bluuebunny/crossref_metadata_2025 huggingface dataset:
MRI measures of perfusion-related changes in human skeletal muscle during progressive contractions
Authors: Wigmore D. M. , Damon B. M. , Pober D. M. , Kent-Braun J. A.
Published: August 2004

Although skeletal muscle perfusion is fundamental to proper muscle function, in vivo measurements are typically limited to those of limb or arterial blood flow, rather than flow within the muscle bed itself. We present a noninvasive functional MRI (fMRI) technique for measuring perfusion-related signal intensity (SI) changes in human skeletal muscle during and after contractions and demonstrate its application to the question of occlusion during a range of contraction intensities. Eight healthy men (aged 20–31 yr) performed a series of isometric ankle dorsiflexor contractions from 10 to 100% maximal voluntary contraction. Axial gradient-echo echo-planar images (repetition time = 500 ms, echo time = 18.6 ms) were acquired continuously before, during, and following each 10-s contraction, with 4.5-min rest between contractions. Average SI in the dorsiflexor muscles was calculated for all 240 images in each contraction series. Postcontraction hyperemia for each force level was determined as peak change in SI after contraction, which was then scaled to that obtained following a 5-min cuff occlusion of the thigh (i.e., maximal hyperemia). A subset of subjects ( n = 4) performed parallel studies using venous occlusion plethysmography to measure limb blood flow. Hyperemia measured by fMRI and plethysmography demonstrated good agreement. Postcontraction hyperemia measured by fMRI scaled with contraction intensity up to ∼60% maximal voluntary contraction. fMRI provides a noninvasive means of quantifying perfusion-related changes during and following skeletal muscle contractions in humans. Temporal changes in perfusion can be observed, as can the heterogeneity of perfusion across the muscle bed.
Thiopentone and Methohexitone Enantiomers Do Not Act Stereoselectively on the Oxidative Response in Human Neutrophils in vitro
Authors: Wittmann S. , Daniels S. , Ittner K.-P. , Fröhlich D.
Published: August 2004

To elucidate potential stereoselective effects of single barbiturate isomers, we compared the inhibitory potency of single thiopentone enantiomers, two isomer-enriched mixtures of methohexitone and racemic mixtures of both barbiturates on the fMLP-induced neutrophil oxidative response. A suppression of the response to 50% compared to control required a 100-fold therapeutic concentration of methohexitone, while therapeutic concentrations of the thiopentone racemate led to a significant inhibition (relative fluorescence of neutrophils 0.46 ± 0.03 compared to fMLP controls). The racemate of thiopentone produced significantly greater inhibition than the single enantiomers. Stereoselectivity in favor of one isomer could not be shown for both barbiturates. The greater inhibition by the thiopentone racemate might suggest two separate binding sites for the enantiomers which are positively coupled.
Central Endothelin-B Receptor Stimulation Does Not Affect Morphine Analgesia in Rats
Authors: Bhalla Shaifali , Matwyshyn George , Gulati Anil
Published: August 2004

Several neurotransmitter mechanisms have been proposed to play a role in the actions of morphine. We reported that centrally administered endothelin A (ETA) receptor antagonists potentiate morphine analgesia in rats. It has also been reported that ETB agonist, IRL1620, has antinociceptive action mediated through opiate receptors in the periphery. The present study was conducted to determine if central ETB receptors are involved in analgesic actions of morphine. The effect of intracerebroventricular (i.c.v.) administration of ETB receptor agonist, IRL1620, on morphine-induced analgesia and hyperthermia was determined in the rat. Morphine (4 mg/kg, s.c.) produced a significant increase (84%) in tail-flick latency compared to the control group and the analgesic response lasted for 4 h. IRL1620 (30 µg, i.c.v.) did not produce any increase (16%) in tail-flick latency over the 5-hour observation period in vehicle-treated rats. Pretreatment with IRL1620 (3, 10, and 30 µg, i.c.v.) did not have any significant effect on the intensity and duration of morphine (4 mg/kg, s.c.)-induced analgesia. Morphine (4 mg/kg, s.c.) administration produced an increase in body temperature compared to the control group. In vehicle-pretreated rats, IRL1620 (30 µg, i.c.v.) did not produce any change in body temperature. The morphine-induced hyperthermic effect was not altered in IRL1620-pretreated rats. These studies demonstrate that IRL1620, a specific ETB receptor agonist, did not affect the morphine-induced analgesic and hyperthermic effect in rats. It can be concluded that central ETB receptors are not involved in modulation of pharmacological actions of morphine.
Effects of Pranidipine, a Novel Calcium Channel Antagonist, on the Progression of Left Ventricular Dysfunction and Remodeling in Rats with Heart Failure
Authors: Wahed Mir Imam Ibne , Watanabe Kenichi , Ma Meilei , Nakazawa Mikio , Takahashi Toshihiro , Hasegawa Go , Naito Makoto , Yamamoto Tadashi , Kodama Makoto , Aizawa Yushifusa
Published: August 2004

