bluuebunny/crossref_metadata_2025 huggingface dataset:
Attention‐deficit/hyperactivity disorder (ADHD): feasibility of linkage analysis in a genetic isolate using extended and multigenerational pedigrees
Authors: Arcos‐Burgos M , Castellanos FX , Lopera F , Pineda D , Palacio JD , Garcia M , Henao GC , Palacio LG , Berg K , Bailey‐Wilson JE , Muenke M
Published: July 2002

Segregation analyses converge in explaining the predisposition to attention‐deficit/hyperactivity disorder (ADHD) as the consequence of a major gene and exclude purely environmental or cultural transmission. As a result of the ADHD phenotype restrictions, collection of extended families or design of linkage studies using families has been extremely difficult and thus currently linkage studies have been performed using only concordant or discordant sib‐pairs rather than large families. On the other hand, intergenerational studies are represented by the transmission disequilibrium test (TDT) using trios. We collected pedigree data on ADHD from the Paisa community from Antioquia, Colombia, a genetic isolate. The goal of this study was to genetically map a putative gene predisposing to ADHD in a set of 27 multigenerational Paisa families. Here we present the results of a power simulation using SIMLINK to detect linkage of ADHD. ADHD was assumed to be a dichotomous trait with incomplete penetrance and a phenocopy rate of 3% in males and 0.2% in females. We simulated cosegregation of the trait and a marker locus in our pedigrees. We assumed Hardy–Weinberg and linkage equilibrium, equally frequent marker alleles and evaluated power at several recombination fractions between the trait and marker loci. Also, the ADHD trait was assumed to be genetically heterogeneous and different functions of age‐dependent penetrance were simulated. We found exceptionally good power to detect linkage (expected LOD > 14 if theta is 0.1 or less), and that the presence of heterogeneity up to 50% does not affect substantially the projected LOD scores even for a theta recombination value of 0.05 (eLOD > 5.87). Having now obtained blood samples and confirmatory interviews in five families (representing 20% of the projected number of families), we performed a new analysis. The expected mean LOD in these five families reached values close to 10 and remained invariant when heterogeneity and different penetrance models were considered. We discuss the relative benefits of using extended and multigenerational families for genetic mapping studies as opposed to using nuclear families, affected sib pairs or sporadic cases which require the collection of over 1000 analytical units to get the same power exhibited by the small number of pedigrees described here.
Familial lethal skeletal dysplasia with cloverleaf skull and multiple anomalies of brain, eye, face and heart: a new autosomal recessive multiple congenital anomalies syndrome
Authors: Sharony R , Kidron D , Amiel A , Fejgin M , Borochowitz ZU
Published: July 2002

We report on a ‘new’ lethal familial short‐limb bone dysplasia associated with multiple anomalies in three sibs born to Arabic‐Muslim consanguineous healthy parents. Clinical abnormalities included short limbs and short hands, cloverleaf skull, frontal bossing, wide anterior fontanel, hypertelorism, bilateral microphthalmia, cataract, low‐set ears, narrow chest, ambiguous genitalia, cardiac ventricular septal defect (VSD) and agenesis of the corpus callosum. Radiological abnormalities included cloverleaf skull, hypoplastic clavicles and scapulae, thin, wavy cupped ribs, flat vertebral bodies with coronal clefting and several unossified vertebral pedicles and hypo‐ossification of the pubic bone. The main changes noted in the long bones consisted of short‐bowed long bones with abnormal metaphyses and unossified epiphyses. Chondro‐osseous morphology documented degenerating chondrocytes with disorganization of the hypertrophied cartilage and short disorganized columns of hypertrophied areas.

An autosomal recessive mode of inheritance seems most likely.
Contact urticaria due to polyethylene gloves
Authors: Sugiura Keiji , Sugiura Mariko , Shiraki Rika , Hayakawa Ritsuko , Shamoto Mikihiro , Sasaki Kazumi , Itoh Akira
Published: July 2002

We report a rare case of contact urticaria due to polyethylene gloves. The patient, a 46‐year‐old cook, had had had chronic urticaria since 1985, and first visited our hospital in June 2000. We began by prescribing antihistamine and antiallergenic drugs for him, but his condition did not improve. From a detailed interview, we established that when he put on polyethylene gloves at work, his condition worsened. We suspected some component of his gloves to be the cause of his symptoms. Prick and scratch tests with a solution extracted from his gloves showed a wheal‐and‐flare reaction at 15 min. We advised him to wear a cotton shirt under his clothes in daily life, and to put on cotton gloves under his polyethylene gloves while at work. Subsequently, the size and the number of wheals were markedly smaller and the subject's symptoms were reduced.
Parthenium dermatitis presenting as photosensitive lichenoid eruption
Authors: Verma Kaushal K. , Sirka C. S. , Ramam M. , Sharma V. K.
Published: July 2002

