bluuebunny/crossref_metadata_2025 huggingface dataset:
Will Directed Parathyroidectomy Utilizing the Gamma Probe or Intraoperative Parathyroid Hormone Assay Replace Bilateral Cervical Exploration as the Preferred Operation for Primary Hyperparathyroidism?
Authors: Burkey Shelby H. , van Heerden Jon A. , Farley David R. , Thompson Geoffrey B. , Grant Clive S. , Curlee Kathleen J.
Published: October 2002

The trend toward limited exploration for primary hyperparathyroidism (1° HPT) has stemmed from advances in sestamibi scanning, gamma probe technology, and intraoperative parathyroid hormone monitoring (iPTH). Prior to widespread application,
directed parathyroidectomy must be shown to meet the high standards of conventional cervical exploration. In this prospective, nonrandomized trial, results of parathyroidectomy utilizing the gamma probe, iPTH, or neither technique were evaluated. Altogether, 150 patients underwent parathyroidectomy utilizing the gamma probe (n = 50),
iPTH (n = 50), or neither technique
(n = 50). Each group was evaluated for operating time,
length of hospitalization, cure rate, morbidity, mortality, and cost. A telephone survey was conducted with 25 patients in each group
(n = 75) to address patient satisfaction. The gamma probe localized the abnormal gland in 66% of patients and confirmed cure intraoperatively in 84%. In the iPTH group, a more than 50%
decrease from baseline occurred in 98%. The mean operating times were
76, 84, and 90 minutes, respectively (p = 0.16); and the mean length of hospitalization was 1 day. The biochemical cure rates were 98%, 100%, and 96%, respectively (p =
0.17). Total costs were $4476, $3918, and $3905, respectively. A total of 96% of patients in all three groups were "very satisfied" with their surgical procedure. Directed parathyroidectomy utilizing the gamma probe or iPTH assay does not significantly alter the operating time, length of hospitalization, cure rate, morbidity, mortality, or patient satisfaction when compared to conventional exploration in our practice. The probe is more expensive and was not a consistently reliable tool for localizing parathyroid pathology. PTH monitoring reliably predicts cure intraoperatively.
Laparoscopic Adrenalectomy for Pheochromocytoma
Authors: Cheah W. Keat , Clark Orlo H. , Horn Jan K. , Siperstein Allan E. , Duh Quan‐Yang
Published: October 2002

aparoscopic adrenalectomy is the standard for most surgical adrenal diseases. The aim of this study was to evaluate the safety and effectiveness of laparoscopic adrenalectomy for patients with pheochromocytoma.
The medical records of 39 consecutive patients who underwent laparoscopic adrenalectomy for pheochromocytomas from 1994 to 2000 at the University of California–San Francisco were reviewed. Three groups of patients were identified. The first group comprised 17 patients with classic symptoms and signs of pheochromocytoma. The second group comprised 17 patients who had minimal symptoms and incidentally discovered pheochromocytoma (i.e.,
"incidentaloma"), almost half of whom inappropriately underwent fine‐needle biopsies before diagnosis. The third group consisted of 5 patients who had acute hypertensive crises and required intensive preoperative preparation. The mean age of the 22 men and 17 women was 46 years (range
20–84 years), and the mean adrenal tumor size was 4.8 cm (range
2–12 cm). A total of 43 laparoscopic adrenalectomies were performed for
35 patients with unilateral tumors and 4 patients with bilateral tumors. The retroperitoneal approach was used in four patients and the lateral transabdominal approach in the remaining patients without conversion to an open operation. There were no intraoperative complications or mortality. The mean duration of hospitalization was 1.7 days. In conclusion, patients with pheochromocytoma have a wide spectrum of presentations, from minimal symptoms to hypertensive crises. When evaluating an incidentaloma, pheochromocytoma should be excluded by metabolic testing, not by needle biopsy. Laparoscopic adrenalectomy is the preferred surgical approach for patients with pheochromocytoma because it is safe and efficacious.
Complications of Neck Dissection for Thyroid Cancer
Authors: Cheah W. Keat , Arici Cumhur , Ituarte Philip H.G. , Siperstein Allan E. , Duh Quan‐Yang , Clark Orlo H.
Published: October 2002

rophylactic and therapeutic neck dissections are used to control or eliminate local nodal disease in patients with thyroid cancer. The purpose of this study was to evaluate the results and complications of neck dissection. From 1992 to 1999 a series of 115 consecutive neck dissections were performed in 74 patients (32 men, 42 women; mean age 48 years)
with thyroid cancer and nodal metastases. Operations included central compartment, lateral modified, and suprahyoid dissection with and without total or completion thyroidectomy. Sixty‐four percent of the patients had papillary,
4% follicular, and 32% medullary thyroid cancer. Complications included transient hypocalcemia (23%) defined by a postoperative serum calcium level of
<2.0 mmol/L (8.0 mg/dl), one neck hematoma (0.9%), and one cardiac death
(0.9%). There were no permanent recurrent nerve palsies. Hypocalcemia occurred more frequently when neck dissection was combined with total thyroidectomy than without it (p <0.005). In this group, the incidence of hypocalcemia was higher after central, than lateral, neck dissection. When neck dissection was performed without thyroidectomy, there was no difference in the rates of hypocalcemia between central, lateral, or central with lateral neck dissection (p = NS).
Hypocalcemia did not increase with repeated neck dissectionsp = NS). Permanent hypoparathyroidism occurred in 0.9%.
There were no complications after suprahyoid dissection. The median duration of hospitalization was 1 day. Therapeutic neck dissection or repeated neck dissection can be performed relatively safely in patients with thyroid cancer.
Hypocalcemia occurs most frequently when neck dissection is combined with total thyroidectomy.
New Point‐of‐care Intraoperative Parathyroid Hormone Assay for Intraoperative Guidance in Parathyroidectomy
Authors: Carneiro Denise M. , Irvin George L.
Published: October 2002