The cardioprotective properties of pranidipine were studied in a rat model of heart failure after autoimmune myocarditis. Twenty-eight days after immunization the surviving rats were divided into three groups and received oral treatment of 0.03 mg/kg/day (group 0.03) or 0.3 mg/kg/day (group 0.3) of pranidipine or vehicle (group V) for 1 month. High-dose pranidipine (group 0.3) improved the survival rate, and significantly reduced heart weight, heart weight to body weight ratio, myocardial fibrosis, central venous pressure and left ventricular end-diastolic pressure than low-dose pranidipine (group 0.03) and group V. Pranidipine at high dose also decreased the left ventricular systolic and diastolic dimensions, and increased fractional shortening compared with group V. The increase in level of TGF-β1 and collagen-III mRNA were suppressed by pranidipine in a dose-dependent manner. Our results indicated that pranidipine has cardioprotective effects on heart failure, and that the beneficial effect can be partly explained by attenuation of fibrotic response through suppression of TGF-β1 and collagen-III mRNA expression, and regression of myocyte hypertrophy.
Radioligand Binding and Functional Characterization of Recombinant Human NmU1 and NmU2 Receptors Stably Expressed in Clonal Human Embryonic Kidney-293 Cells
Authors: Aiyar Nambi , Disa Jyoti , Foley James J. , Buckley Peter T. , Wixted William E. , Pullen Mark , Shabon Usman , Dul Edward , Szekeres Philip G. , Elshourbagy Nabil A. , Sarau Henry M. , Appelbaum Edward , Bolaky Jane
Published: August 2004

Neuromedin U (NmU) is a smooth muscle contracting peptide. Recently, two G-protein-coupled receptors for NmU (NmU1R and NmU2R) have been cloned having approximately 50% homology. They have distinct patterns of expression suggesting they may have different biological functions. This study provides a comprehensive characterization of both NmU receptors expressed in human embryonic kidney 293 cells. [125I]hNmU binding to the recombinant NmU receptors was rapid, saturable, of high affinity and to a single population of binding sites. Exposure of these cells to NmU isopeptides resulted in an increase in intracellular [Ca2+]i release (EC50 value of 0.50 ± 0.10 nmol/l) and inositol phosphate formation (EC50 1.6 ± 0.2 and 1.50 ± 0.4 nmol/l for NmU1R and NmU2R respectively). Furthermore, hNmU inhibited forskolin (3 µmol/l)-stimulated accumulation of cAMP in intact HEK-293 cells expressing either NmU1R or NmU2R. The inhibitory effect was significant for the cells expressing NmU2R with IC50 value of 0.80 ± 0.21 nmol/l. In summary, both NmU1R and NmU2R in HEK-293 cells have similar signaling capability.
Nordihydroguairetic Acid, a Lignin, Prevents Oxidative Stress and the Development of Diabetic Nephropathy in Rats
Authors: Anjaneyulu Muragundla , Chopra Kanwaljit
Published: August 2004

Recent evidences indicate a pivotal role of reactive oxygen species in etiology of diabetic nephropathy, an important microvascular complication of diabetes mellitus. Moreover, oxidative stress leads to an increased production of lipoxygenase derivatives which also play a role in diabetic nephropathy. The present study was thus designed to examine the effect of an antioxidant and a lipoxygenase inhibitor, nordihydroguairetic acid (NDGA), on renal function and oxidative stress in streptozotocin (STZ)-induced diabetic rats. Diabetes was induced by a single intraperitoneal injection of streptozotocin (65 mg/kg) in rats. After the 4th week of STZ injection, NDGA (5 and 10 mg/kg) was given subcutaneously (s.c.) for another 4 weeks to both control and diabetic rats. At the end of the 8th week, diabetic rats exhibited renal dysfunction as evidenced by reduced creatinine and urea clearance along with enhanced albumin excretion rate as compared with control rats. Biochemical analysis of kidneys revealed a marked increase in oxidative stress demonstrated by increased lipid peroxidation and decreased activities of key antioxidant enzymes, glutathione (GSH), superoxide dismutase (SOD) and catalase in diabetic rats. Chronic treatment with NDGA in diabetic rats significantly prevented both renal dysfunction and oxidative stress as compared with vehicle-treated diabetic rats. The kidneys of diabetic rats showed morphological changes such as hyaline casts, glomerular thickening and moderate interstitial fibrosis and arteriolopathy, whereas NDGA administration in diabetic rats markedly prevented renal morphological alterations. These results emphasize the role of oxidative stress in the pathophysiology of diabetic nephropathy and point towards the potential of NDGA as a complementary therapy for the prevention/treatment of diabetic nephropathy.
Itch-Scratch Responses Induced by Lysophosphatidic Acid in Mice
Authors: Hashimoto Terumasa , Ohata Hisayuki , Momose Kazutaka
Published: August 2004

The present investigation was conducted in order to determine whether lysophosphatidic acid (LPA) induces itch-scratch responses (ISRs) in mice. Intradermal administration of LPA induces ISRs; furthermore, the time course for LPA-induced ISRs was similar to that for histamine-induced responses. Comparative study of the pruritogenic activity revealed that histamine possessed a potent effect characterized by a dose-response relationship; however, prostaglandin D2 failed to induce this response. Pretreatment with ketotifen, a histamine H1 receptor antagonist, and capsaicin inhibited LPA-induced ISRs. Additionally, LPA-induced ISRs were abolished by Y-27632, an inhibitor of Rho-associated protein kinase (ROCK). These findings suggest that LPA-induced ISRs are attributable to histamine- and substance-P-mediated pathways. Moreover, the Rho/ROCK-mediated pathway may be involved.
Theorizing the Father-Child Relationship: Mechanisms and Developmental Outcomes
Authors: Paquette Daniel
Published: August 2004

