Impact of effluent of Pulp & Paper industry on the flora of river basin at Jaykaypur, Odisha, India and its ecological implications.

The JK Paper industry located at Rayagada discharges biologically untreated effluent more than the permissible limit prescribed by Pollution Control Board, Odisha in to the environment. The industry is seriously polluting the surrounding aquatic and terrestrial environment. No detailed intensive study was carried out by previous workers on this industry earlier. The present study aims at finding out the impact of effluent on the flora at the contaminated site. The chemically treated effluent (TE) contained significant amount of mercury and cadmium. The TE has high BOD, COD, dissolved solids and suspended solids when compared to normal river water at the site of discharge. The TE deteriorated the natural water bodies changing the physico-chemical properties of natural river water. After meeting the river water the TE was diluted after 1 km distance from the meeting point of the river. Crop plants collected from the contaminated site showed higher level of residual Hg and Cd and significant depletion in pigment was observed. Plants collected from both the sides of the treated effluent canal showed significant amount residue mercury and cadmium in the plant leaves. The plants exposed to the TE, showed variation in chlorophyll and Phaeophytin pigment content when compared to their respective control values in all terrestrial plants collected from the contaminated site. In some plant leaves little increment in the pigment level was noted but the values were not significant. The changes observed in the plant pigment might be due to heavy metal accumulation. The presence of residual Hg and Cd in crop plants and plant leaves grazed by grazing animals after absorption, accumulation and enrichment may lead to a possible biological magnification, warrants attention. Proper biological treatment, treatment of effluent by modern methods and removal of heavy metals from the effluent before discharge by the industry is suggested.

An audit and analysis of different causes of defaults in patients receiving radiation for head and neck cancers: A tertiary regional cancer center experience.

CONTEXT: Strict adherence and timely completion of the external beam radiation therapy (EBRT) schedule is an important prognostic factor in the survival of head and neck cancer patients. However, many patients are unable to complete the radiation treatment due to various reasons resulting in a poor outcome. AIMS: This study aims to study the pattern and various possible causes of defaults for possible intervention. SETTINGS AND DESIGN: A retrospective epidemiological analysis. SUBJECTS AND METHODS: Patients receiving EBRT for head and neck cancers with curative intent from January 2015 to December 2015 but did not complete the prescribed treatment were included. Unplanned treatment breaks in the treatment was not taken into consideration. STATISTICAL ANALYSIS USED: SPSS version 21. RESULTS: Out of 458, 92 (20.08%) patients did not complete the EBRT (P = 0.06). Fifty-six out of total 92 patients (60.9%) who defaulted stopped taking treatment within halfway of the treatment (15 fraction) and 12 out of total 92 patients (13%) just at the 22nd/23rd fraction. Defaulter rates in patients from different places are in the range of 12.8% to 33.0% but was statistically not significant (P = 0.224). There was no particular age (P = 0.966), disease site (P = 0.354) preponderance among defaulters. Use of concurrent chemo-radiation in radical or adjuvant settings was also not related to defaults (P = 0.406). CONCLUSIONS: Radiation-induced acute toxicity, socioeconomic status and distance plays minimal role as a cause of patients who stop taking EBRT. There is no particular relation between age, disease site, treatment received before radiotherapy, intent of treatment, and concurrent chemoradiation-induced acute reactions with defaults among patients. Loss of income and work in the poor population during the treatment may be an important possible cause of defaults.

Regulatory T cells and IL-10 as modulators of chikungunya disease outcome: a preliminary study.

Regulatory T (Treg) cells hold centre stage in regulating the immune responses in most viral infections. However, their involvement in chikungunya infection is unexplored. In the current study, the frequencies and functionality of peripheral Treg and T effector (Teff) cells were assessed during different phases of chikungunya by flow cytometry and in-vitro cytokine assays. Treg cells were also studied in rheumatoid arthritis (RA) patients, whose symptoms closely mimic chronic chikungunya arthritis patients. Frequency of Treg cells was lower in acute and chronic chikungunya arthritis patients than in recovered individuals and controls, and comparable among recovered individuals and controls. Treg/Teff ratio was lower in acute than in chronic chikungunya arthritis patients, recovered individuals and controls. Higher secretion of CHIKV specific IL-10 was observed in recovered individuals than in acute, chronic chikungunya arthritis patients and controls. Frequencies of Treg and Teff cells were higher and Treg/Teff ratio was lower in RA patients than in chronic chikungunya arthritis patients. The results indicate that reduction of Treg cells was associated with ongoing CHIKV infection and normalization of Treg cells with resolution of disease. Contrasting phenotypic data in RA and chronic chikungunya arthritis suggest an altogether different mechanism of Treg-mediated pathology in both arthritis conditions. Overall, our preliminary study, suggesting an association of peripheral Treg cells and IL-10 with recovery from chikungunya, may provide insight into chikungunya disease prognosis and warrants further study.

