Cellular contribution of bone graft to fusion.

Although a number of studies have examined the fate of graft-derived cells during the process of fusion, there remains no consensus regarding their exact contribution to bone formation within the fusion mass. We developed two chimeric mouse isograft fusion models that allowed us to track the fate of graft cells within the host fusion bed. Cortical/cancellous bone graft (1:1 ratio of pelvic to vertebral body bone) from male mice was placed between (a) the tibia and fibula or (b) the coccygeal spine transverse processes of syngeneic female hosts. Both models were characterized histologically and histochemically. Graft-derived cells were then identified by fluorescent in situ hybridization for Y-chromosome sequences present in only the graft (male) cells. When the fusion mass was healing but not yet fused (at 1 and 2 weeks), numerous graft-derived cells were observed throughout the fusion site. The predominant graft-derived cell types included chondrocytes, osteoblasts, and fibroblasts. Chondrocytes arose from precursor cells in the graft de novo. as cartilage was not transplanted during the surgical procedure. By the time a mature fusion mass had formed (at 6 weeks), graft-derived cells persisted as osteocytes within the cortical rim surrounding the fusion mass. These osteocytes likely differentiated from graft-derived precursors that had directly formed bone, because transplanted osteocytes within cortical bone graft fragments were noted to rarely survive even at 1 and 2 weeks. Collectively, our results demonstrate for the first time that bone graft contributes cells that, in conjunction with host cells, directly form bone within the fusion mass during all phases of fusion rather than just the early phases.

Histochemical and molecular analyses of distraction osteogenesis in a mouse model.

A tibial lengthening scheme in the mouse was used to study the molecular and cellular events regulating tissue regeneration during distraction osteogenesis. Here, we report on the surgical technique and frame design and describe the histochemical and molecular aspects of distraction during different phases of treatment. A total of 26 mice were used in this study. The treatment protocol was divided into a latency period of 7 days, a phase of active distraction that lasted 10 days with a distraction rate of 0.42 mm/day, and a maturation phase of 9 days. During latency, the distraction site resembled a stabilized fracture callus on both a histochemical and a molecular level. During active distraction, the gap was characterized by a central fibrous interzone bordered by primary matrix fronts, regenerate bone aligned with the distraction force, parallel columns of vascular sinusoids, and a medullary cavity. Alkaline phosphatase activity was detected in the endosteal and periosteal surfaces of the bone ends. Tartrate resistant acid phosphatase staining revealed that osteoclasts remodeled the bone regenerate as it formed. Collagen type I was expressed in the periosteum and the primary matrix front during distraction, whereas collagen type-II transcripts were localized to discrete regions on the periosteal surfaces, immediately adjacent to the osteotomy ends. Collagen type-II transcripts were not detected in the fibrous interzone. During the maturation phase, cells within the fibrous interzone expressed collagen type I and exhibited abundant alkaline phosphatase activity, suggesting that they had begun to terminally differentiate. Collectively, these data demonstrate the utility of a mouse model to study the molecular and cellular bases for the regeneration and remodeling of tissue.

Neural induction and antero-posterior patterning in the amphibian embryo: past, present and future.

Neural induction and patterning in competent ectoderm occurs during gastrula and early neurula stages in response to signals from dorsal mesoderm. The earliest views of antero-posterior (A-P) patterning were modified beginning in the 1930s, as complexities concerning the timing of the pattern-forming process and potential sources of the patterning signals were revealed. In the 1950s and 1960s several different models for A-P patterning were proposed, all of which, however, bear a number of similarities, including a two-component system for generating A-P axial information in the embryo. Early attempts to identify neural-inducing molecules were largely unsuccessful due to technical limitations in biochemical analyses and concerns about assaying neural responses. The advent of modern molecular genetic technology has permitted more precise tests of a number of classic observations about the timing of A-P patterning and the sources of patterning signals. While some early observations have been confirmed, a number of new concepts have emerged in recent years, particularly concerning the source of patterning signals in the embryo. Striking progress has been made in identifying putative neural-inducing molecules, and recent experiments have begun to suggest how these might contribute to A-P patterning. While the successes in recent years have been revealing, many of the classic issues concerning neural induction and patterning remain essentially as they were when first defined many decades ago. The power of modern molecular genetics, however, should permit many of these issues to be significantly clarified in the decades to come.

