1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.     

Mortality and neurologic morbidity after repair of traumatic aortic disruption

BACKGROUND: Traumatic disruption of the thoracic aorta frequently results in death before operative repair. The determinants of mortality after repair, however, are uncertain. In addition, intraoperative strategies for reducing the incidence of spinal cord injury remain controversial. METHODS: The records of 45 consecutive patients undergoing repair of traumatic disruption of the thoracic aorta at a single institution during a 9-year period were reviewed in a retrospective fashion. Patient age ranged from 15 to 81 years (mean age, 33.9 years). Twenty-two patients (49%) had multiple associated injuries, and 8 (18%) had isolated aortic injuries. Nine patients (20%) experienced preoperative hypotension (systolic blood pressure of less than 90 mm Hg). Repair was performed with partial bypass in 22 patients, a heparinized shunt in 2, and no distal perfusion (clamp and sew technique) in 21. RESULTS: Nine patient (20%) died after operation. Multivariate logistic regression analysis of preoperative and intraoperative variables identified advancing age and preoperative hypotension as independent predictors of operative death. The presence of associated injuries was not an independent predictor of operative death. All 4 patients with injuries proximal to the aortic isthmus died. Ten patients were excluded from analysis of spinal cord injury either because of preoperative neurologic deficit or because of death before postoperative evaluation. Six (17%) of the remaining 35 patients had development of paraplegia: 5 of the 15 patients having the clamp and sew technique, 1 of the 2 with a shunt, and 0 of the 18 patients with bypass (p < 0.05, clamp and sew versus bypass). In the clamp and sew group, patients in whom paraplegia developed had significantly longer aortic clamp times than those without neurologic injury (40.6 +/- 4.4 minutes versus 28.7 +/- 2.9 minutes, respectively; p < 0.05). CONCLUSIONS: Advancing age, preoperative hypotension, and perhaps injury location are important determinants of death after repair of traumatic disruption of the thoracic aorta. Adjunctive perfusion with partial bypass should be used during repair to reduce the incidence of spinal cord injury.     

K-ras gene mutations: an unfavorable prognostic marker in stage I lung adenocarcinoma

Activation of K-ras gene by point mutations, a common finding in lung adenocarcinomas, has been suggested to decrease patient survival. We investigated 109 lung adenocarcinomas, mostly small, peripheral, stage I tumours (81/109) for presence of K-ras gene mutations at codons 12 and 13. Mutations were detected by denaturing gradient gel electrophoresis analysis of specific sequences amplified by polymerase chain reaction from DNA extracted from archival pathological material. Thirty-three of 109 (30.3%) tumours showed mutations at codon 12 (28/33, 84.8%) or 13 (5/33, 15.2%) of the gene. Mutations and type of nucleotide substitutions were differently distributed among cytological subtypes, being more prevalent among less differentiated (G2 and G3) tumours and among bronchial than bronchiolo-alveolar type adenocarcinomas. Survival analysis showed an adverse effect of K-ras mutation on survival, restricted to stage I tumours. Median survival for 81 stage I patients was 30 months for non-mutated tumours versus 20 months for mutated tumours (p = 0.016). Multivariate analysis showed that age of patient (p = 0.001) and K-ras mutation status (p = 0.04) were the only independent factors influencing survival significantly. These data strengthen the hypothesis that K-ras gene mutations may be useful in identifying a subgroup of patients with poor outcome.     

The biological properties of Aerococcus antagonists, representatives of human microbiocenoses

The paper deals with the data on biology of Aerococcus, a slightly studied group of microorganisms. Physiological-biochemical properties of Aerococcus are described, data of their distribution in nature are given. Peculiar attention is paid to the estimate of the role of Aerococcus in human microbiocenoses. As a result of the profound and all-round study of this group of microorganisms the authors have developed new bacterial drug "A-bakterin" based on the aerococcus strain. Data presented about the results of clinical tests of "A-bakterin" are presented, a possibility to use Aerococcus lysate in the elaboration of new drugs is discussed.     

Apoptosis in batch cultures of Chinese hamster ovary cells

One of the main problems in the culture of Chinese Hamster Ovary (CHO) cells continues to be the inability to maintain the viability of the cultures over an extended period of time. The rapid decline in viability at the end of the culture is exacerbated by the absence of serum. In trying to reduce the extent of death in these cultures, we first tried to determine the mode of death. We found that more than 80% of the cells in a standard serum-free batch culture of CHO cells in suspension died via apoptosis--as evidenced by condensed chromatin and the appearance of a characteristic DNA ladder. Furthermore, when protein synthesis was inhibited using cycloheximide, the cells underwent rapid apoptosis indicating that death proteins were present in greater abundance than survival proteins in our CHO cells. Cell lysate from CHO cells showed evidence of cysteine protease (caspase) activity. Caspases of the Interleukin-1-beta-Converting Enzyme (ICE) family, e.g., CPP32, Mch-1, etc., have been implicated in the apoptotic process. Surprisingly, a caspase peptide inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoro-methyl-ketone (z-VAD.fmk), was unable to substantially extend the life of a serum-free batch culture of CHO cells. In addition, z-VAD.fmk was only marginally able to extend viability in response to withdrawal of growth and survival factors, insulin and transferrin. In both these instances, z-VAD.fmk was able to prevent cleavage of caspase substrates, but not protect cells from death. However, we found that bcl-2 expression was able to significantly extend viabilities in CHO batch culture. Bcl-2 expression also substantially extended the viability of cultures in response to insulin and transferrin withdrawal. These results provide interesting insights into the pathways of death in a CHO cell.     

Antitubercular activity of lomefloxacin in an experiment

The effect of lomefloxacin was studied on mice with experimental infection due to Mycobacterium tuberculosis. The antibiotic was administered in doses of 100 and 200 mg/kg. It was shown that the use of lomefloxacin for a month provided a lower death rate of the animals with progressing acute generalized tuberculosis, a lower level of the lesions in the internal organs and a lower number of the Mycobacterium isolates from them. The efficacy of the treatment depended on the drug dose. When lomefloxacin was used in a dose of 200 mg/kg, the survival rate was much higher and the number of the epithelial unicellular granulomas in the tissue of the lung and spleen was markedly decreased while with the lower dose the indices did not differ from those in the control.     

Recombination-induced variants of Clostridium acetobutylicum ATCC 824 with increased solvent production

Three sporulation-specific genes (orfA, sigE, sigG) from clostridium acetobutylicum ATCC 824 are arranged in a cluster, encoding the putative sigma E-processing enzyme, sigma E, and sigma sigma G respectively. When they were transformed into Clostridium acetobutylicum while on a plasmid functional in this organism, transformants did not survive. Three kinds of recombinations were then attempted with nonreplicative plasmids: duplication of orfA and sigE, replacement of all of the three genes, and inactivation of orfA. While the wild-type strain ceased to grow and produce solvents in batch cultures after approximately 24 h, mutant strains were isolated that showed sustained growth for a much longer time and produced a threefold increase in acetone and butanol in test tube cultures. In addition, one of the derived strains showed a significantly higher growth rate. Features of the restriction maps of the recombinants did not correlate with expected maps, indicating possible complications occurring during the recombination events.     

Clinical computed tomographic correlations in children from a group at high risk for schizophrenia

Computed tomography (CT) was performed in 189 individuals: in 44 children of schizophrenic parents (high risk group, HRG), in 39 parents with schizophrenia or with schizophrenic disturbance, in 56 children with schizophrenia, in 50 children with consequences of early organic damages of central nervous system (mental retardation syndrome and generalized tic syndrome). The frequency of CT changes was equal in the mentioned groups but their character was quite different. The widening of brain's liquor system (89.7%), the signs of frontal and temporal atrophia (31%), foci of decreased density of cerebral brain's matter, closer in subcortical ganglia and periventricular zone, and different anomalies of brain were observed in HRG children.     

The molecular structure of green fluorescent protein

The crystal structure of recombinant wild-type green fluorescent protein (GFP) has been solved to a resolution of 1.9 A by multiwavelength anomalous dispersion phasing methods. The protein is in the shape of a cylinder, comprising 11 strands of beta-sheet with an alpha-helix inside and short helical segments on the ends of the cylinder. This motif, with beta-structure on the outside and alpha-helix on the inside, represents a new protein fold, which we have named the beta-can. Two protomers pack closely together to form a dimer in the crystal. The fluorophores are protected inside the cylinders, and their structures are consistent with the formation of aromatic systems made up of Tyr66 with reduction of its C alpha-C beta bond coupled with cyclization of the neighboring glycine and serine residues. The environment inside the cylinder explains the effects of many existing mutants of GFP and suggests specific side chains that could be modified to change the spectral properties of GFP. Furthermore, the identification of the dimer contacts may allow mutagenic control of the state of assembly of the protein.