Discrimination of pollen of New Zealand manuka (Leptospermum scoparium agg.) and kanuka (Kunzea spp.) (Myrtaceae).

The very similar appearance of pollen of the New Zealand Myrtaceous taxa Leptospermum scoparium s.l. (manuka) and Kunzea spp. (kanuka) has led palynologists to combine them in paleoecological and melissopalynological studies. This is unfortunate, as differentiation of these taxa would improve understanding of past ecological change and has potential to add value to the New Zealand honey industry, where manuka honey attracts a premium price. Here, we examine in detail the pollen morphology of the 10 Kunzea species and a number of Leptospermum scoparium morphotypes collected from around New Zealand, using light microscopy, SEM, and Classifynder (an automated palynology system). Our results suggest that at a generic level the New Zealand Leptospermum and Kunzea pollen can be readily differentiated, but the differences between pollen from the morphotypes of Leptospermum or between the species of Kunzea are less discernible. While size is a determinant factor-equatorial diameter of Leptospermum scoparium pollen is 19.08 ± 1.28 µm, compared to 16.30 ± 0.95 µm for Kunzea spp.-other criteria such as surface texture and shape characteristics are also diagnostic. A support vector machine set up to differentiate Leptospermum from Kunzea pollen using images captured by the Classifynder system had a prediction accuracy of ~95%. This study is a step towards future melissopalynological differentiation of manuka honey using automated pollen image capture and classification approaches.

Biosystematics and conservation: a case study with two enigmatic and uncommon species of Crassula from New Zealand.

BACKGROUND AND AIMS: Crassula hunua and C. ruamahanga have been taxonomically controversial. Here their distinctiveness is assessed so that their taxonomic and conservation status can be clarified. METHODS: Populations of these two species were analysed using morphological, chromosomal and DNA sequence data. KEY RESULTS: It proved impossible to differentiate between these two species using 12 key morphological characters. Populations were found to be chromosomally variable with 11 different chromosome numbers ranging from 2n = 42 to 2n = 100. Meiotic behaviour and levels of pollen stainability were both variable. Phylogenetic analyses showed that differences exist in both nuclear and plastid DNA sequences between individual plants, sometimes from the same population. CONCLUSIONS: The results suggest that these plants are a species complex that has evolved through interspecific hybridization and polyploidy. Their high levels of chromosomal and DNA sequence variation present a problem for their conservation.

Expression in hematological malignancies of a glucocorticoid receptor splice variant that augments glucocorticoid receptor-mediated effects in transfected cells.

Glucocorticoids play an important role in the treatment of a number of hematological malignancies, such as multiple myeloma. The effects of glucocorticoids are mediated through the glucocorticoid receptor alpha, the abundance of which can be modulated by alternative splicing of the glucocorticoid receptor mRNA. Two splice variants of the glucocorticoid receptor mRNA have been described: glucocorticoid receptor beta, which reportedly has a dominant negative effect on the actions of the glucocorticoid receptor alpha, and glucocorticoid receptor P, of which the effects are unknown. In this study, we have investigated the expression levels of these two splice variants at the mRNA level in multiple myeloma cells and in a number of other hematological tumors. Although the glucocorticoid receptor beta mRNA was, if at all, expressed at very low levels, considerable amounts (up to 50% of the total glucocorticoid receptor mRNA) glucocorticoid receptor P mRNA was present in most hematological malignancies. In transient transfection studies in several cell types and in multiple myeloma cell lines, the glucocorticoid receptor P increased the activity of the glucocorticoid receptor alpha. These results suggest that the relative levels of the glucocorticoid receptor alpha and the glucocorticoid receptor P may play a role in the occurrence of glucocorticoid resistance in tumor cells during the treatment of hematological malignancies with glucocorticoids.

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria.

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.

Normal HLA class I, II, and MICA gene distribution in uveal melanoma.