Parthenium hysterophorus is the commonest cause of airborne contact dermatitis (ABCD) in India. The disease usually manifests as itchy erythematous, papular, papulovesicular and plaque lesions on exposed areas of the body. Rarely, however, the disease may present as actinic reticuloid or photocontact dermatitis. We have observed a different clinical variant of this disease where certain patients with Parthenium dermatitis have presented with discrete, flat, violaceous papules and plaques on exposed areas of the body closely simulating photosensitive lichenoid eruption. We had 8 patients, 6 males and 2 females between 30 and 62 years of age, with itchy, violaceous, papules and plaques on the face, neck, ears, upper chest and dorsa of the hands for 6 months to 6.5 years. Four of these patients had a history of improvement of the lesions up to 30% in winter and aggravation of lesions on exposure to sunlight. There was no personal or family history of atopy. Cutaneous examination in all patients revealed multiple flat, violaceous, mildly erythematous papules and plaques on the forehead, sides and nape of neck, ears, ‘V’ area of the chest, and extensor aspects of the forearms and hands. Skin biopsies from these lesions showed features of chronic non‐specific dermatitis. Patch testing with standardized plant antigens showed a positive patch test reaction to Parthenium hysterophorus in all patients, with a titre of contact hypersensitivity (TCH) varying from undiluted to 1 : 100. We conclude that Parthenium dermatitis may occasionally present with lesions very similar to the lesions of photosensitive lichenoid eruption in morphology and distribution. This clinical presentation of Parthenium dermatitis needs to be recognized to avoid misdiagnosis.
Genetic changes in hypervariable region 1 as a possible mechanism for biochemical responders with interferon therapy
Authors: Shindo M , Hamada K , Oda Y , Okuno T. , Shindo Michiko , Hamada Kazushige , Oda Yoko , Okuno Tadao
Published: July 2002

Aim:

Approximately 10% of chronic hepatitis C patients who were treated with interferon have a sustained normalization of aminotransferase levels after interferon therapy without HCV RNA clearance. We investigated genetic change in hypervariable region 1 (HVR 1) to see if the mutation in this region is responsible for their sustained biochemical response and relapse thereafter. A total of 250 patients with chronic hepatitis C who were treated with interferon were studied for 8 to 11 years post therapy. Of these, 26 were biochemical responders (BR) whose ALT remained normal without viral clearance for more than 6 months after IFN therapy. HVR 1 was examined by direct sequencing of the PCR products, and found that no significant HVR 1 differences were observed in samples obtained pretreatment, posttreatment or during flares from these patients, suggesting that mutations in this region were not responsible for either the persistence of the HCV RNA or for the ALT relapses thereafter.
Effects of oestrogen replacement on steady and pulsatile haemodynamics in ovariectomized rats
Authors: Lam Kwok‐Keung , Hu Cheng‐Tao , Ou Tein‐Yuan , Yen Mao‐Hsiung , Chen Hsing‐I
Published: July 2002

The effects of ovariectomy (Ovx), menopause and oestrogen replacement on the haemodynamics remain controversial. The present study employed the technique of arterial impedance analysis to measure and calculate the steady and pulsatile haemodynamics. The purpose was to determine the haemodynamic consequence of ovariectomy and oestrogen replacement.

Ovariectomy was carried out under anaesthesia on female Sprague Dawley rats aged 9 weeks. Oestrogen (17 β‐estradiol or E2) replacement started 1 week after ovariectomy for 4 weeks. Ovx increased the body weight (BW), while it greatly reduced the uterus weight. Left ventricular weight (LVW) was slightly increased, but LVW/BW ratio was slightly reduced. These changes were reversed after E2 replacement.

Compared to sham group, Ovx with or without E2 replacement did not significantly affect the systolic, mean and diastolic pressure. In Ovx, pulse pressure (PP) and heart rate were significantly increased, while stroke volume and cardiac output were slightly decreased. Total peripheral resistance (TPR) was largely elevated, indicating Ovx induced systemic vasoconstriction. These changes all returned to close normal values (sham group) after E2 replacement, except PP.

Ovx increased the characteristic input impedance (Zc) and pulse wave reflection, while it decreased arterial compliance. E2 treatment reversed these changes, except Zc.

These results demonstrate that Ovx influences both the resistance and Windkessel functions of the artery. E2 treatment effectively reverses most the effects of Ovx both on the steady and pulsatile haemodynamics.

British Journal of Pharmacology (2002) 136, 811–818. doi:10.1038/sj.bjp.0704762
Molecular modelling of the interactions of carbamazepine and a nicotinic receptor involved in the autosomal dominant nocturnal frontal lobe epilepsy
Authors: Ortells M O , Barrantes G E
Published: July 2002

The normal and a mutant (S248F) human neuronal α4β2 nicotinic receptors, and their interaction with the channel blocker carbamazepine (CBZ) have been modelled. The mutant, responsible for the autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE), has an enhanced sensitivity to and a slower recovery from desensitization, a lower conductance, short open times, reduced calcium permeability, and is 3 fold more sensitive to CBZ, a drug used in the treatment of partial epilepsies.

Mutant channel properties are explained by the physicochemical properties of the two Phe248 side chains, including size and cation‐π interaction, and their dynamic behaviour. A defective mechanism of dehydration might be responsible for the reduced calcium influx.

Phe248 residues are the main component of CBZ binding sites in the mutant, while this is not true for Ser248 in the normal receptor.

A higher number of blocking binding sites and a predicted higher affinity found for CBZ in the mutant account for its differential sensitivity to CBZ.

Aromatic–aromatic interactions between CBZ and the two Phe248 account for the difference in affinity, which is at least 12 times higher for the mutant, depending on the method used for calculating Ki.

Normal vs mutant differences in Ki, enhanced by the higher number of blocking binding sites in the mutant, seem excessive compared to the differential sensitivities to CBZ experimentally found. The negative cooperativity suggested by a predicted overlapping of blocking and non‐blocking binding sites gives an explanation, as overlapping is higher in the mutant.

For both types of receptors we found that the carbamyl group of the best blocking conformers of CBZ forms hydrogen bonds with serine residues, which may explain the fundamental role of that moiety for this molecule to act as antiepileptic drug.

