bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular Mechanisms of the Inhibitory Effects of Bupivacaine, Levobupivacaine, and Ropivacaine on Sarcolemmal Adenosine Triphosphate–sensitive Potassium Channels in the Cardiovascular System
Authors: Kawano Takashi , Oshita Shuzo , Takahashi Akira , Tsutsumi Yasuo , Tomiyama Yoshinobu , Kitahata Hiroshi , Kuroda Yasuhiro , Nakaya Yutaka
Published: July 2004

Background

Sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in the cardiovascular system may be involved in bupivacaine-induced cardiovascular toxicity. The authors investigated the effects of local anesthetics on the activity of reconstituted KATP channels encoded by inwardly rectifying potassium channel (Kir6.0) and sulfonylurea receptor (SUR) subunits.

Methods

The authors used an inside-out patch clamp configuration to investigate the effects of bupivacaine, levobupivacaine, and ropivacaine on the activity of reconstituted KATP channels expressed in COS-7 cells and containing wild-type, mutant, or chimeric SURs.

Results

Bupivacaine inhibited the activities of cardiac KATP channels (IC50 = 52 microm) stereoselectively (levobupivacaine, IC50 = 168 microm; ropivacaine, IC50 = 249 microm). Local anesthetics also inhibited the activities of channels formed by the truncated isoform of Kir6.2 (Kir6.2 delta C36) stereoselectively. Mutations in the cytosolic end of the second transmembrane domain of Kir6.2 markedly decreased both the local anesthetics' affinity and stereoselectivity. The local anesthetics blocked cardiac KATP channels with approximately eightfold higher potency than vascular KATP channels; the potency depended on the SUR subtype. The 42 amino acid residues at the C-terminal tail of SUR2A, but not SUR1 or SUR2B, enhanced the inhibitory effect of bupivacaine on the Kir6.0 subunit.

Conclusions

Inhibitory effects of local anesthetics on KATP channels in the cardiovascular system are (1) stereoselective: bupivacaine was more potent than levobupivacaine and ropivacaine; and (2) tissue specific: local anesthetics blocked cardiac KATP channels more potently than vascular KATP channels, via the intracellular pore mouth of the Kir6.0 subunit and the 42 amino acids at the C-terminal tail of the SUR2A subunit, respectively.
Hemorrhagic Shock in Swine: Nitric Oxide and Potassium Sensitive Adenosine Triphosphate Channel Activation
Authors: Musser Jeffrey B. , Bentley Timothy B. , Griffith Scott , Sharma Pushpa , Karaian John E. , Mongan Paul D.
Published: July 2004

Background

To determine the role of nitric oxide and adenosine triphosphate-sensitive potassium (KATP) vascular channels in vascular decompensation during controlled hemorrhagic shock in swine.

Methods

Thirty instrumented, anesthetized adolescent Yorkshire swine were subjected to controlled isobaric hemorrhage to a mean arterial pressure of 40 mmHg for 2 h (n = 6) or 4 h (n = 10) or 50 mmHg for 4 h (n = 8). An additional six animals were used as anesthetized instrumented time controls. During controlled hemorrhage, plasma and tissue samples were obtained every 30 to 60 min. Before euthanasia, tissue (carotid artery, lung, liver, and aorta) was obtained for analysis of nitrate concentrations and nitric oxide synthase activity. Isolated carotid artery ring reactivity to norepinephrine was also determined with and without glibenclamide.

Results

Animals hemorrhaged to 40 mmHg decompensated earlier than animals hemorrhaged to 50 mmHg. Plasma nitrate concentrations and nitric oxide synthase activity rose consistently throughout hemorrhage in both groups. However, they were substantially higher in the mean arterial pressure 40 group. Constitutive nitric oxide synthase activity was the major contributor to total nitric oxide synthase activity throughout the protocol with only the animals maintained at 40 mmHg for 4 h showing evidence of inducible nitric oxide synthase activity. Profound KATP channel activation and hyporeactivity of isolated vessel rings to norepinephrine was not observed until 4 h after the initiation of hemorrhagic shock. Only those animals with inducible nitric oxide synthase activity showed a decreased response to norepinephrine, and this hyporeactivity was reversed with the KATP channel inhibitor, glibenclamide.

Conclusions

The data indicate that profound KATP activation associated with increased nitric oxide concentrations and inducible nitric oxide synthase induction is a key factor in vascular smooth muscle hyporeactivity characteristic of the late decompensatory phase of hemorrhagic shock in swine.
Temperature-independent Inhibition of L-Type Calcium Currents by Halothane and Sevoflurane in Human Atrial Cardiomyocytes
Authors: Hüneke Rocco , Zitzelsberger Daniela , Fassl Jens , Jüngling Eberhard , Brose Stefan , Buhre Wolfgang , Rossaint Rolf , Lückhoff Andreas
Published: July 2004

Background

Cardiac L-type calcium currents (ICa,L) are affected by volatile anesthetics, possibly contributing to their side effects. Actions of anesthetics on ion channels are usually studied in vitro at room temperature. However, the solubility of anesthetic gases as well as ICa,L are markedly sensitive to the study temperature. Therefore, temperature-dependent effects of halothane and sevoflurane on cardiac ICa,L were analyzed.

