Intestinal IMINO transporter SIT1 is not expressed in human newborns.

The expression of amino acid transporters in small intestine epithelia of human newborns has not been studied yet. It is further not known whether the maturation of imino acid (proline) transport is delayed as in the kidney proximal tubule. The possibility to obtain small intestinal tissue from patients undergoing surgery for jejunal or ileal atresia during their first days after birth was used to address these questions. As control, adult terminal ileum tissue was sampled during routine endoscopies. Gene expression of luminal imino and amino acid transporter SIT1 (SLC6A20) was approximately threefold lower in newborns versus adults. mRNA levels of all other luminal and basolateral amino acid transporters and accessory proteins tested were similar in newborn mucosa compared with adults. At the protein level, the major luminal neutral amino acid transporter B0AT1 (SLC6A19) and its accessory protein angiotensin-converting enzyme 2 were shown by immunofluorescence to be expressed similarly in newborns and in adults. SIT1 protein was not detectable in the small intestine of human newborns, in contrast to adults. The morphology of newborn intestinal mucosa proximal and distal to the obstruction was generally normal, but a decreased proliferation rate was visualized distally of the atresia by lower levels of the mitosis marker Ki-67. The mRNA level of the 13 tested amino acid transporters and accessory proteins was nonetheless similar, suggesting that the intestinal obstruction and interruption of amniotic fluid passage through the small intestinal lumen did not affect amino acid transporter expression. NEW & NOTEWORTHY System IMINO transporter SIT1 is not expressed in the small intestine of human newborns. This new finding resembles the situation in the proximal kidney tubule leading to iminoglycinuria. Lack of amniotic fluid passage in small intestinal atresia does not affect amino acid transporter expression distal to intestinal occlusion.

High salt exacerbates programmed hypertension in maternal fructose-fed male offspring.

BACKGROUND AND AIMS: Consumption of food and drinks containing high fructose (HF), which is associated with hypertension, is increasing steeply. Moreover, increased salt intake significantly increases hypertension risk. We examined whether maternal HF and postnatal high salt (HS) intake had synergistic effects on blood pressure (BP) elevation in adult offspring and determined the underlying mechanisms. METHODS AND RESULTS: Pregnant Sprague-Dawley rats received regular chow or chow supplemented with 60% fructose during the entire pregnancy and lactation periods. Half of the male offspring received 1% NaCl in drinking water from weaning to 3 months of age. Male offspring were assigned to 4 groups (control, HF, HS, and HF + HS) and were sacrificed at 12 weeks of age. Offspring in HF and HS groups developed hypertension, indicating that HF and HS synergistically increased BP. Postnatal HS intake increased Ace expression and decreased Agtr1b and Mas1 expression in the kidneys. Renal mRNA levels of Ace and Agtr1a were significantly higher in HF + HS group than in control group. Renal levels of Na-K-2Cl cotransporter, type 3 sodium hydrogen exchanger, and Na(+)/Cl(-) cotransporter were higher in HS and HF + HS groups than in control group. CONCLUSION: Postnatal HS intake exacerbated prenatal HF-induced programmed hypertension. HF and HS induced programmed hypertension by differentially inducing renin-angiotensin system and sodium transporters in the kidneys. Better understanding of the effect of the relationship between HF and HS on hypertension development will help prevent hypertension in mothers and children exposed to HF and HS.

Cytokine gene-modulated dendritic cells protect against allergic airway inflammation by inducing IL-10(+)IFN-gamma(+)CD4(+) T cells.