The use of the intraoperative parathyroid hormone assay (QPTH) to guide a limited parathyroidectomy in patients with sporadic primary hyperparathyroidism (SPHPT) is well established. The advantage of having this assay performed in the operating room is immediate feedback for (1) confirming the complete excision of all hyperfunctioning parathyroid(s); (2) differential jugular venous sampling for localization; and (3) diagnosing suspected tissue without histopathology. For these reasons, the reliability of the hormone measurement and a short assay turnaround time are essential for surgical guidance. We report our experience using a new
"point‐of‐care" assay for intact parathyroid hormone (iPTH). A new two‐site chemiluminescent immunometric assay was used. The antibodies are inside a microtiter well, where the iPTH is measured by a strip luminometer after incubation for 5 minutes. Sixteen frozen samples were measured simultaneously using the traditional iPTH assay and this new assay for comparison. Fifty‐one patients with SPHPT underwent parathyroidectomy guided by this new assay. The criteria used to predict postoperative normocalcemia was a drop in the hormone level of ≤ 50% from the highest preincision or preexcision levels at 10 minutes after excision of all hypersecreting gland(s). The correlation between the traditional and new assays was 0.98. The assay predicted the postoperative calcium levels in all patients except one (false negative‐delayed drop). The assay turnaround time was 8 minutes. This new point‐of‐care assay is reliable for predicting postoperative calcium levels when used with the described criteria. It has advantages over the traditional assay in that it is faster and easier to perform.
Total chemical synthesis and chemotactic activity of human S100A12 (EN‐RAGE)
Authors: Miranda Les P. , Tao Tony , Jones Alun , Chernushevich Igor , Standing Kenneth G. , Geczy Carolyn L. , Alewood Paul F.
Published: October 2002

Human S100A12 (extracellular newly identified RAGE (receptor for advanced glycosylation end products)‐binding protein), a new member of the S100 family of EF‐hand calcium‐binding proteins, was chemically synthesised using highly optimised 2‐(1H‐benzotriazol‐1‐yl)‐1,1,3,3‐tetramethyluronium hexafluorophosphate/tert‐butoxycarbonyl in situ neutralisation solid‐phase chemistry. Circular dichroism studies indicated that CaCl2 decreased the helical content by 27% whereas helicity was marginally increased by ZnCl2. The propensity of S100A12 to dimerise was examined by electrospray ionisation time‐of‐flight mass spectrometry which clearly demonstrated the prevalence of the non‐covalent homodimer (20 890 Da). Importantly, synthetic human S100A12 in the nanomolar range was chemotactic for neutrophils and macrophages in vitro.
High‐yield expression of a viral peptide vaccine in transgenic plants
Authors: Gil Félix , Brun Alejandro , Wigdorovitz Andrés , Catalá Rafael , Martı́nez-Torrecuadrada Jorge L. , Casal Ignacio , Salinas Julio , Borca Manuel V. , Escribano José M.
Published: October 2002

A high‐yield production of a peptide vaccine in transgenic plants is described here. A 21‐mer peptide, which confers protection to dogs against challenge with virulent canine parvovirus, has been expressed in transgenic plants as an amino‐terminal translational fusion with the GUS gene. Transformants were selected on the basis of their GUS activities, showing expression levels of the recombinant protein up to 3% of the total leaf soluble protein, a production yield comparable to that obtained with the same epitope expressed by chimeric plant viruses. The immunogenicity of the plant‐derived peptide was demonstrated in mice immunized either intraperitoneally or orally with transgenic plant extracts, providing the suitability of the GUS fusions approach for low‐cost production of peptide vaccines.
Expression of glycoconjugates bearing the Lewis X epitope during neural differentiation of P19 EC cells
Authors: Osanai Taka , Chai Wengang , Tajima Youichi , Shimoda Yasushi , Sanai Yutaka , Yuen Chun-Ting
Published: October 2002

The Lewis X (Lex) bearing glycolipids were noticeably increased in amounts during the course of neural differentiation of P19 EC cells induced by retinoic acid (RA, all‐trans form). Applying neoglycolipid technology and in situ TLC‐LSIMS, the oligosaccharide chains of these scarce Lex bearing glycolipids were partially characterized after released by endoglycoceramidase and subsequent conversion into neoglycolipids. In order to understand the enzymatic basis for the expression of Lex bearing glycolipids, we measured glycolipid, glycoprotein and oligosaccharide fucosyltransferase (Fuc‐T) activities using appropriate substrates in P19 EC cells with or without RA treatment. All three Fuc‐Ts were increased after RA treatment and the highest activity was in the differentiated neural cells. We then investigated the two possible Fuc‐T genes that might be responsible for these changes using RT‐PCR analysis. Mouse Fuc‐TIX (mFuc‐TIX) transcript was detected in all cell types but it was only strongly expressed in RA‐induced aggregates and neural cells. In the case of mouse Fuc‐TIV (mFuc‐TIV) gene, its transcript was only detectable in RA‐induced aggregates and not found in either undifferentiated or RA‐induced neural cells. These results strongly support that RA induces only a transient expression of the mFuc‐TIV gene in cell aggregates but a more persistent expression of the mFuc‐TIX gene at the transcription level throughout neural cell differentiation. The mFuc‐TIX gene is probably the main cause for the increased expression of Lex glycoconjugates during neural differentiation of P19 EC cells.
Ceramide generation by two distinct pathways in tumor necrosis factor α‐induced cell death
Authors: Dbaibo Ghassan S , El-Assaad Wissal , Krikorian Armand , Liu Bin , Diab Karim , Idriss Nadine Z , El-Sabban Marwan , Driscoll Timothy A , Perry David K , Hannun Yusuf A
Published: October 2002

