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Unlabelled: Autologous chondrocyte implantation is a new technique for the treatment of chondral defects in the knee. The exact procedure involved is continuously being developed with the ultimate aim of achieving hyaline cartilage regeneration. We present the outcome of our series of 31 patients, focussing on the use of the chondrogide membrane in the implantation process. Assessment is presented both in the form of arthroscopic appearance at approximately 1 year, and in the form of clinical outcome measures at 1 year and at 2 years after the second stage of the procedure., Conclusion: the use of chondrogide membrane in the fixation of cells during the implantation process is associated with satisfactory clinical outcome and does not appear to show evidence of hypertrophy at one-year arthroscopy, as compared to periosteum.

To further our understanding of the regulation of vertebrate globin loci, we have isolated cosmids containing alpha- and beta-globin genes from the pufferfish Fugu rubripes. By DNA fluorescence in situ hybridization (FISH) analysis, we show that Fugu contains 2 distinct hemoglobin loci situated on separate chromosomes. One locus contains only alpha-globin genes (alpha-locus), whereas the other also contains a beta-globin gene (alpha beta-locus). This is the first poikilothermic species analyzed in which the physical linkage of the alpha- and beta-globin genes has been uncoupled, supporting a model in which the separation of the alpha- and beta-globin loci has occurred through duplication of a locus containing both types of genes. Surveys for transcription factor binding sites and DNaseI hypersensitive site mapping of the Fugu alpha beta-locus suggest that a strong distal locus control region regulating the activity of the globin genes, as found in mammalian beta-globin clusters, may not be present in the Fugu alpha beta-locus. Searching the human and mouse genome databases with the genes surrounding the pufferfish hemoglobin loci reveals that homologues of some of these genes are proximal to cytoglobin, a recently described novel member of the globin family. This provides evidence that duplication of the globin loci has occurred several times during evolution, resulting in the 5 human globin loci known to date, each encoding proteins with specific functions in specific cell types.

Hemoglobin switching is a complex process by which distinct globin chains are produced during stages of development. In an effort to characterize the process of hemoglobin switching in the zebrafish model system, we have isolated and characterized several embryonic globin genes. The embryonic and adult globin genes are found in clusters in a head-to-head configuration. One cluster of embryonic and adult genes is localized to linkage group 3, whereas another embryonic cluster is localized on linkage group 12. Several embryonic globin genes demonstrate an erythroid-specific pattern of expression early during embryogenesis and later are downregulated as definitive hematopoiesis occurs. We utilized electrospray mass spectroscopy to correlate globin genes and protein expression in developing embryonic red cells. The mutation, zinfandel, has a hypochromic microcytic anemia as an embryo, but later recovers in adulthood. The zinfandel gene maps to linkage group 3 near the major globin gene locus, strongly suggesting that zinfandel represents an embryonic globin defect. Our studies are the first to systematically evaluate the embryonic globins in the zebrafish and will ultimately be useful in evaluating zebrafish mutants with defects in hemoglobin production and switching.

To date, all of the chromosomal deletions that cause alpha-thalassemia remove the structural alpha genes and/or their regulatory element (HS -40). A unique deletion occurs in a single family that juxtaposes a region that normally lies approximately 18-kilobase downstream of the human alpha cluster, next to a structurally normal alpha-globin gene, and silences its expression. During development, the CpG island associated with the alpha-globin promoter in the rearranged chromosome becomes densely methylated and insensitive to endonucleases, demonstrating that the normal chromatin structure around the alpha-globin gene is perturbed by this mutation and that the gene is inactivated by a negative chromosomal position effect. These findings highlight the importance of the chromosomal environment in regulating globin gene expression.

