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Invasive aspergillosis is a significant cause of morbidity and mortality in patients after stem cell transplantation, in solid organ transplant recipients, and in patients with hematological malignancies. The interactions between human immature dendritic cells (iDCs) and Aspergillus fumigatus antigens are widely uncharacterized. We analyzed the immune response of iDCs to different recombinant A. fumigatus antigens (Aspf1 and Crf1). One of these antigens, the 18-kDa RNase Aspf1, triggered the increased level of expression of genes encoding proinflammatory cytokines and chemokines, and augmented the activation of NFkappaB and the apoptosis of iDCs. Furthermore, by fluorescence microscopy, we could demonstrate that in the first 3 h a major portion of Aspf1 accumulates on the cell surface. Finally, we could show an increased segregation of cytokines and chemokines after the stimulation of iDCs by an Aspf1 deletion mutant strain of A. fumigatus.

The combined blockade of the IL-6R/STAT3 and the MAPK signaling pathways has been shown to inhibit bone marrow microenvironment (BMM)-mediated survival of multiple myeloma (MM) cells. Here, we identify the molecular chaperones heat shock proteins (Hsp) 90alpha and beta as target genes of both pathways. The siRNA-mediated knockdown of Hsp90 or treatment with the novel Hsp90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. Although knockdown of Hsp90beta-unlike knockdown of Hsp90alpha-was sufficient to induce apoptosis, this effect was strongly increased when both Hsp90s were targeted, indicating a cooperation of both. Given the importance of the BMM for drug resistance and MM-cell survival, apoptosis induced by Hsp90 inhibition was not mitigated in the presence of bone marrow stromal cells, osteoclasts, or endothelial cells. These observations suggest that a positive feedback loop consisting of Hsp90alpha/beta and major signaling pathways supports the survival of MM cells. Finally, in situ overexpression of both Hsp90 proteins was observed in most MMs but not in monoclonal gammopathy of undetermined significance (MGUS) or in normal plasma cells. Our results underpin a role for Hsp90alpha and beta in MM pathogenesis.

HSP90's are overexpressed in different cancer types and they probably are required to sustain aberrant signalling in malignant cells. Recently, pharmacological inhibition of HSP90 was found to suppress growth of myeloma cell lines and in primary myeloma cells. Therefore, we wanted to investigate the role of HSP90alpha and HSP90beta in the pathogenesis of malignant myeloma (MM) in more detail. Immunohistochemistry was employed to examine the expression of HSP90alpha and HSP90beta in MM. The importance of HSP90 for survival of MM -cells was investigated by SiRNA-mediated knockdown of HSP90 and blockade of the IL-6R/STAT3 and the MAPK signaling pathways in vitro. HSP90alpha and HSP90beta were overexpressed in majority of investigated MM cases, but not in MGUS or in normal plasma cells. SiRNA-mediated knockdown of HSP90 or treatment with the novel HSP90 inhibitor 17-DMAG attenuated the levels of STAT3 and phospho-ERK and decreased the viability of MM cells. The knockdown of HSP90alpha was sufficient to induce apoptosis. This effect was strongly increased when both HSP90s were targeted, indicating a cooperation of both. HSP90 critically contributes to myeloma survival in the context of its microenvironment and therefore strengthen the potential value of HSP90 as a therapeutic target.

Population-based studies in twins have been of insufficient size to explore the relationship between risk of cerebral palsy and intrauterine growth. Earlier studies in singletons have suggested an optimum size at birth for minimum cerebral palsy risk between the 75th and 90th percentiles of weight for gestational age. We aggregated data from nine European cerebral palsy registers for 1976 to 1990. Using sex-specific fetal growth standards for twins, a z score of weight-for-gestation was derived for each of the 373 twin cases. The rates of cerebral palsy in each z-score band were compared to the rate in the a priori reference band of 0.67 to less than 1.28 (equivalent to the 75th to less than 90th percentiles). In twins born at 32 weeks' gestation or more (92% of all twins), cerebral palsy rates were higher for both light and heavy-for-gestation babies compared to an optimum (i.e., minimum risk) in the reference band. However, the rate ratio for heavy babies (90th percentile or greater) did not reach conventional (95% confidence intervals [CI]) statistical significance (rate ratios = 1.76; 90% CI 1.02-3.03). For twins born at less than 32 weeks, the significantly higher risk for cerebral palsy was observed consistently in all z-score bands less than average compared to the reference band. This multi-center study demonstrates that for twins born at 32 weeks' gestation or more, an increased risk of cerebral palsy is associated with deviations from optimal intrauterine growth at about 1 standard deviation above mean weight, as was earlier reported for singletons. For twins born at less than 32 weeks' gestation, this pattern is only demonstrable for babies weighing below the optimum weight-for-gestation.