PURPOSE: The molecules of the HLA class I and II molecules as well as the MHC class I chain-related gene A (MICA), a polymorphic and stress-induced cell surface molecule, are involved in T-cell and natural killer-cell (NK-cell) mediated immune responses. In this study we looked for any genetic susceptibility contributed by HLA class I, class II, or MICA genes with regard to the development of uveal melanoma. METHODS: Between 1998 and 2001, 159 uveal melanoma patients were typed for HLA class I and II, and 168 uveal melanoma patients were evaluated for MICA by microsatellite typing. The HLA antigen and MICA allele frequencies were compared with control groups of, respectively, 2,440 and 247 healthy Dutch individuals. RESULTS: HLA class I, HLA class II, and MICA gene frequencies in uveal melanoma patients and healthy Dutch controls showed no significant deviations after correction for the number of comparisons. CONCLUSIONS: We conclude that there is no genetic susceptibility or increased risk attributed to any HLA class I, class II, and MICA polymorphism with regard to the development of uveal melanoma.

Differential hormone-dependent transcriptional activation and -repression by naturally occurring human glucocorticoid receptor variants.

The molecular mechanisms underlying primary glucocorticoid resistance or hypersensitivity are not well understood. Using transfected COS-1 cells as a model system, we studied gene regulation by naturally occurring mutants of the glucocorticoid receptor (GR) with single-point mutations in the regions encoding the ligand-binding domain or the N-terminal domain reflecting different phenotypic expression. We analyzed the capacity of these GR variants to regulate transcription from different promoters, either by binding directly to positive or negative glucocorticoid-response elements on the DNA or by interfering with protein-protein interactions. Decreased dexamethasone (DEX) binding to GR variants carrying mutations in the ligand-binding domain correlated well with decreased capacity to activate transcription from the mouse mammary tumor virus (MMTV) promoter. One variant, D641V, which suboptimally activated MMTV promoter-mediated transcription, repressed a PRL promoter element containing a negative glucocorticoid-response element with wild type activity. DEX-induced repression of transcription from elements of the intercellular adhesion molecule-1 promoter via nuclear factor-kappaB by the D641V variant was even more efficient compared with the wild type GR. We observed a general DEX-responsive AP-1-mediated transcriptional repression of the collagenase-1 promoter, even when receptor variants did not activate transcription from the MMTV promoter. Our findings indicate that different point mutations in the GR can affect separate pathways of gene regulation in a differential fashion, which can explain the various phenotypes observed.

Uncoupling protein-3 is a molecular determinant for the regulation of resting metabolic rate by thyroid hormone.

Thyroid hormones increase energy expenditure, partly by reducing metabolic efficiency. The control of specific genes at the transcriptional level is thought to be the major molecular mechanism. However, both the number and the identity of the thyroid hormone-controlled genes remain unknown, as do their relative contributions. Uncoupling protein-3, a recently identified member of the mitochondrial transporter superfamily and one that is predominantly expressed in skeletal muscle, has the potential to be a molecular determinant for thyroid thermogenesis. However, changes in mitochondrial proton conductance and resting metabolic rate after physiologically mediated changes in uncoupling protein-3 levels have not been described. Here, in a study on hypothyroid rats given a single injection of T(3), we describe a strict correlation in terms of time course between the induced increase in uncoupling protein-3 expression (at mRNA and protein levels) and decrease in mitochondrial respiratory efficiency, on the one hand, and the increase in resting metabolic rate, on the other. First, we describe our finding that uncoupling protein-3 is present and regulated by T(3) only in metabolically relevant tissues (such as skeletal muscle and heart). Second, we follow the time course (at 0, 6, 12, 24, 48, 65, 96, and 144 h) of both uncoupling protein-3 mRNA levels and mitochondrial uncoupling protein-3 density in gastrocnemius muscle and heart. In both tissues, the maximal (12-fold) increase in uncoupling protein-3 density was reached at 65 h. The resting metabolic rate [lO(2)(kg(0.75))(-1)h(-1)] showed the same time course, and at 65 h the increase vs. time zero was 45% (1.316 +/- 0.026 vs. 0.940 +/- 0.007; P < 0.001). At the same time point, gastrocnemius muscle mitochondria showed a significantly higher nonphosphorylating respiration rate (nanoatoms of oxygen per min/mg protein; increase vs. time zero, 40%; 118 +/- 4 vs. 85 +/- 9; P < 0.05), whereas the membrane potential decreased by 8% (168 +/- 2 vs. 182 +/- 4; P < 0.05). These data are diagnostic of mitochondrial uncoupling. The results reported here provide the first direct in vivo evidence that uncoupling protein-3 has the potential to act as a molecular determinant in the regulation of resting metabolic rate by T(3).