Attenuation of the cyproterone acetate-induced testicular hypofunction by a novel nutraceutical lycopene: a genomic approach.

This study was designed to explore the cyproterone acetate (CPA)-induced andrological hypofunction and its correction by oral administration of lycopene. In this concern, spermatogenic, biochemical, histological and genomic profiles were studied. Cyproterone acetate administration for 1 month helped to develop infertile model rats. A significant recovery was noted in sperm motility, sperm count, sperm viability, hypo-osmotic swelling tail-coiled spermatozoa; activities of testicular ∆5 , 3ß-hydroxysteroid dehydrogenase (HSD), 17ß-HSD, catalase (CAT) and superoxide dismutase (SOD); and levels of conjugated diene (CD), malondialdehyde (MDA), testicular cholesterol and serum testosterone after the administration of lycopene at 1.5 mg/0.5 ml Tween-80/100 g body weight/day for last 1 month to infertile model rats. Simultaneously, qRT-PCR study of Bax, Bcl-2, caspase-3, ∆5 , 3ß-HSD and 17ß-HSD genes in testicular tissue showed a significant rectification towards the control in CPA-pre-treated cum CPA-lycopene-cotreated rats. Side-by-side histological and histometric studies showed a significant correction in qualitative analysis of spermatogenesis and seminiferous tubular diameter (STD) in CPA-pre-treated cum CPA-lycopene-cotreated rats. Lycopene showed outstanding efficacy in the management of CPA-induced testicular hypofunction with special reference to correction in oxidative stress-induced testicular apoptosis at genomic level.

Altered profile of regulatory T cells and associated cytokines in mild and moderate dengue.

The pathogenesis of dengue is immune-mediated. Regulatory T cells suppress immune response and may contribute to better prognosis. The present study evaluates Tregs and cytokines in dengue patients in the context of disease severity, time of sampling and immune status. The cohort included 90 patients (51 mild, 39 moderate) and 27 healthy controls. Frequencies of Tregs, CD4(+)CD25(-)Foxp3(+) T cells and CD3(+), CD3(+)CD4(+) and CD3(+)CD8(+) T cells were enumerated by flow cytometry. Circulating levels of 15 cytokines/chemokines were measured using Luminex technology and mRNA levels of Foxp3, IL-10 and TGF-ß were assessed by real-time polymerase chain reaction (PCR). Significantly higher frequencies of Tregs were observed in mild cases, especially during post-defervescence. The difference between mild and moderate cases was more evident in secondary infections. Frequencies of T cells were higher in mild cases but during pre-defervescence. On the other hand, the levels of IL-6, IL-7, IL-8, TNF-α and IL-10 were significantly higher in moderate cases. IL-6 and IL-8 levels correlated negatively with Treg frequencies during post-defervescence and in secondary infections. Higher levels of IL-10 and TGF-ß in moderate cases were not reflected by their corresponding mRNA levels. Platelet counts correlated positively with Treg frequencies and TGF-ß levels, and negatively with IL-10 levels. Higher Treg frequencies may favour a beneficial outcome in dengue. Higher cytokine levels may indirectly contribute to disease severity by exerting an inhibitory influence on Tregs. The dichotomy between mRNA and proteins levels for IL-10 and TGF-ß is suggestive of increased translational efficiency.

Feeding stage, species, body part and sex-specific activity of glutathione S-transferase in mosquito.

