bluuebunny/crossref_metadata_2025 huggingface dataset:
Severe community‐acquired pneumonia, shock and multiorgan dysfunction syndrome caused by Chlamydia pneumoniae
Authors: MARIK P. E. , IGLESIAS J.
Published: November 2003

Chlamydia pneumoniae has been increasingly recognized as an important aetiological agent in community‐acquired pneumonia. A case of severe community‐acquired pneumonia and multifunction dysfunction syndrome resulting from Chlamydia pneumoniae infection in a previously healthy adult is presented.
The factor VR506Q mutation causing APC resistance is highly prevalent amongst unselected outpatients with clinically suspected deep venous thrombosis
Authors: SVENSSON P. J. , ZÖLLER B. , MATTIASSON I. , DAHLBÄCK B.
Published: November 2003

Objective. Resistance to activated protein C (APC resistance), caused by a single point mutation in the factor V gene (FV:R506Q), is a major risk factor for venous thrombosis. As the significance of this mutation among unselected outpatients with deep‐vein thrombosis (DVT) is not established, we have studied its prevalence among consecutive outpatients attending the emergency room due to a clinically suspected DVT.

Design, setting and subjects. The FV:R506Q mutation was determined in 223 consecutive Swedish outpatients with clinically suspected DVT, and in 288 healthy controls. Using phlebography, the patients were classified as DVT‐positive or DVT‐negative.

Main outcome measure. The prevalence of FV:R506Q mutation.

Results. The prevalence of the FV∶R506Q mutation was 28% (28/99) in the DVT‐positive subgroup (relative risk: 3.1; 95% CI: 1.7–5.5), and 23% (28/124) in the DVT negative subgroup (relative risk: 2.0; 95% CI: 1.1–3.6), as compared to 11% (32/288) in the control group. In the DVT‐positive subgroup, the FV∶R506Q mutation was most common among younger patients with primary thrombosis (47%) and least common among older patients with secondary thrombosis (19%). The high prevalence of FV:R506Q mutation among DVT‐negative patients was associated with a high frequency of previous venous thrombosis. Thus, 46% (13/28) of the DVT‐negative FV∶R506Q carriers had a history of thrombosis, compared with only 22% (21/96) of the DVT‐negative patients lacking the mutation (P=0.01).

Conclusion. To sum up, the FV:R506Q mutation is present in more than a quarter of Swedish DVT‐positive outpatients with clinically suspected DVT, indicating that APC‐resistance is a major thrombotic risk factor contributing to the high incidence of venous thrombosis in Sweden.
Haemophilia prophylaxis in young patients–a long‐term follow‐up
Authors: LÖFQVIST THOMAS , NILSSON INGA MARIE , BERNTORP ERIK , PETTERSSON HOLGER
Published: November 2003

Objectives. To review long‐term prophylactic factor treatment in young patients with severe haemophilia A and B, focusing on the orthopaedic and radiological outcome.

Design. We received 34 patients with severe haemophilia A (n=29) and B (n=5), aged 7–22 years. Age at start of treatment was 1–4.5 years. Dosages of factor concentrate (F VIII and F IX, respectively) were 25–40 IU/kg body weight, three times a week for haemophilia A and twice a week for haemophilia B. The patients had been checked annually over a 5‐year period (1990–95). Orthopaedic and radiological joint scores were evaluated according to recommendations by the World Federation of Haemophilia.

Setting. All results were obtained at the Department for Coagulation Disorders, University of Lund, Malmö University Hospital, Malmö, Sweden.

Results. Orthopaedic and radiological joint scores were found to have remained unchanged during follow‐up in almost all patients and to be still zero (i.e. no unaffected joints) in 79% (n=27) of the patients.

Conclusion. There is a growing international consensus haemophilic arthropathy can be prevented by administering early high‐dose prophylaxis. The results of the present investigation strongly support this opinion.
Chinese herbs and warfarin potentiation by ‘Danshen’
Authors: YU CHEUK M. , CHAN JULIANA C. N. , SANDERSON JOHN E.
Published: November 2003

Drug interactions with warfarin can be dangerous and although common drug interactions are now well recognized those with Chinese herbs are not widely appreciated. ‘Danshen’ is a herbal medicine often used for various complaints, particularly cardiovascular, in the Chinese community. We report a case of danshen‐induced overcoagulation with severe and dangerous abnormalities of clotting in a patient with rheumatic heart disease.
Do drug costs affect physicians' prescription decisions?
Authors: HART J. , SALMAN H. , BERGMAN M. , NEUMAN V. , RUDNIKI C. , GILENBERG D. , MATALON A. , DJALDETTI M.
Published: November 2003

Objective. To study the impact of the cost of pharmaceuticals on physicians' decisions about drug prescription.

Design. A simulation protocol for the treatment of two patients, one with mild and the other with a severe form of urinary tract infection (UTI), was designed. Thirty family physicians in outpatient clinics and 30 physicians in the internal medicine wards of a Community Hospital participated in the project. They had to prescribe treatment for the patients twice: at phase I, when the drug cost was unknown, and at phase II, after 2 months, when the price of the drugs was brought to their attention. The physicians selected the medication from a list of drugs commonly used for the treatment of UTIs.