The aim of this article is to propose a theorization of the father-child relationship based on our current understanding of attachment, interactions between fathers and their young children, and human-specific adaptations. The comparison of mother-child and father-child interactions suggests that fathers play a particularly important role in the development of children’s openness to the world. Men seem to have a tendency to excite, surprise, and momentarily destabilize children; they also tend to encourage children to take risks, while at the same time ensuring the latter’s safety and security, thus permitting children to learn to be braver in unfamiliar situations, as well as to stand up for themselves. But this dynamic can only be effective in the context of an emotional bond between father and child; this relationship is termed the father-child activation relationship, in contrast to the mother-child attachment relationship aimed at calming and comforting children in times of stress. The activation relationship is developed primarily through physical play. It is postulated, in particular, that father-child rough-and-tumble play encourages obedience and the development of competition skills in children.
Retinal Pigment Epithelial Phagocytosis and Metabolism Differ from Those of Macrophages
Authors: Irschick Eveline U. , Sgonc Roswitha , Böck Günther , Wolf Hugo , Fuchs Dietmar , Nussbaumer Walter , Göttinger Wolfgang , Huemer Hartwig P.
Published: August 2004

The purpose of this study is to compare primary human retinal pigment epithelium (RPE) cells with respect to particle uptake and further processing steps with immunological phagocytes for a better understanding of the possible role of RPE cells in triggering autoimmune diseases in the eye. We investigated the similarities of human RPE and monocytes/macrophages studying the uptake of fluorescein- and europium-labeled synthetic microparticles and microbial pathogens by human and bovine RPE cultures and a permanent RPE cell line (CRL). The uptake was monitored by laser scanning microscopy, flow cytometry and time-resolved fluorescence analysis; for comparison, macrophages and a macrophage-like cell line (MonoMac6) were used. A size-dependent uptake was seen in primary RPE cultures as well as in CRL, showing a preferential uptake of smaller beads followed by Staphylococcus aureus and Escherichia coli. Opsonization with serum caused a modest increase in bacteria uptake, but in contrast to macrophages, the classical complement receptors were not found on RPE cells. Living bacteria were also ingested in a time-dependent manner, but, as no intracellular overgrowth was observed, we further investigated the oxidative ability of RPE as a possible mechanism for microbial suppression. Unlike macrophages/granulocytes, no respiratory burst was detected in RPE cells, but, comparable to MonoMac6, IFN-γ induced neopterin in the human RPE. Interestingly a diurnal rhythm of phagocytosis was observed which was influenced by light exposure suggesting that RPE cells maintain their circadian rhythm also in cell culture to a certain extent. This study further demonstrates that in addition to similar phagocytic properties the RPE still shows substantial metabolic differences in comparison to blood-derived phagocytes.
Glycosaminoglycans in Human Trabecular Meshwork: Age-Related Changes
Authors: Cavallotti Carlo , Feher Janos , Pescosolido Nicola , Sagnelli Paolo
Published: August 2004

Glycosaminoglycans play a central role in maintaining the normal outflow resistance in the human trabecular meshwork. We evaluated the possible morphological, histochemical and morphometrical age-related changes in glycosaminoglycans of the trabecular meshwork. Small human samples were drawn from 24 eyes after exitus from young and old humans. Samples were harvested from the anterior chamber of the eye, without any aesthetic damage for the face. They were divided into three fragments, each used for morphological, histochemical and ultrastructural staining. Quantitative analysis of images was performed to evaluate morphometrical data that were statistically analysed. Our findings demonstrate the following age-related changes: (1) deposition of fibrous granular material in the trabecular meshwork; (2) increased electron density of the structures; (3) strong decrease in the hyaluronic acid content, and (4) increase in sulphated proteoglycans. Glycosaminoglycans of human trabecular meshwork undergo age-related changes, as demonstrated by our morphological, histochemical and morphometrical results.
Transplantation of the Autologous Submandibular Gland to the Lacrimal Basin in Rats
Authors: Liu Wan-Cherng , Hsu Wen-Ming , Lee Shui-Mei , Lee Fenq-Lih , Yen May-Yung , Chou Ching-Kuang , Kao Shu-Ching
Published: August 2004

Purpose: In this study, we designed an animal experiment in which we transferred a part of the autologous submandibular gland without performing a microvascular anastomosis. We studied histological changes and functional effects in the transfer glands. Method: Thirty male adult Sprague-Dawley rats were divided into three groups. In each animal of the three groups, the right eye was the control eye and the left eye the experimental eye. Surgical removal of the extraorbital lacrimal gland from the control eyes was performed to create a condition simulating keratoconjunctivitis sicca. In the experimental eyes of group 1, in addition to the removal of the extraorbital lacrimal gland, a part of the autologous submandibular gland was transferred to the orbit and fixed to the intraorbital lacrimal gland. In the experimental eyes of group 2, in addition to the removal of the extraorbital lacrimal gland, an aseptic silicon rubber was transferred to the orbit and fixed to the intraorbital lacrimal gland. In the experimental eyes of group 3, there was no removal of the extraorbital lacrimal gland but instead a sham operation was performed. The histological changes and innervation pattern in the transferred submandibular gland of group 1 were observed. Tear secretion of each group was measured to study the functional effect. Results: Three months after the transplantation, the transferred submandibular glands were similar to the unoperated submandibular glands both in histology and innervation pattern. In group 1, the tear secretion in the experimental eyes was significantly greater than that of the control eyes at 2 and 3 months following transfer. In group 2, no significant difference was noted between the experimental eyes and control eyes. In group 3, the difference in tear secretion between the experimental eyes and control eyes was significant. Conclusion: The result of this study revealed that although the procedure did not involve vascular anastomosis, the transfer gland showed a normal histological appearance and good reinnervation, even 3 months after the transfer. These transferred submandibular glands continued to secrete tears. This secretion continued to increase up until the end of the experimental period.
Expression of c-Fos and c-Jun in Developing Mouse Lens
Authors: Shirai Kumi , Saika Shizuya , Okada Yuka , Senba Emiko , Ohnishi Yoshitaka
Published: August 2004