Ceramide accumulation in the cell can occur from either hydrolysis of sphingomyelin or by de novo synthesis. In this study, we found that blocking de novo ceramide synthesis significantly inhibits ceramide accumulation and subsequent cell death in response to tumor necrosis factor α. When cells were pre‐treated with glutathione, a proposed cellular regulator of neutral sphingomyelinase, inhibition of ceramide accumulation at early time points was achieved with attenuation of cell death. Inhibition of both pathways achieved near‐complete inhibition of ceramide accumulation and cell death indicating that both pathways of ceramide generation are stimulated. This illustrates the complexity of ceramide generation in cytokine action.
Flavonoid glucuronides are substrates for human liver β‐glucuronidase
Authors: O'Leary Karen A , Day Andrea J , Needs Paul W , Sly William S , O'Brien Nora M , Williamson Gary
Published: October 2002

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme β‐glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited β‐glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell‐free extracts were the most efficient and the activity was completely inhibited by saccharo‐1,4‐lactone (a β‐glucuronidase inhibitor). Furthermore, pure recombinant human β‐glucuronidase hydrolysed various flavonoid glucuronides, with a 20‐fold variation in catalytic efficiency (k
cat/K
m=1.3×103 M−1 s−1 for equol‐7‐O‐glucuronide and 26×103 M−1 s−1 for kaempferol‐3‐O‐glucuronide). Similar catalytic efficiencies were obtained for quercetin O‐glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal β‐glucuronidase from various human cells.
Coenzyme Q blocks biochemical but not receptor‐mediated apoptosis by increasing mitochondrial antioxidant protection
Authors: Alleva Renata , Tomasetti Marco , Andera Ladislav , Gellert Nina , Borghi Battista , Weber Christian , Murphy Michael P. , Neuzil Jiri
Published: October 2002

Generation of free radicals is often associated with the induction and progression of apoptosis. Therefore, antioxidants can prove anti‐apoptotic, and can help to elucidate specific apoptotic pathways. Here we studied whether coenzyme Q, present in membranes in reduced (ubiquinol) or oxidised (ubiquinone) forms, can affect apoptosis induced by various stimuli. Exposure of Jurkat cells to α‐tocopheryl succinate (α‐TOS), hydrogen peroxide, anti‐Fas IgM or TRAIL led to induction of apoptosis. Cell death due to the chemical agents was suppressed in cells enriched with the reduced form of coenzyme Q. However, coenzyme Q did not block cell death induced by the immunological agents. Ubiquinol‐10 inhibited reactive oxygen species (ROS) generation in cells exposed to α‐TOS, and a mitochondrially targeted coenzyme Q analogue also blocked apoptosis triggered by α‐TOS or hydrogen peroxide. Therefore, it is plausible that ubiquinol‐10 protects cells from chemically‐induced apoptosis by acting as an antioxidant in mitochondria. Our results also indicate that generation of free radicals may not be a critical step in induction of apoptosis by immunological agents.
Calcitermin, a novel antimicrobial peptide isolated from human airway secretions
Authors: Cole Alexander M. , Kim Yong-Hwan , Tahk Samuel , Hong Teresa , Weis Peddrick , Waring Alan J. , Ganz Tomas
Published: October 2002

The human airways are protected from pathogenic colonization by a blanket of fluid impregnated with innate antimicrobial effector molecules. Among several previously uncharacterized components, we isolated a peptide that had activity primarily targeting Gram‐negative bacteria. We named the peptide ‘calcitermin’ since its amino acid sequence and mass were equivalent to the 15 C‐terminal residues of the S100 protein, calgranulin C. The antimicrobial activity of calcitermin was enhanced in acidic buffers (pH 5.4) and in the presence of micromolar concentrations of ZnCl2. Analysis revealed a putative zinc‐binding consensus sequence as well as an α‐helical conformation in structure‐promoting solvents.
Functional reconstitution of Arabidopsis thaliana plant uncoupling mitochondrial protein (AtPUMP1) expressed in Escherichia coli
Authors: Borecký Jirı́ , Maia Ivan G. , Costa Alexandre D.T. , Ježek Petr , Chaimovich Hernan , de Andrade Paula B.M. , Vercesi Anı́bal E. , Arruda Paulo
Published: October 2002

The Arabidopsis thaliana uncoupling protein (UCP) gene was expressed in Escherichia coli and isolated protein reconstituted into liposomes. Linoleic acid‐induced H+ fluxes were sensitive to purine nucleotide inhibition with an apparent K
i (in mM) of 0.8 (GDP), 0.85 (ATP), 0.98 (GTP), and 1.41 (ADP); the inhibition was pH‐dependent. Kinetics of AtPUMP1‐mediated H+ fluxes were determined for lauric, myristic, palmitic, oleic, linoleic, and linolenic acids. Properties of recombinant AtPUMP1 indicate that it represents a plant counterpart of animal UCP2 or UCP3. This work brings the functional and genetic approaches together for the first time, providing strong support that AtPUMP1 is truly an UCP.
The potato tuber transcriptome: analysis of 6077 expressed sequence tags
Authors: Crookshanks Meg , Emmersen Jeppe , Welinder Karen G. , Lehmann Nielsen Kåre
Published: October 2002

This is the first report of the biosynthetic potential of a tuber storage organ investigated by expressed sequence tag sequencing. A cDNA library was generated from the mature tuber of field grown potato (Solanum tuberosum var. Kuras). Partial sequences obtained from 6077 clones were assembled into 828 clusters and 1533 singletons. The average read length was 592 bp, and 2254 clones were full length. 5717 clones showed homology to genes from other organisms. Genes involved in protein synthesis, protein destination and cell defense predominated in tuber compared to stolon, shoot and leaf organs. 1063 clones were unique to tuber. Transcripts of starch metabolizing enzymes showed similar relative levels in tuber and stolon.
Specificity in pleckstrin homology (PH) domain membrane targeting: a role for a phosphoinositide–protein co‐operative mechanism
Authors: Maffucci Tania , Falasca Marco
Published: October 2002