Results. Analysis of the findings indicates that an awareness of drug costs affects prescription decisions among physicians in hospital wards, whereas family physicians showed a preference for less expensive drugs even before they were informed about drug costs. An extrapolation of the results shows that knowledge about the cost of the drugs usually administered for treatment of UTI, could save at least IS 112883 ($34207) a month to Kupat Holim Klalit (KHK) the health insurance institution to which the outpatient clinics and the hospital belong.

Conclusions. When economic aspects of healthcare are considered, information on drug costs may be an important factor in physicians' decision‐making processes and for saving pharmaceutical expenses.
Multiple cerebral infarctions in a patient with refractory idiopathic thrombocytopenic purpura
Authors: OTSUKI T. , FUNAKAWA T. , SUGIHARA T. , KANZAKI A. , WADA H. , INOUE T. , YAMADA O. , UEKI A. , YAWATA Y.
Published: November 2003

Multiple cerebral infarctions were observed in a patient with refractory idiopathic thrombocytopenic purpura who was positive for lupus anticoagulant (LAC) when her platelet counts were 2000 μL−1. It is suspected that LAC may have played an important role in the pathogenesis of this patient's cerebral infarctions, although she had severe thrombocytopenia.
The clinical significance of pulmonary embolism: uncertainties and implications for treatment – a debate
Authors: EGERMAYER P. , TOWN G. I.
Published: November 2003

Pulmonary embolism and deep vein thrombosis are different aspects of a single problem, thromboembolic disease, which is frequently asymptomatic. The incidence and clinical significance of thromboembolic disease is uncertain. The mortality of untreated pulmonary embolism is unknown but may be much lower than is generally thought. Investigation of the lower limbs is desirable in most cases, even when there are no symptoms in the legs, in order to evaluate the prognosis and the need for anticoagulant therapy. Not all cases of pulmonary embolism require treatment. The presence or absence of risk factors may be important in determining the appropriate length of treatment.
Haemostatic markers, inflammatory parameters and lipids in male and female patients in the Angina Prognosis Study In Stockholm (APSIS). A comparison with healthy controls
Authors: HELD C. , HJEMDAHL P. , REHNQVIST N. , WALLÉN N. H. , FORSLUND L. , BJÖRKANDER I. , ANGELIN B. , WIMAN B.
Published: November 2003

Objectives.To investigate haemostatic markers (especially fibrinolysis), inflammatory parameters and lipids in patients with stable angina pectoris. Special attention was paid to differences between male and female patients, and to the reactivity to exercise or the diurnal variation of certain parameters.

Subjects. Eight hundred and nine patients (31% females) and a matched healthy control group (n = 50).

Results. The patients had signs of disturbed fibrinolysis, with elevated plasma levels of tissue plasminogen activator (tPA) antigen and plasminogen activator inhibitor (PAI‐1) activity at rest, and attenuated responses of tPA antigen and activity to exercise. Elevated levels of fibrinogen, white blood cell counts and orosomucoid were found, suggesting increased inflammatory activity, as well as a more disturbed lipid profile (higher triglycerides and lower HDL cholesterol levels) than among controls. Female patients had higher HDL cholesterol and lower triglyceride levels than male patients, but higher platelet counts and signs of enhanced platelet activity (β‐thromboglobulin excretion). In addition, female patients had lower white blood cell counts, suggesting lesser inflammatory activity.

Conclusions. Patients with stable angina pectoris have signs of markedly disturbed fibrinolysis both at rest and in response to exercise, as well as signs of enhanced inflammatory activity and dyslipidemia. The observed sex differences suggest that male patients with stable angina pectoris may have a more lipid‐related disease, whereas it may be more dependent on platelet function in females.
Familial amyloidosis in one Chinese family: clinical, immunological, and molecular genetic analysis
Authors: CHOU CHUNG‐TEI , LEE CHENG‐CHUN , CHANG DEH‐MING , BUXBAUM JOEL N. , JACOBSON DANIEL R.
Published: November 2003

Three members of a Taiwanese kindred developed severe, systemic, early onset (<age 25 years), biopsy‐proven amyloidosis. Clinical features included upper and lower extremity sensorimotor neuropathy, abdominal pain, vomiting, corneal ulcerations, cardiomyopathy, and syncope. Immunohistochemical analysis indicated that the deposits consisted of transthyretin. Molecular genetic studies revealed a heterozygous codon 55 point mutation, resulting in a proline for leucine substitution, a mutation previously associated with aggressive familial amyloidosis in a US kindred of Dutch and German descent. The clinical courses and echocardiographic findings are typical for many types of amyloidosis; the pathologic data and genetic studies were necessary to establish a precise diagnosis.
Alcohol consumption, drinking pattern and acute myocardial infarction. A case referent study based on the Swedish Twin Register
Authors: HAMMAR N. , ROMELSJÖ A. , ALFREDSSON L.
Published: November 2003

Objectives. Little is known about the possible influence of different kinds of alcohol drinking pattern on the risk of acute myocardial infarction. In this study the association between average daily alcohol consumption, as well as large intakes of alcohol on single occasions, and myocardial infarction incidence was investigated.

Design. A case referent analysis nested within a prospective cohort study.

Setting. Incident cases of myocardial infarction were identified by using hospital discharge data and deaths. Referents were selected from the study population through a stratified random sample.

Subjects. Individuals of the Swedish Twin Register below 75 years of age living in a region of 10 Swedish counties in 1972–1981 or in Stockholm County in 1972–1987.