Asthma is characterized by allergen-induced airway inflammation orchestrated by Th2 cells. Dendritic cells (DCs) were found to efficiently prime naive T-helper cells. Thus, modification of DC function may be used as an ideal tool to treat allergic asthma by changing CD4(+) T-cell differentiation or suppressing Th2 development. In this study, we examined whether a DC-based vaccine can be applied to DCs modified with interleukin (IL)-10- and IL-12-expressing adenoviruses to prevent ovalbumin (OVA)-induced asthma in mice. Herein, we show that these modified DCs efficiently moderated the characteristics of asthma, including expressions of OVA-specific antibodies, airway hyperresponsiveness, eosinophilic airway inflammation, and Th2 cytokines production. Additionally, IL-10 and IL-12 gene-modified DCs enhanced the development of both T-helper type 1 (Th1) and IL-10(+)IFN-gamma(+) (interferon-gamma) double-positive T cells in vivo. In vitro-generated OVA-specific IL-10(+)IFN-gamma(+)CD4(+) T cells inhibited the proliferation of naive CD4(+) T cells, and this suppressive effect was a cell contact-dependent mechanism. Furthermore, we showed that combined cytokine-modulated DCs could alleviate established allergic airway inflammation. Taken together, these results suggest that IL-10 and IL-12 gene-modulated DCs are effective in suppressing asthmatic airway inflammation through both immune deviation and immune suppression and are a potential therapeutic approach for asthma.

Accelerated resolution of laser-induced bruising with topical 20% arnica: a rater-blinded randomized controlled trial.

BACKGROUND: Dermatological procedures can result in disfiguring bruises that resolve slowly. OBJECTIVES: To assess the comparative utility of topical formulations in hastening the resolution of skin bruising. METHODS: Healthy volunteers, age range 21-65 years, were enrolled for this double (patient and rater) blinded randomized controlled trial. For each subject, four standard bruises of 7 mm diameter each were created on the bilateral upper inner arms, 5 cm apart, two per arm, using a 595-nm pulsed-dye laser (Vbeam; Candela Corp., Wayland, MA, U.S.A.). Randomization was used to assign one topical agent (5% vitamin K, 1% vitamin K and 0·3% retinol, 20% arnica, or white petrolatum) to exactly one bruise per subject, which was then treated under occlusion twice a day for 2 weeks. A dermatologist not involved with subject assignment rated bruises [visual analogue scale, 0 (least)-10 (most)] in standardized photographs immediately after bruise creation and at week 2. RESULTS: There was significant difference in the change in the rater bruising score associated with the four treatments (anova, P=0·016). Pairwise comparisons indicated that the mean improvement associated with 20% arnica was greater than with white petrolatum (P=0·003), and the improvement with arnica was greater than with the mixture of 1% vitamin K and 0·3% retinol (P=0·01). Improvement with arnica was not greater than with 5% vitamin K cream, however. CONCLUSIONS: Topical 20% arnica ointment may be able to reduce bruising more effectively than placebo and more effectively than low-concentration vitamin K formulations, such as 1% vitamin K with 0·3% retinol.

Apoptotic mechanism of paclitaxel-induced cell death in human head and neck tumor cell lines.

BACKGROUND: Paclitaxel (taxol) is clinically used to treat various human tumors. However, the cellular and molecular mechanism regarding apoptotic effect of paclitaxel on head and neck squamous cell carcinoma (HNSCC) remains elusive. METHODS: The apoptotic effect and the mechanism of paclitaxel on FaDu hypopharyngeal cancer cell line, OEC-M1 gingival cancer cell line, and OC3 betel quid chewing-related buccal cancer cell lines were investigated by morphological observations, cell viability assay, flow cytometry assay and Western blotting methods. RESULTS: Rounded-up cell number increased with membrane blebbing as the treatment of paclitaxel (50-500 nM) increased from 24 to 48 h among these cell lines. In cell viability assay, cell surviving rate significantly decreased from 87 to 27% as the dosage and duration of paclitaxel treatment increased (P < 0.05). Flow-cytometry analysis further demonstrated that 50 nM paclitaxel induced G2/M phase cell arrest among these cell lines within 8 h treatment, and then G2/M phase cell fraction significantly decreased as subG1 phase cell fraction significantly increased after 24 h treatment (P < 0.05), suggesting that cells underwent apoptosis. Furthermore, the activated caspases-8, -9, -3, -6 and poly ADP-ribose polymerase cleavage could all be significantly detected in FaDu, OEC-M1 and OC3 cells (P < 0.05). CONCLUSION: Paclitaxel activated cell cycle arrest and caspase protein expressions to induce apoptosis in HNSCC cell lines.

Non-photochemical quenching in the cells of the carotenogenic chlorophyte Haematococcus lacustris under favorable conditions and under stress.