British Journal of Pharmacology (2002) 136, 883–895. doi:10.1038/sj.bjp.0704786
Different actions of protein kinase C isoforms α and ε on gastric acid secretion
Authors: Fährmann Michael , Kaufhold Marc , Rieg Timo , Seidler Ursula
Published: July 2002

The phorbol ester TPA, an activator of protein kinase C (PKC), inhibits cholinergic stimulation of gastric acid secretion but increases basal H+ secretion.

Since these contradictory findings suggest the action of different PKC isozymes we analysed the role of calcium‐dependent PKC‐α, and calcium‐independent PKC‐ε in gastric acid secretion.

Inhibition of PKC‐α by the indolocarbazole Gö 6976 revealed that about 28% of carbachol‐induced acid secretion was inhibited by PKC‐α. In the presence of Gö 6976 approximately 64% of the carbachol‐induced signal transduction is mediated by Ca2+/calmodulin‐dependent protein kinase II (CaMKII), and 14% is conveyed by PKC‐ε as deduced from the inhibition with the bisindolylmaleimide Ro 31‐8220.

Inhibition of carbachol‐induced acid secretion by TPA was accompanied by a decrease in CaMKII activity.

The stimulation of basal acid secretion by TPA was biphasic with a peak at a very low concentration (10 pM), resulting in an activation of the calcium‐sensor CaMKII. The activation was determined with a phosphospecific polyclonal antibody against active CaMKII. The TPA‐induced increase of H+ secretion was sensitive to the cell‐permeable Ca2+‐chelator BAPTA/AM, Ro 31‐8220, and the CaMKII‐inhibitor KN‐62, but not to Gö 6976.

Since TPA induced the translocation of PKC‐ε but not of PKC‐α in resting parietal cells, PKC‐ε seems to be at least responsible for an initial elevation of free intracellular calcium to initiate TPA‐induced acid secretion.

Our data indicate the different roles of two PKC isoforms: PKC‐ε activation appears to facilitate cholinergic stimulation of H+‐secretion likely by increasing intracellular calcium. In contrast, PKC‐α activation attenuates acid secretion accompanied by a down‐regulation of CaMKII activity.

British Journal of Pharmacology (2002) 136, 938–946. doi:10.1038/sj.bjp.0704790
Role of αArg145 and βArg263 in the active site of penicillin acylase of Escherichia coli
Authors: ALKEMA Wynand B.L. , PRINS Antoon K. , de VRIES Erik , JANSSEN Dick B.
Published: July 2002

The active site of penicillin acylase of Escherichia coli contains two conserved arginine residues. The function of these arginines, αArg145 and βArg263, was studied by site-directed mutagenesis and kinetic analysis of the mutant enzymes. The mutants αArg145→Leu (αArg145Leu), αArg145Cys and αArg145Lys were normally processed and exported to the periplasm, whereas expression of the mutants βArg263Leu, βArg263Asn and βArg263Lys yielded large amounts of precursor protein in the periplasm, indicating that βArg263 is crucial for efficient processing of the enzyme. Either modification of both arginine residues by 2,3-butanedione or replacement by site-directed mutagenesis yielded enzymes with a decreased specificity (kcat/Km) for 2-nitro-5-[(phenylacetyl)amino]benzoic acid, indicating that both residues are important in catalysis. Compared with the wild type, the αArg145 mutants exhibited a 3–6-fold-increased preference for 6-aminopenicillanic acid as the deacylating nucleophile compared with water. Analysis of the steady-state parameters of these mutants for the hydrolysis of penicillin G and phenylacetamide indicated that destabilization of the Michaelis—Menten complex accounts for the improved activity with β-lactam substrates. Analysis of pH—activity profiles of wild-type enzyme and the βArg263Lys mutant showed that βArg263 has to be positively charged for catalysis, but is not involved in substrate binding. The results provide an insight into the catalytic mechanism of penicillin acylase, in which αArg145 is involved in binding of β-lactam substrates and βArg263 is important both for stabilizing the transition state in the reaction and for correct processing of the precursor protein.
Perturbation of free oligosaccharide trafficking in endoplasmic reticulum glucosidase I-deficient and castanospermine-treated cells
Authors: DURRANT Christelle , MOORE Stuart E.H.
Published: July 2002

Free oligosaccharides (FOS) are generated both in the endoplasmic reticulum (ER) and in the cytosol during glycoprotein biosynthesis. ER lumenal FOS possessing the di-N-acetylchitobiose moiety at their reducing termini (FOSGN2) are exported into the cytosol where they, along with their cytosolically generated counterparts possessing a single N-acetylglucosamine residue at their reducing termini (FOSGN1), are trimmed in order to be imported into lysosomes for final degradation. Both the ER and lysosomal FOS transport processes are unable to translocate triglucosylated FOS across membranes. In the present study, we have examined FOS trafficking in HepG2 cells treated with the glucosidase inhibitor castanospermine. We have shown that triglucosylated FOSGN2 generated in the ER are transported to the Golgi apparatus where they are deglucosylated by endomannosidase and acquire complex, sialic acid-containing structures before being secreted into the extracellular space by a Brefeldin A-sensitive pathway. FOSGN2 are also secreted from glucosidase I-deficient Lec23 cells and from the castanospermine-treated parental Chinese-hamster ovary cell line. Despite the secretion of FOSGN2 from Lec23 cells, we noted a transient intracellular accumulation (60nmol/g cells) of triglucosylated FOSGN1 in these cells. Finally, in glucosidase I-compromised cells, FOS trafficking was severely perturbed leading to both the secretion of FOSGN2 into the extracellular space and a growth-dependent pile up of triglucosylated FOSGN1 in the cytosol. The possibility that these abnormalities contributed to the severe and rapidly progressive pathology in a patient with congenital disorders of glycosylation type IIb (glucosidase I deficiency) is discussed.
An intron-containing glycoside hydrolase family 9 cellulase gene encodes the dominant 90 kDa component of the cellulosome of the anaerobic fungus Piromyces sp. strain E2
Authors: STEENBAKKERS Peter J.M. , UBHAYASEKERA Wimal , GOOSSEN Harry J.A.M. , van LIEROP Erik M.H.M. , van der DRIFT Chris , VOGELS Godfried D. , MOWBRAY Sherry L. , den CAMP Huub J.M. Op
Published: July 2002