Characterization of chicken syndecan-3 as a heparan sulfate proteoglycan and its expression during embryogenesis.

Syndecan-3 is one of four identified members of a family of transmembrane proteoglycans (the syndecans) that possess highly similar cytoplasmic and transmembrane domains and may function as extracellular matrix receptors and/or low affinity receptors for signaling molecules such as FGF. We previously reported the cloning of a partial cDNA for chicken syndecan-3. Here we report the isolation of a syndecan-3 cDNA containing additional 5' sequence which includes a potential methionine start codon and putative signal sequence. In vitro translation of syndecan-3 cDNA in the presence and absence of microsomes suggests that the putative signal sequence is functional, suggesting that the cDNA may encompass the full coding sequence. We also identify syndecan-3 as a heparan sulfate proteoglycan and report its expression pattern during chicken embryogenesis using polyclonal antibodies raised against a recombinant fusion protein. We detect abundant syndecan-3 expression in the developing brain and neural tube, including a striking expression in the floor plate of the neural tube. During limb development, syndecan-3 is expressed in the distal mesenchymal cells of the limb bud which are undergoing outgrowth in response to the apical ectodermal ridge. Syndecan-3 is also transiently expressed during the formation of the precartilage condensations of the skeletal elements of the limb and subsequently in association with the differentiating osteoblasts of the periosteum. Expression is also observed in several areas of tissue interactions including the developing lens, otic vesicle, genital ridge, sclerotome, and feather buds.

Syndecan 3: a member of the syndecan family of membrane-intercalated proteoglycans that is expressed in high amounts at the onset of chicken limb cartilage differentiation.

A partial cDNA that encodes a newly discovered member of the syndecan family of integral membrane proteoglycans, which we have termed syndecan 3, has been isolated from an embryonic chicken limb bud cDNA library. Syndecan 3 is distinct from but structurally related to syndecan and fibroglycan, two previously characterized members of this family of membrane-intercalated proteoglycans. Syndecan 3 contains a cytoplasmic domain potentially associated with the cytoskeleton that is 85% identical in amino acid sequence to the cytoplasmic domain of syndecan. Syndecan 3 also possesses a hydrophobic transmembrane domain and an extracellular domain containing several clustered potential glycosaminoglycan attachment sites. Like syndecan, the ectodomain of syndecan 3 has a single dibasic protease-susceptible site adjacent to the transmembrane domain, which might be involved in shedding the ectodomain from the cell surface. A striking feature of syndecan 3 is an extensive (182 amino acid) threonine, serine, and proline (T+S+P)-rich domain that closely resembles T+S+P-rich regions in several mucin-like proteins in which O-linked oligosaccharides are bound to the threonine and serine residues. Syndecan 3 is expressed in high amounts during a critical phase of chicken limb chondrogenesis in which limb mesenchymal cells condense, round up, and interact with one another before depositing a cartilage matrix. The multiple functional domains of syndecan 3 provide potential sites for mediating the adhesive cell-matrix interactions and cytoskeletal reorganization involved in this critical condensation process.

A screening programme for hypertension in general practice.

It is recommended that hypertensive patients with a diastolic blood pressure of 100 mmHg be treated. However, it has been proposed that only 50% of hypertensives are diagnosed, and of these, 50% are not treated. We therefore set out to identify hypertensive patients in our practice, who were then actively treated, some in a clinical trial of ketanserin. Of the 3,384 patients (1,667 males and 2,707 females) who were invited to our surgery for a blood pressure measurement, 2,606 patients (77%) attended. The overall prevalence of hypertension in the patients, aged 35-65 years was 2.9%. This prevalence increased with age: from 1.0% in the 35-44 year age group to 3.5% at 45-54 years, and 4.4% at 55-64 years. The mean systolic but not diastolic blood pressure increased with age. There were more male hypertensives than females, except over 55 years of age. Of the 84 hypertensives identified after one visit, the majority remained hypertensive after an additional visit, and 31 agreed to participate in a clinical trial. After a four-week placebo run-in, 22 patients had a diastolic blood pressure above 95 mmHg and were randomly allocated to receive ketanserin 40 mg twice daily or metoprolol 100 mg twice daily. Treatment continued double-blind for three months. There were no significant differences in blood pressure reduction between the treatment groups. The heart rate was reduced more by metoprolol. There were no withdrawals for side effects and no major differences in subjective complaints between the two treatment groups.