Methods

ICa,L were studied at 21 degrees C and 36 degrees C with the patch clamp technique in isolated human atrial cardiomyocytes. Concentrations of anesthetics brought into solution by gassing at both temperatures were determined with gas chromatography.

Results

The aqueous concentrations of halothane and sevoflurane were linearly related to their concentration in the gas phase (1 to 3 minimum alveolar concentration [MAC]). At 21 degrees C, the slope of this relation was 0.52 and 0.12 mm/vol % for halothane and sevoflurane, respectively, and decreased at 36 degrees C to 0.29 and 0.09 mm/vol %, respectively. ICa,L displayed significantly higher current amplitudes at 36 degrees C than at 21 degrees C and significantly accelerated time-dependent inactivation. Halothane (1-2 MAC) and sevoflurane (1-3 MAC) evoked stronger inhibitions of ICa,L at 21 degrees C than at 36 degrees C. In spite of different temperature-dependent current amplitudes, the fractional (percent) inhibition of ICa,L showed the same linear relationship to the concentrations of halothane and sevoflurane in the bath medium at both temperatures, as revealed from present and previous experiments.

Conclusions

Inhibition of ICa,L by halothane and sevoflurane is determined by the aqueous concentration of the anesthetics, independently of the temperature. Increased solubility may explain the stronger effects of the anesthetics at lower temperatures.
Retigabine Stimulates Human KCNQ2/Q3 Channels in the Presence of Bupivacaine
Authors: Punke Mark A. , Friederich Patrick
Published: July 2004

Background

Inhibition of KCNQ2/Q3 channels may cause convulsion in humans. The interaction of bupivacaine with these channels is unknown. The anticonvulsant retigabine activates KCNQ2/Q3 channels and may reverse inhibitory actions of bupivacaine. Potassium channel stimulation may thus constitute a novel approach to treat local anesthetic-induced seizures. The aim of this study was to characterize bupivacaine effects on KCNQ2/Q3 channels and to investigate whether retigabine reverses the effects of the local anesthetic.

Methods

KCNQ2/Q3 channels were transiently expressed in Chinese hamster ovary cells. The effects of bupivacaine and retigabine were studied with the patch-clamp technique.

Results

Bupivacaine inhibited KCNQ2/Q3 channels in a concentration-dependent and reversible manner. The concentration-response curve was described by a Hill equation (IC50 = 173 +/- 7 microm, Hill coefficient = 1.4 +/- 0.1, mean +/- SEM, n = 37). The inhibitory effect did not differ between bupivacaine and levobupivacaine (42 +/- 4%, n = 7, versus 42 +/- 5%, n = 10; P > 0.05). Ropivacaine was four times less potent than bupivacaine. The inhibition of KCNQ2/Q3 channels by bupivacaine resulted in a significant and reversible depolarization of the membrane potential. Retigabine (300 nm-10 microm) reversed the inhibitory action of bupivacaine on KCNQ2/Q3 channels as well as the depolarization of the membrane potential.

Conclusions

The anticonvulsant retigabine at nanomolar concentrations reverses the inhibitory effect of micromolar concentrations of bupivacaine. Our results allow the hypothesis that activation of KCNQ2/Q3 channels by retigabine may offer a novel therapeutic approach for the treatment of bupivacaine-induced seizures.
Lumbar Plexus in Children
Authors: Kirchmair Lukas , Enna Birgit , Mitterschiffthaler Gottfried , Moriggl Bernhard , Greher Manfred , Marhofer Peter , Kapral Stephan , Gassner Ingmar
Published: July 2004

Background

Pediatric regional anesthesia has gained increasing interest over the past decades. The current study was conducted to investigate the lumbar paravertebral region and the lumbar plexus at L3-L4 and L4-L5 by means of sonography to obtain fundamentals for the performance of ultrasound-guided posterior lumbar plexus blocks.

Methods

Thirty-two children (12 boys, 20 girls) with American Society of Anesthesiologists physical status I or II were enrolled in the current study. The lumbar paravertebral region was visualized at L3-L4 and L4-L5 on two corresponding posterior sonograms (longitudinal, transverse). The lumbar plexus had to be delineated, and skin-plexus distances were measured. In a series of five pediatric patients undergoing inguinal herniotomy, ultrasound-guided posterior lumbar plexus blocks at L4-L5 were performed.

Results

The children were stratified into three age groups (group 1: > 3 yr and </= 5 yr; group 2: > 5 yr and </= 8 yr; group 3: > 8 yr and </= 12 yr). The lumbar plexus could be delineated at L3-L4 and L4-L5 in 19 of 20 cases in group 1, in 17 of 20 cases in group 2, in 22 of 24 cases at L3-L4 in group 3, and in 16 of 24 cases at L4-L5 in group 3. In all patients, the lumbar plexus was situated within the posterior part of the psoas major muscle. Skin-plexus distances showed statistical significant differences between groups 1 and 3 and between groups 2 and 3. The strongest positive correlation existed between skin-plexus distances and the children's weight. Ultrasound guidance enabled safe und successful posterior approaches to the lumbar plexus, thus resulting in effective anesthesia and analgesia of the inguinal region.