Main outcome measure. Incidence of acute myocardial infarction.

Results. No difference in myocardial infarction incidence was found between former alcohol drinkers and lifelong abstainers. For men, drinkers had a 40% lower incidence than non‐drinkers, as did those with a drinking pattern involving a large intake on single occasions. Women had on average a very low level of alcohol consumption and there were only small differences in incidence of myocardial infarction between drinkers and non‐drinkers. An increased incidence was indicated for women reporting sometimes drinking comparatively large amounts of alcohol on single occasions.

Conclusions. The results support the suggestion that low and moderate alcohol consumption is protective for myocardial infarction. A drinking pattern involving a large intake of alcohol on single occasions did not seem to substantially influence myocardial infarction incidence except possibly for women with an overall very low level of consumption.
The clinical course of renal function in NIDDM patients with normo‐ and microalbuminuria
Authors: NIELSEN S. , SCHMITZ A. , REHLING M. , MOGENSEN C. E.
Published: November 2003

Objectives. To assess the clinical course of renal function in relation to risk factors in NIDDM patients with normo‐ and microalbuminuria.

Design. Prospective clinical study.

Setting. Outpatient diabetic clinic.

Subjects. Thirty‐two NIDDM patients with normo‐ or microalbuminuria followed for (mean (range)) 5.5 (3.3–7.5) years.

Main outcome measures. Glomerular filtration rate, urinary albumin excretion rate, blood pressure, lipids, glycaemic control.

Results. The mean rate of decline of glomerular filtration rate was −1.2±2.3 (mean±SD) (95% confidence intervals: −2.0–−0.3) mL min−1 1.73 m−2 year−1 (p=0.009). A considerable interindividual variation was observed (range −6.7 to +3.4 mL  min−1 1.73 m−2 year−1). No difference was found between normo‐ and microalbuminuric patients (−1.2±0.5 vs. −1.0±0.7 mL min−1 1.73 m−2 year−1) or between patients with and without antihypertensive treatment (−1.7±0.7 vs. −0.7±0.4  mL min−1 1.73 m−2 year−1). By multiple linear regression analysis the fall rate of glomerular filtration was determined by the mean glomerular filtration rate level (p=0.036). Analysis of patients without antihypertensive treatment revealed that urinary albumin excretion rate and HbA1c levels significantly determined the fall rate of glomerular filtration (P<0.001 and=0.014).

Conclusions. The average decline in renal function of these normo‐ and microalbuminuric NIDDM patients was not increased as compared to the age related fall rate of healthy subjects but varied markedly. Low glomerular filtration rate is associated with a higher fall rate. In patients without antihypertensive treatment higher urinary albumin excretion rate, and poorer glycaemic control are factors associated with an increased fall rate of glomerular filtration.
Effects of short‐term insulin‐like growth factor‐I or growth hormone treatment on bone turnover, renal phosphate reabsorption and 1,25 dihydroxyvitamin D3 production in healthy man
Authors: BIANDA T. , HUSSAIN M. A. , GLATZ Y. , BOUILLON R. , FROESCH E. R. , SCHMID C.
Published: November 2003

Objectives. To find out whether insulin‐like growth factor‐I (IGF‐I) mimics the stimulatory effects of growth hormone (GH) on bone turnover and renal tubular phosphate reabsorption.

Design. Randomized, crossover study.

Setting. University Hospital, Zürich, Switzerland.

Subjects. Seven young healthy male subjects.

Interventions. Each subject was studied three times at 2‐week intervals, treated with saline 0.9% (S), IGF‐I [8 μg kg−1 h−1] by a continuous subcutaneous infusion and finally with GH (6 U. twice daily s.c.) for 5 days.

Main outcome measures. 36 h after the start of treatment, IGF‐I, biochemical markers of bone turnover, calcium, calcium regulating hormones, kidney function and phosphate reabsorption were measured in serum and in 2 h urine in fasting state.

Results. Serum levels of IGF‐I were 26.8±7.3 (S), 119.4±11.4 (IGF‐I) (P<0.02) and 58.4±12.9 nmol L−1 (GH) (P<0.02), respectively. Serum osteocalcin and carboxyterminal propeptide of type I collagen (PICP) as well as the urinary deoxypyridinoline/creatinine and the calcium/creatinine ratios were all significantly higher after IGF‐I (P<0.02) or GH (P<0.02) than after saline treatment. PTH levels did not change in response to treatment. Total albumin‐corrected calcium increased only after GH treatment (P<0.05). The free calcitriol index rose from 2.2.±0.5×10−5 (S) to 2.81±0.25×10−5 (IGF‐I) (P<0.03) and 2.45±0.25×10−5 (GH), respectively. Serum phosphate and maximal tubular reabsorption divided by glomerular filtration rate (TmP/GFR) were significantly raised by GH (P<0.03) but not by IGF‐I as compared to saline 0.9%.