The microalga Haematococcus lacustris (formerly H. pluvialis) is the richest source of the valuable pigment astaxanthin, accumulated in red aplanospores (haematocysts). In this work, we report on the photoprotective mechanisms in H. lacustris, conveying this microalga its ability to cope with a wide range of adverse conditions, with special emphasis put on non-photochemical quenching (NPQ) of the excited chlorophyll states. We studied the changes in the primary photochemistry of the photosystems (PS) as a function of irradiance and the physiological state. We leveraged the transcriptomic data to gain a deeper insight into possible NPQ mechanisms in this microalga. Peculiar to H. lacustris is a bi-phasic pattern of changes in photoprotection during haematocyst formation. The first phase coincides with a transient rise of photosynthetic activity. Based on transcriptomic data, high NPQ level in the first phase is maintained predominantly by the expression of PsbS and LhcsR proteins. Then, (in mature haematocysts), stress tolerance is achieved by optical shielding by astaxanthin and dramatic reduction of photosynthetic apparatus. In contrast to many microalgae, shielding plays an important role in H. lacistris haematocysts, whereas regulated NPQ is suppressed. Astaxanthin is decoupled from the PS, hence the light energy is not transferred to reaction centers and dissipates as heat. It allows to retain a higher photochemical yield in haematocysts comparing to vegetative cells. The ability of H. lacustris to substitute the "classical" active photoprotective mechanisms such as NPQ with optic shielding and general metabolism quiescence makes this organism a useful model to reveal photoprotection mechanisms.

Do circulating leucocytes and lymphocyte subtypes increase in response to brief exercise in children with and without asthma?

BACKGROUND: Exercise can alter health in children in both beneficial (eg reduced long-term risk of atherosclerosis) and adverse (eg exercise-induced asthma) ways. The mechanisms linking exercise and health are not known, but may rest, partly, on the ability of exercise to increase circulating immune cells. Little is known about the effect of brief exercise, more reflective of naturally occurring patterns of physical activity in children, on immune cell responses. OBJECTIVES: To determine whether (1) a 6-min bout of exercise can increase circulating inflammatory cells in healthy children and (2) the effect of brief exercise is greater in children with a history of asthma. METHODS: Children with mild-moderate persistent asthma and age-matched controls (n = 14 in each group, mean age 13.6 years) performed a 6-min bout of cycle-ergometer exercise. Spirometry was performed at baseline and after exercise. Blood was drawn before and after exercise, leucocytes were quantified and key lymphocyte cell surface markers were assessed by flow cytometry. RESULTS: Exercise decreased spirometry only in children with asthma, but increased (p<0.001) most types of leucocytes (eg lymphocytes (controls, mean (SD) 1210 (208) cells/microl; children with asthma, 1119 (147) cells/microl) and eosinophils (controls, 104 (22) cells/microl; children with asthma, 88 (20) cells/microl)) to the same degree in both groups. Similarly, exercise increased T helper cells (controls, 248 (60) cells/microl; children with asthma, 232 (53) cells/microl) and most other lymphocyte subtypes tested. By contrast, although basophils (16 (5) cells/microl) and CD4+ CD45RO+ RA+ lymphocytes (19 (4) cells/microl) increased in controls, no increase in these cell types was found in children with asthma. CONCLUSIONS: Exercise increased many circulating inflammatory cells in both children with asthma and controls. Circulating inflammatory cells did increase in children with asthma, but not to a greater degree than in controls. In fact, basophils and T helper lymphocyte memory transition cells did not increase in children with asthma, whereas they did increase in controls. Even brief exercise in children and adolescents robustly mobilizes circulating immune cells.

Cast wedging: a systematic review of the present evidence.

PURPOSE: The objective of this systematic review was to summarise the outcome after cast wedging due to loss of angulation in conservative fracture treatment of children's fractures. METHODS: Electronic searches were performed using MEDLINE, PubMed, OVID, CENTRAL and EMBASE without language restrictions. RESULTS: Three studies comprising 316 patients (210 radius, 52 forearm/both bone forearm fractures and 54 tibia fractures) were included in the present analysis. Cast wedge failures occurred in 14 of 316 (4.4%) patients. Three patients (0.9%) needed surgical fixation and 11 patients (3.4%) ended up with a healed deformity. Furthermore, eight of 316 (1.8%) patients needed remanipulation and cast change. CONCLUSION: Cast wedging reflects a reliable treatment option for secondary displaced long-bone paediatric fractures.

