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The purpose of this study was to investigate the effects of various solvent systems containing water, ethanol, propylene glycol (PG), and their binary combinations on the ex vivo permeation of zidovudine (AZT) across Sprague-Dawley rat skin using Franz diffusion cells at 37 degrees C. Further, saturation solubility and epidermis/vehicle partition coefficient of AZT in the solvent systems, and their effect on percentage hydration of epidermis using thermogravimetric analysis were determined to understand the mechanisms by which these solvent systems change drug permeability properties. All binary combinations of PG, ethanol and water significantly increased saturation solubility of AZT. Maximum AZT flux was observed with 66.6% ethanol among ethanol-water solvents, with 33.3% PG in PG-water solvents and with 100% ethanol among PG-ethanol combinations. PG-water and PG-ethanol solvents neither reduced the lag time nor increased AZT flux across rat skin. In addition, high concentrations of PG in both water and ethanol reduced steady state flux of AZT. Further, thermogravimetric studies revealed that solvents containing high PG concentrations dehydrate epidermis. Among all the solvent combinations, highest flux and short lag time were achieved with ethanol at 66.6% in water and hence is a suitable vehicle for transdermal delivery of AZT.

Background: Antimitotic chemotherapeutic agents target tubulin, the major protein in mitotic spindles. Tubulin isotype composition is thought to be both diagnostic of tumor progression and a determinant of the cellular response to chemotherapy. This implies that there is a difference in isotype composition between normal and tumor tissues., Methods: To determine whether such a difference occurs in breast tissues, total tubulin was fractionated from lysates of paired normal and tumor breast tissues, and the amounts of beta-tubulin classes I + IV, II, and III were measured by competitive enzyme-linked immunosorbent assay (ELISA). Only primary tumor tissues, before chemotherapy, were examined. Her2/neu protein amplification occurs in about 30% of breast tumors and is considered a marker for poor prognosis. To gain insight into whether tubulin isotype levels might be correlated with prognosis, ELISAs were used to quantify Her2/neu protein levels in these tissues., Results: Beta-tubulin isotype distributions in normal and tumor breast tissues were similar. The most abundant beta-tubulin isotypes in these tissues were beta-tubulin classes II and I + IV. Her2/neu levels in tumor tissues were 5-30-fold those in normal tissues, although there was no correlation between the Her2/neu biomarker and tubulin isotype levels., Conclusion: These results suggest that tubulin isotype levels, alone or in combination with Her2/neu protein levels, might not be diagnostic of tumorigenesis in breast cancer. However, the presence of a broad distribution of these tubulin isotypes (for example, 40-75% beta-tubulin class II) in breast tissue, in conjunction with other factors, might still be relevant to disease progression and cellular response to antimitotic drugs.

Through the sequence analysis of 27 imprinted human genes and a set of 100 control genes we have developed a novel approach for identifying candidate imprinted genes based on the differences in sequence composition observed. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable element (SINE) Alus and mammalian-wide interspersed repeat (MIR) repeat elements, as previously reported. In addition, a significant association between imprinted genes and increased numbers of low-complexity repeats was also evident. Numbers of the Alu classes AluJ and AluS were found to be significantly depleted in some parts of the flanking regions of imprinted genes. A recent study has proposed that there is active selection against SINE elements in imprinted regions. Alternatively, there may be differences in the rates of insertion of Alu elements. Our study indicates that this difference extends both upstream and downstream of the coding region. This and other consistent differences between the sequence characteristics of imprinted and control genes has enabled us to develop discriminant analysis, which can be used to screen the genome for candidate imprinted genes. We have applied this function to a number of genes whose imprinting status is disputed or uncertain.

Translocations occur in a proportion of couples affected by recurrent miscarriages. We describe two such families in which the underlying cause was a cryptic subtelomeric 11p;17p translocation detected only after the birth of an affected child carrying an unbalanced form of the rearrangement. Unbalanced subtelomeric rearrangements are now recognised as a significant cause of mental impairment and we believe that these rearrangements may also be an important cause of recurrent miscarriages. In these two families the translocation is most likely to have arisen from a single ancestral event because all translocation carriers shared almost identical haplotypes around the breakpoints on both chromosomes.

Sequence comparison analysis has been carried out for 31 imprinted mouse genes and a set of 150 control genes. The imprinted genes were found to be associated with significantly reduced numbers of short interspersed transposable elements (SINEs), in particular SINE Alu repeats. This is similar to recent analyses of human imprinted genes and supports the suggestion that there is either active selection against SINE elements in imprinted regions or a reduced rate of insertion of these elements. The reduction in numbers of SINEs was more consistent in paternally expressed genes, whereas for maternally expressed genes significantly reduced numbers of SINE-B2 elements were coupled with increased numbers of SINE-B4 and SINE-ID elements. Paternally expressed genes were also found to be associated with a lower GC content. Discriminant analysis revealed that the two sub-groups of imprinted genes can be cleanly separated from each other on the basis of their genomic sequence characteristics and that they tend to localize to different genomic compartments. The differences between the sequence characteristics of imprinted and control genes have also enabled us to develop a discriminant function that can be used in a genome-wide screen to identify candidate imprinted genes.

