimine: Optimization of the Biocatalyst's Stability
Authors: Torres Rodrigo , Mateo Cesar , Fuentes Manuel , Palomo Jose M. , Ortiz Claudia , Fernández‐Lafuente Roberto , Guisan Jose M.
Published: December 2002

Invertase fromS. cerevisiaehas been immobilized by ionic adsorption on Sepabeads fully coated with PEI. The enzyme was strongly adsorbed on the support (no desorption of the invertase was found under conditions in which all of the enzyme was released from conventional anionic exchanger supports (e.g., DEAE‐agarose)). Nevertheless, the enzyme could still be desorbed after its inactivation, and new fresh enzyme could be adsorbed on the supports without detrimental effects on enzyme loading. This is a multimeric enzyme, its minimal oligomerization active state being the dimer, but under certain conditions of pH and concentration it may give larger multimers. Very interestingly, results suggested that the adsorption of the enzyme on this large and flexible polymeric bed was able to freeze some of the different oligomeric structures of the enzyme. Thus, we have found that the enzyme immobilized at certain pH values (pH 8.5) and high enzyme concentration, in which the main enzyme structure is the tetramer, was more stable than immobilized preparations produced in conditions under which oligomerization was not favorable (dimers at low enzyme concentration) or it was too high (e.g., hexamers‐octamers at low pH value). The optimal enzyme preparation remained fully active after a 15‐day incubation at 50 °C and pH 4.5 (conditions of standard industrial use) and presented an optimal temperature approximately 5 °C higher than that of soluble enzyme.
Strain Improvement to Enhance the Production of Recombinant Penicillin Acylase in High‐Cell‐Density Escherichia coli Cultures
Authors: Lin Y.‐H. , Hsiao H.‐C. , Chou C. P.
Published: December 2002

Using fed‐batch operation for high‐cell‐density cultivation, efforts are frequently made for optimization of culture parameters, particularly feeding strategy. The current study also emphasized the importance of selecting strains for the production of recombinant proteins in high‐cell‐density cultures. With Escherichia coli penicillin acylase (PAC) as a target protein, the host/vector system of MDΔP7 harboring pTrcKnPAC2902 and pKS12 was designed for optimization of fed‐batch cultivation for recombinant protein production. The host, MDΔP7, potentially had a high translational and periplasmic processing efficiency for pac expression. On the other hand, the vector, pTrcKnPAC2902, was genetically constructed for pac overexpression. Coexistence of the other vector, pKS12, significantly enhanced PAC production by improving cell physiology and reducing the amount of inclusion body formation upon pac overexpression. An extremely high volumetric PAC activity at 37,500 U/L was obtained with the use of the developed host/vector system under optimum fed‐batch culture conditions.