The structure of the cytochrome a3-CuB site of mammalian cytochrome c oxidase as probed by MCD and EPR spectroscopy

The nature of the complexes formed between cytochrome c oxidase and the three inhibitory ligands N3-, CN-, and S2- have been investigated by a combination of MCD and EPR spectroscopy. CN- forms a linear bridge between the Fe III a3 and CuB II, suggesting that the distance between these centers in the oxidized enzyme is between 5 and 5.25 A. This distance is too short to permit N3- to form a linear bridge and the evidence suggests this to be bent. In contrast S2- or SH- is unable to form any bridge and it seems likely that two SH- ions are bound by the bimetallic site, one to Fe III a3 and the other to CuB I. The significance of the a3-CuB distance in terms of oxygen binding and reduction is discussed.     

Activity expressed from cloned Anacystis nidulans large and small subunit ribulose bisphosphate carboxylase genes

Summary The ribulose bisphosphate carboxylase/oxygenase (EC4.1.1.39) (RubisCO) large and small subunit genes from Anacystis nidulans have been cloned as a single fragment into M 13mp10 and pEMBL8 and expressed in Escherichia coli. From M 13mp10 a low yield of enzyme with high specific activity was obtained. The molecular weight of the active enzyme was 260 000 Da and of the inactive enzyme approximately 730 000 Da. The small and large subunits cloned separately did not express activity. The RubisCO gene cloned into pEMBL8 expressed activity up to 22 times that from the M 13 cloned RubisCO DNA. The RubisCO protein produced by the pEMBL cloned gene had a normal MW (550 000). Immunoprecipitation and polyacrylamide gel electrophoresis showed the presence of both large and small subunits.     

Further studies of human whole-body radiofrequency absorption rates

Further studies of human whole-body radiofrequency (RF) absorption rates were carried out using a TEM-cell exposure system. Experiments were done at one frequency near the grounded resonance frequency (approximately 40 MHz), and at several below-resonance frequencies. Absorption rates are small for the K and H orientations of the body, even when grounded. For the body trunk in an E orientation, the absorption rate of a sitting person is about half of the rate for the same person standing with arms at the sides; the latter in turn is about half the rate for the same subject standing with arms over the head. Two-body interactions cause no increase in absorption rates for grounded people. They do, however, increase the absorption rates for subjects in an E orientation in free space; the largest interaction occurs when one subject is lambda/2 behind the other (as seen by the incident wave). When these results are applied to practical occupational exposure situations, the whole-body specific absorption rate does not exceed the ANSI limit of 0.4 W/kg for exposures permitted by the ANSI standard (C95.1-1982) at frequencies from 7 to 40 MHz.     

Impact of whooping cough on patients and their families.

The effects of whooping cough were studied in 21 children admitted to hospital with the disease and in their families. The illness caused considerable distress to both child and family. Parents suffered especially from fears for the life and health of their child and from serious loss of sleep. Two months after admission the child''s behaviour was still disturbed, but in most cases the rest of the family had returned to normal. There was much misunderstanding and misinformation about whooping cough among both parents and doctors.     

Cultured fetal rat myoblasts release peptide growth factors which are immunologically and biologically similar to somatomedin

The production of immunologically and biologically active somatomedin activity from isolated myoblasts and fibroblasts from fetal rats of 21 days gestational age was investigated. Myoblast-rich cell populations were derived from primary cultures of dispersed muscle cells by the tendency of myoblasts to become detached from the culture dish in the presence of cytochalasin B. Fibroblasts were obtained from fetal muscle. Culture medium conditioned by exposure to myoblasts for 48 hours produced an increased incorporation of both [35S]sulphate and [3H]thymidine by explants of fetal rat costal cartilage in vitro compared to fresh medium. Myoblast-conditioned medium also contained somatomedin-C-like immunoreactivity which diluted in parallel with partially purified human somatomedin-C (3,271 +/- 446 mU/mg cell protein; mean +/- SEM, seven experiments). Medium conditioned by exposure to fetal rat fibroblasts did not promote isotope uptake by fetal rat cartilage above control values, and contained only low levels of somatomedin-C-like immunoreactivity (343 +/- 89 mU/mg cell protein, three experiments). The release of both somatomedin bioactivity and immunoreactivity into conditioned medium was significantly reduced by the incubation of myoblasts in the presence of rat growth hormone (100 ng/ml and 500 ng/ml). We conclude that fetal rat myoblasts released growth factor activity during culture which exhibited biological and immunologic characteristics of somatomedin. Since the bioactivity was demonstrated on skeletal tissues from rat fetuses of the same gestational age as those that yielded myoblasts such growth factor release may be physiological.     