Conclusions. (i) Similar to GH, IGF‐I rapidly activates bone turnover. (ii) IGF‐I does not mimic the effect of GH on renal phosphate reabsorption in spite of comparable effects on renal blood flow and glomerular filtration rate. (iii) IGF‐I increases free calcitriol index in face of unchanged serum levels of calcium, phosphate and PTH, consistent with a direct stimulatory effect on 25‐OHD‐1a‐hydroxylase.
Internal jugular vein thrombosis after cervical traction
Authors: SIMMERS T. A. , BEKKENK M. W. , VIDAKOVIC‐VUKIC M.
Published: November 2003

A clinically and roentgenographically typical case of internal jugular vein thrombosis is presented in a patient having undergone repeated sessions of Glisson traction for neck and backache. In this mode of therapy, axial traction is applied to the neck by attaching a weight, via a pulley, to a construction of straps encompassing the head. This particular trauma to the neck has not previously been described to be associated with spontaneous internal jugular vein thrombosis.
The additional effects of acipimox to simvastatin in the treatment of combined hyperlipidaemia
Authors: HOOGERBRUGGE N. , JANSEN H. , DE HEIDE L. , ZILLIKENS M. C. , DECKERS J. W. , BIRKENHÄGER J. C.
Published: November 2003

Objectives. Nicotinic acid, an effective drug for treatment of combined hyperlipidaemia, is often not tolerated because of side‐effects. Acipimox is a nicotinic acid like lipid lowering drug with less side‐effects. We studied whether the addition of acipimox to simvastatin improves the lipid profile in patients with a combined hyperlipidaemia.

Design. Randomized double‐blind placebo controlled crossover trial.

Setting. Outpatient lipid clinic of a tertiary referral centre.

Subjects. Eighteen patients with combined hyperlipidaemia treated with diet and 20–40 mg simvastatin for at least 3 months.

Intervention. Acipimox in a daily dose of 3×250 mg for 12 weeks.

Main outcome measures. Effects on the concentration of LDLc, TG, HDLc, Lp(a) and Apolipoprotein B, as well as on LDL‐size and LDL‐resistance to oxidative modification.

Results. Acipimox reduced Lp(a) levels by 8% (P<0.05). A substantial but not statistically significant change in TG (−32%) and HDLc (+6%) levels was seen. All patients were found to have small dense LDL, with a size of 229±4 Å. LDL size and the resistance to oxidation, reflected in the lag phase during in vitro oxidation, were not affected by the addition of acipimox. In a subgroup of 8 patients with the most severe hypertriglyceridaemia (baseline TG<4 mmol L‐[1]), acipimox induced a significant increase in HDLc (+15%, P<0.01). The effects on TG (−41%), LDLc (−10%) and lag phase (+17%) were also more pronounced than in the group with a lower baseline TG, but non of these changes reached the level of significance.

Conclusions. Adding acipimox to simvastatin reduced Lp(a) and substantially but not significantly lowered TG. However, in patients with the highest TG levels, HDLc was also significantly improved.
Dispersion, aberration and deconvolution in multi‐wavelength fluorescence images
Authors: SCALETTAR B. A. , SWEDLOW J. R. , SEDAT J. W. , AGARD D. A.
Published: November 2003

The wavelength dependence of the incoherent point spread function in a wide‐field microscope was investigated experimentally. Dispersion in the sample and optics can lead to significant changes in the point spread function as wavelength is varied over the range commonly used in fluorescence microscopy. For a given sample, optical conditions can generally be optimized to produce a point spread function largely free of spherical aberration at a given wavelength. Unfortunately, deviations in wavelength from this value will result in spherically aberrated point spread functions. Therefore, when multiple fluorophores are used to localize different components in the same sample, the image of the distribution of at least one of the fluorophores will be spherically aberrated. This aberration causes a loss of intensity and resolution, thereby complicating the localization and analysis of multiple components in a multi‐wavelength image. We show that optimal resolution can be restored to a spherically aberrated image by constrained, iterative deconvolution, as long as the spherical aberration in the point spread function used for deconvolution matches the aberration in the image reasonably well. The success of this method is essentially independent of the initial degree of spherical aberration in the image. Deconvolution of many biological images can be achieved by collecting a small library of spherically aberrated and unaberrated point spread functions, and then choosing a point spread function appropriate for deconvolving each image. The co‐localization and relative intensities of multiple components can then be accurately studied in a multi‐wavelength image.
Visualization of proteoglycans and link protein in embryonic chick limb cartilage via cryofixation, freeze‐substitution and immunochemical techniques
Authors: ALLENSPACH A. L. , MAYNARD H. , TSONIS P. A.
Published: November 2003

Chick embryo limb bud cartilage contains a family of proteoglycans, a few of which have been identified ultrastructurally by antibody labelling. Limb bud cartilage from stage 30–34 chick embryos was high‐pressure frozen, freeze‐substituted and embedded in Lowicryl resin. Sections were treated with polyclonal antibodies for core protein and monoclonal antibodies for chondroitin‐6‐sulphate and link protein. Label for core protein was demonstrated on both structural matrix and free within the compartmental space. Quantitative analysis indicates that core protein is preferentially localized on electron‐dense structural matrix, and that this distribution is uniform between stages 30 and 34. The association of protein epitopes on electron‐dense lattice is strongly influenced, rather than a chance observation. Significant quantities of core protein are also located in the free compartments of the cartilaginous lattice. Chondroitin‐6‐sulphate and link protein were localized predominantly within the compartments of the embryonic lattice. Our data provide convincing evidence that the proteoglycans were immobilized within a microcrystalline matrix of the embryonic compartments. A role for core protein as a stabilizer within the lattice and in the free space where it serves to aggregate polymeric proteoglycans is suggested from our results.
Cryofixing single cells and multicellular specimens enhances structure and immunocytochemistry for light microscopy
Authors: BASKIN T. I. , MILLER D. D. , VOS J. W. , WILSON J. E. , HEPLER P. K.
Published: November 2003