Alpha-thalassemia is very common throughout all tropical and subtropical regions of the world. In Southeast Asia and the Mediterranean regions, compound heterozygotes and homozygotes may have anemia that is mild to severe (hemoglobin [Hb] H disease) or lethal (Hb Bart's hydrops fetalis). We have developed a reliable, single-tube multiplex-polymerase chain reaction (PCR) assay for the 6 most frequently observed determinants of alpha-thalassemia. The assay allows simple, high throughput genetic screening for these common hematological disorders. (Blood. 2000;95:360-362)

Aims: (1) To estimate the proportion of subjects with homozygous sickle cell disease who have a benign clinical course, and (2) to assess factors that may be predictive of benign disease., Material: Subjects (n = 280) were participants in a longitudinal cohort study of sickle cell disease. They were classified as benign or control based on clinical history from birth to age 13 years old. Associations with growth, hematology, and an index of social status were investigated., Results: Benign disease occurred in 43 (15%) patients. Neither growth nor social status were related to benign disease. There were only two statistically independent associations: alpha thalassemia status and average steady state fetal hemoglobin (HbF). Patients with a normal complement of alpha globin genes were 2.2 (1.0, 4.9) times more likely to have benign disease than those with gene deletion, and were less likely to have frequent painful crises, dactylitis, and bone necrosis. The odds of having benign disease were 1.09 (1.02, 1.17) times higher for each unit increase in HbF, and 44% of subjects with HbF in the top decile (HbF > 13.8%) of the distribution had benign disease. There was no evidence for a threshold effect of high HbF on benign disease., Conclusion: A benign clinical course of sickle cell disease may occur in Jamaica and is associated with a normal alpha globin gene complement, and high levels of HhF. Ability to predict benign disease at birth is limited.

We have examined the role of the major positive upstream regulatory element of the human alpha-globin gene locus (HS-40) in its natural chromosomal context. Using homologous recombination, HS-40 was replaced by a neo marker gene in a mouse erythroleukemia hybrid cell line containing a single copy of human chromosome 16. In clones from which HS-40 had been deleted, human alpha-globin gene expression was severely reduced, although basal levels of alpha 1 and alpha 2-globin mRNA expression representing less than 3% of the level in control cell lines were detected. Deletion of the neo marker gene, by using FLP recombinase/FLP recombinase target system, proved that the phenotype observed was not caused by the regulatory elements of this marker gene. In the targeted clones, deletion of HS-40 apparently does not affect long-range or local chromatin structure at the alpha promoters. Therefore, these results indicate that, in the experimental system used, HS-40 behaves as a strong inducible enhancer of human alpha-globin gene expression.

We have identified and cloned the major alpha globin locus regulatory element in the mouse (m alpha RE). This element shows a high level of sequence homology to its human counterpart (HS -40) and lies between the same two exons of an upstream, widely expressed gene in both species. Footprinting and band shift studies of the core element show conservation of many (but not all) of the protein binding sites identified as functionally important in HS -40. The functional equivalence of the mouse element was shown by attaching it to a human alpha globin gene and examining expression in transgenic mice. Readily detectable levels of human alpha mRNA were produced in these mice but they were lower than the endogenous gene expression and did not show copy number dependence. These results suggest that sequences additional to this major regulatory element may be necessary to obtain complete regulation of the alpha globin genes in both species.

Erythroid-specific DNase 1 hypersensitive sites have been identified at the promoters of the human alpha-like genes and within the region from 4 to 40 kb upstream of the gene cluster. One of these sites, HS-40, has been shown previously to be the major regulator of tissue-specific alpha-globin gene expression. We have now examined the function of other hypersensitive sites by studying the expression in mouse erythroleukemia (MEL) cells of various fragments containing these sites attached to HS-40 and an alpha-globin gene. High level expression of the alpha gene was observed in all cases. When clones of MEL cells bearing a single copy of the alpha-globin gene fragments were examined, expression levels were similar to those of the endogenous mouse alpha genes and similar to MEL cells bearing beta gene constructs under the control of the beta-globin locus control region. However, there was no evidence that the additional hypersensitive sites increased the level of expression or conferred copy number dependence on the expression of a linked alpha gene in MEL cells.