We carried out immunohistochemistry in embryonic eye-specific antibodies in the mouse to determine the spatial/temporal expression pattern of c-Fos and c-Jun proteins – the main components of the AP-1 transcription factor, in lens epithelial cells during mouse lens morphogenesis. c-Fos protein expression was detected in the equatorial epithelium at E14.5–P25, with a peak at P1–P8. c-Jun protein expression was detected in equatorial lens epithelial cells at E18.5–P8, with a peak at P1. During these intervals, the anterior epithelium lacked expression of c-Fos and c-Jun. They were not detected in an adult mouse lens. The expression pattern of the AP-1 transcription factor in equatorial epithelium may suggest its role in fiber differentiation.
An Alternative Method of Steroid-Induced Lens Opacification in Brown Norway Rat Eyes Applying Systemic Pulse Administration
Authors: Nagai Kouta , Sasaki Hiroshi , Kojima Masami , Sasaki Kazuyuki
Published: August 2004

Purpose: To confirm whether long-term administration of prednisolone sodium succinate (prednisolone) alone is able to induce cataract in rat eyes. Materials and Methods: A 1% solution of prednisolone was administered topically as eye drops to Brown Norway rat eyes, and a systemic pulse administration of 10 mg/kg/day was given via the tail vein. Both administration methods were applied in different combinations. Eighty-three 6-week-old male rats were divided into 8 groups: group 1 = control; group 2 = topical instillation every day; group 3 = single pulse; group 4 = single pulse + eye drops; group 5 = 3 times pulse; group 6 = 3 times pulse + eye drops; group 7 = 3 times pulse per 2 months; group 8 = 3 times pulse per 2 months + eye drops. Observations for changes of lens transparency were made by slitlamp microscopy and documented by an anterior eye segment analysis system (Nidek EAS-1000®) from the onset of drug administration to a maximum period of 16 months. Results: Lens opacity in the shallow anterior and posterior lens layers developed from the tenth month following commencement of prednisolone administration. The incidence of anterior and/or posterior cortical cataract at the sixteenth month was 15% in group 2, 12.5% in group 5, 25% in group 6, 17.9% in group 7 and 35.3% in group 8. The lenses of groups 1, 3 and 4 maintained their transparency throughout the observation period. Light scattering intensity in groups 8 and 7 was the highest, followed by groups 6 and 5, then groups 2, 4, 3 and 1. Conclusion: Cortical cataract was successfully induced in Brown Norway rat eyes by sustained administration of prednisolone succinate alone applied as systemic pulse.
Fluphenazine and Its Toxic Maculopathy
Authors: Lee M.S. , Fern A.I.
Published: August 2004

Ten years ago a 45-year-old female presented with bilateral maculopathy with visual acuity of 6/18 on the right and 6/24 on the left. She had been on fluphenazine for the past 10 years for schizophrenia. Investigation including fluorescein angiogram, colour vision assessment and electrophysiological testing confirmed the nature of the retinal damages. Her maculopathy deteriorated further despite discontinuation of the medication. A previous publication has reported maculopathy with fluphenazine in association with the welding arc injury, but that particular patient had not been exposed to welding flash or other extreme photochemical sources. We believe this is the first reported incidence that fluphenazine has directly caused a maculopathy secondary to its accumulation at the retinal pigment epithelium and its toxic effect.
Occult Tight Filum terminale Syndrome: Results of Surgical Untethering
Authors: Wehby Monica C. , O’Hollaren Patrick S. , Abtin Keyvan , Hume Jenny L. , Richards Benjamin J.
Published: August 2004

The entity of an occult tight filum terminale syndrome, characterized by clinical findings consistent with a tethered cord syndrome, but with the conus ending in a normal position, has been recognized recently. The indications for sectioning the filum terminale in this situation are not well characterized and are controversial. We report a retrospective review of a consecutive series of 60 children (ages 3–18 years) with a diagnosis of occult tight filum terminale syndrome who underwent section of the filum and were followed for more than 6 months (mean 13.9 months). The criteria for surgical intervention were (1) spina bifida occulta, (2) progressive bladder instability unresponsive to conservative measures, (3) urological/nephrological evaluation to confirm or rule out nonneurogenic etiology, and (4) two or more of the following: (a) bowel involvement (fecal incontinence or chronic constipation), (b) lower extremity weakness, (c) gait changes, (d) reflex/tone abnormalities, (e) sensory disturbances, (f) back/leg pain, (g) orthopedic abnormalities/limb length discrepancy, (h) scoliosis/lordosis, (i) recurrent urinary tract infections, (j) abnormal voiding cystourethrogram/ultrasound, (k) syringomyelia, and (l) neurocutaneous stigmata. Postoperatively, urinary incontinence/retention showed complete resolution in 52%, marked improvement (>95% resolution) in 35%, moderate improvement (>75%) in 6%, minimal improvement (> 50%) in 6%, and no improvement (<50%) in 2%. Fecal incontinence completely resolved in 56%, improved in 41%, and was unchanged in 3%. Weakness, sensory abnormalities, and pain improved or resolved in all patients.
Endoscopic Cyst Fenestration Outcomes in Children One Year of Age or Less
Authors: Sikorski Christian W. , Yamini Bakhtiar , Frim David M.
Published: August 2004