Fluorescent DNA-labeling cassettes are designed to have a common absorbing chromophore matched to a single exciting laser wavelength, but up to four different emitters. Experiments reported here have examined the energy-transfer rates and fluorescence polarization characteristics for two different types of cassette, involving three distinct relative orientations of the donor and acceptor transition moments and the axis of the rigid linker. Energy-transfer times range from <200 fs to approximately 20 ps, the fastest transfer times occurring when the transition moments of the donor and acceptor species are aligned parallel to the linker axis. Experimental evidence is presented that supports a through-bond energy-transfer mechanism, in contrast with a commercial DNA-labeling agent, which exhibits much slower transfer times controlled by FRET. These rigid cassettes also exhibit polarized fluorescence from the acceptor species, so that this particular type of DNA-labeling probe has some of the advantages of single-molecule probes such as rhodamine and coumarin dyes.

[reaction: see text] N-Sulfinylimines derived from aromatic or aliphatic aldehydes and ketones react with nitromethane and NaOH in a highly diastereoselective manner under mild conditions. In the presence of TBAF, the reaction rates are strongly increased and the stereoselectivity is inverted. This method provides enantiomerically pure beta-nitroamines derived from enolizable aldimines and ketimines, which so far are hardly accessible by aza-Henry reactions.

Background: A European multicenter study (Surveillance of Cerebral Palsy in Europe, SCPE) was used to describe changes over time in multiple birth rates and cerebral palsy (CP) rates among multiple born infants, to compare CP rates and clinical types between multiples and singletons, and to analyse the influence of birth order in twins., Methods: Data were collected from 12 European population-based CP registers on 6613 children born in 1975-90, as well as demographic data., Results: The rate of multiple birth in the populations increased from 1.9% in 1980 to 2.4% in 1990, and the proportion of multiples among CP infants increased from 4.6% in 1976 to 10% in 1990. Multiples have a four times higher rate of CP than singletons [7.6 vs. 1.8 per 1000 live births, relative risk (RR) 4.36; 95% confidence interval (CI) 3.76-4.97] overall. The risk is marginally higher in multiples with birthweight > 2500 g (RR 1.60; 95% CI 0.95-2.28) and born at term (RR 1.65; 95% CI 0.91-2.40), and there is no difference in the risk for the low-birthweight and preterm groups. Correcting for differences in gestational age and birthweight, the clinical type of CP was the same in multiples and singletons. Twin CP infants are more often second than first born (56% vs. 44%, p < 0.05)., Conclusions: Multiple born infants have a four times higher risk of developing cerebral palsy than singletons, mainly related to the higher risk of preterm birth in multiples. As the rate of multiples doubled through the 1980s, cerebral palsy cases in multiples increased in the same period.

NKG2D is an activating receptor that stimulates innate immune responses by natural killer cells upon engagement by MIC ligands, which are induced by cellular stress. Because NKG2D is also present on most CD8alphabeta T cells, it may modulate antigen-specific T cell responses, depending on whether MIC molecules--distant homologs of major histocompatibility complex (MHC) class I with no function in antigen presentation--are induced on the surface of pathogen-infected cells. We found that infection by cytomegalovirus (CMV) resulted in substantial increases in MIC on cultured fibroblast and endothelial cells and was associated with induced MIC expression in interstitial pneumonia. MIC engagement of NKG2D potently augmented T cell antigen receptor (TCR)-dependent cytolytic and cytokine responses by CMV-specific CD28- CD8alphabeta T cells. This function overcame viral interference with MHC class I antigen presentation. Combined triggering of TCR-CD3 complexes and NKG2D induced interleukin 2 production and T cell proliferation. Thus NKG2D functioned as a costimulatory receptor that can substitute for CD28.

Objective: To examine the relation between breech delivery and cerebral palsy, considering the influence of intrauterine growth, low Apgar score at birth, and mode of delivery., Design: Register-based, case-control study., Population: A cohort of infants with cerebral palsy born between 1979 and 1986 in East Denmark, identified by linkage of the cerebral palsy register with the national birth register. Discharge letters from births of breech infants with cerebral palsy were reviewed., Main Outcome Measures: Presentation, mode of delivery, gestational age, birthweight, Apgar score, type of cerebral palsy, severity of handicap., Results: Breech presentation at term was associated with a borderline significantly higher risk of cerebral palsy than vertex presentation (OR 1.56; 95% CI 0.9-2.4). Breech presentation infants more often had a lower Apgar score (< 7 at 5 minutes) and were smaller for gestational age (SGA < 2 SD) than were those with vertex presentation; infants with a low Apgar score, or who were small for gestational age, had a higher risk of cerebral palsy. After stratification by being small for gestational age the risk of cerebral palsy was not related to presentation. There were no differences between breech and vertex infants with cerebral palsy in terms of low Apgar score, being small for gestational age, mode of delivery, and severity of the handicap. Breech presentation infants were more often classified as diplegic (77.8% versus 42.3% in cephalic infants)., Conclusion: The risk of cerebral palsy among term breech presentation infants does not seem to be related to mode of delivery, but is more likely linked to a higher rate of being small for gestational age in breech infants.

Background: Preterm infants are at 8 times higher risk than term infants for pre- and perinatal brain damage, resulting in cerebral palsy. In this paper we have analysed the influence of prenatal and birth-related risk factors on cerebral palsy in preterm infants., Methods: In a register-based study, 175 preterm singleton infants with cerebral palsy, born in 1982-86, were compared with 687 controls matched by gestational age and year of birth., Results: Statistically significant higher rates in cases were found in parity > or = 3 (22% vs. 16%, p < 0.05), Cesarean section (67% vs. 56%, p < 0.01), and low Apgar scores at 1 minute (45% vs. 36%, p < 0.05). By multivariate analyses, two variables remained statistically significant: parity > or = 3 (adjusted OR = 1.53 (95% CI 1.00-2.34), p < 0.05) and Cesarean section (adjusted OR = 1.57 (95% CI 1.07-2.32), p < 0.05)., Conclusions: Pregnancy complications preceding preterm birth did not imply a higher risk of cerebral palsy. Delivery by Cesarean section was a prognostic factor for developing cerebral palsy, and the predictive value of Apgar scores was highly limited.

We have studied the expression of the 15-lipoxygenase gene in various permanent mammalian cell lines in response to interleukins-4 and -13, and found that none of the cell lines tested expressed 5-, 12- or 15-lipoxygenase when cultured under standard conditions. However, when the lung carcinoma cell line A549 was maintained in the presence of either interleukin for 24 h or more, we observed a major induction of 15-lipoxygenase, as indicated by quantification of 15-lipoxygenase mRNA, by immunohistochemistry, by immunoblot analysis and by enzyme activity assays. This effect was 15-lipoxygenases-specific, since expression of 5- and 12-lipoxygenases remained undetectable. The time course of interleukin-4 treatment indicated maximal accumulation of both 15-lipoxygenase mRNA and functional protein after 48 h. Binding studies revealed that A549 cells express about 2100 high-affinity interleukin-4 binding sites per cell. The interleukin-4 mutant Y124D, which is capable of binding to the interleukin-4 receptor but is unable to trigger receptor activation, counteracted the effect of the wild-type cytokine. Other cell lines, including several epithelial cells and various monocytic cell lines expressing comparable numbers of interleukin-4 receptors, did not express 15-lipoxygenase when stimulated with interleukin-4. These data indicate that A549 cells selectively express 15-lipoxygenase when stimulated with interleukins-4 and -13. The activation of the interleukin-4/13 receptor(s) appears to be mandatory, but not sufficient, for 15-lipoxygenase gene expression.

The colon carcinoma cell line HT-29 was used to explore the potential of interleukin-4 (IL-4) and tumor necrosis factor alpha (TNF-alpha) to modify integrin expression and adhesive functions of tumor cells in vitro and to examine corresponding metastatic effects in vivo. Preincubation of HT-29 cells with 100 U/ml of IL-4 for 48 h downregulated the surface expression of the integrin subunits alpha 2, alpha 3, beta 1 and beta 4 after 48 h, whereas the alpha 1 subunit was upregulated. In contrast, 100 U/ml to TNF-alpha selectively upmodulated the expression of alpha v. Attachment to fibronectin of cells treated with IL-4 increased twofold (63.5% vs 32.4%). Adhesion to fibronectin (54.0% vs 32.4%) and vitronectin (37.9% vs 16.4%) was elevated in the case of TNF-alpha stimulation. Using an experimental metastasis model, HT-29 cells showed a significant reduction of their lung-colonizing potential in nude mice when preincubated with IL-4 for 48 h before intravenous injection. The decrease also observed for TNF-alpha-treated cells was less pronounced. The data indicate that the cytokines IL-4 and TNF-alpha can act as direct regulators of adhesive mechanisms of tumor cells bearing adequate receptors, thus influencing lung-colony formation.

We have previously reported on the stimulation of clonal growth of a glioblastoma cell line by rhSCF (Berdel et al., Cancer Res 1992, 52, 3498-3502). Within an extensive screening programme of haematopoietic growth factor activity on malignant cells, the effects of rhSCF were further tested on the growth of 29 different human cell lines derived from a wide range of solid tumours, among them six lung cancers and five melanomas. RhSCF (0, 1, 10, 100 ng/ml) was tested in a human tumour cloning assay (HTCA) which reliably detects growth modulation of tumour cells by cytokines. Additionally, a tritiated thymidine uptake test was used. Growth of 27 of the 29 cell lines tested was not affected by rhSCF. However, growth of the small cell lung cancer (SCLC) cell line HTB 120 was slightly stimulated (1.5 fold that of controls), and that of the melanoma cell line MeWo was stimulated up to 1.3-fold. This activity was eliminated dose-dependently by the tyrosine kinase inhibitor, genistein. We further analysed the cell lines for expression of the proto-oncogene C-KIT and its ligand SCF. All melanoma and lung cancer cell lines expressed SCF as assessed at the mRNA level. Northern blotting also revealed clear C-KIT mRNA expression in three melanoma (HAS, MeWo, SK-MEL-28), one NSCLC (HTB 53), and four SCLC cell lines (HTB 119, HTB 120, HTB 171, HTB 175). Furthermore, C-KIT protein expression was detected by flow cytometric analysis on the cell surface of MeWo, HTB 119 and HTB 120 cells. Our data indicate that SCF can be operative in growth modulation of non-haematopoietic malignant cells, especially SCLC and melanoma. However, our extensive screening of SCF/tumour cell interaction shows that this interaction is rare and makes potential hazards, such as tumour stimulation upon clinical use of rhSCF in conjunction with chemotherapy in cancer patients, unlikely for the majority of other tumour histologies.

Interleukin-4 (IL-4) plays an important role in activating the immune system against malignant cells. The human interleukin-4 receptor (hIL-4R) is not only expressed by hematopoietic cells but also on a large number of tissue specimens which include colon, breast and lung carcinomas. In this study we report that rhIL-4 has an antiproliferative effect on 2 out of 3 non-small cell lung carcinoma (NSCLC) cell lines in vitro as measured by human tumor cloning assays (HTCA). In comparison, rhIL-4 had no effect on the growth of small cell lung carcinoma cell lines (SCLC) in vitro. The response towards the cytokine is correlated with expression of at least 1500 high affinity receptors/cell for hIL-4 on the responsive cell lines. Xenotransplanting the human lung tumor cell lines into nude mice followed by 12 days of systemic treatment of the mice with rhIL-4 revealed a significant growth retardation of the IL-4R positive NSCLC cell lines when compared with the controls, whereas the growth of the IL-4R negative SCLC cell lines was unaffected also in vivo. Studies of possible mechanisms involved in the antiproliferative effect of rhIL-4 showed that rhIL-4 does not induce apoptosis or modulation of the transcription factor c myc in the responsive NSCLC cell lines. Additionally, the expression of the epidermal growth factor receptor (EGFR), which is discussed as mediating autocrine/paracrine growth stimulation of NSCLC, is unaffected by rhIL-4. However, we have observed that rhIL-4 inhibited G1-S-phase cell cycle progression. We conclude that rhIL-4 has an antiproliferative effect on the growth of some NSCLC in vitro and in vivo. The mechanisms involved remain to be further elucidated.

Interleukin 4, a T cell-derived 20-kDa glycoprotein, plays an important role in regulating the immune response of B cells, T cells, and macrophages against infections and malignant cells. For this reason recombinant human interleukin 4 (rhIL-4) has entered early clinical trials in cancer patients. In the present study we report that rhIL-4 has an antiproliferative effect on five of nine cell lines derived from human colon tumors, head and neck tumors, and glioblastomas as measured by a decrease of colony formation in human tumor cloning assays. All of the cell lines with in vitro responsiveness express at least 100 high-affinity receptors for human interleukin 4 per cell on their cell surface, whereas the nonresponsive tumor cell lines lack expression of high-affinity receptors for human interleukin 4 on their cell surface. In the next series of experiments we have xenotransplanted some of the responsive cell lines into athymic nude mice. Subsequently, the animals were treated s.c. twice daily with 0.5 mg/m2 rhIL-4 or control vehicle for at least 12 days. There was a clear growth inhibition of these xenotransplanted tumors in the mice treated with rhIL-4. Histology of the tumors in both groups revealed no marked infiltration with murine hematopoietic and lymphocytic cells as evaluated by staining with a rat anti-mouse CD45 antibody. We conclude that rhIL-4 has a direct therapeutic activity on the growth of some human epithelial and nonepithelial tumor cell lines which, along with its regulatory function on hematolymphopoietic cells, makes this cytokine an interesting candidate for experimental tumor therapy.

The aim of The Cerebral Palsy Register is to follow the development of cerebral palsy in East Denmark as reported from the children's departments within the area. The prevalence among Danish children born in 1971-1982 is reported. Postnatal cases have been excluded. The live-birth prevalence has increased from 1.7 per 1000 in 1971-1974 to 2.3 per 1000 in 1979-1982. An increased proportion with birthweight under 2500 g and neuroimpairment was found in the latest birth year period. A re-evaluation of diagnostic information confirmed the increasing trend of the prevalence. Preterm births were dominated by perinatal etiology, while term births equally often had a prenatal etiology. It is worrying that the prevalence is rising and that the children have more disabilities. The reason for the increasing trend is most probably due to the decreasing neonatal mortality.

Oxidative phosphorylation (OXPHOS) diseases can be caused by mutations in nuclear genes or mitochondrial DNA (mtDNA) genes. mtDNA mutations include complex mtDNA rearrangements in which large segments of mtDNA are duplicated or deleted and point mutations in which single nucleotide substitutions occur within transfer RNA (tRNA) genes, ribosomal RNA (rRNA) genes, or mitochondrial genes encoding OXPHOS polypeptides. Although over 30 pathogenic mtDNA point mutations and over 60 different types of mtDNA deletions are known (Shoffner and Wallace, 1995; Wallace et al., 1994), only a subset of these mutations are associated with cerebellar ataxia. This review focuses on the clinical, biochemical, and genetic features of OXPHOS diseases caused by mtDNA mutations in which ataxia is a common manifestation.

In this study we report that recombinant human (rh) interleukin-1alpha (IL-1alpha) has direct and dose-dependent growth-modulating effects on human tumor cell lines in vitro as measured by a human tumor cloning assay (HTCA). Colony formation of the melanoma cell line A 375 was inhibited by rhIL-1alpha, whereas colony formation of the glioblastoma cell line HTB 14 was enhanced by this cytokine. Both growth-modulating effects were dose-dependent, however with some saturation. Subsequently, we have tested the activity of recombinant human IL-1 receptor antagonist (rhIL-1ra) on the tumor growth modulation by rhIL-1alpha. Tumor cells were incubated with increasing concentrations of rhIL-1ra and then added to the cultures containing rHIL-1alpha. Concentrations of rhIL-1ra were chosen to achieve a range between 0.01 and 100 ng/ml which includes a 1000-fold molar excess over IL-1alpha. The receptor antagonist was able to block both the inhibition and the stimulation of clonal growth of the respective tumor cell line by rhIL-1alpha. Furthermore, there was a direct dose dependent relationship revealing higher IL-1 antagonism of rhIL-1ra at higher concentrations with maximum efficacy at 1000-molar excess concentrations over IL-1alpha. In addition, rhIL-1ra alone did not reveal major modulation of the growth of A 375, but significantly decreased colony formation of HTB 14. We conclude that rhIL-1ra can counteract modulation of clonal growth of human tumor cells by IL-1alpha in vitro. Since our report provides first evidence that the stimulation of clonal tumor cell growth by IL-1alpha can be blocked by rhIL-1ra, this member of the IL-1 cytokine network should be further studied as a possible candidate for experimental cancer treatment.

Haematopoietic growth factors such as human granulocyte colony-stimulating factor (G-CSF) have been shown to stimulate in vitro growth of some human solid tumour cell lines. Among these is the human colorectal adenocarcinoma cell line HTB 38. The in vivo relevance of this finding was tested by xenografting HTB 38 cells intracutaneously into athymic nu/nu BALB/c mice. Recombinant human (rh) G-CSF was administered as a subcutaneous bolus twice daily from day 1 to 14 after tumour transplantation at a dose level of 312 mug/kg/day. Serum levels of rhG-CSF were within the range required for the in vitro effect. However, the cytokine caused no significant growth modulation of the tumour in vivo.

Cytokines play an important role in activating the immune system against malignant cells. One of these cytokines, interleukin-4 (IL-4) has entered clinical phase I trials because of its immunoregulatory potency. In the present study we report that recombinant human (rh) IL-4 has major direct antiproliferative effects on one human lung cancer cell line (CCL 185) in vitro as measured by a human tumor cloning assay (HTCA), tritiated thymidine uptake, and counting cell numbers and marginal activity in a second cell line (HTB 56) in the HTCA. This activity could be abolished by neutralizing antibody against rhIL-4. The biological response of the tumor cells to the cytokine is correlated with expression of receptors for human IL-4 on both the mRNA level and the protein level. The responsive cell line, CCL 185, secretes IL-6 after being incubated with rhIL-4. On the other hand, neutralizing antibodies against IL-6 showed no influence on the growth modulatory efficacy of rhIL-4 in this cell line. Furthermore, CCL 185 does not show detectable production of IL-1, tumor necrosis factor alpha or interferon gamma after incubation with rhIL-4. Thus, the response to rhIL-4 is not mediated through autocrine production of these cytokines triggered by rhIL-4. In a next series of experiments some of the cell lines were xenotransplanted to BALB/c nu/nu mice. Subsequently, the mice were treated for 12 days with two doses of 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control in two of the non-small cell lung cancer cell lines being responsive in vitro (CCL 185, HTB 56). Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. In contrast, no tumor growth inhibition was found in the small cell lung cancer cell lines (HTB 119, HTB 120) being nonresponsive in vitro. We conclude that rhIL-4 has direct antiproliferative effects on the growth of some human non-small cell lung cancer cell lines in vitro and in vivo, which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment.

In the present study we report that recombinant human (rh) interleukin 4 (IL-4) has direct, dose-dependent antiproliferative effects on the human lung cancer cell line CCL 185 in vitro as measured by a human tumor cloning assay (HTCA). The biological response of the tumor cells to rhIL-4 is correlated with expression of specific binding sites for human (h) IL-4 on the cell surface. Furthermore, we have xenotransplanted CCL 185 into BALB/c nu/nu mice. Subsequently, the mice were treated for 17 days with twice 0.5 mg/m2 rhIL-4 or control vehicle subcutaneously per day. Treatment with rhIL-4 yielded a significant inhibition of tumor growth versus control. Histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of IL-4. We conclude that rhIL-4 has direct antiproliferative effects on the growth of a human lung tumor cell line in vitro and in vivo which together with its regulatory effects on various effector cell populations makes this cytokine a possible candidate for experimental cancer treatment.

The kinetics of ATP-induced Ca2+ uptake by vesicular dispersions of sarcoplasmic reticulum were determined with a time resolution of about 10 ms, depending on the temperature. Ca2+ uptake was initiated by the addition of ATP through the flash photolysis of P3-1-(2-nitrophenyl)-ethyl adenosine 5'-triphosphate utilizing a frequency-doubled ruby laser and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 to 26 degrees C. The Ca2+ ionophore A23187 was used to distinguish those features of the Ca2+ uptake kinetics associated with the formation of a transmembrane Ca2+ gradient. The acid-stable phosphorylated enzyme intermediate, E approximately P, was determined independently with a quenched-flow technique. Ca2+ uptake is characterized by at least two phases, a fast initial phase and a slow phase. The fast phase exhibits pseudo-first-order kinetics with a specific rate constant of 64 +/- 10 s-1 at 23-26 degrees C, an activation energy of 16 +/- 1 kcal mol-1, and a delta S* of approximately 5 cal deg-1 mol-1, is insensitive to the presence of a Ca2+ ionophore, and occurs simultaneously with the formation of the phosphorylated enzyme, E approximately P, with a stoichiometry of approximately 2 mol of Ca2+/mol of phosphorylated enzyme intermediate. The slow phase also exhibits pseudo-first-order kinetics with a specific rate constant of 0.60 +/- 0.09 s-1 at 25-26 degrees C, an activation energy of 22 +/- 1 kcal mol-1, and a delta S* of approximately 16 cal deg-1 mol-1, is inhibited by the presence of a Ca2+ ionophore, and has a stoichiometry of approximately 2 mol of Ca2+/mol of ATP hydrolyzed.

Knowledge of the functional properties of the protein in oriented multilayers, in addition to vesicular dispersions, of membranes such as the isolated sarcoplasmic reticulum (SR), extends the variety of techniques that can be effectively used in studies of the membrane protein's structure or structural changes associated with its function. One technique requiring the use of oriented multilayers to provide more direct time-averaged and time-resolved structural investigations of the SR membrane is x-ray diffraction. Therefore, the kinetics of ATP-induced calcium uptake by isolated SR vesicles in dispersions and hydrated, oriented multilayers were compared. Ca2+ uptake was necessarily initiated by the addition of ATP through flash photolysis of caged ATP, P3-1-(2-nitro)phenylethyl adenosine 5'-triphosphate, with either a frequency-doubled ruby laser or a 200 W Hg arc lamp, and measured with two different detector systems that followed the absorbance changes of the metallochromic indicator arsenazo III, which is sensitive to changes in the extravesicular [Ca2+]. The temperature range investigated was -2 degrees to 26 degrees C. The Ca2+ uptake kinetics of SR membranes in both the vesicular dispersions and oriented multilayers consist of at least two phases, an initial fast phase and a subsequent slow phase. The fast phase, generally believed to be associated with the formation of the phosphorylated enzyme, E approximately P, is kinetically comparable in both SR dispersions and multilayers. The slow phase mathematically follows first-order kinetics with specific rate constants of approximately 0.6 s-1 and approximately 1.2 s-1 for the dispersions at 26 degrees C and multilayers at 21 degrees C, respectively, with the given experimental conditions. The slow phase, generally believed to be associated with the translocation of Ca+2, across the membrane profile, appears to be the same process in SR dispersions and multilayers through their virtually identical rate constants and their identical activation energies of 22 +/-1 kcal mol -1. The stoichiometry of ~2 mol Ca2+/mol ATP hydrolyzed was measured in dispersions for the slow phase of Ca2+ uptake. Photolysis of caged ATP with the lamp and the laser provides comparable results for the Ca2+ uptake kinetics in SR dispersions and multilayers. Laser flash photolysis, however, has the advantages of optimal time resolution and effective synchronization of the ensemble of Ca2+-ATPase molecules in the ATP initiated Ca2+ transport process.