RNase protection assays are commonly thought to be a more sensitive means of detecting and quantitating specific mRNAs than are RNA gel blots (Northern blots). We have directly compared the sensitivity of these two approaches by assaying for known amounts of in vitro synthesized beta-glucuronidase mRNA. With the probes and protocols employed here, the ability to detect a specific mRNA was similar whether RNase protection or RNA gel blot analyses were performed.

Previous work has suggested that the promoter regions of the human embryonic zeta 2 and epsilon globin genes contain negative regulatory regions that could play a role in the repression of these genes in postembryonic erythroblasts. We have examined this possibility by studying the expression of these genes in mouse erythroleukemia cells, an adult erythroid cell line that might be expected to contain repressor molecules that would bind to the putative negative regulatory regions. When attached to appropriate upstream regulatory elements (alpha HS-40 and beta HS1,2) both the zeta and epsilon genes were expressed in these cells at a low level, but no increase in expression was observed when similar constructs lacking the proposed negative regulatory sequences were introduced into these cells. These results cast doubt on the possibility that these sequences play a major role in the developmental repression of the embryonic globin genes, unless they function only in a normal chromosomal organization.

zeta-Globin chain expression in carriers of a number of deletional alpha-thalassemias is investigated by radioimmunoassay. In a few cases, zeta-globin mRNAs are also studied. zeta-Globin chains are detected in (--SEA/), (--MED/), and (--SPAN/) deletions, but not in six other deletional mutations. These results suggest that the DNA element capable of suppressing zeta-globin expression in adult erythroid cells is present within the (--SPAN/) deletion, while the DNA fragment between the 5' breakpoints of the (--SA/) and the (--SEA/) deletions may contain sequences necessary for augmenting zeta-globin expression in adult erythroid cells. Furthermore, zeta-globin chains are shown by an immunocytologic technique to be present in all circulating erythrocytes in carriers of the (--SEA/) and (--MED/) deletions. This simple immunocytologic test is highly sensitive and specific to detect adult carriers of either the (--SEA/) or (--MED/) deletions, and can be used for the detection of couples at risk of pregnancies involving fetuses with homozygous alpha-thalassemia.

A recent survey of foods that constitute the major sources of vitamin C in the American diet yielded information on the total content of this vitamin as well as the amount of its two forms, ascorbic acid and dehydroascorbic acid (DHAA) in these foods. Samples of individual foods showed a surprising large range of vitamin content even for foods collected from the same regions of the country and from the same source. The amount of DHAA in the different foods varied from approximately 10% to 20% of the total vitamin content. The large range of values for the vitamin content in a given food suggests further that in human-diet studies, when the major sources of vitamin C are from a few foods, daily analyses are required for the necessary precision.

We describe an alpha-thalassemia determinant in which alpha-globin expression is silenced by a deletion located 27 kb 5' to the transcription start site of the alpha 2-globin gene. This alpha-thalassemic determinant, (alpha alpha)MM, is a member of a newly described group of thalassemic mutations resulting from deletion of locus-controlling sequences critical to globin gene expression.

We describe a family in which alpha-thalassemia occurs in association with a deletion of 62 kilobases from a region upstream of the alpha globin genes. DNA sequence analysis has shown that the transcription units of both alpha genes downstream of this deletion are normal. Nevertheless, they fail to direct alpha globin synthesis in an interspecific hybrid containing the abnormal (alpha alpha)RA chromosome. It seems probable that previously unidentified positive regulatory sequences analogous to those detected in a corresponding position of the human beta globin cluster are removed by this deletion.

Five Cypriots homozygous for beta +-thalassaemia have inherited deletion or non-deletion forms of alpha-thalassaemia that seem to have modified the usually severe clinical picture to that of mild thalassaemia intermedia. These observations have important implications for the antenatal diagnosis of beta-thalassaemia.