The cellulosome produced by Piromyces sp. strain E2 during growth on filter paper was purified by using an optimized cellulose-affinity method consisting of steps of EDTA washing of the cellulose-bound protein followed by elution with water. Three dominant proteins were identified in the cellulosome preparation, with molecular masses of 55, 80 and 90kDa. Treatment of cellulose-bound cellulosome with a number of denaturing agents was also tested. Incubation with 0.5% (w/v) SDS or 8M urea released most cellulosomal proteins, while leaving the greater fraction of the 80, 90 and 170kDa components. To investigate the major 90kDa cellulosome protein further, the corresponding gene, cel9A, was isolated, using immunoscreening and N-terminal sequencing. Inspection of the cel9A genomic organization revealed the presence of four introns, allowing the construction of a consensus for introns in anaerobic fungi. The 2800bp cDNA clone contained an open reading frame of 2334bp encoding a 757-residue extracellular protein. Cel9A includes a 445-residue glycoside hydrolase family 9 catalytic domain, and so is the first fungal representative of this large family. Both modelling of the catalytic domain as well as the activity measured with low level expression in Escherichia coli indicated that Cel9A is an endoglucanase. The catalytic domain is succeeded by a putative β-sheet module of 160 amino acids with unknown function, followed by a threonine-rich linker and three fungal docking domains. Homology modelling of the Cel9A dockerins suggested that the cysteine residues present are all involved in disulphide bridges. The results presented here are used to discuss evolution of glycoside hydrolase family 9 enzymes.
Transport of phosphatidylserine via MDR1 (multidrug resistance 1)P-glycoprotein in a human gastric carcinoma cell line
Authors: POHL Antje , LAGE Hermann , MÜLLER Peter , POMORSKI Thomas , HERRMANN Andreas
Published: July 2002

The ATP-binding cassette transporter multidrug resistance 1 P-glycoprotein (MDR1 Pgp) has been implicated with the transport of lipids from the inner to the outer leaflet of the plasma membrane. While this has been unambigously shown for the fluorescent lipid analogues [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoyl (C6-NBD)-phosphatidylcholine, -phosphatidylethanolamine, -sphingomyelin and -glucosylceramide, by using a novel approach we have now found significantly increased outward transport also for C6-NBD-phosphatidylserine (C6-NBD-PS) in EPG85-257 human gastric carcinoma cells overexpressing MDR1 (coding for MDR1 Pgp). The increased transport of C6-NBD-PS is mediated by MDR1 Pgp, shown by transport reduction nearly to the level of controls in the presence of MDR1 Pgp inhibitors [PSC 833, cyclosporin A and dexniguldipine hydrochloride (Dex)]. Addition of MK 571, a specific inhibitor of the MDR protein MRP1, does not decrease transport in either of the two cell lines. The plasma-membrane association of FITC-annexin V, a fluorescent protein conjugate binding PS, is significantly increased in MDR1-overexpressing cells as compared with controls, and can be reduced by an MDR1 Pgp inhibitor. This suggests that MDR1 Pgp transports endogenous PS, the lipid exhibiting the most pronounced transverse asymmetry in the plasma membrane.
In vitro selection of fibronectin gain-of-function mutations
Authors: TANI Patricia H. , LOFTUS Joseph C. , BOWDITCH Ron D.
Published: July 2002

Directed protein evolution, which employs a combination of random mutagenesis, phage display, and in vitro selection, was used to identify second-site suppressors of the fibronectin (Fn) cell binding domain mutation Asp1495Ala (RGA). The mutations in the Fn 9th (3fn9) and 10th (3fn10) type III repeats obtained after selection on purified integrins αIIbβ3(D119Y) and α5β1 are reported. The 3fn9–10(D1495A) phage with substitution mutations at Asp1418, which is located within the linker region between 3fn9 and 3fn10, enhanced binding to the integrins αIIbβ3 and α5β1, but not αvβ3. The substitution mutations identified at residue Asp1418 were introduced into the native recombinant 3fn9–10 sequence and found to augment binding to αIIbβ3, demonstrating that the observed gain-of-function phenotype was independent of the multivalent character of the phage. These results support the following conclusions. First, regions of Fn in addition to the RGD loop are in close proximity to αIIbβ3 and α5β1 and are capable of participating in the binding to these integrins. Secondly, the conformational relationship between the 3fn9 and 3fn10 modules may be an important factor in the binding of Fn to these two integrins. Thirdly, other altered properties of Fn-integrin interactions, such as integrin specificity, may also be selected. This is the first description of Fn mutations that augment binding to integrins. The ability to select for particular phenotypes in vitro and the subsequent characterization of these mutations should further our understanding of the molecular details involved in the association of integrins and their ligands. Additionally, these higher-affinity 3fn9–10 ligands provide a starting point for further in vitro evolution and engineering of integrin-specific modules.
Metabolism of the novel Ca2+-mobilizing messenger nicotinic acid–adenine dinucleotide phosphate via a 2′-specific Ca2+-dependent phosphatase
Authors: BERRIDGE Georgina , CRAMER Rainer , GALIONE Antony , PATEL Sandip
Published: July 2002