Extraordinary features in the Chlamydomonas reinhardtii chloroplast genome: (1). rps2 as part of a large open reading frame; (2). A C. reinhardtii specific repeat sequence.

We have determined the DNA sequence of the 3574-bp chloroplast DNA fragment of Chlamydomonas reinhardtii formed by the overlap of BamHI fragment 3 and EcoRI fragment 5. This sequence encodes most of rps18 and orf570, an unidentified open reading frame that contains a 150 amino acid domain with high homology to the N-terminal part of 30 S ribosomal protein S2 of other chloroplast, cyanobacterial and bacterial genomes. Between these two sequences lies a highly repetitive sequence element of 500 bp, that is composed of multiple direct and inverted repeat sequences that occur in rearranged, but highly conserved form in at least 36 locations in the C. reinhardtii chloroplast genome. Among the conserved repeat sequences in the C. reinhardtii chloroplast genome we identified the borders of the inverted repeats near atpB and rps4. This might indicate that the conserved sequence elements are remainders of gene rearrangements in the chloroplast genome that occurred by relocations of the inverted repeats.

Cell cycle-dependent transcriptional and post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii.

The regulated expression of five chloroplast genes in Chlamydomonas reinhardtii during a 24 hour cell cycle (12 hours light, 12 hours dark) was analyzed. Transcription rates of the genes encoding the two reaction center proteins of Photosystem I (psaA, psaB), the subunits alpha and beta (atpA, atpB) of chloroplast ATP synthase and for chloroplast elongation factor tu (EF-tu) were measured during the cell cycle. All genes are maximally transcribed at the beginning of the light period. Transcription was induced before the onset of illumination by a light-independent mechanism. Transcript abundance of the same genes during the cell cycle was determined by quantification of Northern blots hybridized with gene-specific probes. The atpA, atpB and psaB mRNAs were most abundant in the first 6 hours of the light period and decreased to about 15% of maximum in the dark. The abundance of psaA mRNA showed less variation and was maximal around the middle of the cell cycle. The EF-tu mRNA showed a maximum early in the light period, but decreased to almost undetectable levels in the second half of the light period. Because of the similar transcriptional patterns observed, the differential steady state levels of these chloroplast transcripts appeared to be regulated at the post-transcriptional level.

Synthesis of EF-Tu and distribution of its mRNA between stroma and thylakoids during the cell cycle of Chlamydomonas reinhardii.

In Chlamydomonas reinhardii the elongation factor EF-Tu is encoded in the chloroplast DNA. We identified EF-Tu in the electrophoretic product pattern of chloroplast-made proteins and showed that this protein is only synthesized in the first half of the light period in synchronized cells. The newly synthesized EF-Tu contributed little to the almost invariable content of EF-Tu in chloroplasts during the light period of the cell cycle. However, increasing cell volume and the lack of EF-Tu synthesis in the second half of the light period led to a decrease in the concentration of EF-Tu in chloroplasts. At different times in the vegetative cell cycle, the RNA was extracted from whole chloroplasts and from free and thylakoid-bound chloroplast polysomes. The content of mRNA of EF-Tu in chloroplasts and the distribution between stroma and thylakoids were determined. During the light period, the content of the mRNA for EF-Tu varied in parallel to the rate of EF-Tu synthesis. However, in the dark, some mRNA was present even in the absence of EF-Tu synthesis. Most of the mRNA was bound to thylakoids during the whole cell cycle. This suggests that synthesis of EF-Tu is associated with thylakoid membranes.

Differential inhibition of lipolysis by 2-bromopalmitic acid and 4-bromocrotonic acid in 3T3-L1 adipocytes.