Studies in two Jamaican Negro families, including haematological and haemoglobin analysis, haemoglobin synthesis, and globin messenger-RNA assay, have defined two alpha-thalassaemia phenotypes which resemble the severe (alpha-thalassaemia 1) and mild (alpha-thalassaemia 2) forms of the disorder described in Orientals. Genetic analysis suggests that subjects with the alpha-thalassaemia-1 phenotype are homozygous for the alpha-thalassaemia-2 determinant. Restriction-endonuclease mapping shows that alpha-thalassaemia-2 results from the deletion of one of the linked pair of alpha-chain genes. Hence the genotypes of the alpha-thalassaemia heterozygotes and homozygotes in these families are -alpha/alpha alpha and -alpha/-alpha respectively. If these are the usual alpha-thalassaemia genotypes in Negroes, these findings explain the difference in clinical expression of the disorder between Orientals and Negroes--in particular, the absence of haemoglobin Bart's hydrops and the rarity of haemoglobin-H disease in Negroes.

Weanling male rats were fed either a vitamin E-deficient Torula yeast diet or the same diet supplemented with 100 ppm vitamin E for a period of 3 months. One group of animals fed each diet received 250 ppm lead in the drinking water, whereas another group of animals fed each diet received no lead in the water. Vitamin E deficiency per se had little or no effect on hematocrit value, reticulocyte count, spleen weight, or erythrocyte mechanical fragility in rats not poisoned with lead. On the other hand, the decreased hematocrit, increased reticulocyte count, and splenic enlargement due to lead poisoning were much more pronounced in vitamin E-deficient rats than in rats supplemented with vitamin E. The resistance to mechanical trauma of red blood cells from vitamin E-deficient lead-poisoned rats was much less than that of red cells from vitamin E-adequate lead-poisoned rats. Dietary vitamin E status had no significant influence on the increased mechanical fragility of erythrocytes from nonpoisoned rats caused by exposure to lead in vitro. These results suggest that vitamin E deficiency enhances the susceptibility of animals to the in vivo hemolytic effect of lead poisoning.

alpha Thalassemia modifies the hematologic expression of homozygous sickle cell (SS) disease, resulting in increased total hemoglobin and HbA2 and decreased HbF, mean cell volume, reticulocytes, irreversibly sickled cells, and bilirubin levels. The age at which these changes develop in children with SS disease is unknown. Ascertainment of globin gene status in a large representative sample of children with SS disease has afforded an opportunity to study the hematologic indices in nine children homozygous for alpha thalassemia 2 (two-gene group), 90 children heterozygous for alpha thalassemia 2 (three-gene group), and 167 children with a normal alpha globin gene complement (four-gene group). The two-gene group had significantly lower mean cell volumes from birth, higher red cell counts from one month, lower reticulocytes from three months, and higher HbA2 levels from one year, as compared with the four-gene group. Children with three genes had intermediate indices but resembled more closely the four-gene group. Differences in total hemoglobin or in fetal hemoglobin between the groups were not apparent by eight years of age. The most characteristic differences of the two-gene group were the raised proportional HbA2 level and low mean cell volume, the latter having some predictive value for alpha thalassemia status at birth.

A form of alpha zero-thalassemia found in subjects of Mediterranean origin has been analyzed by gene mapping and DNA sequencing. Homozygotes have the hemoglobin Bart's hydrops fetalis syndrome, while compound heterozygotes for this defect and alpha+-thalassemia have hemoglobin H disease. It results from a deletion that removes 20.5 kilobases of DNA from within the alpha-globin gene cluster. Sequence data from the regions adjacent to the breakpoint indicate that the recombination event that caused this deletion occurred between the alpha 1-gene and an unusual AluI sequence located between the embryonic zeta genes.

To determine the influence of wide variations in dietary levels of calcium, zinc and phytic acid (as sodium phytate) on growth and cataract incidence, juvenile chinook salmon held at 10-11 degrees C were fed daily to satiation for 105 d one of nine purified diets containing one of three levels (grams/kilogram) of calcium (averaged 4.8, 17.7, 50.2), zinc (averaged 0.05, 0.15, 0.39) and phytic acid (1.62, 6.46, 25.8). Diets were formulated to have a calcium-phosphorus ratio of close to unity when considering phosphorus sources other than sodium phytate. High dietary phytic acid concentration (25.8 g/kg) depressed chinook salmon growth, food and protein conversion [protein efficiency ratio (PER)] and thyroid function, increased mortality, promoted cataract formation (zinc at 0.05 g/kg) and induced anomalies in pyloric cecal structure. Calcium at 51 g/kg (or phosphorus) exacerbated the effects of high dietary phytate and low dietary zinc on cataract incidence. Moreover, high dietary levels of calcium (48-51 g/kg) coupled with phosphorus significantly impaired the growth and appetite of low phytic acid (1.62 g/kg) groups and led to nephrocalcinosis in low and high phytic acid groups. Plasma zinc levels were directly related to dietary zinc concentration and inversely related to dietary phytic acid level. Calcium (51 g/kg) and/or phosphorus reduced zinc bioavailability when the diet concurrently contained 0.05 g zinc and 25.8 g of phytic acid per kilogram. It is concluded that zinc is essential for normal eye development in juvenile chinook salmon. Further, zinc deficiency could not be induced in chinook salmon fed diets with high ratios of calcium (or phosphorus) to zinc alone. This required the simultaneous presence of a strong mineral (zinc)-binding agent.

In a previous study, we described a form of nondeletion alpha-thalassemia (alpha T Saudi alpha) found in subjects of Saudi Arabian origin. In the current study, using synthetic oligoprobe hybridization and restriction enzyme analysis, we have demonstrated that the molecular basis of alpha T Saudi alpha is due solely to a single base mutation (AATAAA----AATAAG) in the polyadenylation signal of the alpha 2 gene and that the frameshift mutation in codon 14 of the linked alpha 1 gene is the result of a cloning artefact. The alpha 2 polyadenylation signal mutation occurs in other Middle Eastern and the Mediterranean populations and is responsible for the clinical phenotype of Hb H disease in some Saudi Arabian individuals with five alpha genes (alpha T Saudi alpha/(alpha alpha alpha)T Saudi). Evidence suggests that the (alpha alpha alpha)T Saudi haplotype has arisen as a result of a recombination between two misaligned chromosomes bearing the alpha T Saudi alpha defect.

Simple expressions are given for the dispersion and the travel times of samples in simple flow-injection analysis systems. The sum of these two quantities is the total residence time of the sample in the system. The expressions are based on numerical solutions of the diffusion-convection equation. Preliminary experiments are in agreement with the derived simple expressions, as are peak curve shapes. Diffusion coefficients can be obtained in a straightforward manner.

Haemoglobin Bart's was detected in cord blood samples from 81% of 217 infants born in Madang on the north coast of Papua New Guinea. Analysis of the alpha globin genes of 30 infants and adults from the same region showed that all but 3 were heterozygous or homozygous for the deletion form of alpha + thalassaemia. None of 18 cord blood samples from infants born in Goroka in the Eastern Highlands Province had haemoglobin Bart's, and in each case the alpha globin genes were normal. Preliminary geographical and linguistic analyses of both groups suggest that the prevalence of alpha thalassaemia may be related to altitude rather than to linguistic grouping and hence that resistance to malaria may be at least one reason why alpha thalassaemia is so common in some populations.

