Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.     

Evidence for retarded kidney growth in sudden infant death syndrome

The aim of the study was to compare the growth rate of the kidneys of infants who died of sudden infant death syndrome (SIDS) and control babies under 1 year; 227 infants who died in St. Petersburg from 1983 to 1990 and who met the criteria for SIDS were included in the study; 138 infants who died suddenly of respiratory infections within the same period constituted a control group. The infants did not have signs of dehydration, malformations, tumours or intrauterine infections. Morphologically the kidneys were intact. Factors which might influence the weight of the kidneys at the time of death were: the cause of death (whether SIDS or not), gender, gestational age, weight, length and ponderal index at birth, age, weight and length at death. Stepwise (forward) linear regression analysis identified three variables which in combination most accurately and independently influenced the predicted weight of the kidneys. These were the cause of death, gender and weight at the time of death. The weight of the kidneys increased by 6.0 g for each increase in total body weight of 1,000 g [95% confidence interval (CI) 5.0-7.0 g], in boys the kidney weight was 3.3 g (95% CI 1.6-5.0 g) higher than in girls and in the SIDS babies kidney weight was 2.5 g (95% CI 0.8-4.2 g) less than controls. Delayed kidney growth may be an indicator of increased risk of SIDS in infants under 1 year and may contribute in some cases.     

The use of monoclonal antibodies for detecting the influenza virus

The possibilities of using influenza A (Leningrad) 385/80 (H3N2) virus matrix protein-specific FITC-labeled D8 monoclonal antibodies in immunofluorescence assays were investigated. The virus antigen accumulation was detected in chorioallantoic cells of chick embryos. Exhibiting the type-specific properties, the fluorescent antibodies stain the perinuclear space, cytoplasmic membrane, and granular structures in the cytoplasm of infected cells. The haemagglutination test tires in the corresponding specimens were at least 1:16.     

The phosphorylation of eukaryotic initiation factor eIF4E in response to phorbol esters, cell stresses, and cytokines is mediated by distinct MAP kinase pathways

Initiation factor eIF4E binds to the 5'-cap of eukaryotic mRNAs and plays a key role in the mechanism and regulation of translation. It may be regulated through its own phosphorylation and through inhibitory binding proteins (4E-BPs), which modulate its availability for initiation complex assembly. eIF4E phosphorylation is enhanced by phorbol esters. We show, using specific inhibitors, that this involves both the p38 mitogen-activated protein (MAP) kinase and Erk signaling pathways. Cell stresses such as arsenite and anisomycin and the cytokines tumor necrosis factor-alpha and interleukin-1beta also cause increased phosphorylation of eIF4E, which is abolished by the specific p38 MAP kinase inhibitor, SB203580. These changes in eIF4E phosphorylation parallel the activity of the eIF4E kinase, Mnk1. However other stresses such as heat shock, sorbitol, and H2O2, which also stimulate p38 MAP kinase and increase Mnk1 activity, do not increase phosphorylation of eIF4E. The latter stresses increase the binding of eIF4E to 4E-BP1, and we show that this blocks the phosphorylation of eIF4E by Mnk1 in vitro, which may explain the absence of an increase in eIF4E phosphorylation under these conditions.     

Stochastic complexity measures for physiological signal analysis

Traditional feature extraction methods describe signals in terms of amplitude and frequency. This paper takes a paradigm shift and investigates four stochastic-complexity features. Their advantages are demonstrated on synthetic and physiological signals; the latter recorded during periods of Cheyne-Stokes respiration, anesthesia, sleep, and motor-cortex investigation.     

Incremental redistribution of protein kinase C underlies the acquisition curve during in vitro associative conditioning in Hermissenda

Although thoracoabdominal injuries are uncommon in the athlete, they can be catastrophic if unrecognized or if diagnosis and treatment are delayed. This article reviews thoracic, intrathoracic, abdominal, and groin injuries in the athlete, and how they can be diagnosed and managed.     

The correlation of the vasopressin- and oxytocin-producing activities of different nuclei in the hypothalamo-hypophyseal neurosecretory system]