Pleckstrin homology (PH) domains are protein modules found in proteins involved in many cellular processes. The majority of PH domain‐containing proteins require membrane association for their function. It has been shown that most PH domains interact directly with the cell membrane by binding to phosphoinositides with a broad range of specificity and affinity. While a highly specific binding of the PH domain to a phosphoinositide can be necessary and sufficient for the correct recruitment of the host protein to the membrane, a weaker and less specific interaction may be necessary but not sufficient, thus probably requiring alternative, co‐operative mechanisms.
Function of nucleophosmin/B23, a nucleolar acidic protein, as a histone chaperone
Authors: Okuwaki Mitsuru , Matsumoto Ken , Tsujimoto Masafumi , Nagata Kyosuke
Published: October 2002

We previously identified and purified a nucleolar phosphoprotein, nucleophosmin/B23, as a stimulatory factor for replication from the adenovirus chromatin. We show here that nucleophosmin/B23 functions as a histone chaperone protein such as nucleoplasmin, TAF‐I, and NAP‐I. Nucleophosmin/B23 was shown to bind to histones, preferentially to histone H3, to mediate formation of nucleosome, and to decondense sperm chromatin. These activities of B23 were dependent on its acidic regions as other histone chaperones, suggesting that B23/nucleophosmin is a member of histone chaperone proteins.
Zinc regulates the function and expression of the iron transporters DMT1 and IREG1 in human intestinal Caco‐2 cells
Authors: Yamaji Sachie , Tennant Jason , Tandy Sarah , Williams Mark , Singh Srai Surjit Kaila , Sharp Paul
Published: October 2002

Trace metals influence the absorption of each other from the diet and it has been suggested that the divalent metal transporter (DMT1) represents a common uptake pathway for these important micronutrients. However, compelling evidence from our laboratory suggests that DMT1 is predominantly an iron transporter, with lower affinity for other metals. Several studies have shown that increasing dietary iron downregulates DMT1. Interestingly, our current data indicate that zinc upregulates DMT1 protein and mRNA expression and also pH‐dependent iron uptake. Transepithelial flux of iron was also increased and was associated with a rise in IREG1 mRNA expression.
Multiple glutathione S‐transferase isoforms are present on male germ cell plasma membrane
Authors: Rao A.V.S.Kondala , Shaha Chandrima
Published: October 2002

Phase II detoxification enzymes, the glutathione S‐transferases (GSTs) of 24 kDa are known to be cytosolic enzymes. This study shows that multiple GST isoforms that are 24 kDa in size are present on the extracellular side of the plasma membrane of rat male germ cells. The GST activity of male germ cell plasma membranes is several folds higher than somatic cell plasma membrane GST activity. Isoform composition of the germ cell plasma membrane and the cytosolic pool differ, GSTM5 and GSTPi being absent on the plasma membranes. The molecular masses of the common isoforms are comparable between the two pools and both pools show GST and glutathione peroxidase activity.
Activation of the JAK/STAT pathway by ceramide in cultured human fibroblasts
Authors: Mazière Cécile , Conte Marie-Alix , Mazière Jean-Claude
Published: October 2002

Endogenous ceramide (CER) was generated by treatment of cultured fibroblasts with sphingomyelinase (SMase) from Bacillus cereus. A 30 min treatment with 0.1–0.3 U/ml SMase induced a dose‐dependent increase in the intracellular level of CER. The activation of the transcription factors signal transducer and activator of transcription (STAT) 1 and STAT3 by SMase was investigated by determination of the phosphorylation state by immunoblot, and of DNA binding activity by electrophoretic mobility shift assay. SMase treatment induced a dose‐dependent Tyr‐phosphorylation of STAT1/3. SMase also enhanced STAT1/3 DNA binding activity in a dose‐dependent manner. Concomitantly, SMase enhanced the Tyr‐phosphorylation of Janus kinase (JAK) 2, a Tyr‐kinase localized upstream of STATs in the JAK/STAT pathway. The Tyr‐kinase inhibitor genistein and the JAK inhibitor AG490 both prevented JAK2 Tyr‐phosphorylation, together with STAT1 and STAT3 Tyr‐phosphorylation and binding activity. The SMase‐induced increase in STAT1/3 binding activity was prevented by methyl‐β‐cyclodextrin, a cholesterol binding agent that causes a loss of compartmentalization of the molecules located in caveolae. This increase was also prevented by the MEK inhibitor PD98059, thus demonstrating the role of the MEK/ERK pathway in this system. Besides ERK, SMase activated other signaling kinases such as JNK and p38. Exogenous natural CER also activated STAT1/3 binding activity, which indicates that most probably, endogenous CER is the second messenger involved in the effect of SMase. These results describe a crosstalk between the SMase/CER and the JAK/STAT signaling pathways and include JAK2 within the range of CER‐activated intracellular kinases.
Equilibrium between monomeric and dimeric mitochondrial F1–inhibitor protein complexes
Authors: Domı́nguez-Ramı́rez L. , Mendoza-Hernandez G. , Carabez-Trejo A. , Gómez-Puyou A. , Tuena de Gómez-Puyou M.
Published: October 2002

Mg‐ATP particles from bovine heart mitochondria have more than 95% of their F1 in complex with the inhibitor protein (IF1). The F1–IF1 complex was solubilized and purified. The question addressed was if this naturally occurring complex existed as monomers or dimers. Size exclusion chromatography and electron microscopy showed that most of the purified F1–IF1 complex was a dimer of two F1–IF1. As determined by the former method, the relative concentrations of dimeric and monomeric F1–IF1 depended on the concentration of protein that was applied to the column. Apparently, there is an equilibrium between the two forms of F1–IF1.
Identification and role of ionizing functional groups at the active center of Rhodotorula gracilisD‐amino acid oxidase
Authors: Pollegioni Loredano , Harris Christopher M. , Molla Gianluca , Pilone Mirella S. , Ghisla Sandro
Published: October 2002