Cryofixation is widely held to be superior to chemical fixation for preserving cell structure; however, the use of cryofixation has been limited chiefly to electron microscopy. To see if cryofixation would improve sample structure or antigenicity as observed through the light microscope, we cryofixed Nicotiana alata and Lilium longiflorum pollen tubes and Tradescantia virginina stamen hairs by plunge freezing. After freeze‐substitution, and embedding in butylmethylmethacrylate, we found using the light microscope that the superiority of cryofixation over chemical fixation was obvious. Cryofixation, unlike chemical fixation, did not distort cell morphology and preserved microtubule and actin arrays in a form closely resembling that of living cells.

Additionally, to test further the usefulness of cryofixation for light microscopy, we studied the appearance of cells and the retention of antigenicity in a plunge‐frozen multicellular organ. Roots of Arabidopsis thaliana were either chemically fixed or plunge frozen, and then embedded in the removable methacrylate resin used above. We found that plunge freezing preserved cell morphology far better than did chemical fixation, and likewise improved the appearance of both actin and microtubule arrays. Plunge‐frozen roots also had cells with more life‐like cytoplasm than those of chemically fixed roots, as assessed with toluidine‐blue staining or high‐resolution Nomarski optics. Damage from ice crystal formation could not be resolved through the light microscope, even in the interior of the root, 40–75 μm from the surface. We suggest that plunge freezing would enhance many investigations at the light microscope level, including those of multicellular organs, where damage from ice crystals may be less severe than artefacts from chemical fixation.
STM of metal embedded and coated DNA and DNA–protein complexes
Authors: MÜLLER‐REICHERT T. , BUTT H.‐J. , GROSS H.
Published: November 2003

Bare and Pt/Ir/C‐coated DNA has been analysed using scanning tunnelling microscopy (STM). To achieve reproducible imaging of bare DNA on mica ethanol/air‐dried molecules were embedded in Pt/C. By peeling the metal film off the mica, the previously mica‐exposed side of the Pt/C‐film with the embedded DNA molecules was accessible for STM analysis. By applying this replica/anchoring technique only hollow trenches in the metal film, and not the DNA itself, could be visualized. The gaps averaged 3.1 nm (± 0.9 nm) wide and 1 nm (± 0.5 nm) deep. Using scanning force microscopy it could be confirmed that the DNA remained in the Pt/C film during the peel‐off procedure. For STM, DNA fragments were also coated with 0.7–1 nm Pt/Ir/C. Owing to the high Z‐resolution the STM samples were coated at a high elevation angle (65°), thereby minimizing the problem of self‐shadowing. Coating by Pt/Ir/C allowed routine imaging and quantitative analysis of both ethanol/air‐ and freeze‐dried DNA under atmospheric conditions. After ethanol/air drying measured values for DNA width and height were 5.1 nm (± 1.8 nm) and 0.9 nm (± 0.2 nm), respectively. Freeze‐dried DNA averaged 4.2 nm (± 1.3 nm) wide and 1.1 nm (± 0.1 nm) high. A Pt/Ir/C‐coating was also applied to visualize DNA–protein interaction using STM.
High‐resolution confocal microscopy using synchrotron radiation
Authors: VAN DER OORD C. J. R. , JONES G. R. , SHAW D. A. , MUNRO I. H. , LEVINE Y. K. , GERRITSEN H. C.
Published: November 2003

A confocal scanning light microscope coupled to the Daresbury Synchrotron Radiation Source is described. The broad spectrum of synchrotron radiation and the application of achromatic quartz/CaF2 optics allows for confocal imaging over the wavelength range 200–700 nm. This includes UV light, which is particularly suitable for high‐resolution imaging. The results of test measurements using 290‐nm light indicate that a lateral resolution better than 100 nm is obtained. An additional advantage of the white synchrotron radiation is that the excitation wavelength can be chosen to match the absorption band of any fluorescent dye. The availability of UV light for confocal microscopy enables studies of naturally occurring fluorophores. The potential applications of the microscope are illustrated by the real‐time imaging of hormone traffic using the naturally occurring oestrogen coumestrol. (The IUPAC name for coumestrol is 3,9‐dihydroxy‐6H‐benzofuro[3,2‐c][1]benzopyran‐6‐one (Chem. Abstr. Reg. No. 479‐13‐0). The trivial name will be used throughout this paper.)
Early mineralization of matrix vesicles in the epiphyseal growth plate
Authors: PLATE U. , TKOTZ T. , WIESMANN H. P. , STRATMANN U. , JOOS U. , HÖHLING H. J.
Published: November 2003