In the present study, the feeding stage, body parts, development and sex specific activity of Glutathione S-transferases (GSTs) were observed in different mosquito species (Aedes aegypti, Culex quinquefasciatus, Anopheles stephensi, An. culicifacies, An. annularis, An. subpictus, An. vagus). GST activity was assayed spectrophotometrically at 23°C, using a UV Max microplate Reader, to measure the rate of conjugation of GSH to CDNB. A significant species-specific difference in the activity of GST was noticed, highest being in unfed Ae. aegypti (41.2 nmol/min/mg) followed by unfed Cx. quinquefasciatus (7.9 nmol/min/mg) and the least in unfed An. stephensi (5.8 nmol/min/mg). In all the species the GST activity was found to be significantly higher in fully fed and gravid stages compared with the unfed, while the enzyme activity was reduced after egg laying either to the level of unfed animals or well below its level in all the experimental species. The GST activity was found to be higher in the abdominal region of all the experimental species in comparison with the other body parts (head and thorax). The GST activity of An. stephensi increased gradually through the larval stages and reached the maximum level in the pupae and remained at that level in the newly emerged adults. However, its activity declined markedly (10 fold) with ageing from 5 to 40 days. A significant sex-related difference in the specific activity of GST was found in An. stephensi where approximately 3.5 fold lower activity was observed in males compared with its females, whereas no significant variation was noticed in Ae. aegypti and Cx. quinquefasciatus. The study corroborates the fact that GSTs are differentially regulated by multiple mechanisms in response to xenobiotics modulation in situation-specific manner such as species, sex, feeding and developmental stage. The knowledge of situation-specific modulation of GST will provide a better understanding of GST based insecticide resistance mechanism which is essential for the design of sensitive monitoring methods and for an effective insecticide resistance management. The findings are significant in terms of the methods used to control mosquito vector population.

Hydrocele fluid: can it be used for immunodiagnosis of lymphatic filariasis?

BACKGROUND & OBJECTIVES: Diagnosis of lymphatic filariasis using serum has been established but the utility of hydrocele fluid for the purpose is not exactly known. Since, hydrocele is a chronic form of the disease manifestation in a variety of situations and often poses difficulty in diagnosing its origin, we have evaluated the usefulness usage of hydrocele fluid for diagnosis of filarial origin of hydrocele in this study. METHODS: Paired samples of serum and hydrocele fluid from 51 individuals with hydrocele, living in an endemic area of Wuchereria bancrofti were assessed. Circulating filarial antigen, filarial specific antibody and cytokine assay were performed in both serum and hydrocele fluid of patients. RESULTS: Og4C3 assay detected circulating filarial antigen (CFA) in serum and corresponding hydrocele fluids. The level of IgG, IFN-γ and IL-10 were found to be high in CFA-negative, while IgM and IgE were high in CFApositive hydrocele fluid and serum samples associated with hydrocele. On the other hand neither CFA-positive nor CFA-negative hydrocele fluid and serum samples associated with hydrocele showed any difference in IgG4 level. INTERPRETATION & CONCLUSION: This study showed that the filaria related antigens and antibodies found in serum can be detected with equal sensitivity in hydrocele fluid. Therefore, it can be used as an alternative to serum for immunodiagnosis of filariasis, and help monitoring the filarisis elimination programme.

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self-limiting hepatitis E.

Hepatitis E virus (HEV) is a major cause of self-limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(-) Foxp3(+) and rise in IL-10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real-time PCR and by flow cytometry, (ii) the levels of antigen-specific IL-10 and TGF-ß by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg-associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3(+) cells in both patient groups were significantly elevated. The levels of IL-10 and TGF-ß in the acute patients and TGF-ß in the recovered individuals were elevated. Significantly higher expression of CTLA-4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg-mediated suppression.

Peripheral T regulatory cells and cytokines in hepatitis E infection.

This study addresses the involvement of regulatory T cells in hepatitis E (HE) infection. The study population comprised 77 acute viral HE patients, 52 recovered individuals (overall, 129 individuals with HE) and 53 healthy controls. Peripheral CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) frequencies by flow cytometry and HE-specific cytokines/chemokines quantitation were carried out. The median percentage of CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) T cells in acute patients were significantly higher compared to controls and recovered individuals. Both of the T regulatory (Treg) subset populations in overall HE were significantly elevated compared to controls. Comparisons of cytokines/chemokines revealed that the levels of IL-10 were elevated in: (a) acute viral hepatitis E (AVH-E) versus recovered individuals and controls, and (b) HE versus controls. Overall, the elevation of CD4(+)CD25(+)Foxp3(+) and CD4(+)CD25(-)Foxp3(+) frequencies and the rise in IL-10 suggest that Treg cells might be playing a pivotal role in hepatitis E virus (HEV) infection.

Epidemic of hepatitis B with high mortality in India: association of fulminant disease with lack of CCL4 and natural killer T cells.

An explosive outbreak of Hepatitis B with high mortality was reported in 2009, in Modasa, Gujarat, India. Mortality was associated with basal core promoter and precore mutant hepatitis B virus (HBV). The current study addresses the role of immunological parameters in the progression to fulminant hepatitis. The study population comprised of 22 acute HBV patients, 13 fulminant HBV liver failure patients and 54 healthy controls. Hepatitis B surface antigen-induced CTL responses by enzyme-linked immunosorbent spot (ELISPOT), cytokine and chemokine quantitation by Bioplex assay, peripheral NK, natural killer T (NKT), CD4 and CD8 T-cell frequencies by flow cytometry were carried out. The median percentage of NK cells in the lymphocytes of the acute and fulminant liver failure patients were significantly lower compared to controls. Acute and fulminant liver failure patients had significantly high and comparable NKT cells compared to controls, respectively. Importantly, NKT cells were significantly lower in fulminant HBV liver failure than acute HBV patients. Circulating peripheral CD4/CD8 T-cell subsets among the patient categories and controls were comparable. In acute HBV patients, a significant increase in IFN-γ release was recorded (ELISPOT) by the unstimulated, antigen-stimulated and mitogen-stimulated cells when compared to controls. Comparisons of cytokines and chemokines among the disease categories revealed significantly lower levels of CCL4 in fulminant liver failure patients. NKT cells and CCL4 might be playing a pivotal role in limiting HBV infection among the patients investigated.

Viral load, antibody titers and recombinant open reading frame 2 protein-induced TH1/TH2 cytokines and cellular immune responses in self-limiting and fulminant hepatitis e.

OBJECTIVES: Hepatitis E virus (HEV) is the predominant cause of water-borne epidemics, sporadic acute viral hepatitis (AVH) in adults and fulminant hepatic failure (FHF) among pregnant women and other adults in India. This preliminary study was designed to examine the association of viral load and certain host immune responses with uneventful recovery or progression to FHF. METHODS: Viral load, anti-HEV antibody titers, rORF2p-induced Th1/Th2 cytokines levels and cellular immune responses were assessed in 47 patients with self-limiting hepatitis E and 14 FHF-E cases. The controls included 16 anti-HEV-IgM and IgG-negative healthy individuals. RESULTS: In AVH category, the viral load was 2.4 x 10(4) +/- 1.92 x 10(4) copies/ml while except for one, all FHF patients were negative for HEV RNA; anti-HEV-IgM and IgG titers were higher in the FHF group. Lymphocyte proliferative response to rORF2p was comparable in both groups. As compared to AVH, significantly higher levels of both Th1 (IFN-gamma, IL-2 and TNF-alpha) and Th2 (IL-10) cytokines were recorded in FHF patients. Analysis of sequential samples differentiated FHF recovered and fatal patients with respect to IFN-gamma and IL-12. CONCLUSION: The results document increased Th1/Th2 responses and anti-HEV titers in FHF patients that warrant in-depth immunological studies.

Immune responses to polyethylenimine delivered plasmid DNA encoding a Fasciola gigantica fatty acid binding protein in mice and rabbits.

Fasciola gigantica fatty acid binding protein (FABP) was evaluated for evoking an effective immune response in mice and rabbits, when delivered as a DNA vaccine in muscle cells. Polyethylenimine (PEI), 25 kDa, branched cationic polymer was used as a delivery vehicle for this DNA in the muscle cells of mice and rabbits. Naked DNA evoked mixed Th1 and Th2 responses in mice. PEI condensed DNA, at amine nitrogen over DNA phosphate (N/P) ratios of 4, 6 and 8 and with various DNA concentrations, failed to evoke a significantly higher antibody response compared to naked DNA in mice. Similarly, the humoral immune response to naked DNA administration in rabbit thigh muscles was poor and no boosting of this antibody response on administration of DNA complexed to PEI was observed. On metacercarial challenge, rabbits failed to show any significant protective immune response in both the naked DNA and PEI-DNA immunized groups. Administration of PEI alone (12.5 mug) in mouse thigh muscles caused significant muscle cytotoxicity but condensation of DNA with PEI had less of a toxic effect on muscle cells, which was inversely related to the N/P ratio. Delivery of plasmid DNA encoding F. gigantica FABP by high molecular weight polyethylenimine (branched, 25 kDa) did not boost the effective immune response in both the animal species, which could either be attributed to cytotoxicity associated with this cationic polymer or muscle cells being unsuitable target cells for PEI condensed DNA delivery.

Role of 'B-b' knob-hole interactions in fibrin binding to adsorbed fibrinogen.

BACKGROUND: The formation of a fibrin clot is supported by multiple interactions, including those between polymerization knobs 'A' and 'B' exposed by thrombin cleavage and polymerization holes 'a' and 'b' present in fibrinogen and fibrin. Although structural studies have defined the 'A-a' and 'B-b' interactions in part, it has not been possible to measure the affinities of individual knob-hole interactions in the absence of the other interactions occurring in fibrin. OBJECTIVES: We designed experiments to determine the affinities of knob-hole interactions, either 'A-a' alone or 'A-a' and 'B-b' together. METHODS: We used surface plasmon resonance to measure binding between adsorbed fibrinogen and soluble fibrin fragments containing 'A' knobs, desA-NDSK, or both 'A' and 'B' knobs, desAB-NDSK. RESULTS: The desA- and desAB-NDSK fragments bound to fibrinogen with statistically similar K(d)'s of 5.8 +/- 1.1 microm and 3.7 +/- 0.7 microm (P = 0.14), respectively. This binding was specific, as we saw no significant binding of NDSK, which has no exposed knobs. Moreover, the synthetic 'A' knob peptide GPRP and synthetic 'B' knob peptides GHRP and AHRPY, inhibited the binding of desA- and/or desAB-NDSK. CONCLUSIONS: The peptide inhibition findings show both 'A-a' and 'B-b' interactions participate in desAB-NDSK binding to fibrinogen, indicating 'B-b' interactions can occur simultaneously with 'A-a'. Furthermore, 'A-a' interactions are much stronger than 'B-b' because the affinity of desA-NDSK was not markedly different from desAB-NDSK.

Ibandronate reduces skeletal morbidity in patients with breast cancer.

Three phase III studies have assessed the efficacy of intravenous (IV) and oral ibandronate over 96 weeks for metastatic bone disease in patients with breast cancer. The primary endpoint for each trial was the skeletal morbidity period rate, defined as the number of 12-week periods with new bone complications, adjusted for the time spent on study. Both IV ibandronate 6 mg every 3 to 4 weeks and oral ibandronate 50 mg once daily significantly reduced the skeletal morbidity period rate compared with placebo ( P = .004 in each case). The studies were not powered to detect statistical significance on individual components of the skeletal morbidity period rate. Nevertheless, IV ibandronate significantly reduced vertebral fractures and the need for radiotherapy, while oral ibandronate led to significantly fewer bone events needing radiotherapy or surgery than placebo. Using a multivariate Poisson regression model, the mean reduction in the relative risk of new bone events compared with placebo was 40% with IV ibandronate 6 mg ( P = .0033), and 38% with oral ibandronate 50 mg ( P <.001). The clinical equivalence of IV and oral ibandronate was confirmed by a post-hoc Anderson-Gill analysis of time to multiple skeletal events. These results show that IV and oral ibandronate effectively reduce skeletal morbidity in breast cancer patients with bone metastases.

Breast feeding and childhood hematological malignancy.

Breast milk is known to have anti-infective and immunomodulating effects on infants, but its association with childhood cancer has not been well studied. Artificial feeding may affect the immune response in carcinogenesis. In this communication the authors have reviewed different articles describing the association between breast feeding (BF) and subsequent development of childhood hematological malignancy. It appears that BF may have a protective effect on childhood cancer, both the duration of BF as well as the quantity of milk ingested is probably critical to the beneficial immunological effects of BF against childhood cancer if any.

Removal of the pro-domain does not affect the conformation of the procaspase-3 dimer.

We have investigated the oligomeric properties of procaspase-3 and a mutant that lacks the pro-domain (called pro-less variant). In addition, we have examined the interactions of the 28 amino acid pro-peptide when added in trans to the pro-less variant. By sedimentation equilibrium studies, we have found that procapase-3 is a stable dimer in solution at 25 degrees C and pH 7.2, and we estimate an upper limit for the equilibrium dissociation constant of approximately 50 nM. Considering the expression levels of caspase-3 in Jurkat cells, we predict that procaspase-3 exists as a dimer in vivo. The pro-less variant is also a dimer, with little apparent change in the equilibrium dissociation constant. Thus, in contrast with the long pro-domain caspases, the pro-peptide of caspase-3 does not appear to be involved in dimerization. Results from circular dichroism, fluorescence anisotropy, and FTIR studies demonstrate that the pro-domain interacts weakly with the pro-less variant. The data suggest that the pro-peptide adopts a beta-structure when in contact with the protein, but it is a random coil when free in solution. In addition, when added in trans, the pro-peptide does not inhibit the activity of the mature caspase-3 heterotetramer. On the other hand, the active caspase-3 does not efficiently hydrolyze the pro-domain at the NSVD(9) sequence as occurs when the pro-peptide is in cis to the protease domain. Based on these results, we propose a model for maturation of the procaspase-3 dimer.

TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2.

Like other members of the medically important phylum Apicomplexa, Toxoplasma gondii is an obligate intracellular parasite that secretes several classes of proteins involved in the active invasion of target host cells. Proteins in apical secretory organelles known as micronemes have been strongly implicated in parasite attachment to host cells. TgMIC2 is a microneme protein with multiple adhesive domains that bind target cells and is mobilized onto the parasite surface during parasite attachment. Here, we describe a novel parasite protein, TgM2AP, which is physically associated with TgMIC2. TgM2AP complexes with TgMIC2 within 15 min of synthesis and remains associated with TgMIC2 in the micronemes, on the parasite surface during invasion and in the culture medium after release from the parasite plasma membrane. TgM2AP is proteolytically processed initially when its propeptide is removed during transit through the golgi and later while it occupies the parasite surface after discharge from the micronemes. We show that TgM2AP is a member of a protein family expressed by coccidian parasites including Neospora caninum and Eimeria tenella. This phylogenic conservation and association with a key adhesive protein suggest that TgM2AP is a fundamental component of the T. gondii invasion machinery.

A novel fluorescence assay to study propeptide interaction with gamma-glutamyl carboxylase.

The vitamin K-dependent gamma-glutamyl carboxylase catalyzes the posttranslational modification of select glutamate residues of its vitamin K-dependent substrates to gamma-carboxyglutamate. In this report, we describe a new fluorescence assay that is sensitive and specific for the propeptide binding site of active carboxylase. We employed the assay to make three important observations: (1) A tight binding fluorescein-labeled consensus propeptide can be used to quantify the active fraction of the enzyme. (2) The off-rate for a fluorescein-labeled factor IX propeptide was 3000-fold slower than the rate of carboxylation, a difference that may explain how carboxylase can carry out multiple carboxylations of a substrate during the same binding event. (3) We show evidence that substrate binding to the active site modifies the propeptide binding site of carboxylase. The significant (9-fold) differences in off-rates for the propeptide in the presence and absence of its co-substrates may represent a release mechanism for macromolecular substrates from the enzyme. Additionally, sedimentation velocity and equilibrium experiments indicate a monomeric association of enzyme with propeptide. Furthermore, the carboxylase preparation is monodisperse in the buffer used for our studies.

Two domains in dihydropyridine receptor activate the skeletal muscle Ca(2+) release channel.

The II-III cytoplasmic loop of the skeletal muscle dihydropyridine receptor (DHPR) alpha(1)-subunit is essential for skeletal-type excitation-contraction coupling. Single channel and [(3)H]ryanodine binding studies with a full-length recombinant peptide (p(666-791)) confirmed that this region specifically activates skeletal muscle Ca2+ release channels (CRCs). However, attempts to identify shorter domains of the II-III loop specific for skeletal CRC activation have yielded contradictory results. We assessed the specificity of the interaction of five truncated II-III loop peptides by comparing their effects on skeletal and cardiac CRCs in lipid bilayer experiments; p(671-680) and p(720-765) specifically activated the submaximally Ca2+-activated skeletal CRC in experiments using both mono and divalent ions as current carriers. A third peptide, p(671-690), showed a bimodal activation/inactivation behavior indicating a high-affinity activating and low-affinity inactivating binding site. Two other peptides (p(681-690) and p(681-685)) that contained an RKRRK-motif and have previously been suggested in in vitro studies to be important for skeletal-type E-C coupling, failed to specifically stimulate skeletal CRCs. Noteworthy, p(671-690), p(681-690), and p(681-685) induced similar subconductances and long-lasting channel closings in skeletal and cardiac CRCs, indicating that these peptides interact in an isoform-independent manner with the CRCs.