Conclusions

Sonography of the lumbar plexus in children proved to be feasible. Skin-plexus distances correlated with the children's weight rather than with their age. The sonographic findings were fundamental for the performance of successful ultrasound-guided posterior approaches in a small group of pediatric patients.
Changes in Tissue pH and Temperature after Incision Indicate Acidosis May Contribute to Postoperative Pain
Authors: Woo Young Cheol , Park Soo Seog , Subieta Alberto R. , Brennan Timothy J.
Published: July 2004

Background

Incisional pain is a common form of acute pain. Previously, the authors studied persistent pain behaviors caused by incisions, using animal models for postoperative pain. In this study, the authors measured tissue pH and hind paw temperature before and after incision to understand factors that may activate and sensitize nociceptors in the incision.

Methods

Rats underwent a plantar incision, a gastrocnemius muscle incision, or a cutaneous paraspinal incision. For the hind paw incision, pain behaviors were measured. Tissue pH was measured using a pH-sensitive needle electrode in halothane-anesthetized rats. The pH in the incision was compared to a corresponding control site on the contralateral side of the rat or to the sham-operated group.

Results

Plantar tissue pH was 7.16 +/- 0.04 in sham-operated rats. Ten minutes after plantar incision, tissue pH was decreased to 6.91 +/- 0.20 (P < 0.05), and this decrease was sustained through 60 min after incision, when pH was 6.99 +/- 0.06 (P < 0.05). Tissue pH values were 6.95, 6.90, 6.89, and 6.95 (P < 0.05 vs. sham) 4 h and 1, 2, and 4 days after incision, respectively. On postoperative day 7, when plantar pH was same as for the control side (7.13 +/- 0.05), guarding behavior, heat responses, and responses to mechanical stimuli recovered. Outside the incised area in the hind paw, tissue pH was normal. Tissue pH was significantly correlated with all pain behaviors. In the gastrocnemius muscle, tissue pH was 7.14 +/- 0.7 in the sham-operated side. Ten minutes after incision, tissue pH was 6.54 +/- 0.12 (P < 0.05), and muscle pH remained decreased through 60 min after gastrocnemius incision when pH was 6.76 +/- 0.17 (P < 0.05). Tissue pH was also significantly decreased (P < 0.05) on day 1 (6.96 vs. 7.20) and day 4 (7.06 vs. 7.18) after gastrocnemius incision but was not reduced on postoperative day 8 (7.11 vs. 7.15). A paraspinal incision also decreased tissue pH in the hairy skin of the rat compared with the preincision value. Hind paw skin temperature did not change after incision.

Conclusion

A decrease in pH occurs immediately after incision and is sustained for at least 4 days. During the period of decreased tissue pH, pain behaviors are evident. When the tissue pH returns to normal, pain behaviors are diminished. The decreased pH is localized at the incision site and not to areas surrounding the incision. Decreased pH likely contributes to nociceptor sensitization and pain related behaviors after incision. The magnitude of the pH change varies among tissues. An increase in hind paw skin temperature does not play a role in these pain-related behaviors.
Technology integration
Authors: Larpsiri Ravipa , Speece Mark
Published: July 2004

Sales force automation (SFA) is increasingly used to support customer relationships, and provides a good example of technology application on the supplier‐customer interface. While research has begun to examine SFA, understanding it from the customer viewpoint is still vague. This paper reports results of exploratory qualitative research to investigate perceptions of SFA among both salespeople and their customers in the insurance industry in Thailand. Results suggest that customers want technology to be integrated into interpersonal relationships, not to replace them. Most customers, even those with extensive IT background, are not willing to interact with technology‐based self service only. Much of the relationship with the company comes from trust in the salesperson, which is built up through the development of the relationship. This is probably more important in the relationship‐based cultures of Asia, which are strongly familiar with interpersonal service.
The relationship of cultural values and message strategies in service advertising
Authors: Susan Tai H.C.
Published: July 2004

Differences in cultural values may result in perceptual differences and thus necessitate the use of different advertising message strategies in different cultures. This paper tests the relationship between cultural values and advertising message strategies employed in service advertising using the affective response approach, rather than content analysis. The results show that Hong Kong advertisers use significantly more transformational messages than in US services advertising. Advertising message strategies and cultural values often relate in a non‐random way. Informational message strategy and realistic culture are found to be strongly associated. When societies are more materialistic, advertisers are more likely to use transformational messages due to the high correlation between materialism and brand image. The result also shows that masculine culture, and cultures with long‐term orientation, idealism or low individualism are more likely to adopt transformational message strategy.
Research of Bulgarian companies' marketing effectiveness
Authors: Stefanov Velev Mladen , Todorov Marinov Ivo
Published: July 2004

Focuses on the results of a study to define the level of Bulgarian enterprise marketing effectiveness. The main aim is not only to assess and analyze the marketing effectiveness of a great number of Bulgarian companies, but also to compare companies from diverse branches of the economy, search for the relation between their marketing effectiveness and companies’ financial results and present an analysis of basic weaknesses and their causes. The results clarify the real potential of enterprises to face market challenges and thus help complete the overall picture of the peculiarities of economies in transition. They will help researchers working on these problems, investors and business people (especially foreign) and Bulgarians may find out about the state of their companies and their management and direct their efforts in this area.
Studies of luminescence property of long afterglow Eu2+, Dy3+ activated Sr2MgSi2O7 phosphor
Authors: Mirhabibi A.R. , Moztarzadeh F. , Aghajani Bazazi A. , Solati M. , Maghsoudipour A. , Sarrafi M.H.
Published: July 2004