A 3-year-old boy presented with alpha-thalassaemia, dysmorphic features, and mental handicap. His younger sister is also mentally retarded, but haematologically normal. High resolution cytogenetic analysis revealed a normal karyotype in all family members. However, a combination of DNA analysis and in situ hybridisation demonstrated that the mother has a previously unsuspected balanced reciprocal translocation between the tips of the short arms of chromosomes 1 and 16, and that the alpha-globin gene complex (which maps to the tip of chromosome 16) is included in the translocated segment. Both of her children have inherited one of the translocation chromosomes in an unbalanced fashion: the boy has the derived chromosome 16, and therefore has alpha-thalassaemia, whilst the girl has the derived chromosome 1. Such cytogenetically invisible subtelomeric translocations are probably an important and hitherto unrecognised cause of genetic disease.

A sensitive and specific radioimmunoassay (RIA) for human embryonic zeta-globin chains was used to study normal fetal blood and newborn cord blood as well as cord blood from newborns with alpha-thalassemias. From 17 weeks until 37 weeks of gestation, zeta-globin chains were present in almost all fetal and cord blood samples (0.27% +/- 0.15% in samples of weeks 17 through 30; 0.14% +/- 0.11% in samples of weeks 31 through 37). zeta-Globin chains were present in greater than 80% of cord blood hemolysates from normal, full-term newborns (0.15% +/- 0.11%) as well as from 16 near-term newborns of diabetic mothers (0.13% +/- 0.13%). zeta-Globin chains were not detected in normal infants aged 3 months to 2 years. In cord blood hemolysates from alpha-thalassemic newborns, the levels of zeta-globin chain content varied from very high to undetectable levels. Gene mapping of the zeta-alpha-globin gene cluster was performed in 12 newborns in whom cord blood zeta-globin chains had been determined. Newborns who were carriers of alpha-thalassemia-1 due to the (--SEA/) deletion had very high levels of zeta-globin chains (greater than 1.5%).

Hypochromic anaemia is very common among the island populations of Vanuatu in the South-West Pacific. Results of a large-scale survey show that, unexpectedly, this form of anaemia is seldom due to iron deficiency or coexistent parasitic disease. Rather, it results from a previously unsuspected high incidence of alpha-thalassaemia which has been identified only by application of DNA analysis to the populations studied. These findings suggest that hypochromic anaemia in tropical or subtropical populations should not necessarily be attributed to iron deficiency; detailed studies of iron status should be carried out before major dietary changes or fortification of food with iron are implemented.

Fetal hemoglobin (HbF) levels vary widely among individuals with sickle cell anemia (SS). Previous studies have suggested that HbF levels in SS individuals with alpha-thalassemia (two or three functional alpha-globin genes) are lower than HbF levels in SS individuals with four normal alpha-globin genes. Using immunocytochemical techniques, we studied F cell production as measured by % F reticulocytes, the amount of HbF per F cell, and the preferential survival of F cells versus non-F cells in 51 subjects with four alpha genes, 32 subjects with three alpha genes, and 18 subjects with two alpha genes. Comparison between alpha-globin gene groups was performed for the total sample as well as for a subset of 82 individuals who had replicate samples and a further subset of 39 age-matched individuals. %HbF levels were 6.8, 4.9, and 4.5 percent for the total four-, three-, and two-alpha-globin-gene groups, respectively. The percentage of F reticulocytes, percentage HbF per F cell, and the enrichment ratio (% F cell/% F reticulocytes) did not change significantly with alpha-globin gene number. Moreover, no correlation existed between alpha-globin gene number and the absolute number of F cells in any group studied. However, there was a strong inverse correlation (r = -0.407, P = .0001) between non-F cell levels (1.7 +/- 2, 2.2 +/- 5, 3.0 +/- 1.0 X 10(12)/L) and decreasing alpha-globin gene number. These data suggest that falling HbF levels among SS individuals with lessened numbers of alpha-globin genes reflect prolonged survival of non-F cells and are not due to intrinsic differences in F cell production or in the amount of HbF per F cell. The improved survival of non-F cells in SS alpha-thalassemia is presumed to be due to the lower MCHC observed in such individuals.