Matrix vesicles (MVs) induce the primary mineralization in collagen‐rich hard tissues such as bone, mineralizing cartilage and dentine. Calcium and phosphate ions accumulate at the inner MV membrane. This accumulation takes place in association with phospholipids alone and/or in association with Annexin V, which displays Ca ion channel activity when inserted in membranes; consequently, Annexin V may be involved in Ca uptake by matrix vesicles. The first crystal nuclei are formed at these macromolecules of the MV inner membrane. They grow to stable nanometre‐sized particles, dots, which coalesce to form chains of dots along the macromolecules of the MV inner membrane. At the same time, or shortly afterwards, chains of these Ca phosphate dots also develop inside the MVs. The measured centre‐to‐centre distances between these dots represent approximately the distances between the nucleating sites, called active sites, along the MV matrix molecules. The mineralization does not stop at the MV membrane but expands continuously into the extravesicular region in radial directions to form nodules. These radiating Ca phosphate chains, which coalesce to form needles, are composed of such primary dots, which have developed at the nucleating sites of the corresponding macromolecules.
Electron spectroscopic imaging of antigens by reaction with boronated antibodies
Authors: QUALMANN B. , KESSELS M. M. , KLOBASA F. , JUNGBLUT P. W. , SIERRALTA W. D.
Published: November 2003

Two small homogeneous markers for electron spectroscopic imaging (ESI) containing eight dodecaborane cages linked to a poly‐α,ε‐l‐lysine dendrimer were synthesized; one of these was made water soluble by the attachment of a polyether. The markers were coupled to the sulfhydryl group of (monovalent) antibody fragments (Fab′) by a homobifunctional cross‐linker. While the coupling ratios of the poorly water‐soluble compound did not exceed 20%, the polyether‐containing variant reacted quantitatively. Its suitability for immunolabelling was tested in a study of the mechanism of the transcellular transport of an administered heterologous protein (bovine serum albumin, BSA) through ileal enterocytes of newborn piglets by endocytotic vesicles in comparison to conventional immunogold reagents. The post‐embedding technique was employed. The boronated Fab′ gave rise to considerably higher tagging frequencies than seen with immunogold, as could be expected from its form‐ and size‐related physical advantages and the dense packing of BSA in the vesicles. The new probe, carrying the antigen‐combining cleft at one end and the boron clusters at the opposite end of the oval‐shaped conjugate, add to the potential of ESI‐based immunocytochemistry.
Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy‐filtered imaging
Authors: HAN K. F. , GUBBENS A. J. , SEDAT J. W. , AGARD D. A.
Published: November 2003

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron–specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy‐loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy‐filtered microscope and to assess its utility for thick‐section microscopy. We found that for imaging thick biological specimens (> 0.5 μm) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy‐filtering at the most probable energy loss (in this case at the first plasmon energy‐loss peak). Furthermore, the use of zero‐loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero‐loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.
Micrometallurgy: a technique for examining the structure of binary‐element thin films over a wide range of composition
Authors: EGERTON R. F. , BENNETT J. C.
Published: November 2003

We describe a procedure for producing thin‐film TEM specimens containing a thickness and/or composition gradient, utilizing an out‐of‐contact mask at an appropriate distance from the substrate. Imaging and diffraction capabilities of the TEM are used to examine the local structure of the film; EELS or EDX analysis provides the local elemental composition. The procedure is illustrated by results obtained from two binary‐alloy systems: Se–Te (which displays a complete range of solid solubility) and Sn–Ge (where mutual solubility is very low).
Image‐EELS for in situ estimation of the phosphorus content of RNP granules
Authors: ABOLHAS SANI‐DADRAS S. , VÁZQUEZ‐NIN G. H. , ECHEVERRÍA O. M. , FAKAN S.
Published: November 2003

Electron energy‐loss spectroscopic imaging is performed on ultrathin sections, in order to approach the in‐situ distribution of individual elements in the cellular constituents. In a previous ultrastructural study of salivary gland cells of Chironomus thummi and Ch. tentans by means of electron spectroscopic imaging and cytochemistry, we have described a new type of phosphorus‐rich small granular ribonucleoprotein (RNP) component in the nucleoplasm of these cells; an attempt is made in the present work to estimate the phosphorus content of this component by the above mentioned method, using ribosomes as internal standard.  Results show that these RNP granules contain, on average 3600 phosphorus atoms per granule. Another nuclear constituent also studied, the Balbiani ring granule, contains, on average, 43 000 phosphorus atoms per granule.
Measuring fractal dimension and complexity — an alternative approach with an application
Authors: SANDAU K. , KURZ H.
Published: November 2003

Fractal dimension has often been applied as a parameter of complexity, related to, for example, surface roughness, or for classifying textures or line patterns. Fractal dimension can be estimated statistically, if the pattern is known to be self‐similar. However, the fractal dimension of more general patterns cannot be estimated, even though the concept may be retained to characterize complexity. We here show that the usual statistical methods, e.g. the box counting method, are not appropriate to measure complexity. A recently developed approach, the extended counting method, whose properties are closer to what fractal dimension means, is considered here in more detail. The methods are applied to geometric and to blood vessel patterns. The weak assumptions about the structure, and the lower variance of the estimate, suggest that the extended counting method has beneficial properties for comparing complexity of naturally occurring patterns.
Automated detection of macromolecules from electron micrographs using advanced filter techniques
Authors: Stoschek A. , Hegerl R.
Published: November 2003

We have addressed the problem of automated detection of biological macromolecules from electron micrographs by designing a detection filter which combines conventional correlation technique with a synthetic discriminant function (SDF) in conjunction with the constraint to minimize the energy in the correlation plane and that of noise. Combining the constrained SDF filter with circular harmonic expansion results in rotational invariance of the detection process. We define measures to evaluate filter performance by test calculations and discuss the choice of parameters for filter development. Compared with the conventional matched filter, improved recognition and discrimination of objects to be detected is achieved. Applications of the filter to detect biological macromolecules in electron micrographs are presented, demonstrating the improvements in object detection as well as the limitations of the method.
Confocal fluorescence microscopy using spectral and lifetime information to simultaneously record four fluorophores with high channel separation
Authors: CARLSSON K. , LILJEBORG A.
Published: November 2003