Radioiodine long has proven to be a safe and effective treatment for thyroid disease. Nonetheless, persisting concerns regarding radiogenic stochastic risks (e.g., carcinogenesis) to patients, their families, and the general public have led regulators to establish criteria for release of 131I-containing patients from medical confinement, with limits ranging from as low as 2 mCi in some parts of Europe to as high as 30 mCi in the United States. To optimize clinical efficacy and cost-effectiveness of 131I therapy, such regulations should be based on logical dosimetric considerations. The thyroidal absorbed dose, proportional to maximum uptake and effective half-life and inversely proportional to mass, is typically approximately 1,500 rad/mCi of 131I administered to a euthyroid adult (based on a thyroid maximum uptake of 25%, effective half-life equivalent to the physical half-life of 131I (8.04 days), and mass of 20 g). As thyroid uptake increases from 0% to 100%, extrathyroidal absorbed doses range from a minimum of 0.15 to 0.5 rad/mCi for breast and gonads to a maximum of 1.5 to 2 rad/mCi for stomach and salivary glands; the absorbed doses of the urinary bladder wall, in contrast, decrease with increasing thyroid uptake, from 2 to 0.6 rad/mCi. In hyperthyroid patients (approximately 15%) with a small iodine pool (so-called small patients), the short effective half-life of radioiodine in the thyroid and high serum concentrations of long-lived protein-bound 131I result in a standard 7,000-rad absorbed dose for treatment of Graves' disease requiring an administered activity of 28 mCi of 131I and yielding a prohibitively high blood absorbed dose of 150 rad. Importantly, once the fetal thyroid begins to function and accumulate radioiodine at a gestational age of 10-12 weeks, fetal thyroid absorbed doses as large as 5,000 rad/mCi of 131I administered to the mother can result. Thus, pregnancy is an absolute contraindication to administration of 131I because of the risk of radiogenic cretinism. Based on actual measurements of thyroid activity and of external absorbed dose, the total thyroid and mean extrathyroidal absorbed doses to adult family members from immediately released 131I-treated patients are approximately 0.01 and approximately 0.02 rad/mCi administered, respectively, yielding an effective dose of approximately 0.02 rem/mCi. A maximum permissible effective dose of 0.5 rem for adults therefore is consistent with a release criterion of 30 mCi of retained 131I. Lower-activity release criteria therefore may be unnecessarily restrictive.     

Comparative immunoreactivity of CD-68 and HMB-45 in malignant melanoma, neural tumors and nevi

The monoclonal antibody CD 68 (KP 1) reacts with fibrohistiocytic and some epithelial neoplasms; its reactivity compared with that of HMB 45 in malignant melanoma (MM) and neural tumors needs further elucidation. Using a streptavidin-biotin-immunoperoxidase procedure, we examined the reactivity of 65 MM (46 conventional, 1 polypoid, 6 desmoplastic [DMM], and 12 metastatic), 21 neurofibromas, 1 neurofibrosarcoma, 10 schwannomas, 1 perineurioma, 2 neurothekeomas, and 14 blue and 26 other nevi for CD-68, HMB-45-defined antigen, S 100 and neurofilament protein. A positive staining for CD 68 was observed in 38 of 42 primary, 5 of 6 DMM, and 11 of 12 metastatic melanomas; 6 of 10 schwannomas; 5 of 10 nevi with junctional component and all 14 blue nevi. All 21 neurofibromas, 1 each neurofibrosarcoma and perineurioma, both neurothekeomas, and all 12 nevi with dermal component were CD 68-negative. HBM 45 was expressed by all 44 primary, none of 6 DMM, and 7 of 12 metastatic melanomas; by none of 10 schwannomas, 6 neurofibromas, 1 neurofibrosarcoma, 1 perineurioma and 2 neurothekeomas. Both junctional nevi, 8 of 10 nevi with junctional components, 1 of 10 dermal components of junctional nevi, and 11 of 13 blue nevi were also HMB 45 positive. Except for 1 perineurioma, S 100 decorated all tumors examined. NF was immunoreactive in 1 of 45 conventional melanomas, 2 of 21 neurofibromas, 2 of 10 schwannomas, and 3 of 10 blue nevi; it was non-reactive in all polypoid, desmoplastic and metastatic melanomas; neurofibrosarcoma, perineurioma, neurothekeoma and other nevi. We conclude that the CD-68-reactivity in primary melanomas, neurofibromas, neurofibrosarcomas, perineuriomas, and nevi was similar to that of HMB 45. The significantly higher CD 68-positivity than of HMB 45 in metastatic and desmoplastic melanomas and schwannomas may be of diagnostic value.     

Ozone therapy of diffuse peritonitis in the early postoperative period

The functional status of the oxidative-antioxidative system was studied in 72 patients after vast cancer operations. Traditional surgical treatment and its combination with intraoperative irradiation were shown to lead to tense antioxidative defense and to suppressed T-cell immunity and to call for antioxidative and immunomodulating therapy. High intraoperative blood loss complicated by hemorrhagic shock injured the oxidative-antioxidative system greatly. The magnitude of this damage correlated with the rate of prehypoxia. Addition of the potent antioxidant Ceruloplasmin to the drug regimen normalized a recovery period, helped to correct posthypoxic multiorgan insufficiency, to recover oxidative-antioxidative balance, and to decrease the incidence of pyoinflammatory complications. Patients with endogenous intoxication showed activated lipid peroxidation, decreased functional activity of antioxidative defense components and of T-cell immunity in homeostasis. The use of Ceruloplasmin and Laprot had pronounced antiinflammatory and detoxifying effects on the patient's body and activated its antioxidative defense.