Two inhibitors of fatty acid oxidation, 2-bromopalmitic acid (Br-C16) and 4-bromocrotonic acid (Br-C4) were examined for their effect on lipolysis in 3T3-L1 adipocytes. Both agents inhibited in a dose-dependent manner the rate of oxidation of exogenously added [1-14C]palmitate with similar time-courses, reaching a plateau at 3-9 h. While Br-C16 at 50 microM and 100 microM inhibited palmitate oxidation by approximately 40% and 60%, respectively, pretreatment with both concentrations inhibited lipolysis in washed cells in an almost identical manner. The magnitude of inhibition increased with time of pretreatment. On the other hand, like inhibition of fatty acid oxidation, inhibition of lipolysis by Br-C4 pretreatment was dose-dependent with maximal inhibition reached after 3 h pretreatment. The finding that isoproterenol- and dibutyryl cAMP-stimulated lipolysis were similarly suppressed by either Br-C4 or Br-C16 pretreatment, suggesting that a step distal to cAMP formation was involved. In addition, while the inhibitory effect of Br-C16 was not significantly influenced, the inhibition of lipolysis caused by Br-C4 was attenuated by pretreating cells with crotonic acid, octanoate, or palmitate. The longer chain-length of the fatty acids the cells were exposed, the stronger attenuation of the inhibition caused by Br-C4 was observed. Moreover, whereas pretreatment with Br-C16 was without effect, pretreatment with Br-C4 significantly decreased hormone-sensitive lipase (HSL) activity in cell extracts, albeit to an extent much smaller than its inhibitory effect on lipolysis. In conclusion, these results indicate that irreversible inhibition of lipolysis by Br-C16 or Br-C4 cannot be attributed to their effect on fatty acid oxidation. Some factor capable of modulating HSL activity seems to be involved.

Alterations in physiologic functions and in brain monoamine content in streptozocin-diabetic rats.

In the present study, we used streptozocin (STZ) to induce diabetes in rats and observed alterations in several physiologic functions and in monoamine content of different brain regions. Rats with STZ diabetes displayed a thermoregulatory deficit in the cold. Both the body temperature and metabolic rate of the diabetic animals were reduced at ambient temperatures below 22 degrees C. These diabetic animals had a higher level of the spontaneous pain threshold, but displayed a reduced sensitivity of analgesic responses to morphine injection. In addition, these diabetic animals had a lower level of spontaneous motor activity, but displayed an increased sensitivity of locomotor stimulant responses to amphetamine administration. Biochemical examination revealed that the diabetic animals had a lower serotonin level in both the hypothalamus and the brainstem without changes in the serotonin levels of the corpus striatum. These diabetic animals also had a lower catecholamine level in the hypothalamus, but a higher catecholamine level in the corpus striatum. The alterations in brain monoamine content and in the above-mentioned physiologic parameters were reversed after insulin replacement therapy. The data suggest that alterations in various autonomic, somatosensory, and motor neural functions of untreated STZ-diabetic rats correlated with a reproducible pattern of monoamine content in various brain regions (a pattern that differed from that observed in healthy control rats), and that both the altered neural function and the altered brain monoamine pattern were reversed after insulin therapy.

Peripheral blood lymphocytes from normal individuals can be induced to secrete immunoglobulin G antibodies against self-antigen thyroglobulin in vitro.

Autoantibodies to the self-antigen thyroglobulin (Tg) are usually not found in sera of normal individuals, but are often present in sera of patients with autoimmune thyroid diseases. To determine if the presence of such autoantibodies could be due to the abnormal appearance of self-reactive B cells, which are otherwise absent in normal subjects, or to an alteration in the mechanisms regulating such B cells, peripheral blood lymphocytes (PBL) from normal individuals and patients with autoimmune thyroid diseases were cultured and stimulated in vitro with the polyclonal stimulant pokeweed mitogen (PWM). A modified plaque assay was used to enumerate cells secreting protein A-binding immunoglobulins (Igs) and specific antibodies against Tg. PBL from all individuals tested, including normal subjects (n = 26), could be induced by PWM to produce antibodies against Tg in vitro and these antibodies were of IgG isotypes. PBL from patients with detectable serum anti-Tg had more inducible cells secreting anti-Tg [27,000 +/- 10,700 (+/- SD)/10(6) PBL] than those from patients with autoimmune thyroid diseases, who had no detectable serum anti-Tg (8,000 +/- 5,000), and those from normal individuals (7,200 +/- 4,200). The demonstration of inducible mature (IgG) anti-Tg-producing cells in normal individuals suggests that subclinical autoimmunity against certain self-antigens may be a normal phenomenon in man and that its escalation into clinical autoimmune conditions is prevented through regulation of the specific self-reactive cells.