We demonstrate the possibility to increase substantially the number of simultaneously detected fluorophores by utilizing both spectral and lifetime information. Using a two‐detector confocal scanning laser microscope, experiments confirm that four different fluorophores can be detected with good channel separation. The signal‐to‐noise ratio (SNR) of the recorded images is investigated both theoretically and experimentally. It is found that in order to obtain a high SNR fluorophore lifetimes should differ by approximately an order of magnitude.
Multicolour analysis and local image correlation in confocal microscopy
Authors: DEMANDOLX D. , DAVOUST J.
Published: November 2003

Multiparameter fluorescence microscopy is often used to identify cell types and subcellular organelles according to their differential labelling. For thick objects, the quantitative comparison of different multiply labelled specimens requires the three‐dimensional (3‐D) sampling capacity of confocal laser scanning microscopy, which can be used to generate pseudocolour images. To analyse such 3‐D data sets, we have created pixel fluorogram representations, which are estimates of the joint probability densities linking multiple fluorescence distributions. Such pixel fluorograms also provide a powerful means of analysing image acquisition noise, fluorescence cross‐talk, fluorescence photobleaching and cell movements. To identify true fluorescence co‐localization, we have developed a novel approach based on local image correlation maps. These maps discriminate the coincident fluorescence distributions from the superimposition of noncorrelated fluorescence profiles on a local basis, by correcting for contrast and local variations in background intensity in each fluorescence channel. We believe that the pixel fluorograms are best suited to the quality control of multifluorescence image acquisition. The local image correlation methods are more appropriate for identifying co‐localized structures at the cellular or subcellular level. The thresholding of these correlation maps can further be used to recognize and classify biological structures according to multifluorescence attributes.
In‐situ SEM observation of the fracture behaviour of titanium carbide/nickel aluminide composites
Authors: PLUCKNETT K. P. , BECHER P. F. , ALEXANDER K. B.
Published: November 2003

A range of experimental titanium carbide (TiC)/nickel aluminide (Ni3Al) composites have been developed. The Ni3Al content has been varied from 15 to 40 vol.%, for two different alloy compositions. The fracture behaviour of these composites was assessed in situ, within the chamber of a field emission gun scanning electron microscope. An applied moment double cantilever beam test geometry was employed, which allowed determination of the materials fracture resistance curve (or R‐curve), while simultaneously monitoring crack–microstructure interactions. All of the tested materials exhibited a pronounced R‐curve, with fracture resistance increasing with increasing crack length, up to a steady‐state plateau value. The highest steady‐state toughness values (up to 15 MPa m−1/2) were obtained for composites prepared with the highest Ni3Al binder content. In addition, these materials exhibited the highest strength and Weibull modulus. In‐situ examination of the fracture process demonstrated considerable crack wake bridging by ductile Ni3Al ligaments, with bridging zone length of >100 μm often observed. The use of a displacement mapping technique has demonstrated that Ni3Al plastic deformation is highly localized around the crack, typically within one TiC grain width (i.e. ≈3–5 μm) away from the crack.
Three‐dimensional reconstruction of colon carcinoma metastases in liver
Authors: Griffini P. , Smorenburg S. M. , Verbeek F. J. , Van Noorden C. J. F.
Published: November 2003

Resection of liver metastases in patients with colon cancer increases survival but success depends on removal of all tumour tissue. For this purpose, understanding of spatial relationships between metastases and liver architecture is essential. Because metastatic cancer growth is essentially a three‐dimensional (3D) event, we decided to apply 3D reconstruction techniques to study these spatial relationships between metastases and liver structures such as blood vessels, stroma and the liver capsule (Glisson’s capsule). Colon carcinoma metastases were experimentally induced in rat liver by injection of colon cancer cells (CC531) into the portal vein. Three weeks later, livers from these animals and control livers were removed and immediately frozen in liquid nitrogen. Thirty‐seven to 110 consecutive sections were used for each 3D reconstruction of 26 metastases in eight livers. Contours of different structures were stained by (immuno)histochemical means, traced in each section and stored in a database. From the contour model, a volume model was generated. Among the 26 metastases, seven were found to grow distantly from the liver capsule. They were small and consisted of well‐differentiated cancer cells that were totally surrounded by a basement membrane and stroma which was always connected with adjacent blood vessels of a portal tract. The remaining 19 metastases showed a more advanced pattern of development. Infiltration of poorly differentiated colon cancer cells progressed through the stroma at various sites and areas of direct contact between cancer cells and hepatocytes were frequently found. This type of outgrowth of cancer cells was only found when metastases had made contact with the liver capsule. However, some areas in sections of these advanced stages still resembled small metastases. On the basis of these findings, we conclude that stroma affects the differentiation pattern of cancer cells and has at least a dual role in tumour growth. On the one hand it limits invasion of cancer cells in the surrounding host tissue. On the other hand, stroma formation at the capsule, which consists mainly of granulation tissue, facilitates outgrowth of the tumours. Furthermore, our 3D reconstructions demonstrate the spatial heterogeneity of larger metastases and the importance of a 3D approach to understand growth and development of metastases in general and colon cancer metastases in the liver in particular.
Quantitative image analysis of birefringent biological material
Authors: Ross S. , Newton R. , Zhou Y.‐M. , Haffegee J. , Ho M.‐W. , Bolton J. P. , Knight D.
Published: November 2003