D‐Amino acid oxidase (DAAO) is a flavoprotein oxidase that catalyzes the oxidation of amino acids and produces ketoacids and H2O2. The rate of product release from reduced DAAO from Rhodotorula gracilis is pH dependent and reflects a pK
a of ∼9.3. Binding of benzoate and 3,3,3‐trifluoro‐D‐alanine to wild‐type and Y238F–DAAO is also pH dependent (pK
a=9.8±0.1 and 9.05±0.1, respectively for benzoate binding). However, binding of benzoate to Y223F–DAAO is pH independent, indicating the pK
a is due to Y223–OH. This latter residue is thus involved in substrate binding, and probably is the group that governs product release. In contrast to this, the second active site tyrosine, Y238, has little influence on ligand binding.
Functional analysis of the propeptides of subtilisin E and aqualysin I as intramolecular chaperones
Authors: Takagi Hiroshi , Koga Mihoko , Katsurada Saori , Yabuta Yukihiro , Shinde Ujwal , Inouye Masayori , Nakamori Shigeru
Published: October 2002

Several proteases require propeptides for the correct folding of their own protease domain. We have recently found that the propeptide from a thermostable subtilisin homolog aqualysin I can refold subtilisin BPN′ when added in trans. Here, we constructed chimeric genes with subtilisin E and aqualysin I to attempt the in cis folding of subtilisin E by means of the propeptide of aqualysin I. Our results indicate that the propeptide of aqualysin I can to some extent chaperone the intramolecular folding of the denatured subtilisin E. These results suggest that propeptides in the subtilisin family, despite their sequence diversity, have similar functions. Further, some enzymatic properties of some chimeras in which the subtilisin mature domain is partly swapped with that of aqualysin I were shown to be more similar to those of aqualysin I.
CD30L up‐regulates CD30 and IL‐4 expression by T cells
Authors: Rossi Francesca Maria , Degan Massimo , Mazzocut-Zecchin Linda , Di Francia Raffaele , Aldinucci Donatella , Pinto Antonio , Gattei Valter
Published: October 2002

CD30L is frequently expressed on acute myeloid leukemia (AML) blasts. Its presence is associated with the co‐expression of interleukin‐4 (IL‐4) receptor and with the expansion of specific T‐helper 2 (Th2) cell subsets producing IL‐4 and expressing CD30. Recombinant CD30L‐bearing cells up‐regulated the expression of surface CD30 and increased the production of IL‐4 and soluble (s) CD30 by co‐cultured T cells. These findings were confirmed with AML blasts expressing surface CD30L, where blocking anti‐CD30 antibodies completely abolished the release of sCD30 and reduced the production of IL‐4. Our data indicates a direct role of CD30L+ neoplastic cells in driving the immune response toward a Th2‐polarized non‐protective state.
Conformational and functional significance of residue proline 17 in chicken muscle adenylate kinase
Authors: Sheng Xiangrong , Pan Xianming , Wang Chu , Zhang Yu , Jing Guozhong
Published: October 2002

The effect of mutation proline 17 on the multiple conformations and catalytic function in chicken muscle adenylate kinase (AK) has been studied. The substitution of proline 17 with glycine or valine altered the distribution of multiple conformations. Compared with the wild‐type enzyme, the P17G and P17V mutants contained decreased fraction of minor conformer from 18% to 9% and 11%, respectively. Due to the mutation, the enzyme showed lower secondary structural content, poorer affinity to substrates or substrate analogues, and reduced catalytic efficiency. The results revealed the significance of proline 17 in the conformation and function of AK.
Crystal structure of a cyclic form of bovine pancreatic trypsin inhibitor
Authors: Botos Istvan , Wu Zhibin , Lu Wuyuan , Wlodawer Alexander
Published: October 2002

The crystal structure of a cyclic form of a mutant of bovine pancreatic trypsin inhibitor has been solved at 1.0 Å resolution. The protein was synthesized by native chemical ligation and its structure is almost indistinguishable from the previously described recombinant form of the same mutant; however, the new loop containing the former termini became much better ordered.
A role for N‐WASP in invasin‐promoted internalisation
Authors: McGee Karen , Zettl Markus , Way Michael , Fällman Maria
Published: October 2002

Phagocytosis of Yersinia pseudotuberculosis occurs through interaction of the bacterial protein invasin with β1‐integrins. Here we report that N‐WASP plays a role in internalisation of an invasin‐expressing, avirulent strain of Y. pseudotuberculosis. Ectopic expression of N‐WASP mutants, which affect recruitment of the Arp2/3 complex to the phagosome, reduces uptake of Yersinia. In addition, expression of the Cdc42/Rac‐binding (CRIB) region of N‐WASP has an inhibitory effect on uptake. Using GFP‐tagged Rho GTPase mutants, we provide evidence that Rac1, but not Cdc42, is important for internalisation. Furthermore, activated Rac1 rescues Toxin B, CRIB and Src family kinase inhibitor PP2‐mediated impairment of uptake. Our observations indicate that invasin‐mediated phagocytosis occurs via a Src and WASP family‐dependent mechanism(s), involving the Arp2/3 complex and Rac, but does not require Cdc42.
Overproduced p73α activates a minimal promoter through a mechanism independent of its transcriptional activity
Authors: Takagi Shinji , Ueda Yoshihide , Hijikata Makoto , Shimotohno Kunitada
Published: October 2002

p73, the gene for a protein related to the tumor suppressor p53, encodes several variants which bear distinct carboxy‐terminal structures as a result of alternative splicing. We and others showed that these splicing variants have different transcriptional effects on promoters with a p53‐binding consensus sequence (p53BCS). Here we show that when transiently overexpressed, p73α but not p73β activated several minimal promoters without the p53BCS, while p73γ and p73ϵ activated them to a much lesser extent than p73α, and p53 suppressed the promoters without p53BCS as reported previously. Moreover, the results of RNase protection and RNA transfection assays suggested that this activation occurred at the transcriptional level. Deletion analysis of p73α revealed that the transactivation domain of p73 was not involved in this activity and the C‐terminal region of p73α which is a specific structure of this variant was essential, suggesting that this phenomenon occurs independent of the transactivation activity of p73α and that the C‐terminal extension of p73α may affect the basal level of transcription.
Characterisation of a concentrative type of adenosine transporter from Arabidopsis thaliana (ENT1,At)
Authors: Möhlmann Torsten , Mezher Zeina , Schwerdtfeger Gabriele , Neuhaus H.Ekkehard
Published: October 2002