Interperson variability but intraperson stability of baseline plasma cortisol concentrations, and its relation to feedback sensitivity of the hypothalamo-pituitary-adrenal axis to a low dose of dexamethasone in elderly individuals.

In the present study, we investigated whether the negative feedback action of glucocorticoids (GCs) on the hypothalamo-pituitary-adrenal (HPA) axis changes with age. We performed a 1-mg dexamethasone (DEX) suppression test in 216 healthy elderly individuals. To investigate individual variability of feedback sensitivity in more detail, 2.5 yr later a 0.25-mg DEX suppression test was carried out in 164 of the same individuals. We investigated whether there was an effect of age or gender on both basal and post-DEX cortisol levels, as well as on the concentration of DEX. Furthermore, we examined whether the reactions to the two doses of DEX differed, and whether indications for an intraperson stability of baseline cortisol levels could be found. Neither the pre- nor the post-1-mg DEX plasma cortisol concentrations showed a correlation with age, and there were no differences between men and women. The same was true for the pre- and post-0.25-mg DEX cortisol concentrations. In reaction to 1 mg DEX, over 90% of the subjects investigated showed a cortisol suppression to levels below 50 nmol/L. After the administration of 0.25 mg DEX, there was a much wider range in post-DEX cortisol concentrations. After the administration of 1 mg DEX, there was a significant correlation between liver function parameters and plasma DEX concentrations in males, and there was a correlation between body mass index and plasma DEX concentration in females. Plasma DEX concentrations after the administration of 1 mg and 0.25 mg DEX were closely correlated within subjects (P < 0.001). There was an intraindividual stability of serum cortisol levels determined at an interval of 2.5 yr. Furthermore, the individuals with the highest baseline cortisol concentrations also had the highest post-0.25-mg DEX cortisol concentrations, indicating a close relationship between basal cortisol levels and the feedback sensitivity of the HPA axis to a low dose of DEX. These observations suggest a genetic influence on the set point of the HPA axis. Aging does not seem to lead to a change in HPA activity as measured by early morning total cortisol levels. Also, no changes in the sensitivity of the feedback system to DEX were observed with age. DEX metabolism is influenced by liver function (in males) and by body mass index (in females).

Five patients with biochemical and/or clinical generalized glucocorticoid resistance without alterations in the glucocorticoid receptor gene.