A previously described optical technique for enhancing interference colour contrast in polarized light microscopy is adapted for quantitative image analysis. This is based on a linear relationship for colour intensity vs. molecular birefringence and degree of phase alignment, derived using nematic liquid crystal approximations, and verified experimentally. The image analysis is described and illustrated on a section of the marginal rib of the dogfish egg case.
Plasmon coupling and finite size effects in metallic multilayers
Authors: NEYER T. , SCHATTSCHNEIDER P. , BOLTON J. P. R. , BOTTON G. A.
Published: November 2003

This work uses electron energy loss spectroscopy (EELS) to study the excitation of interface and bulk plasmons in artificial, nonmagnetic metallic multilayers. We obtain EELS spectra for W/Si and Ta/Si multilayers with layer periodicity d using an off‐axis acquisition technique and compare the results with dispersion relations and energy loss probability distributions predicted from dielectric theory. Our energy loss spectra, taken for a fixed out‐of‐plane wavevector kz = π/d and low in‐plane wavevectors ky < 1/d, contain features which are identified as coupled interface modes by solving the appropriate superlattice dispersion relation. These interface modes are accompanied by bulk plasmons of the individual media. We find that dielectric theory greatly overemphasizes these bulk modes. This behaviour is interpreted phenomenologically as a finite size effect and is modelled by introducing a cut‐off for the long‐wavelength bulk modes.
Centenary of Gaule and Lewin, pioneers in cell counting methodology
Authors: Hedreen J. C. , Vonsattel J. P.
Published: November 2003

We commemorate the one hundredth anniversary of the publication of a pioneering paper on cell counting, by Gaule and Lewin. Their paper describes a new method for counting cells in tissue sections. First they found the mean number of cell profiles per cell by examining 50 selected cells in serial sections. Then they counted the total number of cell profiles in the ganglion, and finally divided this total profile number by the mean number of profiles per cell. They thought this method more accurate than counting cells by counting only profiles that showed the nucleolus because they noted that a cell's nucleolus sometimes appeared in more than one section and that a single cell could have more than one nucleolus.
The role of specimen‐induced spherical aberration in confocal microscopy
Authors: Török P. , Hewlett S. J. , Varga P.
Published: November 2003

We present an overview of recent theories for describing specimen‐induced spherical aberration in confocal microscopy. One of these theories is used to compute numerically the role of spherical aberration in general confocal, and especially in biological confocal, microscopy for a variety of three‐layer specimen structures. In particular, we study the effect of specimen‐induced spherical aberration on the maximum value of the overall confocal point spread function, the accompanying focal shift and the size of the optical probe in both fluorescence and brightfield confocal microscopy.
Transmission X‐ray microscopy of intact hydrated PtK2 cells during the cell cycle
Authors: Methe O. , Spring H. , Guttmann P. , Schneider G. , Rudolph D. , Trendelenburg M. F. , Schmahl G.
Published: November 2003

Transmission X‐ray microscopy makes it possible to investigate biological specimens, i.e. cells and cell organelles, in their natural wet environment. The main processes determining the contrast in X‐ray microscopy are photoelectric absorption and phase shift. X‐ray microscopic experiments can therefore be carried out in both amplitude and phase contrast. The Göttingen X‐ray microscope at the BESSY storage ring in Berlin is described. PtK2 cells were examined during different stages of the cell cycle. All major constituents of the mitotic apparatus, e.g. chromosomes, centromeres, microtubules and centrosomes, could be visualized, as well as the main structural compartments and organelles of the interphase cell, e.g. nuclear membrane, interphase chromatin, nucleolus and cytoplasmic mitochondria, as well as parts of the cytoskeletal apparatus. In this way new information can be obtained with regard to the ultrastructure of the constituents of intact and unstained cells at a resolution which bridges the gap between light microscopy and electron microscopy. The prospects for the future application of transmission X‐ray microscopy in biomedical research are discussed.
A physical approach to the radiation damage mechanisms induced by X‐rays in X‐ray microscopy and related techniques
Authors: Cazaux J.
Published: November 2003

A physical approach is used to analyse the various mechanisms induced by the absorption of X‐ray photons of energies in the 0.2–20 keV range. At the atomic scale, besides the (Auger and photo) electron transport in the bulk or the ejection into the surrounding media, special attention is devoted to the specific consequences of the initial Auger decay mechanism. At the macroscopic scale, the decisive role of the poor electronic conductivity of the radiation‐sensitive materials is outlined and it is shown that the damaging effects occur in irradiated insulators because the lack of conduction electrons prevents the initial charge of the excited atoms being quickly restored. Correlating irradiation conditions and physical properties of the specimen, various expressions are proposed for the first time to quantify these effects. Some are neither dose‐ nor dose‐rate‐dependent and the influence of the surrounding medium is also considered. The fundamental mechanisms investigated here hold for a wide variety of specimens or components investigated in X‐ray microscopy. Their consequences can be easily transposed to other techniques using transmitted X‐rays.