A placebo-controlled crossover study of ketanserin in elderly hypertensive patients.

The results of the European Working Party for Hypertension in the Elderly Study showed that treatment of high blood pressure reduced the morbidity and mortality from strokes and myocardial infarction and reduced the incidence of heart failure in elderly patients. The largest number of hypertensive patients are elderly, and it is in this group of patients that the maximum benefit of treatment might be expected. The present study was designed to study in detail the efficacy and tolerability of ketanserin in an elderly population. Seventeen elderly (greater than 70 years) patients with a lying systolic blood pressure of 160 mmHg and/or a diastolic blood pressure of greater than or equal to 90 mmHg were included in the study. For the 12 patients who completed the study, the mean blood pressure was significantly reduced on ketanserin compared with placebo (p less than 0.001) in the supine and erect positions. The mean net changes in blood pressure after 8 weeks were 21/17 mmHg and 23/16 mmHg erect. Heart rate was also significantly reduced (p less than 0.001) by a mean of 8 beats/min lying and 9 beats/min erect. Analysis of ambulatory 24-hour ECG tapes showed no significant effect of ketanserin on heart rhythms. Ketanserin therapy had no significant effect on routine hematology, plasma electrolytes, biochemistry, or urinalysis. Total exchangeable sodium and potassium and body weight were also unchanged. On ketanserin treatment, the overall quality of life score was significantly improved (p = 0.002; analysis of variance on log transformed data) compared with the placebo phase.

Could the increased antihypertensive efficacy of ketanserin in the elderly be due to altered pharmacokinetics?

Ketanserin is a serotonin S2-receptor antagonist that is an effective antihypertensive agent with a greater blood pressure reduction in older patients. We have reviewed the data from two studies of ketanserin pharmacokinetics in elderly patients, one in general practice (GP) and one in hospital patients. We compared these data with the results from two of our previous studies in young volunteers. The purpose was to determine whether the enhanced efficacy of ketanserin in elderly hypertensive patients could be due to altered pharmacokinetics. After a single dose of ketanserin, elderly hypertensives showed about a 60% increase in bioavailability compared with young volunteers. This increase is likely to be explained by a reduced metabolism of ketanserin on first pass through the liver. The elimination half-life of ketanserin was found to be longer in elderly hospital outpatients, but not in our elderly subjects in general practice. This prolongation of the elimination half-life of ketanserin appears to be unrelated to age, since the hospital outpatient elderly and elderly subjects in general practice were of similar ages. The elimination half-life of ketanserinol was longer in the hospital elderly subjects. This probably reflects a slight diminution of renal function in the elderly hospital outpatients, resulting in reduced clearance of ketanserinol. The peak and trough ketanserin concentrations were similar in young and elderly subjects during chronic treatment, and it is therefore unlikely that the increased efficacy of ketanserin in elderly patients is due to altered pharmacokinetics.

The effects of ketanserin on ventricular ectopic activity in humans.

Ketanserin is a serotonin S2-receptor antagonist that lowers blood pressure and inhibits platelet aggregation. Ketanserin treatment is also associated with prolongation of the corrected QT interval. The recently reported Prevention of Atherosclerotic Complications with Ketanserin (PACK) trial confirmed this prolongation of QT and also revealed a significant excess of deaths in patients receiving ketanserin together with potassium-losing diuretics. The investigators suggested that this excess of deaths may have been attributable to exacerbation of hypokalemia-induced ventricular arrhythmias by the repolarization-prolonging effect of ketanserin. However, drugs that prolong the QT interval may affect ventricular ectopic activity beneficially, and our study was designed to evaluate the effects of ketanserin on ventricular ectopic activity. Twenty patients (18 male, 2 female) aged 42-73 years were studied, each having at least 15 ventricular ectopic beats/hour. The study design was a double-blind, crossover comparison of ketanserin, 40 mg twice daily, and placebo, both given for 1 week. Ventricular ectopic activity was assessed by 48-hour Holter electrocardiogram (ECG) tapes at the end of each treatment period. Ketanserin treatment was associated with prolongation of repolarization, as reflected by the significant mean increases in both QT interval (+30 ms; p less than 0.001) and corrected QT interval (+20 ms; p less than 0.05). The mean overall degree of ventricular ectopic activity, as represented by a score based on the Lown classification, was significantly reduced (p less than 0.05). This was associated with a concordant improvement in the individual indices of ectopic activity. Our results show that ketanserin significantly suppressed ventricular ectopic activity in our normokalemic patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin antagonism reduces the adverse symptoms of beta blockade.

Beta-adrenoceptor antagonists (beta blockers) are a well-established first-line treatment for hypertension, but they have been associated with unwanted symptoms including cold extremities, lethargy, and nightmares. Ketanserin is a serotonin S2-receptor antagonist that has previously been shown to reduce blood pressure in hypertensive patients by reducing systemic vascular resistance. Hypertensive patients whose sitting diastolic blood pressure was greater than or equal to 95 mmHg, despite at least 4 weeks therapy with an optimal dose of beta blocker, were selected for the study. The beta-blocker dose remained constant throughout the study, but patients were randomly allocated to receive ketanserin 20 mg twice daily, ketanserin 40 mg twice daily, or bendrofluazide 5 mg each morning plus placebo at night in addition to the beta-blocker therapy. One hundred and forty two patients completed the symptom questionnaire at randomization and after 12 weeks treatment. The treatment groups were well matched for age, sex, weight, and blood pressure. Blood pressure was reduced significantly by all treatments, and there were no between-group differences. Bendrofluazide adversely affected alertness (p less than 0.05) and concentration (p less than 0.01) whereas ketanserin had no significant effect and the ketanserin 20 mg twice daily group had better concentration than the bendrofluazide group (p less than 0.05). Ketanserin treatment reduced the incidence of nightmares (p less than 0.05 for 20 mg twice daily and 40 mg twice daily) and was an improvement over bendrofluazide treatment in this respect (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study of ketanserin and metoprolol in essential hypertension.

The efficacy and tolerability of ketanserin was compared with metoprolol in a double-blind parallel group study. After a 4-week placebo run-in on no treatment patients with a diastolic blood pressure (BP) of 95 mmHg or more received ketanserin 40 mg (n = 16) or metoprolol 100 mg (n = 17) twice daily. Blood pressure was measured in duplicate using a Hawksley random zero sphygmomanometer. Both blood pressure and heart rate were recorded after 5 min supine and 1 min standing. Patients visited after 2, 4, 8 and 12 weeks of treatment. Systolic, diastolic and mean arterial BPs, both supine and standing, were significantly reduced from week 2 by both treatments (P less than 0.05, Student's t-test). The mean (+/- s.e.m.) changes in supine BP at 3 months compared with baseline were -15.7 (3.6) mmHg systolic and -13.9 (2.7) mmHg diastolic in the ketanserin group and -26.6 (7.9) mmHg systolic and -15.2 (2.7) mmHg diastolic in the metoprolol group. There was a tendency for the fall in systolic BP to be greater in the metoprolol group, but this did not reach statistical significance except for the standing systolic BP at 1 month. Metoprolol caused a significant fall in heart rate compared with baseline values throughout the study, and the metoprolol group was significantly different from the ketanserin group at 2 months for the supine heart rate and at all time points for standing heart rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Polysaccharides in germination. Xyloglucans ( amyloids') from the cotyledons of white mustard.

Two xyloglucan fractions have been isolated from the cotyledons of resting white-mustard seeds, the first by extraction with hot EDTA, and the second by subsequent extraction with alkali or lithium thiocyanate. Although both appear to have the ;amyloid' type of structure in which chains of (1-->4)-linked beta-d-glucopyranose residues carry d-xylose-rich side chains through position 6, these side chains are rather different in structure in the two polysaccharide fractions, and the second or ;insoluble' xyloglucan has fewer of them. The side chains in both polysaccharides are also different from those in other seed amyloids, especially in having xylose linked through positions 3 and 4 (instead of through position 2 as usual) and in containing fucose residues. Both polysaccharides show the characteristic blue ;amyloid' colour with iodine in the presence of sodium sulphate, and it is suggested that this arises by the interaction of iodine molecules and possibly iodide ions within the interstices between aggregated xyloglucan chains. ;Soluble' xyloglucan is metabolized during germination and is presumed to have a reserve function. ;Insoluble' xyloglucan is metabolized less completely over the period studied but its lack of turnover during cell-wall differentiation indicates that it also is a reserve. These and other beta-(1-->4)-linked reserve polysaccharides of seeds might also have a structural function which is of particular value for the survival of the dormant seed.