Cortisol resistance (CR) is a rare disease characterized by a generalized reduced sensitivity of end-organs to the actions of glucocorticoids (GCs). GC effects are mediated by the GC receptor (GR). The molecular alterations in CR described thus far were located in the hormone-binding domain of the GR gene. Recent reports of a considerable prevalence of abnormalities in the GR in patients attending the endocrine clinic prompted us to carry out further investigations with respect to GR protein and GR gene in patients attending the endocrine clinic for a broad spectrum of complaints and biochemical evidence suggesting a CR. In the present study, we describe five patients with biochemical and clinical CR. All patients showed a diurnal rhythm of serum cortisol concentrations (albeit at a high level), an insufficient suppression of serum cortisol concentration in reaction to 1 mg dexamethasone (DEX), and variable degrees of androgen overproduction, in the absence of clinical signs and symptoms of Cushing's syndrome. Three of the four female patients presented with complaints of androgen overproduction, two of them in combination with fatigue. The other female patient had severe steroid-resistant asthma. The only male patient and his son were asymptomatic. In four patients, we investigated receptor protein characteristics on mononuclear leukocytes in a whole cell DEX binding assay and studied the ability of DEX to inhibit mitogen-induced cell proliferation in mononuclear leukocytes in vitro. In all patients investigated, we found alterations in receptor number or ligand affinity and/or the ability of DEX to inhibit mitogen-induced cell proliferation. To investigate the molecular defects leading to the clinical and biochemical pictures in these patients, we screened the GR gene using PCR/single-strand conformational polymorphism/sequence analysis. No GR gene alterations were found in these patients. In conclusion, the five patients described had clinical and biochemical evidence of CR, but no abnormalities were demonstrated in the GR gene. Probably, as yet undefined alterations somewhere in the cascade of events starting with ligand binding to the GR protein, and finally resulting in the regulation of the expression of GC responsive genes, or postreceptor defects or interactions with other nuclear factors form the pathophysiologic basis of CR in these patients.

A polymorphism in the glucocorticoid receptor gene may be associated with and increased sensitivity to glucocorticoids in vivo.

We investigated whether a polymorphism at nucleotide position 1220, resulting in an asparagine-to-serine change at codon 363 in the glucocorticoid receptor (GR) gene is associated with an altered sensitivity to glucocorticoids. In a group of 216 elderly persons, 13 heterozygotes for the N363S polymorphism were identified by PCR/single strand conformation polymorphism analysis. In 2 dexamethasone (DEX) suppression tests (DSTs), using 1 and 0.25 mg DEX, the circulating cortisol and insulin concentrations were compared between N363S carriers and controls. In the 1-mg DST, there were no differences between N363S carriers and controls, with respect to adrenal suppression, but there was a significantly higher (P < 0.05) insulin response in N363S carriers. In the 0.25-mg DST, a significantly larger (P < 0.05) cortisol suppression and higher (P < 0.05) insulin response were seen in N363S carriers. Comparison of blood pressure, body mass index (BMI), and bone mineral density (BMD) between the N363S carriers and controls showed that N363S carriers had a higher (P < 0.05) BMI but normal blood pressure. There was an obvious trend towards lower age-, BMI-, and sex-adjusted BMD in the lumbar spine in N363S carriers. GR characteristics measured in 41 controls and 9 N363S carriers in peripheral mononuclear leucocytes showed no differences between N363S carriers and controls, with respect to GR number and ligand binding affinity. However, there was a trend towards greater sensitivity to DEX in the carriers' lymphocytes, in a mitogen-induced cell proliferation assay. In transfection assays, the capacity of the codon 363 variant to activate mouse mammary tumor virus promotor-mediated transcription in COS-1 cells was unaltered, when compared with the wild-type GR. We conclude that in 6.0% of our study population, a polymorphism in codon 363 of the GR gene was found. Individuals carrying this polymorphism seemed healthy at clinical examination but had a higher sensitivity to exogenously administered glucocorticoids, with respect to both cortisol suppression and insulin response. Life-long exposure to the mutated allele may be accompanied by an increased BMI and a lowered BMD in the lumbar spine but does not affect blood pressure.

Human adrenocorticotropin-secreting pituitary adenomas show frequent loss of heterozygosity at the glucocorticoid receptor gene locus.

Corticotropinomas are characterized by a relative resistance to the negative feedback action of cortisol on ACTH secretion. In this respect there is a similarity with the clinical syndrome of cortisol resistance. As cortisol resistance can be caused by genetic abnormalities in the glucocorticoid receptor (GR) gene, we investigated whether the insensitivity of corticotropinomas to cortisol is also caused by de novo mutations in the GR gene. We screened for the GR gene in leukocyte and tumor DNA from 22 patients with Cushing's disease for mutations using PCR/single strand conformation polymorphism analysis. In a previous study, we identified 5 polymorphisms in the GR gene in a normal population. These polymorphisms were used as markers for the possible occurrence of loss of heterozygosity (LOH) at the GR gene locus. Except for 1 silent point mutation, we did not identify novel mutations in the GR gene in leukocytes or corticotropinomas from these patients. Of the 22 patients, 18 were heterozygous for at least 1 of the polymorphisms. In 6 of these patients, LOH had occurred in the tumor DNA. Of 21 patients examined for LOH on chromosome 11q13, only 1, with a corticotroph carcinoma, showed allelic deletion. As controls we studied 28 pituitary tumors of other subtypes (11 clinically nonfunctioning, 8 prolactinomas, and 9 GH-producing adenomas) and found evidence for LOH in only 1 prolactinoma. In six patients LOH was found at the GR gene locus (chromosome 5) in DNA derived from adenoma cells. Our observations indicate for the first time that LOH at the GR gene locus is a relatively frequent phenomenon in pituitary adenomas of patients with Cushing's disease. This might explain the relative resistance of the adenoma cells to the inhibitory feedback action of cortisol on ACTH secretion. The specificity of the GR LOH to corticotropinomas supports this concept. Somatic mutations of the GR are not a frequent cause of relative cortisol resistance in these cells.

Skeletal muscle mitochondrial free-fatty-acid content and membrane potential sensitivity in different thyroid states: involvement of uncoupling protein-3 and adenine nucleotide translocase.

The effect of triiodothyronine (T3) on mitochondrial efficiency could be related to an increase in the concentrations of some proteins, such as uncoupling proteins (UCPs). Free fatty acids (FFA) seem to be a cofactor essential for the uncoupling activity of UCP3. In this paper, we report that the hypothyroidism-hyperthyroidism transition is accompanied by increases: (i) in the endogenous levels of mitochondrial FFA and (ii) in the sensitivity to FFA shown by the mitochondrial respiration rate and membrane potential, which correlated with the level of UCP3 protein. The level of the mRNA for adenine-nucleotide translocase-1 (ANT) was not affected by the thyroid state, while the ANT contribution to FFA-induced changes in mitochondrial uncoupling was low in the hypothyroid and euthyroid states but became more relevant in the hyperthyroid state at the highest concentration of FFA.

Regulation of plant gene expression by antisense RNA.

Regulation of gene expression by antisense RNA was first discovered as a naturally-occurring phenomenon in bacteria. Recently natural antisense RNAs have been found in a variety of eukaryotic organisms; their in vivo function is, however, obscure. Deliberate expression of antisense RNA in animal and plant systems has lead to successful down-regulation of specific genes. We will review the current status of antisense gene action in plant systems. The recent discovery that 'sense' genes are able to mimic the action of antisense genes indicates that (anti)sense genes must operate by mechanisms other than RNA-RNA interaction.

An unexpected effect of matching for HLA-A9 in renal transplantation.

The observation of elevated levels of HLA class I molecules in sera of HLA-A9-positive individuals, and their potential role in the regulation of the immune response, motivated us to study the effect of the presence of HLA-A9 in either kidney donor or recipient on graft survival. Analysis of data from unrelated first transplants performed within the Eurotransplant area revealed that in the group of patients who were not treated with cyclosporine (n = 2051), transplants with no HLA-DR mismatches in which donors (D) and recipients (R) shared the HLA-A9 antigen (D+R+), had significantly poorer graft survival (P = 0.0001) than all other combinations, reaching a 20% difference at 5 years posttransplantation. This effect, which was not found in the CsA-treated patient group (n = 7297), was specific for HLA-A9. The implications of this findings are discussed in relation to the mechanisms of the alloimmune response.