Here we report on the isolation of an Arabidopsis thaliana cDNA that is able to complement a Saccharomyces cerevisiae mutant unable to synthesise adenine. This cDNA encodes a highly hydrophobic protein (ENT1,At) of 428 amino acids, showing high similarity to the human nucleoside transporter hENT1. Yeast cells expressing ENT1,At are able to grow on adenosine‐containing media, adenosine import exhibited an apparent affinity (K
M) of 3.6 μM, and led to accumulation of this nucleoside within the yeast cell. Transport is inhibited by various nucleosides. Typical inhibitors of ENT‐type nucleoside transporters do not inhibit 3H‐adenosine import. The presence of protonophores abolished adenosine import, indicating that ENT1,At catalyse a proton‐dependent adenosine transport. This is the first functional characterisation of a plant nucleoside transport protein.
Use of molecular mechanics for secondary structure prediction. Is it possible to reveal α‐helix?
Authors: Kilosanidze Gelena T , Kutsenko Alexey S , Esipova Natalia G , Tumanyan Vladimir G
Published: October 2002

A new approach to predicting protein standard conformations is suggested. The idea consists in modeling by molecular mechanics tools a continuous α‐helical conformation for the whole protein. The profile of energy along the model α‐helix reveals minima corresponding to real α‐helical segments in the native protein. The 3/10‐helices and β‐turns including a local α‐helical conformation may be detected as well. All α‐helical segments in the test sample are delineated; mean residue by residue accuracy Q
3α is 79%. This non‐statistical approach can shed light on the physical grounds of α‐helix formation.
Replacement of the methionine axial ligand in cytochrome c550 by a lysine: effects on the haem electronic structure
Authors: Louro Ricardo O , de Waal Ellen C , Ubbink Marcellus , Turner David L
Published: October 2002

The prosthetic group of low‐spin haem proteins is an iron porphyrin with two axial ligands, typically histidine, methionine or lysine. Determining the geometry of the axial ligands is an important step in structural characterisation, particularly in the paramagnetic oxidised forms. This work extends earlier studies of the hyperfine nuclear magnetic resonance (NMR) shifts of haem substituents in bis‐His and His–Met cytochromes to His–Lys co‐ordination in the M100K mutant of Paracoccus versutus cytochrome c
550. The electronic structure of the His–Lys haem is shown to be similar to that produced by His–cyanide co‐ordination, such that NMR can be used to determine the geometry of the His ligand.
NF‐κB and ERK cooperate to stimulate DNA synthesis by inducing ornithine decarboxylase and nitric oxide synthase in cardiomyocytes treated with TNF and LPS
Authors: Tantini Benedetta , Pignatti Carla , Fattori Monia , Flamigni Flavio , Stefanelli Claudio , Giordano Emanuele , Menegazzi Marta , Clô Carlo , Caldarera Claudio Marcello
Published: October 2002

We previously reported that tumor necrosis factor‐α (TNF) and lipopolysaccharide (LPS) stimulate DNA synthesis in chick embryo cardiomyocytes (CM) via nitric oxide and polyamine biosynthesis. Here we show an involvement of nuclear factor‐κB (NF‐κB) in the induction of nitric oxide synthase (NOS) and ornithine decarboxylase (ODC), the key enzyme in polyamine biosynthesis. In addition NF‐κB activation appears to favor survival of CM by reducing caspase activation. TNF and LPS also stimulate phosphorylation of extracellular signal‐regulated kinase (ERK), which is required for the changes in ODC and caspase activity, but not for NOS induction or NF‐κB activation. In conclusion, these results indicate that NF‐κB, in cooperation with ERK, plays a pivotal role in the growth stimulating effects of TNF and LPS, leading to the induction of both ODC and NOS and to the reduction of caspase activity.
Different effects of calnexin deletion in Saccharomyces cerevisiae on the secretion of two glycosylated amyloidogenic lysozymes
Authors: Song Youtao , Azakami Hiroyuki , Shamima Begum , He Jianwei , Kato Akio
Published: October 2002

Both glycosylated amyloidogenic lysozymes I55T/G49N and D66H/G49N were expressed in wild‐type and calnexin‐disrupted Saccharomyces cerevisiae. The secretion amounts of mutant I55T/G49N were almost similar in both wild‐type and calnexin‐disrupted S. cerevisiae. In contrast, the secretion of mutant D66H/G49N greatly increased in calnexin‐disrupted S. cerevisiae, while the secretion was very low in the wild‐type strain. In parallel, the induction level of the molecular chaperones BiP and PDI located in the endoplasmic reticulum (ER) was investigated when these glycosylated amyloidogenic lysozymes were expressed in wild‐type and calnexin‐disrupted S. cerevisiae. The mRNA concentrations of BiP and PDI were evidently increased when mutant lysozyme D66H/G49N was expressed in calnexin‐disrupted S. cerevisiae, while they were not so increased when I55T/G49N mutant was expressed. This observation indicates that the conformation of mutant lysozyme D66H/G49N was less stable in the ER, thus leading to the higher‐level expression of ER molecular chaperones via the unfolded protein response pathway. This suggests that glycosylated amyloidogenic lysozyme I55T/G49N may have a relatively stable conformation in the ER, thus releasing it from the quality control of calnexin compared with mutant lysozyme D66H/G49N.
Participation of peroxynitrite in oxidative modification of LDL by aqueous extracts of cigarette smoke
Authors: Yamaguchi Yu , Kagota Satomi , Haginaka Jun , Kunitomo Masaru
Published: October 2002