Paternal duplications of distal 11p result in Beckwith Wiedemann syndrome (BWS), whereas maternal duplications have not, to our knowledge, been reported previously in the literature. We present three unrelated patients with maternal duplications of distal 11p. Patient 1 is a 31-year-old female with a de novo inverted duplication of distal 11p, i.e. inv dup del(11)(qter-->p15.5::p15.5-->15.3); this rearrangement was shown to be maternal in origin by microsatellite analysis and methylation-specific polymerase chain reaction. Patient 2 is a 4-year-old female with a derived chromosome 20, which arose from adjacent 1 malsegregation of a maternal t(11;20)(p15.3;q13.33). Patient 3 presented as an intrauterine death with trisomy for the majority of chromosome 11p as a result of 3:1 segregation of a maternal t(11;15)(p11.2;q11.2). In view of the imprinted status of this region, it is pertinent that none of our patients showed features of BWS; indeed, all had growth retardation, in contrast to the overgrowth characteristic of BWS. It is of note that, of the living patients, Patient 1 went into early puberty at 9.5 years and Patient 2 showed breast development in infancy. Both patients shared some dysmorphological features, namely short palpebral fissures, a prominent nasal tip, a short philtrum and 5th finger clinodactyly.

During spermatogenesis in human adults, testicular germ cells proliferate, differentiate, and die by apoptosis. However, little is known about the temporal or spatial nature of this programmed cell death. Such information may be useful for understanding prenatal developmental biology as well as spermatogenesis during adulthood, particularly in the context of germ cell disorders. We undertook this study to determine 1) whether apoptosis occurred in a cell-specific fashion in the germ cell population and the supporting somatic cells; and 2) whether apoptosis varied with gestational age. We examined human fetal testicular tissues obtained from 17 karyotypically and structurally normal fetuses of mothers who underwent spontaneous or induced abortions. Three gestational ages were defined as follows: group A, 12-13 wk gestation (n = 5); group B, 20-22 wk gestation (n = 7); and group C, 37-40 wk gestation (n = 5). Morphology in conjunction with in situ end labeling was used to identify and quantify apoptotic nuclei in fetal gonadal tissues. The results of this study suggest that gonadal apoptosis occurred in germ cells, Sertoli cells, and Leydig cells at all gestational ages. Apoptotic death was highest in the Leydig cells, followed by germ cells and Sertoli cells. There was a significant positive correlation between the apoptosis of germ cells and Sertoli cells (P < 0.01) and a negative correlation between healthy germ cells and Sertoli cells (P < 0.001). There was also a negative correlation between the intratubular cell number and the gestational age. Specifically, the proportion of Sertoli cells decreased with gestational age, although there was no significant change in the germ cell in relation to gestational age. No such relationship was found in the Leydig cell population, all of which reside outside the seminiferous tubules. These results are the first to suggest that fetal testicular apoptosis begins in the first trimester, occurs in the three major cell types, and continues throughout pregnancy. Our data also suggest that in the fetal gonad, germ and Sertoli cell proliferation and death may be controlled by a genetic program distinct from that of the Leydig cells. This information is relevant to the understanding of abnormal spermatogenesis associated with infertility and to germ cell tumors in adult life.

The objective of this investigation was to study the effect of different terpenes on IMH permeation in EtOH:W (2:1) system. Permeation studies of IMH were carried out with unjacketed Franz diffusion cells through rat skin. The flux of IMH with terpenes was found to be significantly higher than that in control (EtOH:W, 2:1) (P<0.05). Amongst all studied terpenes, menthol, terpineol, cineole and menthone were found to be effective permeation enhancers for IMH. It was found that the contribution of diffusivity in enhanced permeation of IMH was much higher in comparison to partitioning of IMH in skin with terpene treatment. Results of this study were explained with the help of H-bond breaking potential and self-association of terpenes. In order to elucidate the effect of terpenes on stratum corneum barrier FT-IR was used.

Survival signals elicited by cytokines include the activation of phosphatidylinositol 3-kinase (PI3K), which in turn promotes the activation of protein kinase B (PKB). Recently, PKB has been demonstrated to phosphorylate and inactivate forkhead transcription factor FKHR-L1, a potent inducer of apoptosis. To explore the mechanisms underlying the induction of apoptosis after cytokine withdrawal or FKHR-L1 activation, we used a cell line in which FKHR-L1 activity could be specifically induced. Both cytokine withdrawal and FKHR-L1 activation induced apoptosis, which was preceded by an upregulation in p27KIP1 and a concomitant decrease in cells entering the cell cycle. Induction of apoptosis by both cytokine withdrawal and activation of FKHR-L1 correlated with the disruption of mitochondrial membrane integrity and cytochrome c release. This was preceded by upregulation of the pro-apoptotic Bcl-2 family member Bim. Ectopic expression of an inhibitory mutant of FKHR-L1 substantially reduced the levels of apoptosis observed after cytokine withdrawal. Activation of PKB alone was sufficient to promote cell survival, as measured by maintenance of mitochondrial integrity and the resultant inhibition of effector caspases. Furthermore, hematopoietic stem cells isolated from Bim-/- mice exhibited reduced levels of apoptosis upon inhibition of PI3K/PKB signaling. These data demonstrate that activation of FKHR-L1 alone can recapitulate all known elements of the apoptotic program normally induced by cytokine withdrawal. Thus PI3K/PKB--mediated inhibition of this transcription factor likely provides an important mechanism by which survival factors act to prevent programmed cell death.