The interaction between the Lewis blood group system and HLA-matching in renal transplantation.

A retrospective study was initiated to investigate the influence of recipients' Lewis subtype and HLA-matching on cadaveric kidney graft outcome. A total of 1111 patients receiving a first cadaveric kidney graft were analyzed. No difference in one-year graft survival was found between Lewis-negative (73%, n = 133) and Lewis-positive (73%, n = 978) recipients. Further subdivision of the study group into HLA-A,-B well-matched (0 or 1 mismatches [MM]) and poorly matched (2, 3, or 4 MM) revealed a strong deleterious effect of HLA-A,-B mismatching in the Lewis-negative group only. One year graft survival in Lewis-negative HLA-A,-B poorly matched (2, 3, or 4 A,B MM) patients was 60% (n = 67) versus 86% (n = 66) in the Lewis-negative HLA-A,-B well-matched (0 or 1 A,B MM) group (P = 0.004). For the Lewis-positive group the one-year graft survival rates were 72% (2, 3, or 4 A,B MM; n = 498) and 74% (0 or 1 A,B MM; n = 480), respectively (P = n.s.). The additional beneficial effect of HLA-DR matching again turned out to be strongest in the Lewis-negative group. In Lewis-negative, HLA-DR (0 MM) and -A,-B well-matched recipients (n = 36) graft survival was 94% versus only 64% in the Lewis-negative, DR matched, A,-B mismatched (2, 3, or 4 A,B MM) group (n = 25; P = 0.09). In the Lewis-positive, HLA-DR 0 mismatched group the one-year survival rates were 78% (0 or 1 A,B MM; n = 240) and 73% (2, 3, or 4 A,B MM; n = 253), respectively (P = 0.05). Our data suggest that donor recipient selection should not be based on Lewis matching per se. However, since Lewis-negative patients are at high risk of graft failure when receiving HLA mismatched kidneys, they should preferentially receive optimally HLA matched grafts.

Evidence that the difference in kidney graft survival in DRw6+ and DRw6- recipients may be explained by a blood transfusion policy that is disadvantageous for DRw6+ recipients.

Kidneys transplanted to HLA-DRw6+ recipients have been shown to have an inferior graft survival compared with DRw6- patients. Because pretransplant blood transfusions influence kidney graft survival, we investigated whether the number of blood transfusions contributes to the observed poor graft survival in DRw6+ patients. We have found that the difference in graft survival in DRw6+ and DRw6- recipients may be explained by a blood transfusion policy that is disadvantageous for DRw6+ recipients. Thus, graft survival in DRw6+ recipients was excellent for those who had received only a single transfusion. More transfusions resulted in a gradual decrease in graft survival. When the number of transfusions exceeded 5, graft survival improved again. By contrast, DRw6- recipients showed an improvement in graft survival with an increasing number of transfusions. DRw6+ recipients therefore display inferior graft survival only when they receive 3-5 transfusions. This finding provides a possible explanation as to why the "DRw6 effect" is a controversial issue, and it suggests that DRw6+ recipients should be given a different pre-transplant transfusion protocol than DRw6- patients.

Cross-reactive group matching does not lead to a better allocation and survival of donor kidneys.

BACKGROUND: In cadaveric renal transplantation HLA-A, -B, -DR matching of donor and recipient is beneficial for graft survival. However, allocation based on HLA matching seems to favor recipients with more frequently occurring HLA antigens. In this study we investigated whether matching on the basis of cross-reactive groups (CREGs), defined according to the United Network for Organ Sharing (UNOS), would be a good alternative for the allocation of kidneys without negatively influencing graft survival. Theoretically, this approach would provide more recipients with an immunologically well-matched donor organ. METHODS: The influence of CREG matching on graft survival was studied in univariate analyses using the Eurotransplant database. RESULTS: No beneficial effect of CREG matching was observed, whereas a significant HLA matching effect was observed in the 0 CREG mismatched donor/ recipient combinations. Only in the small subgroup with 1 MM for HLA-A, -B and 0 MM for HLA-DR, a significantly better survival was observed, when this mismatch belonged to the 0 or 1 MM CREG group versus two or more MM CREG group. However, this subgroup concerns only 8% of the transplants performed. CONCLUSIONS: In contrast to other reports, our study showed that HLA matching is by far more beneficial than CREG matching. In the homogenous Eurotransplant population, adjusting the matching criteria toward CREG matching would not lead to an improved graft survival.