The use of endoscopic fenestration (EF) is becoming an increasingly common treatment for symptomatic intracranial cysts. Very little data exist regarding outcomes for this procedure in children 1 year of age or younger. We retrospectively reviewed the clinical outcomes of 8 children 1 year of age or less treated at our institution with endoscopic cyst fenestration. The mean follow-up was roughly 2.5 years. These data were combined with 17 other cases obtained from the published literature. EF was successful in rendering patients shunt-free or minimizing the number of ventricular catheters in 18 of 26 operations. There were 8 outright failures – two in 1 patient. Given the risks and complications of cerebrospinal fluid shunting in children less than 1 year of age, we advocate the consideration of EF as initial treatment of symptomatic intracranial cysts.
Thoracic Meningocele, Meningomyelocele or Myelocystocele? Diagnostic Difficulties, Consequent Implications and Treatment
Authors: Arts M.P. , de Jong T.H.R.
Published: August 2004

Spina bifida cystica is a closing disorder of the neural tube which infrequently occurs in the thoracic region. A rare lesion called myelocystocele is a variant of spina bifida cystica and is associated with syringomyelia, Chiari type 2 malformation and hydrocephalus. Usually the patient has no neurological deficit, but future deterioration can occur due to posterior tethering of the spinal cord by adhesions. The prenatal diagnosis by ultrasound study can be misleading and in order to attain the correct diagnosis, especially if abortion is considered, a prenatal MRI scan should be done before the parents are counselled, and should be repeated prior to operative treatment. Surgical correction of myelocystocele is not only for cosmetic reasons, but also to untether the spinal cord prophylactically to prevent future neurological deterioration. In this case report, we present a child born with a thoracic myelocystocele, the diagnostic difficulties, consequent implications and surgical treatment.
Split Cord Malformation as aCause of Tethered Cord Syndrome in a 78-Year-Old Female
Authors: Pallatroni Henry F. , Ball Perry A. , Duhaime Ann-Christine
Published: August 2004

A 78-year-old woman presented for evaluation of back pain, urinary dysfunction, leg weakness and progressive equinovarus foot deformity. She reported that shortly after her birth in 1924, she underwent resection of a subcutaneous ‘cyst’ in the lower lumbar area. Seven years prior to evaluation at our institution, she had undergone bilateral total knee arthroplasty for osteoarthritis. After the procedure, she began to experience severe low back pain that radiated into her legs. Weakness of the foot inverters, urinary dysfunction and worsening bilateral equinovarus foot deformity developed in the years following the surgery. MRI revealed a split cord malformation with a tethered spinal cord. Because of the patient’s age and poor medical condition, her symptoms were managed conservatively. This case demonstrates symptomatic deterioration in an elderly patient with a tethered spinal cord after many years of clinical stability.
Properties of Human Trabecular Bone Cells from Elderly Women: Implications for Cell-Based Bone Engraftment
Authors: Mayer Hubert
Published: August 2004

Tissue-engineered bone regeneration offers new therapeutic options in the treatment of patients with fractures. Due to the changes in the hormone levels, elderly women are most affected by bone fractures and thus constitute one of the future target groups of cell-based bone therapy. For designing cell-based therapy approaches, a better understanding of individual-dependent variations in bone-derived cells of the host is required. In this study, a simple, high-yield procedure is described for the collection of cells from bone tissue of a high number of elderly women. The cultured cells display stem cell characteristics indicated by the presence of aCD13+, CD44+, CD90+, CD147+, CD14–, CD34–, CD45– and CD144– cell populations and by a stable undifferentiatedphenotype as well as by the ability to proliferate extensivelywhile retaining the potential to differentiate along the osteoblastic lineage even after 27 cell doublings. A high variability in the number of cell-forming units (CFUs) within a donor population of 34 samples, in the morphology within 50 donors, in the expression of alkaline phosphatase within 15 samples and in the responsiveness to BMP-2 was evident, but no age-related correlation could be found. In conclusion, the data indicate that individual variations in cell number, cell morphology and in the osteogenic potential of progenitor cells of the patient may be relevant for a successful treatment of bone fractures in the elderly by cell-based therapy approaches.
Evaluation of Mineralized Collagen and α-Tricalcium Phosphate as Scaffolds for Tissue Engineering of Bone Using Human Mesenchymal Stem Cells
Authors: Niemeyer Philipp , Krause Ulf , Fellenberg Jörg , Kasten Philip , Seckinger Anja , Ho Anthony D. , Simank Hans-Georg
Published: August 2004