Fetal haemopoietic cells continually circulate and migrate into tissues, and thus may have specialized homing capabilities. In this study we investigated the in vitro features of haemopoietic cells in fetal blood and liver which are relevant to homing and engraftment. Fetal cells were examined for long-term culture-initiating cell (LTC-IC) and progenitor content, adhesion molecule expression, cell cycle behaviour and transendothelial migratory activity. The LTC-IC content of fetal CD34+ cells is similar to that of CD34+ cells from cord and adult mobilized blood. In contrast to adult and cord blood CD34+ cells, fetal CD34+ cells were actively cycling (11.0 +/- 1.7% and 28 +/- 1.1% of fetal blood and liver CD34+ cells, respectively, in S+G2M, P < 0.001, compared with cord and adult cells). The striking finding was that fetal haemopoietic cells (both LTC-ICs and committed progenitors) displayed significantly higher levels of migration across endothelium (P < 0.05 compared with cord, P < 0.01 compared with adult blood and bone marrow CD34+ cells), which were further increased by chemokines and growth factors. The superior migratory activity of fetal haemopoietic cells may underlie a more efficient homing ability, in keeping with their physiological role.

This study investigated the phenotypic manifestations of interstitial duplications of chromosome 15 that involve the Prader-Willi/Angelman syndrome critical region (PWACR). Twenty-one affected individuals from six families were evaluated in detail, using standardized and semi-standardized measures of intelligence, psychopathology, and physical anomalies. Special attention was placed on determining the prevalence of autism spectrum disorders as well as the relationship between the parental origin of the duplication and the phenotypic effects. Assessments of the affected individuals were compared with evaluations of the unaffected relatives from the same families. Results indicated that duplications in the region were associated with variable degrees of intellectual impairments and motor coordination problems. Four of the subjects received a diagnosis of pervasive developmental disorder. Three of these cases were probands and only one met criteria for classic autism. There was very little evidence of the duplication cosegregating with autism spectrum disorder diagnosis. Paternally inherited duplications were significantly less likely to give rise to phenotypic effects. The findings indicate that duplications in the PWACR give rise to developmental delay but not necessarily autism spectrum disorders. They also suggest that phenotypic expression is dependent on the parental origin of the duplication and implicate maternally active genes in the pathogenesis of the developmental impairments. Further research will be required to clarify the range and basis of the phenotypic manifestations., (Copyright 2001 Wiley-Liss, Inc.)

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-beta, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-kappaB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G(0) to G(1). Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.

Attempts to identify genetic contributors to human meiotic nondisjunction have met with little, if any, success. Thus, recent reports linking Down syndrome to maternal polymorphisms at either of two folate metabolism enzymes, methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR), have generated considerable interest. In the present report, we asked whether variation at MTHFR (677C-->T) or MTRR (66A-->G) might be associated with human trisomies other than trisomy 21. We analyzed maternal polymorphisms at MTHFR and MTRR in 93 cases of sex-chromosome trisomy, 44 cases of trisomy 18, and 158 cases of autosomal trisomies 2, 7, 10, 13, 14, 15, 16, 18, or 22, and compared the distributions of genotypes to those of control populations. We observed a significant increase in the MTHFR polymorphism in mothers of trisomy 18 conceptuses but were unable to identify any other significant associations. Overall, our observations suggest that, at least for the sex chromosomes and for a combined set of autosomal trisomies, polymorphisms in the folate pathway are not a significant contributor to human meiotic nondisjunction.

Oestradiol increases the protein expression of connexin43 (Cx43) gap junctions in myometrium but the effect of oestriol on gap junction expression has not been described previously. Oestriol is the most abundant free oestrogen in pregnant women and there is a marked surge in oestriol concentrations before term and idiopathic preterm labour. In order to determine whether oestriol may have a physiological action on the myometrium, cultured human myometrial cells obtained from non-pregnant hysterectomy specimens were exposed to 10 nmol/l oestradiol or oestriol. Intercellular communication between myometrial cells was investigated by microinjection of confluent cultured cells with the gap junction-permeant tracer Cascade Blue. There was a progressive increase in coupling after exposure to oestradiol or oestriol (P < 0.0005). An increase in Cx43 protein expression was demonstrated by immunocytochemistry after 1 h (P < 0.01) and 3 days (P < 0.01) exposure, and by Western blotting after 1 h (P < 0.01) and 3 days (P < 0.05) exposure, to both oestradiol and to oestriol. We conclude that oestriol increases gap junction communication in human myometrium by increasing gap junction expression. Elevated oestriol concentrations may thus play a role in the initiation of labour in women, by increasing cell-cell communication in the myometrium.

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27(Kip1) to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.

The effect of the solvent systems water, ethanol (EtOH), propylene glycol (PG) and their binary combinations was studied on the ex vivo permeation profile of the opioid receptor antagonist, naloxone, through rat skin. Fourier transform-infrared (FT-IR) spectroscopic studies were done to investigate the effect of enhancers on the biophysical properties of the stratum corneum (SC), in order to understand the mechanism of permeation enhancement of naloxone by the solvent systems used. The flux of naloxone was found to increase with increasing concentrations of EtOH, upto 66% in water, and PG upto 50% in water. The maximum flux of 32.85 microg cm(-2) h(-1) was found with 33% PG in EtOH. The FT-IR spectra of SC treated with EtOH showed peak broadening at 2920 cm(-1) at all concentrations of EtOH studied indicating that EtOH increases the translational freedom (mobility) of lipid acyl chains. Theoretical blood levels well above the therapeutic concentration of naloxone can be achieved with the solvent system comprising 33% PG in EtOH and hence, provides flexibility in choice of patch size depending on the addiction status of the patient to be treated.