In vitro CTL precursor frequencies do not reflect a beneficial effect of cross-reactive group (CREG) matching.

Adjustment of histocompatibility-based allocation criteria in kidney transplantation from HLA matching to matching on the basis of cross-reactive groups (CREG), was recently suggested to be a good alternative to transplant with more "well-matched" kidneys, without negatively influencing graft survival. Because graft rejection is often mediated by cytotoxic T cells (CTLs), we investigated whether a beneficial effect of CREG matching is reflected in vitro by lower CTL precursor frequencies (CTLpf). Therefore, CTLpf were determined in a group of healthy individuals and analyzed with respect to the number of HLA and CREG mismatches. A clear correlation was found between the number of HLA mismatches and the CTLpf, that is, the lowest mean frequency in case of 0 HLA-A, B mismatches (66 CTL precursors per 10(6) cells) and the highest in combinations with 4 HLA mismatches (mean = 303 CTLp/10(6) cells). The situation was different in the case of CREG mismatches. Although the highest frequency was found in the group of 4 CREG mismatches, no significant differences were observed between 0, 1, and 2 CREG mismatches. High CTLpf, up to 430/10(6), were even seen in the case of 0 CREG mismatches. Also within a well-defined group of single HLA-A or HLA-B mismatches no difference in CTLpf were observed between the subgroups with 0 vs. 1 CREG mismatches. The present study showed that in vitro the CTLpf correlates better with HLA than with CREG matching. These data are consistent with findings reported by several groups that matching for the CREG does not benefit transplant outcome.

Validation of haplotype frequency estimation methods.

In order to investigate the performance of haplotype frequency estimation methods using unrelated individuals, we compared the results of three estimation methods with those from the haplotypes deduced from family pedigrees. To that end we used the HLA phenotypes of the parents of 1040 families as data for the estimation methods and the full pedigree information as data for the deductive method. We evaluated the results of the following estimation methods: the method using two by two tables described by Mattiuz et al., the maximum likelihood method described by Yasuda and Tsuji and a crude method that uses the information on homozygosity in the phenotypes. All estimation methods generate reliable haplotype frequencies for the more frequent haplotypes, but are unreliable for the less frequent haplotypes. The maximum likelihood estimation method shows the best overall correlation with the results of the deductive method.

Natural variants of the beta isoform of the human glucocorticoid receptor do not alter sensitivity to glucocorticoids.

The beta isoform of the human glucocorticoid receptor, hGRbeta, is a product of alternative splicing of the hGR gene. The physiological function of this isoform is unknown up to now. Recent data are contradictory in that they either favor or argue against a role of hGRbeta as a repressor of the functional hGRalpha isoform. In the present study hGRbeta did not inhibit transcriptional activation of the MMTV-driven luciferase reporter gene by dexamethasone-activated hGRalpha in COS-1 cells. In addition, two naturally occurring variants of the hGRbeta isoform associated with altered sensitivity to glucocorticoids, termed hGRbeta-R23K and hGRbeta-N363S, did not repress hGRalpha, even when overexpressed 10-fold. We conclude that the hGRbeta isoform, as well as two of its natural variants, do not act as dominant negative inhibitors of hGRalpha function and that the beta isoform does not appear to play a role in the regulation of glucocorticoid sensitivity.