Owing to their plasticity and high proliferation capacity in vitro, mesenchymal stem cells (MSC) isolated from human bone marrow are promising candidates for use in tissue engineering approaches for the repair or replacement of mesenchymal tissues such as bone, cartilage or tendon. In keeping with the tissue engineering concept, these cells are cultivated on three-dimensional (3D) scaffolds to replace 3D tissue defects. Among the scaffolds tested for tissue engineering of bone, those containing phosphorus and calcium, as natural bone does, are the most promising candidates for this purpose. In this study, MSC from five patients were isolated from bone marrow. After in vitro expansion, cells were cultivated and differentiated towards the osteogenic lineage on mineralized collagen sponges and α-tricalcium phosphate (α-TCP). To analyze how appropriate these scaffolds would be for tissue engineering purposes, we established an in vitro characterization system to describe seeding efficiency, cell distribution and proliferation behavior on each scaffold. Real-time reverse transcriptase polymerase chain reaction quantification of important genes involved in osteogenic differentiation [e.g. bone sialoprotein (BSP), bone morphogenic protein 2, alkaline phosphatase and osteocalcin] was used to monitor the differentiation process of cells seeded on mineralized collagen and α-TCP. Using this in vitro characterization, we were able to demonstrate effective 3D growth of MSC on both scaffolds investigated. Improved osteogenic differentiation was observed on the scaffolds as compared to control monolayers. Of the two matrices, mineralized collagen was superior to α-TCP with regard to seeding efficacy (98 vs. 67%, p = 0.0003), increase in osteogenic marker genes (BSP expression on day 24, pcollagen vs. TCP = 0.046) and 3D cell alignment (cell infiltration up to 500 vs. 200 µm). In conclusion, our data suggest that mineralized collagen is a promising candidate for use as a scaffold in tissue engineering of bone.
Developing Mouse Sertoli Cells in vitro: Effects on Developing Ovaries in Co-Culture and Production of Anti-Müllerian Hormone
Authors: Mackay Sarah , Willerton Louise , Ballingall Christine L. , Henderson Nicola J.S. , Smith Robert A.
Published: August 2004

Previous work in our laboratory showed that pre-Sertoli cells adopt an epithelial phenotype when cultured in the presence of reconstituted basement membrane (RBM), and so cultures were established with and without this substrate. Biological activity of isolated developing mouse Sertoli cells maintained in vitro was assessed in the current study by utilising a co-culture approach, to determine whether the cells were capable of affecting ovarian differentiation. Developing Sertoli cells isolated at embryonic day (E) 12.5 exerted a deleterious effect on E12.5 ovaries in co-culture, inducing a loss of germ cells. However, when cells were isolated a day later and co-cultured with E13.5 ovaries (after entry to meiosis has begun), germ cells survived and showed evidence of meiosis, although ovigerous cords in co-cultures were masculinised compared to those of control cultured ovaries. Thus, both stages examined showed biological effects; cultured pre-Sertoli cells explanted at E12.5 showed a negative effect on female germ cells, whereas those explanted at E13.5 masculinised ovigerous cords. The functional status of isolated developing mouse Sertoli cells in vitro was further assessed by immunocytochemistry to investigate the expression of anti-Müllerian hormone, an early product of pre-Sertoli cells. Positive immunostaining was seen in developing Sertoli cells in vitro, particularly where cells had been explanted to an RBM substrate, demonstrating that good epithelial morphology is associated with improved function. Our culture system is therefore well suited for investigating factors produced by developing Sertoli cells, their role in influencing testicular morphogenesis and their potential to perturb ovarian differentiation. We believe that this in vitro approach provides a more physiological assessment compared with the knockout mouse model, where global effects of genes with housekeeping functions can compromise overall development.
The Reliability of Haplotyping Inference in Nuclear Families: Misassignment Rates for SNPs and Microsatellites
Authors: Lindholm Eva , Zhang Junying , Hodge Susan E. , Greenberg David A.
Published: August 2004

Single nucleotide polymorphisms (SNPs) are widely used when investigators try to map complex disease genes. Although biallelic SNP markers are less informative than microsatellite markers, one can increase their information content by using haplotypes. However, assigning haplotypes (i.e., assigning phase) correctly can be problematic in the presence of SNP heterozygosity. For example, a doubly heterozygous individual, with genotype 12, 12, could have haplotypes 1-1/2-2 or 1-2/2-1 with equal probability; in the absence of additional information, there is no way to determine which haplotype is correct. Thus an algorithm that assigns haplotypes to such an individual will assign the wrong one 50% of the time. We have studied the frequency of haplotype misassignments, i.e., haplotypes that are misassigned solely because of inherent marker ambiguity (not because of errors in genotyping or calculation). We examined both SNPs and microsatellite markers. We used the computer programs GENEHUNTER and SIMWALK to assign the haplotypes. We simulated (a) families with 1–5 children, (b) haplotypes involving different numbers of marker loci (3, 5, 7 and 10 loci, all in linkage equilibrium), and (c) different allele frequencies. Misassignment rates are highest (a) in small families, (b) with many SNP loci, and (c) for loci with the greatest heterozygosity (i.e., where both alleles have frequency 0.5). For example, for triads (i.e., one-child families with both parents genotyped), misassignment rates for SNPs can reach almost 50%. Family sizes of 4–5 children are required in order to ensure a misassignment frequency of ≤5% for ten-SNP haplotypes with allele frequencies of 0.25–0.5. For microsatellites, a family size of at least 2–3 children is necessary to keep haplotyping misassignments ≤5%. Finally, we point out that it is misleading for a computer program to yield haplotype assignments without indicating that they may have been misassigned, and we discuss the implications of these misassignments for association and linkage analysis.
Association Analysis of the Plasminogen Activator Inhibitor-1 4G/5G Polymorphism in Hispanics and African Americans: The IRAS Family Study
Authors: Bensen Jeannette T. , Hsu Fang-Chi , Brown W. Mark , Sutton Beth S. , Norris Jill M. , Tracy Russell P. , Jenny Nancy S. , Saad Mohammed F. , Haffner Steven , Bowden Donald W. , Langefeld Carl D.
Published: August 2004

Objective: Plasminogen activator inhibitor type-1 (PAI-1) plays a central role in fibrolysis and has recently been hypothesized to influence components of the insulin resistance syndrome. We consider whether the 4G/5G polymorphism influences components of insulin resistance and obesity solely through PAI-1 protein levels or also though a secondary pathway. In addition, we explore whether transforming growth factor (TGF-β1), a key regulator of PAI-1 expression, modifies the influence of the PAI-1 4G/5G polymorphism on these traits. Methods and Results: The Insulin Resistance and Atherosclerosis (IRAS) Family Study genotyped 287 African American (18 pedigrees) and 811 Hispanic American (45 pedigrees) individuals for the 4G/5G PAI-1 and two TGF-β1 polymorphisms (R25P, C-509T). Individuals were recruited from three clinical centers located in San Antonio (urban Hispanic), San Luis Valley (rural Hispanic) and Los Angeles (African American). The presence of the 4G PAI-1 allele was positively associated with PAI-1 protein level (combined sample p < 0.0001). Hispanic Americans average 65% higher PAI-1 protein levels than African Americans (p < 0.0001). Consistently across ethnic groups, increased PAI-1 protein levels were associated with increased insulin resistance and overall and central obesity (p value < 0.0001, combined sample). Adjusting for PAI-1 protein levels, there was evidence of an association of PAI-1 genotype (4G) with insulin sensitivity (p < 0.002) and subcutaneous fat (p < 0.01). These associations were not influenced by TGF-β1 genotypes. Conclusions: PAI-1 protein is a strong correlate of insulin resistance (IR) and obesity in Hispanics and African Americans. However, PAI-1 4G/5G polymorphism appears to influence insulin resistance and obesity beyond its direct influence on serum PAI-1 protein levels.
Comparative in vitro Expression Study of Four Fabry Disease Causing Mutations at Glutamine 279 of the α-Galactosidase A Protein
Authors: Dominissini Silvia , Cariati Roberta , Nevyjel Marco , Guerci Veronica , Ciana Giovanni , Bembi Bruno , Pittis Maria Gabriela
Published: August 2004

Fabry disease is an X-linked lysosomal storage disorder caused by the deficiency of α-galactosidase A that results in the accumulation of neutral sphingolipids. We report a novel point mutation in exon 6, Q279K, carried by an asymptomatic child with a family history of classic Fabry disease. Moreover, we comparatively study the in vitro expression and enzyme activity of Q279K and three other already described mutants in glutamine 279. The Q279K, Q279H and Q279R mutants transfected in COS-1 cells expressed no activity while the residual enzyme activity of the Q279E mutant represented 10% of wild type value. Western blot analysis demonstrated a differential behavior of the mutant proteins: Q279K and Q279H persisted as the inactive 50-kD precursor, indicating that these mutations may affect the normal processing of the enzyme, while the Q279R mutant was not detected probably due to an unstable protein which is rapidly degraded. The in vitro expression studies of the novel Q279K mutation were confirmed by Western blot analysis performed in the patient’s lymphocytes which revealed the α-galactosidase A precursor of 50 kD but not the processed form.
Evidence of Admixture from Haplotyping in an Epidemiological Study of UK Caucasian Males: Implications for Association Analyses
Authors: Chen Xiao-he , Rodríguez Santiago , Hawe Emma , Talmud Philippa J. , Miller George J. , Underhill Peter , Humphries Stephen E. , Day Ian N.M.
Published: August 2004

Objective: Cohort and case-control genetic association studies offer the greatest power to detect small genotypic influences on disease phenotypes, relative to family-based designs. However, genetic subdivisions could confound studies involving unrelated individuals, but the topic has been little investigated. We examined geographical and interallelic association of SNP and microsatellite haplotypes of the Y chromosome, of regions of chromosome 11, and of autosomal SNP genotypes relevant to cardiovascular risk traits in a UK-wide epidemiological survey. Results: We show evidence (p = 0.00001) of the Danelaw history of the UK, marked by a two-fold excess of a Viking Y haplotype in central England. We also found evidence for a (different) single-centre geographical over-representation of one haplotype, both for APOC3-A4-A5 and for IGF2. The basis of this remains obscure but neither reflect genotyping error nor correlate with the phenotypic associations by centre of these markers. A panel of SNPs relevant to cardiovascular risks traits showed neither association with geographical location nor with Y haplotypes. Conclusion: Combinations of Y haplotyping, autosomal haplotyping, and genome-wide SNP typing, taken together with phenotypic2 associations, should improve epidemiological recognition and interpretation of possible confounding by genetic subdivision.
Selecting Tagging SNPs for Association Studies Using Power Calculations from Genotype Data
Authors: Hu Xiaolan , Schrodi Steven J. , Ross David A. , Cargill Michele
Published: August 2004

Recent studies have indicated that linkage disequilibrium (LD) between single nucleotide polymorphism (SNP) markers can be used to derive a reduced set of tagging SNPs (tSNPs) for genetic association studies. Previous strategies for identifying tSNPs have focused on LD measures or haplotype diversity, but the statistical power to detect disease-associated variants using tSNPs in genetic studies has not been fully characterized. We propose a new approach of selecting tSNPs based on determining the set of SNPs with the highest power to detect association. Two-locus genotype frequencies are used in the power calculations. To show utility, we applied this power method to a large number of SNPs that had been genotyped in Caucasian samples. We demonstrate that a significant reduction in genotyping efforts can be achieved although the reduction depends on genotypic relative risk, inheritance mode and the prevalence of disease in the human population. The tSNP sets identified by our method are remarkably robust to changes in the disease model when small relative risk and additive mode of inheritance are employed. We have also evaluated the ability of the method to detect unidentified SNPs. Our findings have important implications in applying tSNPs from different data sources in association studies.
The distribution of B chromosomes across species
Authors: Palestis B.G. , Trivers R. , Burt A. , Jones R.N.
Published: August 2004

In this review we look at the broad picture of how B chromosomes are distributed across a wide range of species. We review recent studies of the factors associated with the presence of Bs across species, and provide new analyses with updated data and additional variables. The major obstacle facing comparative studies of B chromosome distribution is variation among species in the intensity of cytogenetic study. Because Bs are, by definition, not present in all individuals of a species, they may often be overlooked in species that are rarely studied. We give examples of corrections for differences in study effort, and show that after a variety of such corrections, strong correlations remain. Several major biological factors are associated with the presence of B chromosomes. Among flowering plants, Bs are more likely to occur in outcrossing than in inbred species, and their presence is also positively correlated with genome size and negatively with chromosome number. They are no more frequent in polyploids than in diploids, nor in species with multiple ploidies. Among mammals, Bs are more likely to occur in species with karyotypes consisting of mostly acrocentric chromosomes. We find no evidence for an association with chromosome number or genome size in mammals, although the sample for genome size is small. The associations with breeding system and acrocentric chromosomes were both predicted in advance, but those with genome size and chromosome number were discovered empirically and we can offer only tentative explanations for the very strong associations we have uncovered. Our understanding of why B chromosomes are present in some species and absent in others is still in its infancy, and we suggest several potential avenues for future research.
Distribution and stability of supernumerary microchromosomes in natural populations of the Amazon molly, Poecilia formosa
Authors: Lamatsch D.K. , Nanda I. , Schlupp I. , Epplen J.T. , Schmid M. , Schartl M.
Published: August 2004

In animals, supernumerary chromosomes and their evolution have mostly been studied in sexual reproducing species. In the present study, for the first time, the natural distribution and stability of supernumerary microchromosomes were investigated in the unisexual fish species Poecilia formosa. Natural habitats throughout the range of P. formosa were screened for the presence of microchromosomes over several years. A high frequency of microchromosomes was found in the Río Purificación river system. Evidence points to the presence of the same microchromosome lineage over many generations. No supernumerary chromosomes were found elsewhere than in the Río Purificación representing a significant difference in the distribution of microchromosome-bearing individuals between the Río Purificación and all other collection sites.
B chromosomes in Amazonian cichlid species
Authors: Feldberg E. , Porto J.I.R. , Alves-Brinn M.N. , Mendonça M.N.C. , Benzaquem D.C.
Published: August 2004

B chromosomes are reported in three different Amazonian cichlid species. One to three supernumerary microchromosomes were detected in the peacock bass Cichla monoculus (4 out of 28 specimens) and Cichla sp. (4 out of 13 specimens), and pike cichlids Crenicichla reticulata (2 out of 5 specimens), with no similar standard chromosomal morphology. C-banding revealed that B chromosomes are totally heterochromatic. We suggest two scenarios for the origin of these B chromosomes either by chromosomal breakdowns due to mutagenic action of methyl mercury present in the aquatic environment or by interspecific origin due to hybridization events.
The B chromosomes in Brachycome
Authors: Leach C.R. , Houben A. , Timmis J.N.
Published: August 2004

This review presents a historical account of studies of B chromosomes in the genus Brachycome Cass. (synonym: Brachyscome) from the earliest cytological investigations carried out in the late 1960s though to the most recent molecular analyses. Molecular analyses provide insights into the origin and evolution of the B chromosomes (Bs) of Brachycome dichromosomatica, a species which has Bs of two different sizes. The larger Bs are somatically stable whereas the smaller, or micro, Bs are somatically unstable. Both B types contain clusters of ribosomal RNA genes that have been shown unequivocally to be inactive in the case of the larger Bs. The large Bs carry a family of tandem repeat sequences (Bd49) that are located mainly at the centromere. Multiple copies of sequences related to this repeat are present on the A chromosomes (As) of related species, whereas only a few copies exist in the A chromosomes of B. dichromosomatica. The micro Bs share DNA sequences with the As and the larger Bs, and they also have B-specific repeats (Bdm29 and Bdm54). In some cases repeat sequences on the micro Bs have been shown to occur as clusters on the A chromosomes in a proportion of individuals within a population. It is clear that none of these B types originated by simple excision of segments from the A chromosomes.
B chromosomes in Sternorrhyncha (Hemiptera, Insecta)
Authors: Maryańska-Nadachowska A.
Published: August 2004

In the hemipteroid insects of the suborder Sternorrhyncha, B chromosomes are relatively common in comparison with other suborders of Hemiptera. However, the occurrence of supernumerary chromosomes is restricted, in most cases, to several genera or closely related species. At least in some species of Psylloidea with the XY sex determination system, a mitotically stable B chromosome integrated into an achiasmatic segregation system with the X, and became fixed as a Y chromosome. In some Aphidoidea with a multiple X system of sex determination, B chromosomes appear to be in fact non-functional X chromosomes. Supernumerary chromosomes thus probably play an important role in the evolution of sex determination systems in Sternorrhyncha.