Nicotinic acid—adenine dinucleotide phosphate (NAADP) is a newly described Ca2+-mobilizing nucleotide that appears to target intracellular Ca2+-release channels distinct from those sensitive to inositol trisphosphate or ryanodine/cyclic ADP-ribose. Little, however, is known concerning the regulation of cellular NAADP levels. In the present study, we have characterized the metabolism of NAADP by brain membranes. From HPLC and MS analyses we show that loss of NAADP was associated with the appearance of a major product that is likely to be nicotinic acid—adenine dinucleotide (NAAD), the dephosphorylated form of NAADP. Dephosphorylation of NAADP, but not 3′-NAADP, was dramatically attenuated by Ca2+ chelators and stimulated by Ca2+ over a physiological range in a calmodulin-insensitive manner. In contrast, NADP was metabolized predominantly to ADP-ribose phosphate via glycohydrolase activity, although slower Ca2+-dependent dephosphorylation of both NADP and 2′-AMP could also be demonstrated. This is the first report describing a Ca2+-regulated 2′-specific phosphatase which is probably the major pathway for the inactivation of NAADP in brain. Our data provide a potential feedback mechanism for limiting NAADP-induced Ca2+ release within cells through stimulation of NAADP metabolism by Ca2+ and strongly support a signalling role for this novel nucleotide in the brain.
Activation of glucokinase gene expression by hepatic nuclear factor 4α in primary hepatocytes
Authors: ROTH Ulrike , JUNGERMANN Kurt , KIETZMANN Thomas
Published: July 2002

Glucokinase (GK) is a key enzyme for glucose utilization in liver and shows a higher expression in the perivenous zone. In primary rat hepatocytes, the GK gene expression was activated by HNF (hepatic nuclear factor)-4α via the sequence −52/−39 of the GK promoter. Venous pO2 enhanced HNF-4 levels and HNF-4 binding to the GK—HNF-4 element. Thus, HNF-4α could play the role of a regulator for zonated GK expression.
Lipid modification of proteins in Archaea: attachment of a mevalonic acid-based lipid moiety to the surface-layer glycoprotein of Haloferax volcanii follows protein translocation
Authors: KONRAD Zvia , EICHLER Jerry
Published: July 2002

Once the newly synthesized surface (S)-layer glycoprotein of the halophilic archaeaon Haloferax volcanii has traversed the plasma membrane, the protein undergoes a membrane-related, Mg2+-dependent maturation event, revealed as an increase in the apparent molecular mass and hydrophobicity of the protein. To test whether lipid modification of the S-layer glycoprotein could explain these observations, H. volcanii cells were incubated with a radiolabelled precursor of isoprene, [3H]mevalonic acid. In Archaea, isoprenoids serve as the major hydrophobic component of archaeal membrane lipids and have been shown to modify other haloarchaeal S-layer glycoproteins, although little is known of the mechanism, site or purpose of such modification. In the present study we report that the H. volcanii S-layer glycoprotein is modified by a derivative of mevalonic acid and that maturation of the protein was prevented upon treatment with mevinolin (lovastatin), an inhibitor of mevalonic acid biosynthesis. These findings suggest that lipid modification of S-layer glycoproteins is a general property of halophilic archaea and, like S-layer glycoprotein glycosylation, lipid-modification of the S-layer glycoproteins takes place on the external cell surface, i.e. following protein translocation across the membrane.
Detection of drug use in a methadone maintenance clinic: sweat patches versus urine testing
Authors: Taylor John R. , Watson Ian D. , Tames Frank J. , Lowe Derek
Published: July 2002

Aims. To evaluate the novel use of sweat patches in outpatients attending a methadone maintenance clinic. Design. Assessment of inter‐patch reliability and validity of patch results compared to urine tests at the start and end of the patch period. Semi‐structured questionnaire on patients' opinion of the patches. Randomized cross‐over trial comparing illicit drug use during the week that the patch was worn with a control period. Setting. Methadone maintenance outpatient clinic in a deprived urban area. Participants. Forty‐eight patients with a diagnosis of opiate addiction prescribed methadone for a median of 5 years. Measurements. Analysis of urine and patch tests by standard methods for methadone, opiates, morphine (heroin metabolite) and benzoylecgonine (cocaine metabolite). Findings. There was good inter‐patch reliability between arm and side patches for methadone (all positive), opiates ( k = 0.8) and morphine ( k = 1.0) but only moderate agreement for benzoylecgonine ( k = 0.49). There was good agreement between the sweat patches and urine tests for methadone (all positive), opiates ( k = 1.00) and morphine ( k = 0.8), but again only moderate agreement for benzoylecgonine ( k = 0.50). The patches were well tolerated and the main side effect was minor irritation. The majority of men preferred a urine test while more women preferred a sweat patch. There was no evidence of reduced use of illicit drugs during the period that the patch was worn . Conclusion. Sweat patches are reliable and give valid results for patients on maintenance methadone. There are few side effects and it is practical to use them in busy outpatient clinics. They are preferred by some patients but there was no evidence that they altered behaviour.
A comparison of the prevalence of HIV infection and injecting risk behaviour in urban and rural samples in Scotland
Authors: Haw Sally , Higgins Kathryn
Published: July 2002