Biological activity of autoantibodies associated with Graves' dermopathy.

To test the hypothesis that Graves' dermopathy is due to cross-reactivity of thyroid autoantibody(ies) with a cellular target in pretibial skin, we tested the serum and serum immunoglobulin fraction of 20 such patients for their effects on the metabolic activities of cultured thyrocytes (rat FRTL cells), human pretibial skin fibroblasts, and human fibroblasts of other origins. The incorporation of 3H-labeled thymidine, amino acids, and glucosamine into DNA, protein, and glycosaminoglycans, respectively, was measured. TSH and the serum of each of the 20 patients with Graves' dermopathy markedly stimulated the synthesis of DNA, protein, and glycosaminoglycans by FRTL cells, but not by fibroblasts, whereas assays of serum from 38 of 40 patients with Graves' disease without dermopathy did not stimulate these processes in FRTL cells more than normal serum. Stimulatory activity was associated with immunoglobulins. Serum dermopathy-associated antibodies disappeared with the disappearance of the skin lesions. These results suggest that the serum of patients with dermopathy contains antibodies that recognize a component of the TSH receptor different from that recognized by serum of Graves' patients without dermopathy, the former acting in some manner to induce lesions in pretibial skin. The skin target remains unidentified.

Helper and suppressor activities of lymphocyte subsets on antithyroglobulin production in vitro.

We studied the regulatory activities of T cells on specific antithyroglobulin (anti-Tg) and nonspecific immunoglobulin secretion in cultures of peripheral blood lymphocytes (PBL) of five patients with autoimmune thyroid diseases and high levels of serum anti-Tg. PBL were separated into a non-T population, including B-cells and monocytes, and a T-cell population by rosetting with sheep red cells. T-Cells were further separated into T helper (Th) and T suppressor (Ts) subsets by a panning technique using the monoclonal antibodies anti-Leu 3a and anti-Leu 2a, respectively. The three sets of cells, i.e. B, Th, and Ts, from patients and from normal individuals were cocultured in various combinations and stimulated with the polyclonal stimulant pokeweed mitogen. A sensitive plaque assay was used to enumerate cells producing anti-Tg and protein A-binding immunoglobulins. The PBL of both patients and normal individuals had Tg-specific suppressor cells. Ts-cells from patients in syngeneic or allogeneic combinations with B- and Th-cells at a ratio of 1:1:1 suppressed the pokeweed mitogen-induced anti-Tg response to 41 +/- 8% (+/-SE) and 50 +/- 20% of the control value, respectively, while Ts from normal individuals suppressed the response to 7 +/- 3% of the control value. The suppressive effect of the Ts-cells from patients and normal individuals on nonspecific immunoglobulin secretion was similar (reduced to 10-15% of control). Thus, there appeared to be a deficiency in Tg-specific suppressor activity in PBL of patients. On the other hand, Th-cells from patients (syngeneic or allogeneic) cocultured with patient B-cells produced a greater anti-Tg response than Th-cells from normal individuals. The helper activities of Th-cells of patients and normal individuals on nonspecific immunoglobulin secretion were similar. Thus, there appeared to be an increase in Tg-specific helper activity in PBL of patients.

Monoclonal antithyroglobulin antibodies derived from immunizations of mice with human eye muscle and thyroid membranes.