Immunoelectron microscopic localization of alpha-actinin and actin in embryonic hamster heart cells

Myofibrillogenesis in developing cardiac cells of the Syrian hamster from early embryonic stages through newborn was studied by electron microscopy, immunofluorescence microscopy and immunoelectron microscopy. alpha-Actinin and actin were localized at light and electron microscopic levels in embryonic heart cells which had been fixed in a periodate-lysine-paraformaldehyde or a glutaraldehyde-formaldehyde mixture, and embedded in Lowicryl K4M. Indirect staining methods were used for immunofluorescence staining of thick sections and immunoferritin staining of thin sections. The earliest evidence of myofibrillogenesis in embryonic myocardial cells was the presence of many randomly arranged thin (6 nm) filaments and a few scattered thick filaments (15 nm) near the plasma membrane. alpha-Actinin was detected in a semi-continuous, diffuse layer in some portions of the cell just beneath the plasma membrane in association with the filamentous collections. Later in development, alpha-actinin coalesced into Z-plaques at the membrane as the filaments arranged into parallel arrays. Actin was localized in the thin filaments as expected. In later stages of development, alpha-actinin was observed at the Z-lines and intercalated discs of the mature myofibrils while actin was localized at both the I-band and Z-line. Our results suggest that myofibrillogenesis is initiated at the plasma membrane and that Z-plaques are precursors of myofibrillar Z-bands and may serve as organizing centers for myofibrillogenesis in developing cardiomyocytes.     

Kinetic studies on the chymotrypsin Aα-catalyzed hydrolysis of a series of N-acetyl-l-phenylalanyl peptides

A series of N-acetyl-l-phenylalanyl peptides of general formula Ac-Phe-(Gly)_n-NH₂ (n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A_α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH₂, AcPheAlaGlyNH₂, and AcPheGlyAlaNH₂. Values of k_cat and K_m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.     

Incidence of antibiotic-resistant Escherichia coli in milk produced in the west of Scotland

Antibiotic-resistant Esch. coli were found in 10.6% of milk samples collected from 998 farms in the west of Scotland. The incidence of both Esch. coli and antibiotic-resistant Esch. coli in milk was higher when the cattle were housed day and night than when they were outdoors. Of the 1125 Esch. coli isolates tested 22.2% were antibiotic resistant and of these 42.4% were resistant to more than one antibiotic. Escherichia coli carrying up to six resistance determinants were isolated. The possible implications to animal and human health are discussed.     

Detection and enumeration of virulent Yersinia enterocolitica in food by DNA colony hybridization.

A portion of a 44-megadalton plasmid found in Yersinia enterocolitica 8081 was used as a genetic probe to differentiate virulent and nonvirulent strains of the species. A DNA colony hybridization technique was employed. Three BamHI restriction endonuclease fragments labeled with 32P by nick translation were hybridized to lysed colonies of pure cultures, mixtures of virulent and nonvirulent cells, and portions of a food sample artificially contaminated with virulent Y. enterocolitica. The results of the colony hybridization test for virulence were the same as those obtained by the autoagglutination and suckling mouse tests. DNA colony hybridization detects pathogenic Y. enterocolitica in foods without the need for enrichment or pure cultures.