This study investigated the influence of expression of proteins of the INK4 family, particularly p16, on the growth and self-renewal kinetics of hematopoietic cells. First, retrovirus-mediated gene transfer (RMGT) was used to restore p16(INK4a) expression in the p16(INK4a)-deficient lymphoid and myeloid cell lines BV173 and K562, and it was confirmed that this inhibited their growth. Second, to sequester p16(INK4a) and related INK4 proteins, cyclin-dependent kinase 4 (CDK4) was retrovirally transduced into normal human CD34(+) bone marrow cells and then cultured in myeloid colony-forming cell (CFC) assays. The growth of CDK4-transduced colonies was more rapid; the cell-doubling time was reduced; and, upon replating, the colonies produced greater yields of secondary colonies than mock-untransduced controls. Third, colony formation was compared by marrow cells from p16(INK4a-/-) mice and wild-type mice. The results from p16(INK4a-/-) marrow were similar to those from CDK4-transduced human CFCs, in terms of growth rate and replating ability, and were partially reversed by RMGT of p16(INK4a). Lines of immature granulocytic cells were raised from 15 individual colonies grown from the marrow of p16(INK4a-/-) mice. These had a high colony-forming ability (15%) and replating efficiency (96.7%). The p16(INK4a-/-) cell lines readily became growth factor-independent upon cytokine deprivation. Taken together, these results demonstrate that loss of INK4 proteins, in particular p16(INK4a), increases the growth rate of myeloid colonies in vitro and, more importantly, confers an increased ability for clonal expansion on hematopoietic progenitor cells.

Tuberculosis affects more than 8 million people and has serious repercussions on economic, psychologic, and social status. Since its declaration as a global emergency in 1993 by the World Health Organization, significant development in the treatment and control of tuberculosis has been the implementation of the short-course directly observed treatment along with fixed dose combinations of existing drugs. However, the currently available therapeutic regimens have inherent disadvantages of long treatment duration, patient noncompliance, and risk of drug resistance. Hence, new antituberculosis drugs that are potent, are active against resistant strains and latent forms, and reduce the treatment period are needed to combat this disease. In this review, the authors discuss new chemical entities that in their opinion have a potential to become new antituberculosis drugs. This article emphasizes the role of biopharmaceutics and pharmacokinetics as an indispensable part in the development of new antituberculosis drugs to overcome the common hurdles such as exorbitant cost, time, and attrition rate involved in the process.

A member of the retinoblastoma family of cell cycle regulatory proteins, p107, is not normally expressed in non-cycling cells. We demonstrate here that p107 is expressed in monocytes as they differentiate into dendritic cells under the influence of granulocyte-macrophage colony-stimulating factor and interleukin 4, a process shown not to involve cellular proliferation. We show that p107 is localized to the nucleus of these cells and is active, in that it binds an E2F-DNA binding site, together with E2F transcription factors. These findings suggest a hitherto unknown role for p107 in non-proliferating dendritic cells that warrants further investigation.

Human trisomy is attributable to many different mechanisms and the relative importance of each mechanism is highly chromosome specific. The association between altered recombination and maternal non-disjunction is well documented: reductions in recombination have been reported for maternal meiosis I (MI) errors involving chromosomes 15, 16, 18 and 21 and increased recombination has been reported for meiosis II (MII) errors involving chromosome 21. We therefore investigated maternal X chromosome non-disjunction, to determine whether the effects of recombination are unique to the X chromosome or similar to any of the autosomes thus far studied. We genotyped 45 47,XXX females and 95 47,XXY males of maternal origin. Our results demonstrate that 49% arose during MI, 29% during MII and 16% were postzygotic events; a further 7% were meiotic but could not be assigned as either MI or MII because of recombination at the centromere. Among the MI cases, a majority (56%) had no detectable transitions and so absent recombination is an important factor for X chromosome non-disjunction. However, similar to trisomy 15 and unlike trisomy 21, we observed a significant increase in the mean maternal age of transitional MI errors compared with nullitransitional cases. In our studies of MII errors, recombination appeared normal and there was no obvious effect of maternal age, distinguishing our results from MII non-disjunction of chromosomes 18 or 21. Thus, surprisingly, the risk factors associated with both MI and MII non-disjunction appear to be different for virtually every chromosome that has been adequately studied.

Interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor regulate the survival, proliferation, and differentiation of hematopoietic lineages. Phosphatidylinositol 3-kinase (PI3K) has been implicated in the regulation of these processes. Here we investigate the molecular mechanism by which PI3K regulates cytokine-mediated proliferation and survival in the murine pre-B-cell line Ba/F3. IL-3 was found to repress the expression of the cyclin-dependent kinase inhibitor p27(KIP1) through activation of PI3K, and this occurs at the level of transcription. This transcriptional regulation occurs through modulation of the forkhead transcription factor FKHR-L1, and IL-3 inhibited FKHR-L1 activity in a PI3K-dependent manner. We have generated Ba/F3 cell lines expressing a tamoxifen-inducible active FKHR-L1 mutant [FKHR-L1(A3):ER*]. Tamoxifen-mediated activation of FKHR-L1(A3):ER* resulted in a striking increase in p27(KIP1) promoter activity and mRNA and protein levels as well as induction of the apoptotic program. The level of p27(KIP1) appears to be critical in the regulation of cell survival since mere ectopic expression of p27(KIP1) was sufficient to induce Ba/F3 apoptosis. Moreover, cell survival was increased in cytokine-starved bone marrow-derived stem cells from p27(KIP1) null-mutant mice compared to that in cells from wild-type mice. Taken together, these observations indicate that inhibition of p27(KIP1) transcription through PI3K-induced FKHR-L1 phosphorylation provides a novel mechanism of regulating cytokine-mediated survival and proliferation.

We have used polymorphisms within the Xp/Yp pseudoautosomal region (PAR 1) to determine the frequency and location of recombination in 80 paternally derived 47, XXY males. Of 64 informative results, there were 10 single cross-overs, one double cross-over and 53 without a cross-over. Therefore 2/3 of 47, XXY males of paternal origin result from meiosis in which the X and Y chromosomes fail to recombine. This failure was not associated with the presence of an increase in recombination in the smaller Xq/Yq pseudoautosomal region (PAR 2) or with the presence of microdeletions within PAR 1.

With the synergistic and multiplicative interactions of rational drug design, recombinant biotechnology, combinatorial chemistry and high-throughput screening, millions of compounds are being synthesized by chemists. However, development of these drug candidates has often been impeded, if not terminated, due to biopharmaceutic and/or pharmacokinetic constraints. This has resulted in delays in development time and escalation of cost in the drug research programmes. So, the present emphasis is to reduce development time and cost, which is analogous to added patent life besides the enormous reduction in human suffering. In this compilation the important biopharmaceutic and pharmacokinetic approaches are discussed, which will help in the development of safe and more efficacious drugs with reduced development time and cost.

Dendritic cells may be generated ex vivo from CD34+ progenitor cells or peripheral blood mononuclear cells. Initial reports suggested that monocyte-derived dendritic cells (MoDCs) arise from a proliferating precursor and several groups subsequently reported successful retroviral transduction of these cells, again implying that cell division occurs. As this is of importance in the development of immunotherapy protocols, we investigated whether monocytes proliferate as they differentiate into MoDCs and also their susceptibility to retroviral transduction. During MoDC differentiation, there was a 51 +/- 12% reduction in cell number, 98% of cells were in G0/G1, no DNA synthesis was detectable and the cell cycle regulatory proteins pRb and p130 were in the hypophosphorylated forms observed in non-cycling cells. As expected from these results, MoDCs were refractory to transduction with a GALV1 pseudotyped Moloney murine leukaemia virus (MoMLV)-based retroviral vector. In contrast, generation of DCs from purified CD34 progenitors was accompanied by rapid entry into the cell cycle and a 41.1-fold cell expansion at the end of 14 d culture.

Farnesol preferentially inhibits proliferation and induces apoptosis of tumour-derived but not non-transformed cell lines. We investigated whether farnesol induces apoptosis of blasts from patients with acute myeloid leukaemia (AML) and leukaemic cell lines, as compared with normal, human primary haemopoietic cells. We show that 30 microM farnesol causes apoptosis of leukaemic cell lines of T- and B-lymphocyte, myeloid or erythroid lineages and primary blasts obtained from patients with AML. However, the same concentration did not kill primary monocytes, or quiescent or proliferating T-lymphocytes. We conclude that farnesol selectively kills AML blasts and leukaemic cell lines in preference to primary haemopoietic cells.

Cyclin D2 is the only D-type cyclin expressed in mature mouse B-lymphocytes, and its expression is associated with retinoblastoma protein (pRB) and pRB-related protein phosphorylation and induction of E2F activity, as B-cells enter the cell cycle following stimulation via surface IgM and/or CD40. Cyclin D-dependent kinase activity is required for cell proliferation, yet cyclin D2(-/-) mice have normal levels of mature B-lymphocytes. Here we show that B-lymphocytes from cyclin D2(-/-) mice can proliferate in response to anti-IgM and anti-CD40, but the time taken to enter S-phase is longer than for the corresponding cyclin D2(+/+) cells. This is due to the compensatory induction of cyclin D3, but not cyclin D1, which causes pRb phosphorylation on CDK4-specific sites. This is the first demonstration that loss of a D-type cyclin causes specific expression and functional compensation by another member of the family in vivo and provides a rationale for the presence of mature B-lymphocytes in cyclin D2(-/-) mice.

Both asthma and atopy run families indicating a strong genetic component. To investigate possible candidate gene regions, we have recruited 131 families without reference to atopy and asthma (random sample) and 60 extended families with two or more members affected by asthma (multiplex sample). Using both candidate and genome screen approaches, we have been able to provide evidence supporting the presence of candidate genes on chromosome 11q (E237G) and 12q but we have been unable to confirm reports of linkage and association for asthma on 5q. Our experience to date suggests that larger numbers of families are needed to increase the confidence of gene localisation and there is a need to improve the phenotypic description of asthma. Finally, it is essential that claims for linkage or association are confirmed in different populations using the same markers.

A 3-year-old female referred with developmental delay, hypotonia and seizures was found to have a cryptic interstitial duplication of the Prader-Willi/Angelman critical region (PWACR). Her clinical features form part of a common phenotype characteristic of PWACR duplications including developmental delay, behavioural problems and speech difficulties. Microsatellite analysis showed that the duplication had arisen de novo, was maternal in origin and involved the entire 4-Mb PWACR between the common deletion breakpoints. The existence of cryptic rearrangements emphasises the need for molecular tests alongside conventional cytogenetics when investigating abnormalities involving this imprinted region.

An efficient test of deviation from Hardy-Weinberg frequencies with one degree of freedom was applied to 44 marker loci in a genome scan, and 7 loci had a significant excess of apparent homozygotes (chi2 (1) > 6) suggestive of typing error. In this example evidence for linkage did not increase when outliers were censored. Statistical quality control is an essential part of genotyping, and the effect of mistyping and map error should be considered in evaluating any genome scan.

When cord blood is used as a source of haemopoietic stem cells for transplantation, fewer cells are required per kg of recipient. This greater engraftment efficiency of cord blood cells may relate to an increased ability to traverse sinusoidal endothelium, a crucial step in the homing of stem cells. We report that freshly isolated cord blood progenitors migrated more efficiently than mobilized adult cells. Cord blood progenitors responded rapidly to growth factor stimulation with an increase in migratory ability within 24 h whereas mobilized adult cells responded only after 72 h (P < 0.01). Cord blood cells also exited G0/G1 rapidly; after 24 h of growth factor exposure, 20.2 +/- 1.2% of cord blood CD34+ cells were in S + G2/M compared to 6.9 +/- 1.2% of adult CD34+ cells (P < 0.01). Proliferating CFC migrated more efficiently (13.3 +/- 3.4% for GM-CFC) than non-proliferating CFC (1.4 +/- 0.5%, P < 0.01) as determined using a 3H-thymidine suicide assay. Cord blood progenitor cells also demonstrated a greater transmigratory response to chemokine stimulation compared with adult cells; this was manifested as a differential response of freshly isolated cells to SDF-1, and of growth factor activated cells to MIP-3beta. Finally, cord blood CD34+ cells express higher levels of the chemokine receptor for SDF-1, CXCR4, when compared with mobilized adult CD34+ cells (P < 0. 05).

The ability of the CBP (CREB binding protein) coactivator to stimulate transcription has previously been shown to be stimulated by treatment of neuronal cells with nerve growth factor (NGF). This effect is dependent upon activation of the p42/p44 MAPK (mitogen activated protein kinase) pathway. Here we show that both CBP and the related p300 protein directly associate with the p42/p44 MAPK enzymes both prior to and following their activation by NGF and that CBP is phosphorylated following NGF treatment. These results indicate that phosphorylation of CBP itself by the p42/p44 MAPK pathway is likely to be critical for its role in NGF-mediated stimulation of gene expression.

The pocket protein-E2F complexes are convergence points for cell cycle signaling. In the present report, we identified and monitored the pocket protein-E2F complexes in human primary B-lymphocytes after activation by phorbol 12-myristate 13-acetate. Consistent with previous data from human and mouse fibroblasts and T-lymphocytes, E2F4 and DP1 form the predominant E2F heterodimers both in G0 and G1 phases of the human B-lymphocyte cell cycle, whereas E2F1 and -3 are first detected in late G1, and their expression levels increase towards S phase. Intriguingly, the major E2F complex that we detected in quiescent human B-lymphocytes is consisted of pRB, E2F4, and DP1. Though the levels of DP1 and -2 increase when cells progress from G0 to S, the proportion of DP1 to DP2 remains relatively constant during the cell cycle. We also observed an increase in electrophoretic mobility of the predominant E2F components, DP1 and E2F4, as B-lymphocytes progressed from G0 into early G1. This increase in mobility was attributable to dephosphorylation, as lambda phosphatase treatment could convert the slower migrating forms into the corresponding faster mobility forms. We further demonstrated that this change in phosphorylation status correlates with a decrease in DNA binding activity. This modulation of DNA binding activity mediated through the dephosphorylation of DP1 and E2F4 could help to explain the lack of in vivo DNA footprinting in late G1 and S phases of gene promoters negatively regulated through E2F sites and suggests a novel mechanism for controlling E2F transcriptional activity during the transition from quiescence to proliferation.

Background: Using quantitative phenotype scores, we have genotyped four markers close to the FcepsilonRI-beta gene on chromosome 11q and 17 markers on chromosome 12. We have also determined the frequency of the I181L/V183L and E237G polymorphisms in our population., Methods: 131 randomly ascertained families and 109 families with an asthmatic proband were recruited. Written and video questionnaires, bronchial challenge, and skinprick tests were administered and IgE levels measured. Phenotype scores were derived using principal-component analysis. The asthma score incorporated the questionnaire data reduced to two variables (wheeze and video), the ratio of predicted to observed FEV1, and bronchial hyperresponsiveness calculated as the slope of the dose-response curve. Total IgE, with the highest heritability of the atopy variables, was used as the atopy score. I181L/V183L polymorphism was determined by sequencing and E237G polymorphism by the amplification refractory mutation system. The data were analyzed using the BETA programme., Results: No examples of the I181L/V183L polymorphism were identified. The E237G polymorphism was identified with a frequency of 3.5% with weak evidence for linkage (lod 1.522) to asthma. Linkage was found to markers on chromosome 12 and asthma. The largest single locus lod was achieved for D12S366 and wheeze (lod 3. 307). Using multipoint analysis, a maximum lod score of 2.29 centres around D12S97 at location 173.5 cM for the asthma score., Conclusion: The linkage results for chromosome 12 justify further interest in this region. Future endeavours will be directed towards fine mapping in the hope of identifying novel candidate genes.

X-linked myotubular myopathy (XLMTM) is a congenital muscle disorder mainly affecting newborn males. Neonatal muscle weakness and hypotonia usually leads to a rapid demise. The responsible gene, MTM1, was isolated in 1996, and mutational data derived from 90 patients have been published. We report on our findings in a further 53 patients, using genomic DNA and mRNA screening protocols. Thirty-four novel mutations were identified in 37 cases, and six known mutations found in 10 other patients. The 34 new mutations include five large deletions, eight nonsense, six frameshift, five missense, and eight splice-site mutations, whereas two intronic variants causing partial exon skipping represent the first report on such a mechanism in MTM1. Two deletions, one involving exon 1, and the second exon 15, are the first defects to be identified in these exons. The heterogeneity of the mutations, their mutational origins, and the varied ethnic backgrounds of the patients, indicate that the majority of XLMTM families are affected by unique MTM1 mutations.

We report eight females with small deletions of the short arm of the X chromosome, three of whom showed features of autism. Our results suggest that there may be a critical region for autism in females with Xp deletions between the pseudoautosomal boundary and DXS7103. We hypothesise that this effect might be due either to the loss of function of a specific gene within the deleted region or to functional nullisomy resulting from X inactivation of the normal X chromosome.

We present evidence of linkage between markers on chromosome 12 and asthma using the BETA program for nonparametric single- and multipoint linkage analysis. We have used quantitative scores as our phenotypic variables, combining data into indices for asthma and atopy and maximizing heritability. The largest single-locus LODs were achieved for asthma: D12S342 and asthma score (LOD 2.255), D12S324 and asthma affection (LOD 2.214), and D12S366 and wheeze (LOD 3.307). The region of interest identified using multipoint analysis, with a maximum LOD of 2.29, centers around D12S97 at location 173.5 cM with a standard error of 6.5 for the asthma score and close agreement for asthma affection and wheeze. Such evidence merits further investigation of this area in an attempt to define the region with greater precision with a view to identifying candidate genes. We hope that the methods presented will encourage researchers to use phenotypic information in a way that encourages meta-analysis., (Copyright 1998 Academic Press.)

We have determined how the phosphorylation of the retinoblastoma family (pRb, p107, and p130) is governed in individual cell cycle phases of Daudi B-cells during cell cycle exit triggered by alpha-interferon (alpha-IFN). alpha-IFN causes dephosphorylation of pRb and loss of p130 phosphorylated Form 3. However, the change in p130 phosphorylation in response to alpha-IFN occurs before dephosphorylation of pRb is complete because loss of p130 Form 3 occurs throughout the cell cycle prior to complete arrest in G1, whereas pRb is dephosphorylated only in G1. In contrast, p107 is dephosphorylated and is then depleted from cells as they exit the cell cycle. p130, predominantly in Form 1, and hypophosphorylated pRb bind an E2F DNA binding site; p130 complexes E2F-4, whereas pRb binds both E2F-4 and E2F-1. The phosphorylated forms of E2F-4 that bind to the E2F DNA site are different from hyperphosphorylated E2F-4, which predominates in primary hemopoietic cells in G0. We conclude that although cell cycle arrest induced by alpha-IFN may be mediated in part by formation of a complex containing p130 and E2F-4, alpha-IFN does not induce hyperphosphorylation of E2F-4, which characterizes primary hemopoietic cells in G0.

We have undertaken a clinical and molecular study of 25 females with deletions of the short arm of the X chromosome. We have determined the deletion breakpoints, the parental origin and the activation status of the deleted X chromosomes. Genotype-phenotype correlations suggest that the presence of a single copy of the DFFRX gene, previously postulated as a gene involved in the ovarian failure seen in Turner syndrome, may be compatible with normal ovarian function, and that there may be a gene for Turner-like features located in distal Xp22.3.

Herpes simplex virus (HSV) has often been suggested as a vector for gene delivery to the nervous system although it is also capable of infecting many other cell types. HSV also has the ability to package large genetic insertions so the expression of multiple genes from a single virus is possible. Here we show that a green fluorescent protein (GFP) expressing HSV1 vector can transduce two primary human cell types--quiescent human CD34+ hematopoietic progenitor cells and dendritic cells--which are both hard to transduce by other means. We also show that GFP is an effective marker when expressed from an HSV vector in vivo in the mouse brain. When GFP is expressed together with a second gene (in this case lacZ) from a single virus, transduced GFP-positive CD34+ hematopoietic progenitor cells or dendritic cells can both be generated at an effective efficiency of 100% for the second gene. Here transduction with the vector is combined with flow cytometry allowing GFP-positive cells to be sorted from the untransduced population. Such completely transduced populations of quiescent CD34+ hematopoietic progenitor and dendritic cells cannot easily be achieved by other means, and might thus allow experimental or therapeutic protocols to be carried out requiring high-level transduction which would not otherwise be possible. Such an approach using HSV vectors might also be applicable to other cell types for which transduction is as yet unreliable or of low efficiency.

The A-Myb transcription factor is structurally related to the c-myb proto-oncogene and is involved in the control of proliferation and/or differentiation of mature B lymphocytes. We have shown previously by PCR analysis that A-myb is preferentially expressed in CD38+ CD39- sIgM- mature B cells. We demonstrate here, using in situ hybridization, that A-myb expression is restricted to the dark zone of human tonsils and lymph nodes. Furthermore, we show that A-Myb expression is cell cycle regulated both in tonsillar B cells and in Burkitt's lymphoma cell lines, being detectable only in the S and G2/M phases of the cell cycle and not in G0/G1 phase. Strong proliferation of resting human B cells induced in vitro by a variety of physiologic signals, including anti-mu, CD40 ligand, IL-2, IL-4, IL-6, IL-13, IFN-gamma, TNF-alpha, anti-CD19, and anti-CD20, failed to induce A-myb expression, suggesting that proliferation alone is not sufficient for A-myb expression in the absence of induction of a true centroblast phenotype. Finally, we show that differentiation of germinal center B cells in vitro toward either memory or plasma cells is accompanied by rapid down-regulation of A-myb expression. We conclude that A-myb is a marker of centroblasts generated in vivo.

Indirect molecular diagnosis of X linked hypohidrotic ectodermal dysplasia (XLHED), a congenital disorder of hair, teeth, and eccrine sweat glands, has been possible by linkage analysis. Direct mutation detection would enable carrier detection in female relatives of sporadic cases, as well as help distinguish XLHED from the rarer, clinically indistinguishable, autosomal recessive disorder ARHED. Recently, a candidate gene for XLHED has been identified. Genomic DNA from 162 affected males and 21 females, who were either obligate carriers or had manifestations of the disorder, were screened by SSCP analysis. A subset of the patients had been previously screened for large genomic deletions and had limited screening of a single exon by SSCP analysis. The two known exons were amplified using flanking primers. Approximately 7% of patients, all males, had putative mutations identified within exon 1, but no variants were found within exon 2. Ten different putative mutations and four probable polymorphisms were identified. Both of the known exons were sequenced in 10 patients who had no detectable SSCP changes, but no additional mutations were found. No correlation between phenotype and genotype was evident between either affected subjects or subjects with or without detectable mutations. The results of the study indicate that only a small minority of affected males can be diagnosed by direct mutation analysis, and that the remainder of the patients are likely to have mutations in as yet unidentified exons of the EDA gene. Linkage analysis, in informative situations, therefore remains the only practical diagnostic option available.

To date, the strongest linkage claims for genes underlying asthma and atopy have been for chromosomes 5 and 11. Chromosome 5q contains the cytokine cluster and the beta2 adrenoceptor, and the beta chain of the high-affinity IgE receptor (FCepsilonRI-beta) is encoded on chromosome 11q13. We have attempted to replicate these findings in two distinct sample populations from the United Kingdom. Allelic associations were identified in both regions, but there was no significant evidence for linkage. Although we could not substantiate the existence of the nucleotide changes reported within exon 6 of the FCepsilonRI-beta gene, an amino acid substitution in exon 7 was strongly linked to asthma and atopy. We have also identified positive linkage and allelic associations to several markers on chromosome 12q in both our UK populations. Independent evidence from another study also supports linkage to 12q, so although our data could not confirm linkage to chromosomes 5 or 11, we have identified an additional region of the genome that could be important for the genetic predisposition to asthma and atopy.

From the whole plant of Caraluma umbellata, three new C-21 steroidal glycosides, named as carumbellosides III-V, were isolated and their structures elucidated by extensive spectroscopic experiments, devoid of any derivatisation, as caralumagenin 3-O-beta-D-glucopyranosyl(1-->4)-beta-D-digitalopyranoside-20-O-be ta- D-glucopyranoside, caralumagenin 3-O-beta-D-glucopyranosy(1-->4)- beta-D-digitalopyranoside-20-O-(2-O- benzoyl)-beta-D-glucopyranoside and caralumagenin 3-O-[6-O-benzoyl-beta-D-glucopyranosyl(1-->4)]-beta-D- digitalopyranoside-20-O-(2-O-benzoyl)-beta-D-glucopyranoside. The determination of the absolute configuration of the aglycone as (20 R), the conformations of the sugars and the unambiguous assignments of their NMR spectroscopic signals were achieved by a combination of 2D-NMR techniques. The isolates were devoid of significant cytotoxity in the UIC human cancer cell panel.

Background: The beta2-adrenoceptor polymorphisms occurring at amino acid positions 16 (arginine to glycine) and 27 (glutamine to glutamate) are known to be functionally relevant and also disease-modifying in subjects with asthma. However, the contribution of these polymorphisms to the development of the asthmatic phenotype or other markers for allergic disease remains to be established., Objective: This large family study examines the contributions of these polymorphisms in determining the heritable component of markers for allergic disease in asthmatic families., Methods: Three hundred twenty-four individuals from 60 families multiplex for asthma selected by means of an asthmatic proband were characterized for the following markers of allergic disease: asthma, atopy, and serum IgE. The polymerase chain reaction was used to generate a 234 base pair fragment spanning the region of interest, and the beta2-adrenoceptor polymorphism was then defined by allele-specific oligonucleotide hybridization. Segregation analysis was then performed., Results: We found a significant association (p = 0.009) between the glutamine 27 beta2-adrenoceptor polymorphism and elevated levels of IgE, which was supported by the observation of linkage between IgE and beta2-adrenoceptor polymorphisms at locus 27 (p = 0.037). However, there was no association between either the arginine-glycine 16 or the glutamine-glutamate 27 beta2-adrenoceptor polymorphism and an increased risk of asthma or atopy per se., Conclusion: The glutamine 27 beta2-adrenoceptor polymorphism appears to contribute to IgE variability in families with asthma. However, it seems that although both amino acid 16 and 27 beta2-adrenoceptor polymorphisms are disease-modifying in subjects with asthma, they do not contribute markedly to the development of the asthmatic phenotype.

Mobilized peripheral blood progenitors (CD34+ cells) have been shown to be either in the G0 or G1 phase of the cell cycle. In this study, it is shown that they are small cells with low protein content suggestive of G0. Support for this is provided by showing that the principal E2F complex consists of hypophosphorylated p130, E2F-4, and DP-1. The E2F-4 is more highly phosphorylated than in quiescent T cells. In response to cytokines in vitro, the CD34+ cells start to enter G1 within 8 hours and enter S-phase at about 48 hours. As cells enter G1, E2F-4 is dephosphorylated to several hypophosphorylated forms and three new DNA-binding complexes appear, including one containing E2F-4, DP-1, and p107. We suggest that mobilized CD34+ cells may be maintained in G0 by p130, E2F-4, and DP-1 and the coordinate dephosphorylation of E2F-4 and hyperphosphorylation of p130 may be central to the initiation of proliferation.

Insights into the complex genetic factors underlying asthma and atopy have arisen from investigating genes whose functions are likely to be involved in the pathogenesis of these disorders. Candidate genes have now been studied on 13 human autosomes, and genetic linkage analysis and association studies have identified potentially important genes on chromosomes 5, 6, 11 and 14. However, these four regions do not account for all the heritability of these disorders, and additional regions are now being identified by systematic searches of the whole genome.