Aqueous extracts of cigarette smoke (CSE) can oxidatively modify plasma low‐density lipoprotein (LDL). The aim of the present study was to elucidate the participation of peroxynitrite in LDL oxidation. When LDL was incubated with CSE, its oxidative modification was dependent on time and concentration. It could be effectively prevented by vitamin E, partially by superoxide dismutase, but hardly by catalase, mannitol and metal chelators. CSE also increased the 3‐nitrotyrosine content in LDL. A similar increase of 3‐nitrotyrosine occurred after incubation of LDL with a peroxynitrite generating agent, 3‐morpholinosydnonimine, thus suggesting that prominent pro‐oxidants in CSE are peroxynitrite‐generating species.
Starvation‐induced degradation of yeast hexose transporter Hxt7p is dependent on endocytosis, autophagy and the terminal sequences of the permease
Authors: Krampe Stefanie , Boles Eckhard
Published: October 2002

The yeast high‐affinity glucose transporters Hxt6p and Hxt7p are rapidly degraded during nitrogen starvation in the presence of high concentrations of fermentable carbon sources. Our results suggest that degradation is mainly due to the stimulation of general protein turnover and not caused by a mechanism specifically triggered by glucose. Analysis of Hxt6p/7p stability and cellular distribution in end4, aut2 and apg1 mutants indicates that Hxt7p is internalized by endocytosis, and autophagy is involved in the final delivery of Hxt7p to the vacuole for proteolytic degradation. Internalization and degradation of Hxt7p were blocked after truncation of its N‐terminal hydrophilic domain. Nevertheless, this fully functional and stabilized hexose transporter could not maintain fermentation capacity of the yeast cells under starvation conditions, indicating a regulatory constraint on glucose uptake.
The advantages of cDNA microarray as an effective tool for identification of reproductive organ‐specific genes in a model legume, Lotus japonicus
Authors: Endo Makoto , Matsubara Hitoshi , Kokubun Takao , Masuko Hiromi , Takahata Yoshihito , Tsuchiya Tohru , Fukuda Hiroo , Demura Taku , Watanabe Masao
Published: October 2002

To understand the molecular mechanisms intrinsic to reproductive organ development a cDNA microarray, fabricated from flower bud cDNA clones, was used to isolate genes, which are specifically expressed during the development of the anther and pistil in Lotus japonicus. Cluster analysis of the microarray data revealed 21 and 111 independent cDNA groups, which were specifically expressed in immature and mature anthers, respectively. RT‐PCR was performed to provide a direct assessment of the accuracy and reproducibility of our approach. Confirmation of our results suggests that cDNA microarray technology is an effective tool for identification of novel reproductive organ‐specific genes.
Artificial in vivo antigen presentation by the APCs and subsequent T‐cell activation: a feasibility analysis
Authors: Jain Siddhartha
Published: October 2002

Inappropriate antigen presentation by the antigen‐presenting cells (APCs) is a cause of various diseases. One of the ways to combat these diseases is to immobilize the APCs near the infected tissue or a tissue which is susceptible to an antigen. The antigen is presented by the APCs present in the immobilized form on an implant and these upon binding to TH‐cells result in triggering of a cascade of events as part of the natural immune response leading to the destruction of the antigen. This system has been modeled as a dialysis bag containing immobilized receptors inside the bag and the ligand diffusing out of the bag. The simulations show that by using the implant, the concentration of the ligand that has diffused into the tissue matrix can be substantially reduced and by suitably choosing the coupler size, the TH‐cells can also effectively be activated.
Leukotactin‐1/CCL15‐induced chemotaxis signaling through CCR1 in HOS cells
Authors: Ko Jesang , Kim In Sik , Jang Sung-Wuk , Lee Young Han , Shin Soon Young , Min Do Sik , Na Doe Sun
Published: October 2002

Leukotactin‐1 (Lkn‐1)/CCL15 is a recently cloned CC‐chemokine that binds to the CCR1 and CCR3. Although Lkn‐1 has been known to function as a chemoattractant for neutrophils, monocytes and lymphocytes, its cellular mechanism remains unclear. To understand the mechanism of Lkn‐1‐induced chemotaxis signaling, we examined the chemotactic activities of human osteogenic sarcoma cells expressing CCR1 in response to Lkn‐1 using inhibitors of signaling molecules. Inhibitors of Gi/Go protein, phospholipase C (PLC) and protein kinase Cδ (PKCδ) inhibited the chemotactic activity of Lkn‐1 indicating that Lkn‐1‐induced chemotaxis signal is transduced through Gi/Go protein, PLC and PKCδ. The activities of PLC and PKCδ were also enhanced by Lkn‐1 stimulation. Chemotactic activity of Lkn‐1 was inhibited by the treatment of cycloheximide and actinomycin D suggesting that newly synthesized proteins are needed for chemotaxis. Nuclear factor‐κB (NF‐κB) inhibitor reduced chemotactic activity of Lkn‐1. DNA binding activity of NF‐κB was also enhanced by Lkn‐1 stimulation. These results suggest that Lkn‐1 transduces the signal through Gi/Go protein, PLC, PKCδ, NF‐κB and newly synthesized proteins for chemotaxis.
NMR studies of the aggregation of glucagon‐like peptide‐1: formation of a symmetric helical dimer
Authors: Chang Xiaoqing , Keller Danielle , O'Donoghue Seán I. , Led Jens J.
Published: October 2002