A mouse hybridoma clone secreting an immunoglobulin M monoclonal antibody (K-5-4), which reacted with human thyroglobulin (Tg), was obtained from spleen cells of mice immunized with crude membranes of human eye muscle tissues (Em). Its binding to Tg could be inhibited by another monoclonal anti-Tg (F1-11-1) derived from spleen cells of mice immunized with human thyroid cell membranes, but K-5-4 did not inhibit the binding of F1-11-1 to Tg. This finding suggests that K-5-4 may react with a site on the Tg molecule which is susceptible to conformational changes, such as that induced by binding of another anti-Tg antibody at another site on Tg. K-5-4 reacted with human, mouse, rat, guinea pig, bovine, and porcine Tg. Binding and immunohistological staining experiments failed to detect binding of K-5-4 to Em tissue. The very low frequency of one Tg-reacting hybridoma from 6 X 10(8) spleen cells fused after Em immunization contrasts with the relative ease with which monoclonal anti-Tgs were generated from spleen cells of mice immunized with crude human thyroid membranes. In the latter case, 1 anti-Tg hybridoma was generated for every 100,000 spleen cells fused, and an extensive library of monoclonal anti-Tgs was collected. Some of these antibodies were specific for human Tg only, while others cross-reacted with Tg of other animal species. None had the species reativity pattern of K-5-4. The anti-Tgs were used to affinity purify human Tg directly from supernatant of thyroid homogenate; the purified Tg was, in turn, used to affinity purify human polyclonal but monospecific anti-Tg directly from serum of patients in a simple and rapid procedure. We conclude that the monoclonal anti-Tgs are useful reagents in isolating and purifying Tg and anti-Tg.

Activation of indices of cell-mediated immunity in bipolar mania.

BACKGROUND: Evidence supports that macrophages as well as lymphocytes and their products may be involved in the pathophysiology of psychiatric disorders. Whether patients with bipolar disorder have activation or reduction of immunity during a manic episode remains unclear. METHODS: The purpose of this case-control study was to investigate the lymphocyte proliferation to phytohemagglutinin (PHA), concanavalin A, and pokeweed mitogen, and plasma levels of soluble interleukin-2 receptor (sIL-2R) and sIL-6R in patients with bipolar mania (DSM-III-R). The subjects were 23 physically healthy patients with Young Mania Rating Scale (YMRS) scores > or = 26 as well as aged < or = 45 years and 23 age- and gender-matched normal control subjects. The above immune variables were measured in acute mania and consequent remission (YMRS scores < or = 12) among bipolar patients. RESULTS: The lymphocyte proliferation to PHA and the plasma sIL-2R levels, but not sIL-6R, of bipolar patients were significantly higher in acute mania than in consequent remission. These elevations were not due to differences in medication status. Only in acute mania were the plasma sIL-2R levels of patients significantly higher than control subjects. A positive correlation between the changes of manic severity and plasma sIL-2R levels was observed. Remitted bipolar patients and normal control subjects did not differ in any of these measures. CONCLUSIONS: Cell-mediated immunity activation in bipolar mania was demonstrated and may be through a specifically state-dependent immune response.

Transcription and translation of the chloroplast atpB-gene and assembly of ATP synthase subunit beta.

In vitro transcription and subsequent translation of the cloned Chlamydomonas chloroplast atpB gene was used to study assembly of ATP synthase. Translation in the presence of thylakoids resulted in association of the beta subunit with the membrane. The in vitro synthesized polypeptide bound to the membrane copurified with CF1 on sucrose gradients. This provides more evidence for the self-assembly of CF1.

The C. reinhardtii CF1 with the mutation betaT168S has high ATPase activity.

We have generated the mutation T168S in the beta subunit of the chloroplast ATP synthase complex of Chlamydomonas reinhardtii by site directed mutagenesis and chloroplast transformation. CF1 and the alpha3beta3gamma complex of this mutant strain were isolated and their enzymatic activities were characterized and compared to those of the corresponding wild type complexes. Without activation the mutant CF1 exhibits MgATPase activity with at least 10 times higher rates than the wild type enzyme. The MgATPase activity could be stimulated to some extent by methanol, but less by ethanol and octylglucoside. The alpha3beta3gamma complex had an even higher MgATPase activity, which was only slightly enhanced by ethanol or methanol. The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg2+. Free ADP inhibited neither the mutant nor the wild type ATPase significantly. Azide, which strongly inhibited the ATPase activity of the wild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesting that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast ATPase activity. The mutant cells grew photoautotrophically at a growth rate of about 50%. Similar to the wild type the cells survived on minimal medium in the dark. Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically.