Ensuring continuous TB treatment across Asian borders.

BACKGROUND: Mid-treatment cross-border migration of patients with TB increases the risk of treatment interruption. OBJECTIVE: To establish a cross-border referral process for patients with TB in Japan, and enhance their access to health facilities and treatment outcomes. DESIGN: This prospective cohort study describes and assesses the process of foreign-born patients with TB who returned to their home countries during treatment, focusing on their access to healthcare facilities and treatment outcomes. RESULTS: We enrolled 135 foreign-born patients with TB, and confirmed that 112 (83.0%) were referred to and accessed healthcare facilities after returning to their home countries. Of 102 patients due to complete treatment as of July 2023, 87 (85.3%) completed their treatment. We did not identify significant differences in the treatment success rate among patient characteristics, except between the patients with confirmed access to a healthcare facility and those without (P < 0.001). We confirmed that 49/87 (56.3%) patients had completed treatment with official data. CONCLUSION: The access and treatment success rates of the cross-bordered patients with TB from Japan were >80%; however, we should further improve this proportion by confirming the treatment outcomes with official data.

CONTEXTE: La migration transfrontalière en milieu de traitement des patients atteints de TB augmente le risque d'interruption du traitement. OBJECTIF: Etablir un processus d'orientation transfrontalière pour les patients atteints de TB au Japon et à améliorer leur accès aux établissements de santé et les résultats de leur traitement. CONCEPTION: Cette étude de cohorte prospective décrit et évalue le processus des patients atteints de TB et nés à l'étranger qui sont retournés dans leur pays d'origine pendant le traitement, en se concentrant sur leur accès aux établissements de santé et sur les résultats du traitement. RÉSULTATS: Nous avons recruté 135 patients atteints de TB et nés à l'étranger et confirmé que 112 (83,0%) ont été orientés vers des établissements de santé et y ont accédé après leur retour dans leur pays d'origine. Des 102 patients qui devaient terminer leur traitement en juillet 2023, 87 (85,3%) l'ont terminé. Nous n'avons pas identifié de différences significatives dans le taux de réussite du traitement en fonction des caractéristiques des patients, sauf entre les patients ayant un accès confirmé à un établissement de santé et ceux qui n'en ont pas (P < 0,001). Nous avons confirmé que 49 (56,3%) des 87 patients avaient terminé leur traitement à l'aide des données officielles. CONCLUSION: Les taux d'accès et de réussite du traitement des patients transfrontaliers atteints de TB en provenance du Japon étaient >85% ; cependant, nous devrions encore améliorer cette proportion en confirmant les résultats du traitement à l'aide de données officielles.

Use of a 23-gauge continuous spinal catheter for labor analgesia: a case series.

Seven women received labor analgesia with 0.125% bupivacaine and fentanyl 2 µg/mL delivered through a new generation of over-the-needle 23-gauge spinal catheters. The first patient was managed with intermittent bolus injections but inadequate pain control prompted a conversion to a continuous infusion for subsequent patients. One patient developed a postdural puncture headache following catheterization for 5 h, but there were no headaches in those who had an indwelling catheter for 8h or longer. In one patient the catheter was also used to provide anesthesia for cesarean delivery with 0.5% bupivacaine and fentanyl 20 µg. The largest drop in mean arterial blood pressure was 34% which occurred during the intermittent dosing period in the first patient. The mean blood pressure decrease was <25% in the remaining patients. One patient with labor lasting over 17 h developed pain and paresthesia that resolved in 24 h without treatment. Two patients had motor block that necessitated a temporary reduction in rate or discontinuation of the infusion. The continuous spinal catheter appeared to be acceptable to patients but the optimal choice of drugs, concentration, and mode of administration remains to be determined.

Irradiation-induced rescue of thymocyte differentiation and V(D)J recombination in mice lacking the catalytic subunit of DNA-dependent protein kinase.

Scid mice express a truncated form of the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) and are unable to properly rearrange their Ig and TCR genes, resulting in a severe combined immunodeficiency that is characterized by arrested differentiation of B and T lymphocytes. Treatment of scid mice with low doses of gamma irradiation rescues rearrangements at several TCR loci and promotes limited thymocyte differentiation. The machinery responsible for sensing DNA damage and the mechanism by which irradiation compensates for the scid defect in TCR recombination remain unknown. Because DNA-PKcs is present in scid thymocytes, it may mediate some or all of the irradiation effects. To test this hypothesis, we examined the effects of irradiation on DNA-PKcs-deficient (slip) mice. Our data provide the first evidence that DNA-PKcs is not required for limited rescue of thymocyte differentiation or TCR rearrangements.

Activation of androgen receptor in epidermal growth factor modulation of fetal mouse sexual differentiation.

Previous studies from this laboratory indicated a role for epidermal growth factor (EGF) in androgen-dependent male sexual differentiation. The mechanism by which EGF modulates male sexual differentiation has not been determined and investigation has been made to assess the role for androgen receptor (AR) in mediating the EGF-induced effect. We report that EGF, like androgen, stabilized the Wolffian duct in the 13-day female specimen, grown in organ culture. Anti-AR, flutamide and cyproterone acetate blocked the Wolffian duct-stabilizing effect of EGF. EGF also induced cell proliferation of the fetal reproductive tract in a dose-dependent manner and a combination of physiological dosages of EGF and androgen-induced cell proliferation synergistically, suggesting an interactive effect of these two drugs. Cyproterone acetate blocked both EGF-induced normal cell proliferation and the synergistic cell proliferation induced by combination of EGF and androgen suggesting a role of AR in the effects of EGF. The role of AR was further assessed by determining the effect of EGF on AR binding directly. It was shown that EGF stimulated androgen binding activity of the male fetal reproductive tract cells significantly by increasing the number of binding sites by 3-fold with slight decrease in binding affinity. Thus, it appears that AR plays a role in mediating EGF-modulation of sexual differentiation.

The role of growth hormone in fetal mouse reproductive tract differentiation.

Although GH plays a key role in postnatal growth and differentiation, its role in fetal differentiation is not clear at the present. The aim of the present study was to investigate whether GH plays a role in fetal sexual differentiation, and we used in vitro organ culture assay of sexual differentiation to determine this. The results showed that anti-rGH antibody blocked Wolffian duct differentiation specifically in the presence of fetal testes. Exogenous GH supplemented in the above experiment reversed the blocking effect of anti-GH. Among the other related products, insulin-like growth factor I was highly effective in reversing the anti-GH effect, insulin-like growth factor II was partially effective, but PRL was unable to reverse the anti-GH effect. GH itself was found to produce some masculinizing effect, as demonstrated by its ability to stabilize the Wolffian duct in female fetuses. The role of GH was further demonstrated by the observation that GH-immunoreactive material of the size of authentic GH was detected in the 18-day fetal reproductive tract, and the concentration of this material increased in response to progression of sexual differentiation. Determination of androgen-binding activity using Scatchard analysis on the cells isolated from the 18-day fetal reproductive tract indicated that androgen-binding activity increased after GH treatment of the cells. Thus, it may be concluded that GH influences male sexual differentiation and alters the androgen-binding activity of the fetal reproductive tract.

Amino acid residues that influence the binding of manganese or calcium to photosystem II. 1. The lumenal interhelical domains of the D1 polypeptide.

To identify amino acid residues that ligate the manganese and calcium ions of photosystem II or that are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate, histidine, and tyrosine residues in the lumenal interhelical domains of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R. J. (1994) Biochemistry 33, 6137-6149]. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that Asp-59 and Asp-61 may ligate calcium, that Asp-59, Asp-61, Glu-65, and His-92 influence the properties of the manganese cluster without significantly affecting its stability or ability to assemble, that Glu-189 plays an important structural role in maintaining the catalytic efficiency of the Mn cluster and partly influences the cluster's stability or ability to assemble, that His-92 and Glu-189 influence the binding of calcium, and that His-190 strongly influences the redox properties of the secondary electron donor, YZox, and either ligates manganese or serves as a crucial base or hydrogen bond donor. In addition, we conclude that Asp-170 may ligate manganese, but that its replacement with Val, Leu, or Ile causes structural perturbations that partly compensate for the loss of the carboxylate moiety.

Amino acid residues that influence the binding of manganese or calcium to photosystem II. 2. The carboxy-terminal domain of the D1 polypeptide.

To identify amino acid residues that ligate the manganese and calcium ions of photosystem II or are otherwise crucial to water oxidation, site-directed mutations were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803 at all conserved carboxylate and histidine residues in the carboxy-terminal domain of the D1 polypeptide. Mutants with impaired photoautotrophic growth or oxygen evolution were characterized in vivo by measuring changes in the yield of variable chlorophyll a fluorescence after a saturating flash or brief illumination given in the presence of an electron-transfer inhibitor or following each in a series of saturating flashes given in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R.J. (1994) Biochemistry 33, 6137-6149]. Mutants were also characterized after propagation in media having other cations substituted for calcium. We conclude that His-332 Glu-333, His-337, and Asp-342 influence the assembly and/or stability of the manganese cluster, that His-332, Glu-333, and His-337 may ligate manganese, that Asp-342 may ligate manganese, calcium, or both, that Glu-333 and Asp-342 may play important structural roles, and that His-332, Glu-333, and His-337 influence the binding of calcium, although Glu-333 is unlikely to ligate Ca2+ directly. Several His-332, Glu-333, His-337, and Asp-342 mutants were very light sensitive, possibly because toxic activated oxygen species were released from altered or partly assembled manganese clusters. Finally, mutations at Asp-342 do not prevent posttranslational cleavage of the carboxy-terminal extension of the D1 polypeptide's precursor form in vivo.

A difference infrared study of hydrogen bonding to the Z. tyrosyl radical of photosystem II.

Photosystem II, the photosynthetic water oxidizing complex, contains two well characterized redox active tyrosines, D and Z. D forms a stable radical of unknown function. Z is an electron carrier between the primary chlorophyll donor and the manganese catalytic site. The vibrational difference spectra associated with the oxidation of tyrosines Z and D have been obtained through the use of infrared spectroscopy (MacDonald, G. M., Bixby, K.A., and Barry, B.A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11024-11028). Here, we examine the effect of deuterium exchange on these vibrational difference spectra. While the putative C-O vibration of stable tyrosine radical D. downshifts in 2H2O, the putative C-O vibration of tyrosine radical Z. does not. This result is consistent with the existence of a hydrogen bond to the phenol oxygen of the D. radical; we conclude that a hydrogen bond is not formed to the Z. radical. In an effort to identify the amino acid residue that is the proton acceptor for Z, we have performed global 15N labeling. While significant 15N shifts are observed in the vibrational difference spectrum, substitution of a glutamine for a histidine that is predicted to lie in the environment of tyrosine Z has little or no effect on the difference infrared spectrum. There is also no significant change in the yield or lineshape of the Z. EPR signal under continuous illumination in this mutant. Our results are inconsistent with the possibility that this residue, histidine 190 of the D1 polypeptide, acts as the sole proton acceptor for tyrosine Z.

Site-directed photosystem II mutants with perturbed oxygen-evolving properties. 1. Instability or inefficient assembly of the manganese cluster in vivo.

Several site-directed photosystem II mutants with substitutions at Asp-170 of the D1 polypeptide were characterized by noninvasive methods in vivo. In several mutants, including some that evolve oxygen, a significant fraction of photosystem II reaction centers are shown to lack photooxidizable Mn ions. In this fraction of reaction centers, either the high-affinity site from which Mn ions rapidly reduce the oxidized secondary electron donor, YZ+, is devoid of Mn ions or the Mn ion(s) bound at this site are unable to reduce YZ+. It is concluded that the Mn clusters in these mutants are unstable or are assembled inefficiently in vivo. Mutants were constructed in the unicellular cyanobacterium Synechocystis sp. PCC 6803. The in vivo characterization procedures employed in this study involved measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination given in the presence of the electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, or following each of a series of saturating flashes given in the absence of this inhibitor. These procedures are easily applied to mutants that evolve little or no oxygen, facilitate the characterization of mutants with labile oxygen-evolving complexes, permit photosystem II isolation efforts to be concentrated on mutants having the stablest Mn clusters, and guide systematic spectroscopic studies of isolated photosystem II particles to mutants of particular interest.

Site-directed photosystem II mutants with perturbed oxygen-evolving properties. 2. Increased binding or photooxidation of manganese in the absence of the extrinsic 33-kDa polypeptide in vivo.

Several site-directed photosystem II mutants with substitutions at Asp-170 or in the carboxyterminal region of the D1 polypeptide were characterized in vivo in the absence of the extrinsic 33-kDa polypeptide. Site-directed mutations were constructed in the cyanobacterium Synechocystis sp. PCC 6803. The 33-kDa polypeptide was removed by insertional inactivation of the Synechocystis psbO gene. Mutants were characterized by measuring changes in the yield of variable chlorophyll a fluorescence following a saturating flash or brief illumination in the presence of an electron-transfer inhibitor or following each of a series of saturating flashes in the absence of inhibitor [Chu, H.-A., Nguyen, A. P., & Debus, R. J. (1994) Biochemistry (preceding paper in this issue)]. In the presence of the extrinsic 33-kDa polypeptide, many site-directed mutants contained a significant fraction of photosystem II reaction centers that lacked photooxidizable Mn ions. This fraction decreased dramatically in the absence of the extrinsic 33-kDa polypeptide, even in mutants having a significantly perturbed high-affinity Mn binding site (e.g., in the mutants D170A and D170T). These results show that, in vivo, the extrinsic 33-kDa polypeptide directly or indirectly governs the occupancy of the high-affinity Mn binding site by Mn ions or the ability of bound Mn ions to reduce YZ+.

Removal of stable tyrosine radical D+ affects the structure or redox properties of tyrosine Z in manganese-depleted photosystem II particles from Synechocystis 6803.

Photosystem II contains two redox-active tyrosines, D and Z. To understand the function of the dark stable tyrosine radical, D+, we have characterized two site-directed mutations at the D tyrosine residue in the transformable cyanobacterium, Synechocystis sp. PCC 6803, through the use of purified photosystem II particles (Noren, G. H., Boerner, R. J., and Barry, B. A. (1991) Biochemistry 30, 3943-3950). In manganese-depleted mutant particles, a light-induced EPR signal is observed. This signal contains a stable component, due to a chlorophyll radical, and an unstable component. The lineshape of the unstable, oxidized component, which we call M+, is obtained by subtraction; it has a lineshape different from tyrosine Z+/D+ and a g value of 2.004. Up to one M+ spin per reaction center can be photooxidized. The characteristic light-induced EPR signal ascribed to Z+ is not detected; under the same conditions, Z+ is detected in control preparations. The M+ radical lineshape is similar to the light-induced photosystem II radical identified in a site-directed mutant in the D1 polypeptide (YF161D1) (Noren, G. H., and Barry, B. A. (1992) Biochemistry 31, 3335-3342). Optical measurements on manganese-depleted photosystem II particles from control and D2 mutant preparations show that charge recombination kinetics between Q-A and an oxidized redox-active component are similar, to within a factor of two, in all three preparations. We conclude that lack of the stable tyrosine D+ alters the structure or redox properties of tyrosine Z in manganese-depleted preparations.

Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex.

To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.

Regulation of calbindin-D28K gene expression by 1,25-dihydroxyvitamin D3 is correlated to receptor occupancy.

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3) induces the synthesis of calcium binding protein (calbindin-D28K) and calbindin-D28K-mRNA in the chick intestine. We have examined the relationship between nuclear uptake of 1,25-(OH)2D3, 1,25-(OH)2D3 receptor occupancy levels, and transcription of the calbindin-D28K gene and found all three parameters to be highly correlated. All three events occur rapidly (within 15-30 min) following a single dose of 6.5 nmol of 1,25-(OH)2D3 to vitamin D-deficient chicks, reaching peak values by 1-2 h; by 4 h, values of all three parameters start to decline. Calbindin-D28K-mRNA begins to accumulate by 3-5 h but does not peak until 12 h following hormone administration. The levels of calbindin-D28K start to increase by 5-8 h and do not peak until 48 h after the 1,25-(OH)2D3 dose. These observations suggest that post-transcriptional regulatory mechanisms may be involved. Measurements of basal levels of calbindin-D28K gene transcription show that there is virtually no transcription in the vitamin D-deficient chick intestine, and a 12-fold induction in the intestine of vitamin D-replete chicks. This basal level of transcription in vitamin D-replete chick intestine is not inhibited by cycloheximide pretreatment. These results confirm the thesis that a major component of the mechanism of action of 1,25-(OH)2D3 is functioning as a steroid hormone, effected through the direct action of the seco-steroid-receptor complex on the initiation of transcription of specific genes.

Cyclic regulation of growth hormone gene transcription in vivo and in vitro.

The in vivo thyroid and glucocorticoid hormone regulation of GH gene transcription was compared with that found in cultured GH rat pituitary tumor cells. The GH cell lines have been widely used to study GH gene expression, but their relevance to the in vivo regulation of the gene has not been well established. The in vivo studies described here utilized rats that were both thyroparathyroidectomized and adrenalectomized to remove the organ sources of these hormones. The in vitro studies described utilized GC cells hormonally deinduced in medium lacking the hormones. Continuous administration of glucocorticoid or thyroid hormones to either system induced multiple cycles of GH transcriptional activation and deactivation. These cycles were accompanied by cycles of increasing and decreasing GH messenger RNA. In both systems, a brief transcription cycle occurred within hours of thyroid or glucocorticoid hormone addition, and a second broad occurred between 3 and 11 days later. These cycles were independent of changes in receptor levels. The similarities in the responses found in vivo and in cell culture suggest that the molecular mechanisms regulating expression of the GH gene appropriately function in GC cells, despite their transformed phenotype and prolonged maintenance in culture. Thus, these cell lines appear to be appropriate model systems for studies of thyroid and glucocorticoid hormone action.

Relationship between thyroid and glucocorticoid hormone receptor occupancy, growth hormone gene transcription, and mRNA accumulation.

The temporal relationship between the occupancy of the thyroid hormone (3,5,3'-triiodo-L-thyronine (T3)) and glucocorticoid (dexamethasone) receptors and the rate of growth hormone (GH) gene transcription and mRNA accumulation were investigated. The GC line of cultured rat pituitary tumor cells was used in these studies. The rate of GH gene transcription was measured by elongation of in vivo initiated RNA chains in isolated nuclei using radioactive precursors and hybridization of labeled transcripts to immobilized GH gene sequences. T3 receptor occupancy was directly proportional to the rate of GH gene transcription during the initial phase of hormone induction. These results suggest that a single occupied receptor is sufficient to fully activate the gene and that the unoccupied receptor resides close to the gene regulatory site. A decrease in GH gene transcription rapidly followed the initial increase in activity. This decrease occurred in the absence of new protein synthesis and was not accompanied by a proportional decrease in the level of occupied T3 receptor. An analysis of dexamethasone activation of GH gene transcription found maximum stable binding of occupied receptor to nuclei within 5 min of hormone addition, while transcription of the gene did not become fully activated until 30 to 60 min later. Transcription of the gene then declined to a rate 2 to 3 times that observed before addition of dexamethasone. These results suggest that at least one relatively slow molecular step precedes glucocorticoid hormone-receptor enhancement of gene transcription and that a second molecular event later attenuates the response. The simultaneous addition of both hormones produced two peaks of enhanced transcription, each apparently due to the separate effects of the hormones. GH mRNA levels were closely linked to the rate of transcription of the gene under all induction conditions. Comparison of the rates of decay of GH mRNA in cells cultured in the presence or the absence of hormones suggests that GH mRNA is much more stable when T3 is present. The glucocorticoid responses of the gene were consistently obtained only in the presence of T3. These results and the transcriptional synergism of the two hormones suggest a direct interaction of the occupied receptors at their regulatory sites. The transcriptional effects of glucocorticoids were observed only in about half the experiments performed in the absence of T3. When transcriptional activation did not occur, GH mRNA usually still increased 24 h after hormone treatment was begun, but not early in the induction.(ABSTRACT TRUNCATED AT 400 WORDS)