Nuclear magnetic resonance (NMR) spectroscopy reveals that higher‐order aggregates of glucagon‐like peptide‐1‐(7–36)‐amide (GLP‐1) in pure water at pH 2.5 are disrupted by 35% 2,2,2‐trifluoroethanol (TFE), and form a stable and highly symmetric helical self‐aggregate. NMR spectra show that the helical structure is identical to that formed by monomeric GLP‐1 under the same experimental conditions [Chang et al., Magn. Reson. Chem. 37 (2001) 477–483; Protein Data Bank at RCSB code: 1D0R], while amide proton exchange rates reveal a dramatic increase of the stability of the helices of the self‐aggregate. Pulsed‐field gradient NMR diffusion experiments show that the TFE‐induced helical self‐aggregate is a dimer. The experimental data and model calculations indicate that the dimer is a parallel coiled coil, with a few hydrophobic residues on the surface that may cause aggregation in pure water. The results suggest that the coiled coil dimer is an intermediate state towards the formation of higher aggregates, e.g. fibrils.
Cav2.3 (α1E) Ca2+ channel participates in the control of sperm function
Authors: Sakata Yu , Saegusa Hironao , Zong Shuqin , Osanai Makoto , Murakoshi Takayuki , Shimizu Yasufumi , Noda Tetsuo , Aso Takeshi , Tanabe Tsutomu
Published: October 2002

To know the function of the Ca2+ channel containing α12.3 (α1E) subunit (Cav2.3 channel) in spermatozoa, we analyzed Ca2+ transients and sperm motility using a mouse strain lacking Cav2.3 channel. The averaged rising rates of Ca2+ transients induced by α‐D‐mannose–bovine serum albumin in the head region of Cav2.3−/− sperm were significantly lower than those of Cav2.3+/+ sperm. A computer‐assisted sperm motility assay revealed that straight‐line velocity and linearity were greater in Cav2.3−/− sperm than those in Cav2.3+/+ sperm. These results suggest that the Cav2.3 channel plays some roles in Ca2+ transients and the control of flagellar movement.
Abnormal glycosylation and altered Golgi structure in colorectal cancer: dependence on intra‐Golgi pH
Authors: Kellokumpu Sakari , Sormunen Raija , Kellokumpu Ilmo
Published: October 2002

Abnormal glycosylation of cellular glycoconjugates is a common phenotypic change in many human tumors. Here, we explore the possibility that an altered Golgi pH may also be responsible for these cancer‐associated glycosylation abnormalities. We show that a mere dissipation of the acidic Golgi pH results both in increased expression of some cancer‐associated carbohydrate antigens and in structural disorganization of the Golgi apparatus in otherwise normally glycosylating cells. pH dependence of these alterations was confirmed by showing that an acidification‐defective breast cancer cell line (MCF‐7) also displayed a fragmented Golgi apparatus, whereas the Golgi apparatus was structurally normal in its acidification‐competent subline (MCF‐7/AdrR). Acidification competence was also found to rescue normal glycosylation potential in MCF‐7/AdrR cells. Finally, we show that abnormal glycosylation is also accompanied by similar structural disorganization and fragmentation of the Golgi apparatus in colorectal cancer cells in vitro and in vivo. These results suggest that an inappropriate Golgi pH may indeed be responsible for the abnormal Golgi structure and lowered glycosylation potential of the Golgi apparatus in malignant cells.
Evidence that the putative α‐glucosidase of Thermotoga maritima MSB8 is a pNP α‐D‐glucuronopyranoside hydrolyzing α‐glucuronidase
Authors: Suresh Cuddapah , Rus'd Ahmed Abu , Kitaoka Motomitsu , Hayashi Kiyoshi
Published: October 2002

The gene (agu) encoding p‐nitrophenyl α‐D‐glucuronopyranoside (pNP‐GUA) hydrolyzing α‐glucuronidase of the hyperthermophilic bacterium Thermotoga maritima was cloned and expressed in Escherichia coli. The recombinant enzyme was purified and characterized. The gene previously designated as putative α‐glucosidase was found to code for a protein that had no α‐glucosidase activity. It showed a rare activity profile with its ability to hydrolyze pNP‐GUA, an activity not known in the α‐glucuronidases from microbial sources. This is the first report on the occurrence of an α‐glucuronidase which belongs to the family 4 of glycosyl hydrolases.
Sphingomyelin content conditions insertion of daunorubicin within phosphatidylcholine monolayers
Authors: Lecompte Marie-France , Laurent Guy , Jaffrézou Jean-Pierre
Published: October 2002

Cell death induced by the chemotherapeutic drug daunorubicin (DNR) implicates an apoptotic pathway originating at the plasma membrane and characterized by sphingomyelin (SM) hydrolysis and ceramide generation. The mechanisms by which such a drug (hypothetically passively diffusing across a structural membrane) can trigger SM hydrolysis is unknown, but raises the question of the precise interaction between DNR and membrane lipid constituents. In this initial study, using alternative current polarography together with voltammetry, we report that after a first step of adsorption, insertion of DNR within a condensed phosphatidylcholine monolayer was significantly facilitated by SM content.
The hppA gene of Helicobacter pylori encodes the class C acid phosphatase precursor
Authors: Godlewska Renata , Bujnicki Janusz M. , Ostrowski Jerzy , Jagusztyn-Krynicka Elżbieta K.
Published: October 2002

Screening of the Helicobacter pylori genomic library with sera from infected humans and from immunized rabbits resulted in identification of the 25 kDa protein cell envelope (HppA) which exhibits acid phosphatase activity. Enzyme activity was demonstrated by specific enzymatic assays with whole‐cell protein preparations of H. pylori strain N6 and from Escherichia coli carrying the hppA gene (pUWM192). HppA showed optimum activity at pH 5.6 and was resistant to inhibition by EDTA. Bioinformatics analysis and site‐directed mutagenesis of two putative active site residues (D73 and D192) provide further insight into the sequence–structure–function relationships of HppA as a member of the DDDD phosphohydrolase superfamily.