Long afterglow Sr2MgSi2O7 (SMS) phosphor was prepared by Douby's methods at high temperature and reductive atmosphere. The excitation and emission spectra of this phosphor showed that both had broad bands and that the main emission peak at 469 nm was due to 4f‐5d transitions of Eu+2 and implied that the luminescence centres Eu+2 occupied the Sr+2 sites in the Sr2MgSi2O7 host. The phosphor doped only with Eu ions did not demonstrate the long afterglow phenomenon, but when co‐doped with Dy+3 ions in the SMS matrix, significant long afterglow was observed.
High performance anticorrosive paint formulations based on phosphate pigments
Authors: Abd El‐Ghaffar M.A. , Youssef E.A.M. , Ahmed N.M.
Published: July 2004

Historically, paints designed to protect steel and other metals have been formulated using anticorrosive chromate pigments, which are currently under environmental restrictions. During the investigation reported here, various phosphate compounds. The pigments prepared were characterised using a variety of chemical and spectrophotometric methods of analysis including emission atomic absorption, transmission electron microscope, X‐ray diffraction, in addition to thermal gravimetric analysis. The pigments were also evaluated according to relevant international standard testing methods. The phosphates prepared were incorporated into anticorrosive paint formulations, to replace the imported zinc phosphate pigment, containing medium oil alkyd resin, and melamine formaldehyde resin. Paint films obtained were tested in artificial seawater for 28 days for anticorrosion properties. The results indicated that the paint films had good anticorrosive protection properties that could be attributed to the pigments prepared and the resins used.
Ink‐jet technology: status quo and future – relevance to surface coatings
Authors: Lin L. , Bai X.
Published: July 2004

Recent years have seen a rapid development of ink‐jet printing technology. This paper reviews the state‐of‐the‐art in ink‐jet printing technology and gives an overview of ink‐jet printing into the immediate future. The focus is placed on various applications of jet printing technology. The potential of applying jetting technology in the conventionally surface coating dominated applications will also be explored.
Synthesis and characterisation of hole transporting soluble polyschiff base containing triarylamine unit in the backbone
Authors: Niu H. , Lin L. , Bai X. , Huang Y. , Li X.
Published: July 2004

A soluble polyschiff base containing triarylamine unit in backbone was synthesised by condensation polymerisation. The hole transport properties of such polyschiff base were studied. The mobility of the hole carrier in the polyschiff base film was also measured and found to be μ=1.68×10−4 cm2/V s by means of time of flight technique. A polymer electroluminescence device was prepared with the polyschiff base used as hole transporting moiety by spin coated.
Do‐not‐call as the US Government's improvement to telemarketing efficiency
Authors: Rotfeld Herbert Jack
Published: July 2004

Under what might be one of the most well‐known new consumer protection activities in the USA, people can register their phone numbers with the “Do‐not‐call” registry that assures to block most commercial telemarketing calls. While it is hard to find a member of the general public who is opposed to the program, the telemarketing companies claim that it would cause a massive loss of jobs and some legal scholars assert that it violates provisions of the US Constitution. However, the regulation could actually improve telemarketing practice and the general efficiency of the business, making it more profitable.
The effects of aging on brand attitude measurement
Authors: Jayanti Rama K. , McManamon Mary K. , Whipple Thomas W.
Published: July 2004

Memory impairments in the elderly have been widely studied in the past. This study focuses on the effects of these memory impairments on the ability of mature consumers to respond to brand attitude scales. An experimental study investigates the impact of age and type of measurement scale on responses to brand attitude scales. Groups of seniors within the elderly market (55‐65, 66‐75, and over 75) are investigated as opposed to contrasting two extreme points on the continuum, namely the elderly versus the young. Three commonly used attitude scales were manipulated to determine how age interacts with the form of scale to generate response bias. Three types of response bias; extremity response, acquiescence, and item non‐response were investigated. Results indicate a significant interaction between age and type of scale. Implications of these results for those involved in marketing to seniors are highlighted.
A cross‐national study on children's purchasing behavior and parental response
Authors: Wimalasiri Jayantha S.
Published: July 2004

Children use various influencing tactics to persuade the parents to comply with their requests. Parents' responses vary from outright denial to total acceptance. A sample of 255 parents selected from the Fiji Islands, Tonga and the Cook Islands were interviewed using a structured questionnaire to measure the effects of children's influence on the parental decision making process. This preliminary investigation suggests that the children in the Pacific Islands have not moved from parent‐centered family environment to the modern, mostly Western, child‐centered family environment. The second part of the research was designed to identify the demonstrated influence tactics used by the children in the island nations. The findings indicate that the children are less demanding and more persuasive in their attempt to obtain parental approval. Details of statistical analysis of the study are given. The implications of the findings for marketing management are also discussed in the paper.
Participation in tertiary study abroad programs: the role of personality
Authors: Bakalis Steve , Joiner Therese A.
Published: July 2004

The increasing trend for the globalisation of business has highlighted the need for a better understanding of the factors that influence levels of intercultural awareness within organisations. Within the higher education sector, one initiative that aims to address this issue is student study abroad programs. This paper reports on a study that investigates factors that influence Australian students' propensity to engage in these programs. Using both qualitative and quantitative analysis, we examined the role of students' personality in the decision to participate in study abroad programs. The implications for education in international business are discussed.
In search of a consensus team decision budget agreement
Authors: Chong Philip S. , Runyon Lowell R.
Published: July 2004

In searching for a new budget formula for the College of Business at California State University at Long Beach, a major university in the west, several rational budget formulas were explored. This report develops an explanation in quantitative terms of the reasoning process pursued by the department chairs in arriving at the compromise budget allocation model that is currently in place in the college. It shows that in a group decision‐making process concerning the allocation of resources, compromises are made between decision‐makers in order to come to some common agreement, if one in fact exists. Rational models based on some formula are introduced, and resources can be allocated based on the formula. However, among the models presented using a decision matrix, the model that will eventually be selected is the one that has the minimal variance in ranking regrets and monetary regrets if the highest‐ranking model is not chosen. The ranking regret provides a good guide and quick identification of the “most‐likely‐to‐succeed” compromise model. However, the monetary regret appears to be the final compromise determinant.
Consumer confusion: reduction strategies in higher education
Authors: Drummond Graeme
Published: July 2004

This paper highlights the increasingly important topic of consumer confusion. Drawing parallels with experiences in the private sector, the concept of consumer confusion is explored within the higher education sector; what causes the phenomenon, how do consumers react to it and how can it be negated/minimised? The expansion and commercialisation of higher education has seen the wide‐scale adoption of marketing techniques within the sector. Such actions generate increased capacity for consumer confusion, with consumers being overwhelmed with information and potentially making sub‐optimum decisions. Given that the selection of a degree course is normally a life changing event, careful consideration needs to be given, by all parties, to whether marketing helps or hinders this process. While focusing on higher education, the issues considered are equally applicable to any public sector body adopting a more market driven approach.
Baldrige theory into practice: a working model
Authors: Arif Mohammed , Smiley Frederick M.
Published: July 2004

Malcolm Baldrige Awards has established a framework of operations in order to encourage accountability, transparent decision making, and optimal use of available resources. In 2001 the University of Wisconsin at Stout was the first higher educational institute to receive this award. Their operation has been prioritized into the factors of: leadership; strategic planning; customer and market focus; information and analysis; human resource focus; process management; and, business results. This article traces the progress of UW‐Stout's implementation of the above factors in their day‐to‐day operations on their students' learning. The implications of this article are that this Wisconsin university has revamped their curriculum using Malcolm Baldrige framework, and that their successes can be used as a model of operations for colleges and universities.
A review of low‐power wireless sensor microsystems for biomedical capsule diagnosis
Authors: Arshak K. , Jafer E. , Lyons G. , Morris D. , Korostynska O.
Published: July 2004

The development of a sensor microsystems containing all the components of data acquisition system, such as sensors, signal‐conditioning circuits, analog‐digital converter, interface circuits and embedded microcontroller (MCU), has become the focus of attention in many biomedical applications. A review of the microsystems technology is presented in this paper, along with a discussion of the recent trends and challenges associated with its developments. A basic description of each sub‐system is also given. This includes the different front end, mixed analog‐digital, power management, and radio transmitter‐receiver circuits. These sub‐system designs are presented and discussed in a comparative study and final remarks are made. The performance of each sub‐system is assessed regarding many aspects related to the overall system performance.
Screen‐printed sensors with graphite electrodes – comparison of properties and physical method of sensitivity enhancement
Authors: Krejčí Jan , Prášek Jan , Fujcik Lukáš , Khatib Sameh , Hejátková Edita , Jakubka Luboš , Giannoudi Louisa
Published: July 2004

Screen‐printed electrodes are widely used in the construction of sensors. The use of graphite material is preferred due to its simple technological processing and low cost. Different graphite pastes are compared for hydrogen peroxide detection. The slope of the calibration curve, linearity and limit of detection have been compared for different pastes and technologies of graphite electrode preparation. The influence of the structure of the paste on response is discussed. Physical methods of sensitivity enhancement are proposed. All results are compared with platinum electrode as technological reference.
Global analysis of three‐state protein unfolding data
Authors: Harder Mark E. , Deinzer Max L. , Leid Mark E. , Schimerlik Michael I.
Published: July 2004

A new method for analyzing three‐state protein unfolding equilibria is described that overcomes the difficulties created by direct effects of denaturants on circular dichroism (CD) and fluorescence spectra of the intermediate state. The procedure begins with a singular value analysis of the data matrix to determine the number of contributing species and perturbations. This result is used to choose a fitting model and remove all spectra from the fitting equation. Because the fitting model is a product of a matrix function which is nonlinear in the thermodynamic parameters and a matrix that is linear in the parameters that specify component spectra, the problem is solved with a variable projection algorithm. Advantages of this procedure are perturbation spectra do not have to be estimated before fitting, arbitrary assumptions about magnitudes of parameters that describe the intermediate state are not required, and multiple experiments involving different spectroscopic techniques can be simultaneously analyzed. Two tests of this method were performed: First, simulated three‐state data were analyzed, and the original and recovered thermodynamic parameters agreed within one standard error, whereas recovered and original component spectra agreed within 0.5%. Second, guanidine‐induced unfolding titrations of the human retinoid‐X‐receptor ligand‐binding domain were analyzed according to a three‐state model. The standard unfolding free energy changes in the absence of guanidine and the guanidine concentrations at zero free‐energy change for both transitions were determined from a joint analysis of fluorescence and CD spectra. Realistic spectra of the three protein states were also obtained.
Probing folding and fluorescence quenching in human γD crystallin Greek key domains using triple tryptophan mutant proteins
Authors: Kosinski‐Collins Melissa S. , Flaugh Shannon L. , King Jonathan
Published: July 2004

Human γD crystallin (HγD‐Crys), a major component of the human eye lens, is a 173‐residue, primarily β‐sheet protein, associated with juvenile and mature‐onset cataracts. HγD‐Crys has four tryptophans, with two in each of the homologous Greek key domains, which are conserved throughout the γ‐crystallin family. HγD‐Crys exhibits native‐state fluorescence quenching, despite the absence of ligands or cofactors. The tryptophan absorption and fluorescence quenching may influence the lens response to ultraviolet light or the protection of the retina from ambient ultraviolet damage. To provide fluorescence reporters for each quadrant of the protein, triple mutants, each containing three tryptophan‐to‐phenylalanine substitutions and one native tryptophan, have been constructed and expressed. Trp 42‐only and Trp 130‐only exhibited fluorescence quenching between the native and denatured states typical of globular proteins, whereas Trp 68‐only and Trp 156‐only retained the anomalous quenching pattern of wild‐type HγD‐Crys. The three‐dimensional structure of HγD‐Crys shows Tyr/Tyr/His aromatic cages surrounding Trp 68 and Trp 156 that may be the source of the native‐state quenching. During equilibrium refolding/unfolding at 37°C, the tryptophan fluorescence signals indicated that domain I (W42‐only and W68‐only) unfolded at lower concentrations of GdnHCl than domain II (W130‐only and W156‐only). Kinetic analysis of both the unfolding and refolding of the triple‐mutant tryptophan proteins identified an intermediate along the HγD‐Crys folding pathway with domain I unfolded and domain II intact. This species is a candidate for the partially folded intermediate in the in vitro aggregation pathway of HγD‐Crys.
Evolutionary constraints associated with functional specificity of the CMGC protein kinases MAPK, CDK, GSK, SRPK, DYRK, and CK2α
Authors: Kannan Natarajan , Neuwald Andrew F.
Published: July 2004

Amino acid residues associated with functional specificity of cyclin‐dependent kinases (CDKs), mitogen‐activated protein kinases (MAPKs), glycogen synthase kinases (GSKs), and CDK‐like kinases (CLKs), which are collectively termed the CMGC group, were identified by categorizing and quantifying the selective constraints acting upon these proteins during evolution. Many constraints specific to CMGC kinases correspond to residues between the N‐terminal end of the activation segment and a CMGC‐conserved insert segment associated with coprotein binding. The strongest such constraint is imposed on a “CMGC‐arginine” near the substrate phosphorylation site with a side chain that plays a role both in substrate recognition and in kinase activation. Two nearby buried waters, which are also present in non‐CMGC kinases, typically position the main chain of this arginine relative to the catalytic loop. These and other CMGC‐specific features suggest a structural linkage between coprotein binding, substrate recognition, and kinase activation. Constraints specific to individual subfamilies point to mechanisms for CMGC kinase specialization. Within casein kinase 2α (CK2α), for example, the binding of one of the buried waters appears prohibited by the side chain of a leucine that is highly conserved within CK2α and that, along with substitution of lysine for the CMGC‐arginine, may contribute to the broad substrate specificity of CK2α by relaxing characteristically conserved, precise interactions near the active site. This leucine is replaced by a conserved isoleucine or valine in other CMGC kinases, thereby illustrating the potential functional significance of subtle amino acid substitutions. Analysis of other CMGC kinases similarly suggests candidate family‐specific residues for experimental follow‐up.
Double‐stranded DNA bacteriophage prohead protease is homologous to herpesvirus protease
Authors: Cheng Hua , Shen Nan , Pei Jimin , Grishin Nick V.
Published: July 2004

Double‐stranded DNA bacteriophages and herpesviruses assemble their heads in a similar fashion; a pre‐formed precursor called a prohead or procapsid undergoes a conformational transition to give rise to a mature head or capsid. A virus‐encoded prohead or procapsid protease is often required in this maturation process. Through computational analysis, we infer homology between bacteriophage prohead proteases (MEROPS families U9 and U35) and herpesvirus protease (MEROPS family S21), and unify them into a procapsid protease superfamily. We also extend this superfamily to include an uncharacterized cluster of orthologs (COG3566) and many other phage or bacteria‐encoded hypothetical proteins. On the basis of this homology and the herpesvirus protease structure and catalytic mechanism, we predict that bacteriophage prohead proteases adopt the herpesvirus protease fold and exploit a conserved Ser and His residue pair in catalysis. Our study provides further support for the proposed evolutionary link between dsDNA bacteriophages and herpesviruses.
The C‐terminal domain of dimeric serine hydroxymethyltransferase plays a key role in stabilization of the quaternary structure and cooperative unfolding of protein: Domain swapping studies with enzymes having high sequence identity
Authors: Bhatt Anant Narayan , Khan M. Yahiya , Bhakuni Vinod
Published: July 2004

The serine hydroxymethyltransferase from Bacillus subtilis (bsSHMT) and B. stearothermophilus (bstSHMT) are both homodimers and share ∼77% sequence identity; however, they show very different thermal stabilities and unfolding pathways. For investigating the role of N‐ and C‐terminal domains in stability and unfolding of dimeric SHMTs, we have swapped the structural domains between bs‐ and bstSHMT and generated the two novel chimeric proteins bsbstc and bstbsc, respectively. The chimeras had secondary structure, tyrosine, and pyridoxal‐5′‐phosphate microenvironment similar to that of the wild‐type proteins. The chimeras showed enzymatic activity slightly higher than that of the wild‐type proteins. Interestingly, the guanidium chloride (GdmCl)–induced unfolding showed that unlike the wild‐type bsSHMT, which undergoes dissociation of native dimer into monomers at low guanidium chloride (GdmCl) concentration, resulting in a non‐cooperative unfolding of enzyme, its chimera bsbstc, having the C‐terminal domain of bstSHMT was resistant to low GdmCl concentration and showed a GdmCl‐induced cooperative unfolding from native dimer to unfolded monomer. In contrast, the wild‐type dimeric bstSHMT was resistant to low GdmCl concentration and showed a GdmCl‐induced cooperative unfolding, whereas its chimera bstbsc, having the C‐ terminal domain of bsSHMT, showed dissociation of native dimer into monomer at low GdmCl concentration and a GdmCl‐induced non‐cooperative unfolding. These results clearly demonstrate that the C‐terminal domain of dimeric SHMT plays a vital role in stabilization of the oligomeric structure of the native enzyme hence modulating its unfolding pathway.
Insights into the DNA repair process by the formamidopyrimidine‐DNA glycosylase investigated by molecular dynamics
Authors: Amara Patricia , Serre Laurence , Castaing Bertrand , Thomas Aline
Published: July 2004

Formamidopyrimidine‐DNA glycosylase (Fpg) identifies and removes 8‐oxoguanine from DNA. All of the X‐ray structures of Fpg complexed to an abasic site containing DNA exhibit a common disordered region present in the C‐terminal domain of the enzyme. However, this region is believed to be involved in the damaged base binding site when the initial protein/DNA complex is formed. The dynamic behavior of the disordered polypeptide (named Loop) in relation to the supposed scenario for the DNA repair mechanism was investigated by molecular dynamics on different models, derived from the X‐ray structure of Lactococcus lactis Fpg bound to an abasic site analog‐containing DNA and of Bacillus stearothermophilus Fpg bound to 8‐oxoG. This study shows that the presence of the damaged base influences the dynamics of the whole enzyme and that the Loop location is dependent on the presence and on the conformation of the 8‐oxoG in its binding site. In addition, from our results, the conformation of the 8‐oxoG seems to be favored in syn in the L. lactis models, in agreement with the available X‐ray structure from B. stearothermophilus Fpg and with a possible catalytic role of the flexibility of the Loop region.
Proteome‐wide functional classification and identification of prokaryotic transmembrane proteins by transmembrane topology similarity comparison
Authors: Arai Masafumi , Okumura Kosuke , Satake Masanobu , Shimizu Toshio
Published: July 2004

We propose a new method for classifying and identifying transmembrane (TM) protein functions in proteome‐scale by applying a single‐linkage clustering method based on TM topology similarity, which is calculated simply from comparing the lengths of loop regions. In this study, we focused on 87 prokaryotic TM proteomes consisting of 31 proteobacteria, 22 gram‐positive bacteria, 19 other bacteria, and 15 archaea. Prior to performing the clustering, we first categorized individual TM protein sequences as “known,” “putative” (similar to “known” sequences), or “unknown” by using the homology search and the sequence similarity comparison against SWISS‐PROT to assess the current status of the functional annotation of the TM proteomes based on sequence similarity only. More than three‐quarters, that is, 75.7% of the TM protein sequences are functionally “unknown,” with only 3.8% and 20.5% of them being classified as “known” and “putative,” respectively. Using our clustering approach based on TM topology similarity, we succeeded in increasing the rate of TM protein sequences functionally classified and identified from 24.3% to 60.9%. Obtained clusters correspond well to functional superfamilies or families, and the functional classification and identification are successfully achieved by this approach. For example, in an obtained cluster of TM proteins with six TM segments, 109 sequences out of 119 sequences annotated as “ATP‐binding cassette transporter” are properly included and 122 “unknown” sequences are also contained.
Orientation and helical conformation of a tissue‐specific hunter‐killer peptide in micelles
Authors: Plesniak Leigh A. , Parducho Jonathan I. , Ziebart Angie , Geierstanger Bernhard H. , Whiles Jennifer A. , Melacini Guiseppe , Jennings Patricia A.
Published: July 2004

Hunter‐killer peptides are chimeric synthetic peptides that selectively target specific cell types for an apoptotic death. These peptides, which are models for potential therapeutics, contain a homing sequence for receptor‐mediated interactions and a pro‐apoptotic sequence. Homing domains have been designed to target angiogenic tumor cells, prostate cells, arthritic tissue and, most recently, adipose tissue. After a receptor‐mediated internalization, the apoptotic sequence, which contains D‐enantiomer amino acids, initiates apoptosis through mitochondrial membrane disruption. We have begun structure and functional studies on a peptide (HKP1) that specifically targets angiogenic tumor cells for apoptosis. As a model for mitochondrial membrane disruption, we have examined peptide‐induced leakage of a calcein fluorophore from large unilamellar vesicles. These experiments demonstrate more potent leakage activity by HKP1 than the peptide lacking the homing domain. Circular dichroism and 2D homonuclear NMR experiments demonstrate that this tumor‐specific HKP adopts a left‐handed amphipathic helix in association with dodecylphosphorylcholine micelles in a parallel orientation to the lipid–water interface with the homing domain remaining exposed to solvent. The amphipathic helix of the apoptotic domain orients with nonpolar leucine and alanine residues inserting most deeply into the lipid environment.
Endurance swimming of European eel
Authors: Van Den Thillart G. , Van Ginneken V. , Körner F. , Heijmans R. , Van Der Linden R. , Gluvers A.
Published: July 2004

A long‐term swim trial was performed with five female silver eels Anguilla anguilla of 0·8–1·0 kg (c. 80 cm total length, LT) swimming at 0·5 body lengths (BL) s−1, corresponding to the mean swimming speed during spawning migration. The design of the Blazka‐type swim tunnel was significantly improved, and for the first time the flow pattern of a swim tunnel for fish was evaluated with the Laser‐Doppler method. The velocity profile over three different cross‐sections was determined. It was observed that 80% of the water velocity drop‐off occurred over a boundary layer of 20 mm. Therefore, swim velocity errors were negligible as the eels always swam outside this layer. The fish were able to swim continuously day and night during a period of 3 months in the swim tunnel through which fresh water at 19° C was passed. The oxygen consumption rates remained stable at 36·9 ± 2·9 mg O2 kg−1 h−1 over the 3 months swimming period for all tested eels. The mean cost of transportation was 28·2 mg O2 kg−1 km−1. From the total energy consumption the calculated decline in fat content was 30%. When extrapolating to 6000 km this would have been 60%, leaving only 40% of the total energy reserves for reproduction after arriving at the spawning site. Therefore low cost of transport combined with high fat content are crucial for the capacity of the eel to cross the Atlantic Ocean and reproduce.
The development of haddock and Atlantic cod sperm cryopreservation techniques and the effect of sperm age on cryopreservation success
Authors: Rideout R. M. , Trippel E. A. , Litvak M. K.
Published: July 2004

Three cryoprotectants [dimethyl sulphoxide (DMSO), propylene glycol (PG) and glycerol], two diluents (sucrose‐ and saline‐based), two sperm collection times, two freezing rates and three times between thaw and activation (0, 30 and 60 min) were tested in order to develop a protocol for the cryopreservation of sperm of haddock Melanogrammus aeglefinus and Atlantic cod Gadus morhua. The faster freezing rate resulted in extremely low post‐thaw motility in comparison to the slower freezing rate, which was successful for sperm from both gadids. In both cases, the use of PG resulted in significantly higher post‐thaw sperm motility‐recovery indices than with DMSO or glycerol, which did not differ significantly from one another. Diluent had no effect on post‐thaw sperm motility for Atlantic cod or haddock. Sperm collected at the end of the spawning season tended to have reduced post‐thaw motility compared to that collected 2 weeks after the start of spawning. A 30 min delay between thaw and activation of haddock and Atlantic cod sperm resulted in a significant decrease in sperm motility. When PG was used as cryoprotectant, sperm motility continued to decrease between 30 and 60 min post‐thaw. With DMSO or glycerol as cryoprotectant, motilities were already very low after 30 min post‐thaw and did not decrease any further after 60 min. Cryoprotectant, diluent and time between thaw and activation had no effect on mean or maximum sperm swimming speeds for either Atlantic cod or haddock sperm. Fertilization success for haddock eggs, like sperm motility, was higher with PG‐frozen sperm than DMSO‐ or glycerol‐frozen sperm. These results constitute the first reported successful cryopreservation of haddock sperm and improve on previous methods used to cryopreserve sperm from Atlantic cod.
Relationship between sperm density, spermatocrit, sperm motility and spawning date in wild and cultured haddock
Authors: Rideout R. M. , Trippel E. A. , Litvak M. K.
Published: July 2004

Semen was collected repeatedly from captive haddock Melanogrammus aeglefinus and the effect of seasonality on various sperm parameters was investigated. No differences in sperm traits were observed for wild and cultured haddock. A highly significant positive relationship existed between spermatocrit and spermatozoa density. A significant increase in mean spermatocrit occurred throughout the spawning season but the amount of variability explained by collection date was low (35·1%) due to variability between males. Each of 10 males sampled repeatedly throughout the spawning season demonstrated an increase in spermatocrit. No relationship existed between spermatocrit and proportion of motile spermatozoa when spermatocrit was ≤70%. Motility was reduced in semen samples with spermatocrits >70%. The proportion of spermatozoa that were motile decreased with time since activation. Some motility was still observed after 60 min in sea water (0·1–15·2%) for sperm collected at all times within the spawning season. Of those spermatozoa that were motile, the proportion that exhibited forward swimming motion decreased and the proportion that had only vibratory movement increased with time post‐activation. The speed of forward swimming spermatozoa showed no significant relationship with spermatocrit at any time between 0 and 60 min after activation. Swimming speed was negatively related to time since activation, decreasing from 174–240 μm s−1 at 0 min to 80–128 μm s−1 at 60 min after activation.