Aims. To compare the prevalence of HIV infection and injecting risk behaviour among injecting drug users from urban and rural areas. Designs. Two samples of injecting drug users were recruited using a multi‐site sampling strategy. Respondents were first interviewed by trained interviewers then specimens of saliva were collected for anonymous testing for antibodies to HIV. Setting. Respondents were recruited from drug treatment services and street sites. Participants. Respondents were eligible for inclusion in the study if they had "ever" injected a drug and were currently using drugs. Measurements. Measurements taken included self‐reported patterns of drug use and injecting risk behaviour. Specimens of saliva were tested for the presence of HIV infection using GACELISA with reactive specimens confirmed by Western blot analysis. Findings. Our data indicate that there are two separate populations which are geographically discrete but broadly similar in profile and current injecting risk behaviour. The prevalence of HIV infection among IDUs from Dundee city was found to be 26.8% (95% CI, 20.2%‐33.0%) compared with 3.7% (95% CI, 0.13%‐15.8%) for IDUs from rural Tayside. This marked difference in prevalence of HIV infection we attribute to a high level of injecting risk among urban IDUs between 1980 and 1984, limited migration from the urban epicentre of infection and a reluctance among rural IDUs to share with IDUs outside their immediate social and kinship networks. Conclusions. Although current levels of injecting risk behaviour are similar in our urban and rural samples, rural IDUs may be less likely to contract HIV from their fellow injectors because of the lower prevalence of HIV infection and more closed sharing networks within the rural population. The implications of this for the development and expansion of drug services are considered.
Individual case studies in clinical research
Authors: Charlton Bruce G. , Walston Florence
Published: July 2002

Case studies have acquired an unmerited reputation as being anecdotal, unscientific and intrinsically inferior to group studies. The subsequent disregarding of individual patients as the focus of investigation has led to the neglect of an extremely useful clinical research method, and has probably impaired the pace of therapeutic innovation. The purpose of this paper is to clarify the scope and nature of case studies and promote their rehabilitation. Case studies can, in principle, be used to test any theory that has implications for individual patients. There are two crucial methodological stages. The first is to identify scientifically plausible general theories and derive from them specific hypotheses or models of sufficient precision to have implications for individual cases. The second is to test these hypothetical models against ‘pure’ cases, selected so as to exclude interfering variables. There are two main types of case study — those made by serendipity (unplanned case observations which challenge an implicit theoretical framework); and formal case studies (designed prospectively to collect pure cases to test a prior hypothesis). The difference between serendipity and planned case studies roughly corresponds to the difference between surveillance and screening. A worked‐example of a formal case study is described here in order to illustrate the method. Individual case studies deserve fresh consideration by researchers, since they are a clinician‐friendly method with a unique potential for incorporation into routine practice.
Staphylococcus aureus peptidoglycan ameliorates cyclophosphamide‐induced impairment of wound healing
Authors: Imegwu Obi , Chang Tsueng H. , Steinberg Jacob J. , Levenson Stanley M.
Published: July 2002

Cyclophosphamide given systemically to rats leads to impaired wound healing, characterized by decreases in the inflammatory reaction, fibroplasia, neovascularization, reparative collagen accumulation, and wound breaking strength. In contrast, the local application of Staphylococcus aureus peptidoglycan at the time of wounding increases all of these processes in normal rats. Accordingly, we hypothesized that inoculation of S. aureus peptidoglycan into wounds of cyclophosphamide‐treated rats would ameliorate the otherwise impaired healing. Dorsal bilateral skin incisions and subcutaneous implantation of polyvinyl alcohol sponges (two on each side) were performed on male Sprague‐Dawley rats receiving either saline or cyclophosphamide (24 mg/kg) intraperitoneally at the time of operation, on postoperative days 1, 2, 3, 4 (for rats killed on postoperative day 7), and also on day 8 (for rats killed on postoperative day 14). The incisions on one side were inoculated at the time of closure with 0.2 ml of saline solution, and the incisions on the other side with 6 mg S. aureus peptidoglycan in 0.2 ml saline solution (860 µg/cm incision). The sponges were instilled with 0.1 ml saline solution on the saline solution‐instilled incision side or with S. aureus peptidoglycan 0.5 mg/sponge) in 0.1 ml saline solution on the other side. In control rats receiving saline solution intraperitoneally, incisions treated with S. aureus peptidoglycan were significantly stronger than saline solution‐treated incisions by a factor of 1.8 at 1 week (p < 0.001); at 2 weeks the increase was small and not significant. Cardiac blood leukocytes and platelets fell markedly (90%) in cyclophosphamide‐ treated rats, and there was a decrease in wound breaking strength of their saline‐treated incisions at both 7 and 14 days compared with saline solution‐treated incisions of control rats. S. aureus peptidoglycan treatment of the wounds completely prevented this effect at 7 days, and partially at 14 days. Polyvinyl alcohol sponge reparative tissue hydroxyproline, 7 days after surgery, was decreased in cyclophosphamide‐treated rats; this was completely prevented by S. aureus peptidoglycan treatment of the sponges. Histologically, the inflammatory response to the wounding, influx of macrophages and fibroblasts, angiogenesis, and collagen accumulation were all reduced at day 7 and 14 after surgery in the sponge reparative tissue of cyclophosphamide‐ treated rats; this was prevented by S. aureus peptidoglycan treatment of the sponges. In conclusion, a single local application of S. aureus peptidoglycan ameliorates cyclophosphamide‐impaired wound healing.
In vitro studies on the antioxidant and growth stimulatory activities of a polyphenolic extract from Cudrania cochinchinensis used in the treatment of wounds in Vietnam
Authors: Van Hien Tran , Hughes Margaret A. , Cherry George W. C.
Published: July 2002

