bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparison of laser interferometry and ultrasound A‐scan in the measurement of axial length
Authors: Goyal R. , North R. V. , Morgan J. E.
Published: July 2003

Aim:  Laser interferometry is a new, non‐contact technique for the measurement of axial length. In this study we compared measurements of axial length obtained with this technique with those obtained with ultrasound (A‐scan). The reproducibility and examiner‐dependency of the two methods were also analysed.

Methods:  Patients presenting at the cataract assessment clinic were invited to participate in the study. Axial length measurements were obtained both by contact ultrasound (A‐scan) and by non‐contact laser interferometry (IOLMasterTM V1.1, Carl Zeiss, Jena, Germany). Intraocular lens powers were calculated using both sets of measurements. The coefficient of variation served as a measure of reproducibility.

Results:  A total of 100 eyes in 100 patients were evaluated after informed consent had been obtained. Although estimates of axial length obtained with the two techniques were highly correlated, axial lengths obtained with the contact method (mean 23.35 mm, SD 1.81 mm) were consistently lower than those obtained with the non‐contact method (mean 23.55 mm, SD 1.76 mm) and the difference was statistically significant (p < 0.001). The coefficient of variation was lower with laser interferometry (0.1%) than with the ultrasound technique (0.49%).

Conclusions:  Different estimates of axial length are obtained using contact and non‐contact techniques, with the latter producing consistently higher measurements than the former. Laser interferometry provides more reproducible results that should improve the accuracy of measurements of axial length in the clinical setting.
Cortical blindness and hepatic encephalopathy
Authors: Ammar T. , Auwzinger G. , Michaelides M.
Published: July 2003

Purpose:  To present a case of permanent cortical blindness (CB) following hepatic encephalopathy.

Methods:  Systematic clinical examination and further investigations to elicit the diagnosis.

Conclusion:  Complete CB is an uncommon neurological disorder and is rarely persistent. In rare cases of hepatic encephalopathy, the visual cortex may be affected and CB may occur, which resolves following treatment of the hepatic encephalopathy. We report a previously unreported complication involving permanent CB following hepatic encephalopathy in a young man.
A new index to measure the accuracy of refractive operations
Authors: Suominen S. , Pietilä J. , Mäkinen P. , Uusitalo H.
Published: July 2003

Purpose:  To create a new index to help evaluate the accuracy of the outcome of refractive surgery.

Methods:  By combining information given by the cross‐cylinder form of surgically induced refractive change (SIRC) and basic methods of vector analysis, we constructed a new index which serves as a tool to evaluate postoperatively the changes achieved by refractive surgery. This new index gives easily understandable numerical values and takes into account changes in both spherical refraction and astigmatism. We demonstrated the use of this index in two study populations consisting of 20 consecutive eyes operated on using two different lasers, Meditec MEL60 and MEL70, respectively.

Results:  Although postoperative uncorrected visual acuity (UCVA) and best corrected visual acuity (BCVA) were better in the MEL70 group, the difference of photoefractive keratectomy (PRK) for combined astigmatism and myopia was not statistically significant. Comparison of the changes in refraction achieved by the two lasers indicated that the MEL60 was more accurate; this was also seen when comparing the total index of error between the two groups.

Conclusions:  We suggest the use of this total index of error (TIE) in clinical praxis because it provides an easy and accurate method of evaluating the accuracy of refractive operations. These inaccuracies might otherwise go unnoticed if the basic values only (e.g. UCVA, BCVA and haze) were used in postoperative evaluation.
Visually impaired children with posterior ocular malformations: pre‐ and neonatal data and visual functions
Authors: Fahnehjelm Kristina Teär , Jacobson Lena , Hellström Ann , Lewensohn‐Fuchs Ilona , Ygge Jan
Published: July 2003

Aim:  To analyse pre‐ and neonatal data and ocular findings in children with visual impairment caused by posterior ocular malformations.

Methods:  Medical records were scrutinized, dried blood spot cards were analysed for virus DNA and ophthalmological assessments were performed in 28 children with optic nerve hypoplasia (ONH) and 10 with optic/chorio‐retinal coloboma.

Results:  Prenatal exposure to possible teratogens was documented in 5/28, herpes simplex virus type 1 DNA was identified in the dried blood spot cards of 1/26 children and neonatal hypoglycaemia in 12/28 children with ONH. The time delay from ocular to endocrinological diagnosis and treatment was 3 years. Children with ONH and severe visual impairment had endocrinopathy more often (11/13) than ONH children with better visual functions (5/15). Prenatal exposure to teratogens or neonatal hypoglycaemia was not identified in any of the children with coloboma.

Conclusion:  Neonatal hypoglycaemia was common in children with ONH. Severe visual impairment predicted endocrinopathy. Analysis of dried blood spot cards could serve as an additional diagnostic tool in children with ocular malformations.
Metastasis‐free survival following treatment for uveal melanoma with either stereotactic radiosurgery or enucleation
Authors: Cohen V. M. L. , Carter M. J. , Kemeny A. , Radatz M. , Rennie I. G.
Published: July 2003

Purpose:  To determine whether metastasis‐free survival following stereotactic radiosurgery is comparable with that following enucleation in a cohort of patients with choroidal and ciliary body melanoma.

Methods:  This was a non‐randomized, retrospective study of 196 patients with uveal melanoma treated between 1990 and 2000. A total of 118 eyes were enucleated and 78 eyes were treated with stereotactic radiosurgery. The following variables were recorded: age and sex of patient; tumour location, volume and height; the presence of retinal detachment, and extrascleral extension. Tumour location was divided into three categories: within the posterior pole; beyond the limits of the posterior pole but not including the ciliary body, and ciliary body location. Analysis of survival time was performed using Kaplan−Meier estimation of survival curves and Cox's proportional hazards regression modelling.

Results:  The 5‐year cumulative metastasis‐free survival rate was 51% in the enucleation treatment group compared to 74% in the stereotactic treatment group. However, in the multivariate analysis there was no statistical difference in survival rates between the two treatment groups. The only variables that influenced survival rates were tumour location (p = 0.002), ciliary body tumours with the worst prognosis, and tumour volume (p = 0.001).

Conclusions:  Tumour size and location at presentation determined metastasis‐free survival. Large ciliary body tumours had the highest risk of metastasis. Metastasis‐free survival after stereotactic radiosurgery was comparable to that after enucleation.
Aqueous humour flow after a single oral dose of isosorbide‐5‐mononitrate in healthy volunteers
Authors: Kotikoski Hanna , Oksala Olli , Vapaatalo Heikki , Aine Esko
Published: July 2003

Purpose:  To investigate whether a nitric oxide donor given as a single oral dose is able to modify aqueous humour flow in healthy volunteers.

Methods:  Ten healthy volunteers participated in a randomized, double‐masked and placebo‐controlled cross‐over study. Aqueous humour flow was assessed by fluorophotometry after intake of isosorbide‐5‐mononitrate (ISMN), 10 mg. Topical timolol maleate, which is known to reduce aqueous humour flow, was used as a positive control. Intraocular pressure (IOP) was measured by applanation tonometry and blood pressure was registered.

Results:  The basal rate of aqueous humour flow did not change significantly after a single oral dose of ISMN. The aqueous humour flow in the timolol‐treated eye was reduced as compared to the contralateral control eye (p = 0.002). Mean IOP 6 hours after placebo and ISMN intake did not differ significantly. Timolol lowered IOP by 4 mmHg (p < 0.001). ISMN did not lower systolic blood pressure, but diastolic blood pressure was reduced by 4 mmHg (p = 0.048).

Conclusion:  A single oral dose of 10 mg ISMN had no significant effect on aqueous humour flow in healthy volunteers.
Physicians' liability in ophthalmology practice
Authors: Mavroforou Anna , Michalodimitrakis Emmanuel
Published: July 2003

Purpose:  The aims of this paper are to point out the importance of coherence in established guidelines in daily practice, highlight the value of signed patient informed consent forms and stress the value of a good understanding of the law, while focusing on issues related to malpractice in ophthalmology.

Methods:  Search of the current relevant literature.

Results:  The most common causes of medical litigation in ophthalmology are related to LASIK surgery, cataract surgery, glaucoma care, radial keratotomy for myopia reduction and failure to diagnose binocular vision anomalies in children and adults in a timely fashion. The litigation raised in most cases is driven by unfavourable outcomes and not by malpractice.

Conclusion:  Maintenance of high standards in daily practice, with continuous training and clear communication along with the appropriate documentation of any procedure carried out, may improve the professional safety of practising eye specialists in the event of medical litigation. Although the use of patient informed consent varies substantially among European countries, it may serve as useful evidence in the physician's defence.
Processing of alpha4 integrin by the proprotein convertases: histidine at position P6 regulates cleavage
Authors: BERGERON Eric , BASAK Ajoy , DECROLY Etienne , SEIDAH Nabil G.
Published: July 2003

The proprotein convertases (PCs) participate in the limited proteolysis of integrin α4 subunit at the H592VISKR597 ↓ ST site (where underlined residues indicate positively charged amino acids important for PC-mediated cleavage and ↓ indicates the cleavage site), since this cleavage is inhibited by the serpin α1-PDX (α1-antitrypsin Portland). Co-expression of α4 with each convertase in LoVo (furin-deficient human colon carcinoma) cells revealed that furin and proprotein convertase 5A (PC5A) are the best pro-α4 convertases. In agreement, processing of endogenous pro-α4 in human lymphoblastoid CEM-T4 cells was enhanced greatly in stable transfectants overexpressing either enzyme. In many leucocyte cell lines, the expression of furin closely correlated with the endogenous processing efficacy, suggesting that furin is a candidate pro-α4 convertase. Mutational analysis showed that replacement of P1 Arg597 with alanine (R597A) abrogated cleavage, whereas the P6 mutant H592R is even better processed by the endogenous convertases of Chinese-hamster ovary CHO-K1 cells. In vitro kinetic studies using synthetic peptides confirmed the importance of a positively charged residue at P6 and showed that wild-type α4 processing is performed best by furin and PC5A at acidic and neutral pHs, respectively. Biosynthetic analysis of pro-α4 and its H592R and H592K mutants in the presence or absence of the weak base, NH4Cl, revealed that the P6 histidine residue renders its processing by furin sensitive to cellular pH. This suggests that pro-α4 cleavage occurs preferentially in acidic compartments. In conclusion, although the accepted furin processing motif is Arg-Xaa-(Lys/Arg)-Arg↓, our data further extend it to include a regulatory histidine residue at P6 in precursors that lack a basic residue at P4.
Induction of glycosylation in human C-reactive protein under different pathological conditions
Authors: DAS Tanusree , SEN Asish , KEMPF Tore , PRAMANIK Sumit R. , MANDAL Chhabinath , MANDAL Chitra
Published: July 2003

As an acute-phase protein, human C-reactive protein (CRP) is clinically important. CRPs were purified from several samples in six different pathological conditions, where their levels ranged from 22 to 342 μg/ml. Small, but significant, variations in electrophoretic mobilities on native PAGE suggested differences in molecular mass, charge and/or shape. Following separation by SDS/PAGE, they showed single subunits with some differences in their molecular masses ranging between 27 and 30.5 kDa, but for a particular disease, the mobility was the same for CRPs purified from multiple individuals or pooled sera. Isoelectric focusing (IEF) also indicated that the purified CRPs differed from each other. Glycosylation was demonstrated in these purified CRPs by Digoxigenin kits, neuraminidase treatment and binding with lectins. The presence of N-linked sugar moiety was confirmed by N-glycosidase F digestion. The presence of sialic acid, glucose, galactose and mannose has been demonstrated by gas liquid chromatography, mass spectroscopic and fluorimetric analysis. Matrix-assisted laser-desorption ionization analysis of the tryptic digests of three CRPs showed systematic absence of two peptide fragments, one at the N-terminus and the other near the C-terminus. Model-building suggested that the loss of these fragments exposed two potential glycosylation sites on a cleft floor keeping the protein–protein interactions in pentraxins and calcium-dependent phosphorylcholine-binding qualitatively unaffected. Thus we have convincingly demonstrated that human CRP is glycosylated in some pathological conditions.
Structural and functional characterization of recombinant mouse annexin A11: influence of calcium binding
Authors: LECONA Emilio , TURNAY Javier , OLMO Nieves , GUZMÁN-ARÁNGUEZ Ana , MORGAN Reginald O. , FERNANDEZ Maria-Pilar , LIZARBE Ma Antonia
Published: July 2003

Annexin A11 is one of the 12 vertebrate subfamilies in the annexin superfamily of calcium/phospholipid-binding proteins, distinguishable by long, non-homologous N-termini rich in proline, glycine and tyrosine residues. As there is negligible structural information concerning this annexin subfamily apart from primary sequence data, we have cloned, expressed and purified recombinant mouse annexin A11 to investigate its structural and functional properties. CD spectroscopy reveals two main secondary-structure contributions, α-helix and random coil (approx. 30% each), corresponding mainly to the annexin C-terminal tetrad and the N-terminus respectively. On calcium binding, an increase in α-helix and a decrease in random coil are detected. Fluorescence spectroscopy reveals that its only tryptophan residue, located at the N-terminus, is completely exposed to the solvent; calcium binding promotes a change in tertiary structure, which does not affect this tryptophan residue but involves the movement of approximately four tyrosine residues to a more hydrophobic environment. These calcium-induced structural changes produce a significant thermal stabilization, with an increase of approx. 14 °C in the melting temperature. Annexin A11 binds to acidic phospholipids and to phosphatidylethanolamine in the presence of calcium; weaker calcium-independent binding to phosphatidylserine, phosphatidic acid and phosphatidylethanolamine was also observed. The calcium-dependent binding to phosphatidylserine is accompanied by an increase in α-helix and a decrease in random-coil contents, with translocation of the tryptophan residue towards a more hydrophobic environment. This protein induces vesicle aggregation but requires non-physiological calcium concentrations in vitro. A three-dimensional model, consistent with these data, was generated to conceptualize annexin A11 structure–function relationships.
Mutational analysis of ribosomal S6 kinase 2 shows differential regulation of its kinase activity from that of ribosomal S6 kinase 1
Authors: PHIN Sopheap , KUPFERWASSER Deborah , LAM Joseph , LEE-FRUMAN Kay K.
Published: July 2003

Ribosomal S6 kinase 2 (S6K2) is a serine/threonine kinase identified as a homologue of p70 ribosomal S6 kinase 1 (S6K1). S6K1 and S6K2 show different cellular localization as well as divergent amino acid sequences in non-catalytic domains, suggesting that their cellular functions and/or regulation may not be identical. Many of the serine/threonine residues that become phosphorylated and contribute to S6K1 activation are conserved in S6K2. In this study we carry out mutational analyses of these serine/threonine residues on S6K2 in order to elucidate the mechanism of S6K2 regulation. We find that Thr-228 and Ser-370 are crucial for S6K2 activity, and the three proline-directed serines in the autoinhibitory domain, Ser-410, Ser-417 and Ser-423, play a role in S6K2 activity regulation in a mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase (MEK)-dependent manner. However, unlike S6K1, changing Thr-388 to glutamic acid in S6K2 renders the kinase fully active. This activity was resistant to the effects of rapamycin or wortmannin, indicating that mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K) regulate S6K2 activity via Thr-388. MEK-dependent phosphorylation of the autoinhibitory serines in S6K2 occurs prior to Thr-388 activation. Combining T388E and T228A mutations inhibited S6K2 activation, and a kinase-inactive phosphoinositide-dependent protein kinase (PDK1) diminished T388E activity, suggesting that the role of Thr-388 is to allow further phosphorylation of Thr-228 by PDK1. Thr-388 fails to become phosphorylated in Ser-370 mutants, suggesting that the role of Ser-370 phosphorylation may be to allow Thr-388 phosphorylation. Finally, using the rapamycin-resistant T388E mutant, we provide evidence that S6K2 can phosphorylate S6 in vivo.
A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2beta)
Authors: DRAKE Paul G. , PETERS Günther H. , ANDERSEN Henrik Sune , HENDRIKS Wiljan , MØLLER Niels Peter H.
Published: July 2003

Islet-cell antigen 512 (IA-2) and phogrin (IA-2β) are atypical members of the receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent studies indicate that IA-2 may be involved in granule trafficking and exocytosis. To further understand their function, we have embarked upon developing low-molecular-mass inhibitors of IA-2 and IA-2β. Previously, we have shown that a general PTP inhibitor, 2-(oxalylamino)benzoic acid (OBA), can be developed into highly selective and potent inhibitors of PTP1B. However, since wild-type IA-2 and IA-2β lack conventional PTP activity, a novel strategy was designed whereby catalytically active species were generated by ‘back-mutating’ key non-consensus catalytic region residues to those of PTP1B. These mutants were then used as tools with which to test the potency and selectivity of OBA and a variety of its derivatives. Catalytically competent IA-2 and IA-2β species were generated by ‘back-mutation’ of only three key residues (equivalent to Tyr46, Asp181 and Ala217 using the human PTP1B numbering) to those of PTP1B. Importantly, enzyme kinetic analyses indicated that the overall fold of both mutant and wild-type IA-2 and IA-2β was similar to that of classic PTPs. In particular, one derivative of OBA, namely 7-(1,1-dioxo-1H-benzo[d]isothiazol-3-yloxymethyl)-2-(oxalylamino)-4,7-dihydro-5H-thieno[2,3-c]pyran-3 -carboxylic acid (‘Compound 6’ shown in the main paper), which inhibited IA-2β(S762Y/Y898P/D933A) (IA-2β in which Ser762 has been mutated to tyrosine, Tyr898 to proline, and Asp933 to alanine) with a Ki value of ≈8 μM, appeared ideal for future lead optimization. Thus molecular modelling of this classical, competitive inhibitor in the catalytic site of wild-type IA-2β identified two residues (Ser762 and Asp933) that offer the possibility for unique interaction with an appropriately modified ‘Compound 6’. Such a compound has the potential to be a highly selective and potent active-site inhibitor of wild-type IA-2β.
Spermidine/spermine N1-acetyltransferase (SSAT) activity in human small-cell lung carcinoma cells following transfection with a genomic SSAT construct
Authors: MURRAY-STEWART Tracy , APPLEGREN Nancy B. , DEVEREUX Wendy , HACKER Amy , SMITH Renee , WANG Yanlin , CASERO Robert A.
Published: July 2003

Spermidine/spermine N1-acetyltransferase (SSAT) activity is typically highly inducible in non-small-cell lung carcinomas in response to treatment with anti-tumour polyamine analogues, and this induction is associated with subsequent cell death. In contrast, cells of the small-cell lung carcinoma (SCLC) phenotype generally do not respond to these compounds with an increase in SSAT activity, and usually are only moderately affected with respect to growth. The goal of the present study was to produce an SSAT-overexpressing SCLC cell line to further investigate the role of SSAT in response to these anti-tumour analogues. To accomplish this, NCI-H82 SCLC cells were stably transfected with plasmids containing either the SSAT genomic sequence or the corresponding cDNA sequence. Individual clones were selected based on their ability to show induced SSAT activity in response to exposure to a polyamine analogue, and an increase in the steady-state SSAT mRNA level. Cells transfected with the genomic sequence exhibited a significant increase in basal SSAT mRNA expression, as well as enhanced SSAT activity, intracellular polyamine pool depletion and growth inhibition following treatment with the analogue N1, N11-bis(ethyl)norspermine. Cells containing the transfected cDNA also exhibited an increase in the basal SSAT mRNA level, but remained phenotypically similar to vector control cells with respect to their response to analogue exposure. These studies indicate that both the genomic SSAT sequence and polyamine analogue exposure play a role in the transcriptional and post-transcriptional regulation and subsequent induction of SSAT activity in these cells. Furthermore, this is the first production of a cell line capable of SSAT protein induction from a generally unresponsive parent line.
Evidence that TRPC1 (transient receptor potential canonical 1) forms a Ca2+-permeable channel linked to the regulation of cell volume in liver cells obtained using small interfering RNA targeted against TRPC1
Authors: CHEN Jinglong , BARRITT Greg J.
Published: July 2003

The TRPC1 (transient receptor potential canonical 1) protein, which is thought to encode a non-selective cation channel activated by store depletion and/or an intracellular messenger, is expressed in a number of non-excitable cells. However, the physiological functions of TRPC1 are not well understood. The aim of these studies was to investigate the function of TRPC1 in liver cells using small interfering RNA (siRNA) to ablate the TRPC1 protein. Treatment of H4-IIE liver cells with siRNA targeted against TRPC1 caused an approx. 50% decrease in expression of the human TRPC1 protein in cells transfected with cDNA encoding human TRPC1, and a 50% decrease in expression of the endogenous TRPC1 protein (assessed by Western blot and immunofluorescence). The decrease in endo-genous TRPC1 protein in cells transfected with TRPC1 siRNA was associated with a greater increase in cell volume (compared with the increase observed in control cells) immediately after cells were placed in a hypotonic medium, and an enhanced regulatory cell volume decrease after exposure to hypotonic medium. Treatment with siRNA targeted against TRPC1 also led to a 25% inhibition of thapsigargin-stimulated Ca2+ inflow, a 40% inhibition of ATP and maitotoxin-stimulated Ca2+ inflow, and a 50% inhibition of maitotoxin-stimulated Mn2+ inflow. The idea that, in liver cells, TRPC1 encodes a non-selective cation channel involved directly or indirectly in the regulation of cell volume is consistent with the results obtained.
Stability and conformational properties of doppel, a prion-like protein, and its single-disulphide mutant
Authors: WHYTE Sheena M. , SYLVESTER Ian D. , MARTIN Stephen R. , GILL Andrew C. , WOPFNER Franziska , SCHÄTZL Hermann M. , DODSON Guy G. , BAYLEY Peter M.
Published: July 2003

Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the α→β transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95–148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced β-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrPC→PrPSc). Furthermore, no direct interaction of either doppel protein with prion protein, either in the α-form or the β-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the α→β transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.
Selective interaction of megalin with postsynaptic density-95 (PSD-95)-like membrane-associated guanylate kinase (MAGUK) proteins
Authors: LARSSON Mårten , HJÄLM Göran , SAKWE Amos M. , ENGSTRÖM Åke , HÖGLUND Anna-Stina , LARSSON Erik , ROBINSON Robert C. , SUNDBERG Christian , RASK Lars
Published: July 2003

Megalin is an integral membrane receptor belonging to the low-density lipoprotein receptor family. In addition to its role as an endocytotic receptor, megalin has also been proposed to have signalling functions. Using interaction cloning in yeast, we identified the membrane-associated guanylate kinase family member postsynaptic density-95 (PSD-95) as an interaction partner for megalin. PSD-95 and a truncated version of megalin were co-immunoprecipitated from HEK-293 cell lysates overexpressing the two proteins, which confirmed the interaction. The two proteins were found to be co-localized in these cells by confocal microscopy. Immunocytochemical studies showed that cells in the parathyroid, proximal tubuli of the kidney and placenta express both megalin and PSD-95. We found that the interaction between the two proteins is mediated by the binding of the C-terminus of megalin, which has a type I PSD-95/Drosophila discs-large/zona occludens 1 (PDZ)-binding motif, to the PDZ2 domain of PSD-95. The PSD-95-like membrane-associated guanylate kinase (‘MAGUK’) family contains three additional members: PSD-93, synapse-associated protein 97 (SAP97) and SAP102. We detected these proteins, apart from SAP102, in parathyroid chief cells, a cell type having a marked expression of megalin. The PDZ2 domains of PSD-93 and SAP102 were also shown to interact with megalin, whereas no interaction was detected for SAP97. The SAP97 PDZ2 domain differed at four positions from the other members of the PSD-95 subfamily. One of these residues was Thr389, located in the αB-helix and part of the hydrophobic pocket of the PDZ2 domain. Surface plasmon resonance experiments revealed that mutation of SAP97 Thr389 to alanine, as with the other PSD-95-like membrane-associated guanylate kinases, induced binding to megalin.
A colourless green fluorescent protein homologue from the non-fluorescent hydromedusa Aequorea coerulescens and its fluorescent mutants
Authors: GURSKAYA Nadya G. , FRADKOV Arkady F. , POUNKOVA Natalia I. , STAROVEROV Dmitry B. , BULINA Maria E. , YANUSHEVICH Yurii G. , LABAS Yulii A. , LUKYANOV Sergey , LUKYANOV Konstantin A.
Published: July 2003

We have cloned an unusual colourless green fluorescent protein (GFP)-like protein from Aequorea coerulescens (acGFPL). The A. coerulescens specimens displayed blue (not green) luminescence, and no fluorescence was detected in these medusae. Escherichia coli expressing wild-type acGFPL showed neither fluorescence nor visible coloration. Random mutagenesis generated green fluorescent mutants of acGFPL, with the strongest emitters found to contain an Glu222→Gly (E222G) substitution, which removed the evolutionarily invariant Glu222. Re-introduction of Glu222 into the most fluorescent random mutant, named aceGFP, converted it into a colourless protein. This colourless aceGFP-G222E protein demonstrated a novel type of UV-induced photoconversion, from an immature non-fluorescent form into a green fluorescent form. Fluorescent aceGFP may be a useful biological tool, as it was able to be expressed in a number of mammalian cell lines. Furthermore, expression of a fusion protein of ‘humanized’ aceGFP and β-actin produced a fluorescent pattern consistent with actin distribution in mammalian cells.
Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence
Authors: KIRWAN Angie F. , BIBBY Ashley C. , MVILONGO Thierry , RIEDEL Heimo , BURKE Thomas , MILLIS Sherri Z. , PARISSENTI Amadeo M.
Published: July 2003

The N-terminal pseudosubstrate site within the protein kinase Cα (PKCα)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCα-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940–8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCα-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Rα39–177: this protein contained the full regulatory domain of human PKCα fused to glutathione S-transferase (GST), but lacked amino acids 1–38 (including the pseudosubstrate sequence) and amino acids 178–270 (including the C2 region)]. GST-Rα39–177 significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCα holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33–86 within GST-Rα39–177 dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159–242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCα-regulatory domain that play a direct role in the inhibition of catalytic domain activity.
Distinctive properties and expression profiles of glutamine synthetase from a plant symbiotic fungus
Authors: MONTANINI Barbara , BETTI Marco , MÁRQUEZ Antonio J. , BALESTRINI Raffaella , BONFANTE Paola , OTTONELLO Simone
Published: July 2003

Nitrogen retrieval and assimilation by symbiotic ectomycorrhizal fungi is thought to play a central role in the mutualistic interaction between these organisms and their plant hosts. Here we report on the molecular characterization of the key N-assimilation enzyme glutamine synthetase from the mycorrhizal ascomycete Tuber borchii (TbGS). TbGS displayed a strong positive co-operativity (n=1.7±0.29) and an unusually high S0.5 value (54±16 mM; S0.5 is the substrate concentration value at which v=½Vmax) for glutamate, and a correspondingly low sensitivity towards inhibition by the glutamate analogue herbicide phosphinothricin. The TbGS mRNA, which is encoded by a single-copy gene in the Tuber genome, was up-regulated in N-starved mycelia and returned to basal levels upon resupplementation of various forms of N, the most effective of which was nitrate. Both responses were accompanied by parallel variations of TbGS protein amount and glutamine synthetase activity, thus indicating that TbGS levels are primarily controlled at the pre-translational level. As revealed by a comparative analysis of the TbGS mRNA and of the mRNAs for the metabolically related enzymes glutamate dehydrogenase and glutamate synthase, TbGS is not only the sole messenger that positively responds to N starvation, but also the most abundant under N-limiting conditions. A similar, but even more discriminating expression pattern, with practically undetectable glutamate dehydrogenase mRNA levels, was observed in fruitbodies. The TbGS mRNA was also found to be expressed in symbiosis-engaged hyphae, with distinctively higher hybridization signals in hyphae that were penetrating among and within root cells.
Fractalkine (CX3CL1) stimulated by nuclear factor kappaB (NF-kappaB)-dependent inflammatory signals induces aortic smooth muscle cell proliferation through an autocrine pathway
Authors: CHANDRASEKAR Bysani , MUMMIDI Srinivas , PERLA Rao P. , BYSANI Sailaja , DULIN Nickolai O. , LIU Feng , MELBY Peter C.
Published: July 2003

Fractalkine (also known as CX3CL1), a CX3C chemokine, activates and attracts monocytes/macrophages to the site of injury/inflammation. It binds to CX3C receptor 1 (CX3CR1), a pertussis toxin-sensitive G-protein-coupled receptor. In smooth muscle cells (SMCs), fractalkine is induced by proinflammatory cytokines [tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)], which may mediate monocyte adhesion to SMCs. However, the mechanisms underlying its induction are unknown. In addition, it is unlear whether SMCs express CX3CR1. TNF-α activated nuclear factor κB (NF-κB) and induced fractalkine and CX3CR1 expression in a time-dependent manner in rat aortic SMCs. Transient transfections with dominant-negative (dn) inhibitory κB (IκB)-α, dnIκB-β, dnIκB kinase (IKK)-γ, kinase-dead (kd) NF-κB-inducing kinase (NIK) and kdIKK-β, or pretreatment with wortmannin, Akt inhibitor, pyrrolidinecarbodithioc acid ammonium salt (‘PDTC’) or MG-132, significantly attenuated TNF-α-induced fractalkine and CX3CR1 expression. Furthermore, expression of dn TNF-α-receptor-associated factor 2 (TRAF2), but not dnTRAF6, inhibited TNF-α signal transduction. Pretreatment with pertussis toxin or neutralizing anti-CX3CR1 antibodies attenuated TNF-α-induced fractalkine expression, indicating that fractalkine autoregulation plays a role in TNF-α-induced sustained fractalkine expression. Fractalkine induced its own expression, via pertussis toxin-sensitive G-proteins, phosphoinositide 3-kinase (PI 3-kinase), phosphoinositide-dependent kinase 1 (PDK1), Akt, NIK, IKK and NF-κB activation, and induced SMC cell–cell adhesion and cellular proliferation. Taken together, our results demonstrate that TNF-α induces the expression of fractalkine and CX3CR1 in rat aortic SMCs and that this induction is mediated by NF-κB activation. We also show that fractalkine induces its own expression, which is mediated by the PI 3-kinase/PDK1/Akt/NIK/IKK/NF-κB signalling pathway. More importantly, fractalkine increased cell–cell adhesion and aortic SMC proliferation, indicating a role in initiation and progression of atherosclerotic vascular disease.
Effect of calcium ions on the irreversible denaturation of a recombinant Bacillus halmapalus alpha-amylase: a calorimetric investigation
Authors: NIELSEN Anders D. , FUGLSANG Claus C. , WESTH Peter
Published: July 2003

The effect of temperature and calcium ions on the denaturation of a recombinant α-amylase from Bacillus halmapalus α-amylase (BHA) has been studied using calorimetry. It was found that thermal inactivation of BHA is irreversible and that calcium ions have a significant effect on stability. Thus an apparent denaturation temperature (Td) of 83 °C in the presence of excess calcium ions was observed, whereas Td decreased to 48 °C when calcium was removed. The difference in thermal stability with and without calcium ions has been used to develop an isothermal titration calorimetric (ITC) procedure that allows simultaneous determination of kinetic parameters and enthalpy changes of the denaturation of calcium-depleted BHA. An activation energy EA of 101 kJ/mol was found for the denaturation of calcium-depleted BHA. The results support a kinetic denaturation mechanism where the calcium-depleted amylase denatures irreversibly at low temperature and if calcium ions are in excess, the amylase denatures irreversibly at high temperatures. The two denaturation reactions are coupled with the calcium-binding equilibrium between calcium-bound and -depleted amylase. A combination of the kinetic denaturation results and calcium-binding constants, determined by isothermal titration calorimetry, has been used to estimate kinetic stability, expressed in terms of the half-life of BHA as a function of temperature and free-calcium-ion concentration. Thus it is estimated that the apparent EA can be increased to approx. 123 kJ/mol by increasing the free-calcium concentration.
Transposon-5 mutagenesis transforms Corynebacterium matruchotii to synthesize novel hybrid fatty acids that functionally replace corynomycolic acid
Authors: TAKAYAMA Kuni , HAYES Barry , VESTLING Martha M. , MASSEY Randall J.
Published: July 2003

Enzymes within the biosynthetic pathway of mycolic acid (C60–C90 α-alkyl,β-hydroxyl fatty acid) in Mycobacterium tuberculosis are attractive targets for developing new anti-tuberculosis drugs. We have turned to the simple model system of Corynebacterium matruchotii to study the terminal steps in the anabolic pathway of a C32 mycolic acid called corynomycolic acid. By transposon-5 mutagenesis, we transformed C. matruchotii into a mutant that is unable to synthesize corynomycolic acid. Instead, it synthesized two related series of novel fatty acids that were released by saponification from the cell wall fraction and from two chloroform/methanol-extractable glycolipids presumed to be analogues of trehalose mono- and di-corynomycolate. By chemical analyses and MS, we determined the general structure of the two series to be 2,4,6,8,10-penta-alkyl decanoic acid for the larger series (C70–C77) and 2,4,6,8-tetra-alkyl octanoic acid for the smaller series (C52–C64), both containing multiple keto groups, hydroxy groups and double bonds. The mutant was temperature-sensitive, aggregated extensively, grew very slowly relative to the wild type, and was resistant to the presence of lysozyme. We suggest that a regulatory protein that normally prevents the transfer of the condensation product back to β-ketoacyl synthase in the corynomycolate synthase system of the wild type was inactivated in the mutant. This will result in multiple Claisen-type condensation and the formation of two similar series of these complex hybrid fatty acids. A similar protein in M. tuberculosis would be an attractive target for new drug discovery.
Relationship between bacterial virulence and nucleotide metabolism: a mutation in the adenylate kinase gene renders Yersinia pestis avirulent
Authors: MUNIER-LEHMANN Hélène , CHENAL-FRANCISQUE Viviane , IONESCU Mihaela , CHRISTOVA Petya , FOULON Jeannine , CARNIEL Elisabeth , BÂRZU Octavian
Published: July 2003

Nucleoside monophosphate kinases (NMPKs) are essential catalysts for bacterial growth and multiplication. These enzymes display high primary sequence identities among members of the family Enterobacteriaceae. Yersinia pestis, the causative agent of plague, belongs to this family. However, it was previously shown that its thymidylate kinase (TMPKyp) exhibits biochemical properties significantly different from those of its Escherichia coli counterpart [Chenal-Francisque, Tourneux, Carniel, Christova, Li de la Sierra, Barzu and Gilles (1999) Eur. J. Biochem. 265, 112–119]. In this work, the adenylate kinase (AK) of Y. pestis (AKyp) was characterized. As with TMPKyp, AKyp displayed a lower thermodynamic stability than other studied AKs. Two mutations in AK (Ser129→Phe and Pro87→Ser), previously shown to induce a thermosensitive growth defect in E. coli, were introduced into AKyp. The recombinant variants had a lower stability than wild-type AKyp and a higher susceptibility to proteolytic digestion. When the Pro87→Ser substitution was introduced into the chromosomal adk gene of Y. pestis, growth of the mutant strain was altered at the non-permissive temperature of 37 °C. In virulence testings, less than 50 colony forming units (CFU) of wild-type Y. pestis killed 100% of the mice upon subcutaneous infection, whereas bacterial loads as high as 1.5×104 CFU of the adk mutant were unable to kill any animals.
Ca2+-independent protein kinase C activity is required for alpha1-adrenergic-receptor-mediated regulation of ribosomal protein S6 kinases in adult cardiomyocytes
Authors: WANG Lijun , ROLFE Mark , PROUD Christopher G.
Published: July 2003

The α1-adrenergic agonist, phenylephrine (PE), exerts hypertrophic effects in the myocardium and activates protein synthesis. Both Ca2+-dependent protein kinase C (PKC, PKCα) and Ca2+-independent PKC isoforms (PKCδ and ε) are detectably expressed in adult rat cardiomyocytes. Stimulation of the α1-adrenergic receptor by PE results in activation of Ca2+-independent PKCs, as demonstrated by translocation of the δ and ε isoenzymes from cytosol to membrane fractions. PE also induces activation of p70 ribosomal protein S6 kinases (S6K1 and 2) in adult cardiomyocytes. We have studied the role of Ca2+-independent PKCs in the regulation of S6K activity by PE. Activation of S6K1/2 by PE was blocked by the broad-spectrum PKC inhibitor bisindolylmaleimide (BIM) I, whereas Gö6976, a compound that only inhibits Ca2+-dependent PKCs, did not inhibit S6K activation. Rottlerin, which selectively inhibits PKCδ, also prevented PE-induced S6K activation. The isoform-specific PKC inhibitors had similar effects on the phosphorylation of eukaryotic initiation factor 4E (eIF4E)-binding protein 1, a translation repressor that, like the S6Ks, lies downstream of the mammalian target of rapamycin (mTOR). Infection of cells with adenoviruses encoding dominant-negative PKCδ or ε inhibited the activation of extracellular-signal-regulated kinase (ERK) by PE, and also inhibited the activation and/or phosphorylation of S6Ks 1 and 2. The PE-induced activation of protein synthesis was abolished by BIM I and markedly attenuated by rottlerin. Our data thus suggest that Ca2+-independent PKC isoforms play an important role in coupling the α1-adrenergic receptor to mTOR signalling and protein synthesis in adult cardiomyocytes.
Modulation of spontaneous transmitter release from the frog neuromuscular junction by interacting intracellular Ca2+ stores: critical role for nicotinic acid-adenine dinucleotide phosphate (NAADP)
Authors: BRAILOIU Eugen , PATEL Sandip , DUN Nae J.
Published: July 2003

Nicotinic acid–adenine dinucleotide phosphate (NAADP) is a recently described potent intracellular Ca2+-mobilizing messenger active in a wide range of diverse cell types. In the present study, we have investigated the interaction of NAADP with other Ca2+-mobilizing messengers in the release of transmitter at the frog neuromuscular junction. We show, for the first time, that NAADP enhances neurosecretion in response to inositol 1,4,5-trisphosphate (IP3), cADP-ribose (cADPR) and sphingosine 1-phosphate (S1P), but not sphingosylphosphorylcholine. Thapsigargin was without effect on transmitter release in response to NAADP, but blocked the responses to subsequent application of IP3, cADPR and S1P and their potentiation by NAADP. Asynchronous neurotransmitter release may therefore involve functional coupling of endoplasmic reticulum Ca2+ stores with distinct Ca2+ stores targeted by NAADP.
Okadaic acid-induced, naringin-sensitive phosphorylation of glycine N-methyltransferase in isolated rat hepatocytes
Authors: MØLLER Michael T. N. , SAMARI Hamid R. , FENGSRUD Monica , STRØMHAUG Per E. , ØSTVOLD Anne C. , SEGLEN Per O.
Published: July 2003

Glycine N-methyltransferase (GNMT) is an abundant cytosolic enzyme that catalyses the methylation of glycine into sarcosine, coupled with conversion of the methyl donor, S-adenosylmethionine (AdoMet), into S-adenosylhomocysteine (AdoHcy). GNMT is believed to play a role in monitoring the AdoMet/AdoHcy ratio, and hence the cellular methylation capacity, but regulation of the enzyme itself is not well understood. In the present study, treatment of isolated rat hepatocytes with the protein phosphatase inhibitor okadaic acid, was found to induce an overphosphorylation of GNMT, as shown by proteomic analysis. The analysis comprised two-dimensional gel electrophoretic separation of 32P-labelled phosphoproteins and identification of individual protein spots by matrix-assisted laser-desorption ionization–time-of-flight mass spectrometry. The identity of GNMT was verified by N-terminal Edman sequencing of tryptic peptides. Chromatographic separation of proteolytic peptides and 32P-labelled amino acids suggested that GNMT was phosphorylated within a limited region, and only at serine residues. GNMT phosphorylation could be suppressed by naringin, an okadaic acid-antagonistic flavonoid. To assess the possible functional role of GNMT phosphorylation, the effect of okadaic acid on hepatocytic AdoMet and AdoHcy levels was examined, using HPLC separation for metabolite analysis. Surprisingly, okadaic acid was found to have no effect on the basal levels of AdoMet or AdoHcy. An accelerated AdoMet–AdoHcy flux, induced by the addition of methionine (1 mM), was likewise unaffected by okadaic acid. 5-Aminoimidazole-4-carboxamide riboside, an activator of the hepatocytic AMP-activated protein kinase, similarly induced GNMT phosphorylation without affecting AdoMet and AdoHcy levels. Activation of cAMP-dependent protein kinase by dibutyryl-cAMP, reported to cause GNMT phosphorylation under cell-free conditions, also had little effect on hepatocytic AdoMet and AdoHcy levels. Phosphorylation of GNMT would thus seem to play no role in regulation of the intracellular AdoMet/AdoHcy ratio, but could be involved in other GNMT functions, such as the binding of folates or aromatic hydrocarbons.