The leaves of Cudrania cochinchinensis, a Vietnamese folk remedy, have been suggested as a beneficial agent for wound healing. Animal studies and clinical observations in Vietnam have shown positive wound healing activity. We studied the effects of a polyphenolic extract from the plant on the proliferation of cells in culture and their response to oxidative damage by hydrogen peroxide. Fibroblasts were incubated with different concentrations of the extract in 0.4% fetal calf serum, and proliferation was monitored by a colorimetric assay. Cell damage was induced by exposure to hydrogen peroxide 7 × 10−5 mol/L for 3 hours. The same colorimetric assay was used to assess cell damage and the protective effect of the extract against oxidative damage. The extract at low concentrations (0.1 to 5 µg/ml) had a stimulatory effect on fibroblast growth. The effect was significant by 7 days after the addition of the extract and was strongest at a concentration of 1 µg/ml of extract (p≤ 0.01). The extract at concentrations of 5 or 50 µg/ml protected fibroblasts and endothelial cells against hydrogen peroxide‐induced damage. Pretreatment with the extract or exposure to extract simultaneously with hydrogen peroxide gave only partial protection against oxidative damage, whereas a combination of the two treatments gave complete protection (p≤ 0.005). Stimulation of fibroblast proliferation and protection of cells against destruction by inflammatory mediators may be ways in which the polyphenolic substances from the plant, Cudrania cochinchinensis, contribute to wound healing.
Bioactive factors affect proliferation and phenotypic expression in progenitor and pluripotent stem cells
Authors: Young Henry E. , Wright Robert P. , Mancini Matthew L. , Lucas Paul A. , Reagan Charles R. , Black Asa C.
Published: July 2002

Progenitor and pluripotent stem cells reside within connective tissue compartments. They are also present in granulation tissue. This study examined the effects of treating these two cell populations with eight bioactive factors. Cells were assayed for DNA content as a measure of proliferation and for tissue‐specific phenotypic markers as measures of lineage progression and lineage commitment. Platelet‐derived endothelial growth factor and insulin‐like growth factor‐II did not induce proliferation in either population. However, dexamethasone, insulin, insulin‐like growth factor‐I, muscle morphogenetic protein, platelet‐derived growth factor‐AA, and platelet‐derived growth factor‐BB stimulated proliferation in one or both cell populations. Platelet‐derived growth factor‐BB was the most potent stimulator of proliferation in either population. Phenotypic expression markers were induced in the progenitor cells by insulin, insulin‐like growth factor‐I, insulin‐like growth factor‐II, dexamethasone, and muscle morphogenetic protein. However, only dexamethasone and muscle morphogenetic protein induced phenotypic expression markers in the pluripotent cells. Platelet‐derived endothelial cell growth factor, platelet‐derived growth factor‐AA, and platelet‐derived growth factor‐BB did not induce phenotypic expression markers in progenitor or pluripotent cells. This study suggests the potential for using progenitor and pluripotent cells as an in vitro model to ascertain the effects of various bioactive factors on stem cells potentially involved in tissue maintenance and repair.
Ultrasound therapy in chronic leg ulceration: a meta‐analysis
Authors: Johannsen Finn , Gam Arne Nyholm , Karlsmark Tonny
Published: July 2002

We reviewed all articles on ultrasound therapy published since 1950 to assess the evidence for an effect of this therapy in the treatment of chronic leg ulcers. Fourteen studies concerning ultrasound therapy, chronic leg ulcers, and wound healing were found. The six studies that fulfilled inclusion criteria for a randomized controlled trial were generally found lacking with respect to description of ulcer etiology‐treated area, ultrasound head area, control of ultrasound apparatus, sham ultrasound apparatus, and follow‐up evaluations. However, all six studies presented their data in a way that made pooling possible with respect to a decrease in ulcer area as a percent of the starting area. A standardized effect size was applied and gave evidence for a significant effect of ultrasound, showing a 16.9% (Cl95: 6.3% to 27.5%, p = 0.011) mean difference in healing after 4 weeks of treatment and a 14.5% (Cl95: 6.6% to 22.3%, p = 0.005) mean difference after 8 weeks of treatment compared with control treatment. In three studies, the number of healed ulcers was assessed, and pooling showed a nonsignificant (p = 0.06) therapeutic gain of 15% (Cl95: 1% to 30%). This analysis would suggest that ultrasound has the best effect being delivered in low doses around the edge of the ulcer, but further studies are required to confirm this possible effect and to evaluate a possible dose‐response relationship.
Dynamics of the matrix metalloproteinases MMP‐1 and MMP‐8 in acute open human dermal wounds
Authors: Nwomeh Benedict C. , Liang Hui‐Xiu , Diegelmann Robert F. , Cohen I. Kelman , Yager Dorne R.
Published: July 2002

Extracellular matrix degradation during dermal wound healing involves multiple levels of regulation by several enzymes of the matrix metalloproteinase family, their activators, and their inhibitors. This study tested the hypothesis that a temporal pattern of interstitial collagenase appearance occurs during normal dermal wound healing, with matrix metalloproteinase‐8 originating from neutrophils appearing earlier than the fibroblast‐derived matrix metalloproteinase‐1. Open (6 mm) full‐thickness dermal wounds, which were covered by transparent occlusive dressings, were made in healthy human volunteers (n = 20). Wound fluids from under the dressings were collected daily through day 8, and wound tissue biopsies were obtained on days 0, 2, 4, 14, and 28. Collagenases were extracted from homogenized tissue biopsies for analysis. Samples were analyzed for the presence of matrix metalloproteinase‐1 and matrix metalloproteinase‐8 by enzyme‐linked immunosorbent assays and by collagenase activity assays using purified types I and III collagen as substrates. In addition, tissue inhibitor of metalloproteinases‐1 and matrix metalloproteinase‐1/tissue inhibitor of metalloproteinases‐1 complexes in wound fluids were measured. Results showed a differential temporal pattern of matrix metalloproteinase‐1 and matrix metalloproteinase‐8 in wound exudates with peak levels of matrix metalloproteinase‐8 occurring on day 4 and matrix metalloproteinase‐1 peak levels on day 7. Maximal levels in tissue for both enzymes occurred on day 2. At all time points examined, levels of matrix metalloproteinase‐8 were statistically higher than matrix metalloproteinase‐1 (100‐fold to 200‐fold). Tissue inhibitor of metalloproteinases‐1 levels declined over time, whereas levels of matrix metalloproteinase‐1/tissue inhibitor of metalloproteinase‐1 complexes increased to a plateau on day 7. This study provides new evidence implicating matrix metalloproteinase‐8 as a major collagenase in healing human dermal wounds. It also shows a temporal pattern in the appearance of the matrix metalloproteinases, tissue inhibitor of metalloproteinase‐1, and matrix metalloproteinase‐1/tissue inhibitor of metalloproteinases‐1 complexes, suggesting that a tightly regulated pattern of expression of matrix metalloproteinases and their inhibitors is essential for normal wound healing in humans.
Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of L‐lactate
Authors: Hehenberger Karin , Heilborn Johan D. , Brismar Kerstin , Hansson Anders
Published: July 2002

Diabetes is accompanied by delayed wound healing and insufficient granulation tissue formation, possibly because of a defect in fibroblast function. We have previously shown that fibroblasts derived from chronic diabetic foot ulcers have lower proliferation compared with those from uninjured skin. The aim of this study was to investigate possible mechanisms explaining the impaired fibroblast proliferation observed in fibroblasts from non‐insulin‐dependent diabetes mellitus chronic wounds and normal fibroblasts cultured in high glucose. Fibroblasts from two groups of patients were studied: nondiabetic patients with chronic venous stasis ulcers and non‐insulin‐dependent diabetes mellitus patients with chronic diabetic wounds. Biopsies from both uninjured skin and wounds were taken from the same patients to serve as sources of fibroblasts. A fluorometric method was used to determine DNA content, and a spectrophotometric lactate oxidase method was used for lactate level analysis. We found a dose‐dependent inhibition of normal fibroblast proliferation when adding conditioned media from non‐insulin‐dependent diabetes mellitus wound fibroblasts. The conditioned medium, from these cells showed elevated L‐lactate levels, 6.3 ± 0.7 mmol/L, compared with media derived from nondiabetic, 2.1 ± 0.3 mmol/L (p < 0.01), and diabetic uninjured skin fibroblasts, 3.5 ± 0.6 mmol/L, and from chronic nondiabetic wound fibroblasts 2.9 ± 0.3 mmol/L. Addition of 6 mmol/L L‐lactate to uninjured normal fibroblasts resulted in decreased DNA content (58 ± 7%, p < 0.01). Previously we have shown that high glucose concentrations inhibit fibroblast proliferation and induce growth factor resistance. When increasing the amount of D‐glucose in the media, L‐lactate levels increased in all cell types. When the uninjured normal cells were treated with β‐hydroxybutyrate, the total DNA content decreased by 42 ± 5% (p < 0.05), with no significant increase in the L‐lactate levels. These observations indicate that L‐lactate production may be of importance for fibroblast proliferation in vitro and may play a role in fibroblast proliferation in vivo.
Sympathetic denervation impairs epidermal healing in cutaneous wounds
Authors: Kim Lincoln R. , Whelpdale Keith , Zurowski Mateusz , Pomeranz Bruce
Published: July 2002

The involvement of peripheral nerves in dermal wound healing, particularly in the inflammatory response has not been extensively studied. Therefore, this study was performed to examine the role of peripheral nerves in the healing of rat skin linear incisions. We report that chemical sympathectomy with 6‐hydroxydopamine significantly impaired wound healing as measured on days 7, 11, and 14 postsurgery (by day 14, 48% of the sympathectomized rats were healed in contrast with 84% of the controls; p = 0.0104). In contrast, neonatal capsaicin treatment, which predominantly destroys sensory afferents, had no effect on wound healing (p > 0.05 on all days). These results support the hypothesis that sympathetic efferents are important for wound healing. Unlike previous research, which showed that peripheral nerves influence ischemic skin flaps, we are the first to demonstrate a role for peripheral nerves in the healing of skin incisions. Because inflammation is an important step in cutaneous wound healing, we propose that a reduction of neurogenic inflammation caused by sympathectomy may explain the impaired wound healing that we observed in our study.