Substituent effects in aqueous solutions of carboxylate salts studied by x-ray absorption spectroscopy at the oxygen K-edge.

The present study aims at probing the influence of different substituents of sodium carboxylate salts R-COO-:Na+ in aqueous solutions, with R = H, CH3, C2H5, CH2Cl, CF3, and C6H5. X-ray absorption spectroscopy was used in the oxygen K-edge region to highlight the effect of R on the energy position of the O1s-to-πCOO* resonance of the carboxylate ion. Ab initio static exchange and ΔSCF calculations are performed and confirm the experimental observations. We qualitatively discuss the results on the basis of the polar properties of these groups as well as on the basis of the πCOO* orbital energy in the ground states, the oxygen 1s orbital ionization energy, and the O1s-to-πCOO* resonance energy.

Pseudo-subarachnoid hemorrhage found in patients with postresuscitation encephalopathy: characteristics of CT findings and clinical importance.

BACKGROUND AND PURPOSE: High-attenuation areas (HDAs) called pseudo-subarachnoid hemorrhages (SAHs) may develop in some patients resuscitated from cardiopulmonary arrest (CPA), though no hemorrhage has occurred. We investigated the imaging characteristics and clinical significance of this phenomenon. MATERIALS AND METHODS: CT images of consecutive patients resuscitated from nontraumatic CPA were reviewed and classified into cases with pseudo-SAH (pseudo-SAH[+] group, n = 9), those without pseudo-SAH (pseudo-SAH[-] group, n = 28), and those with true SAH (SAH-CPA group, n = 8). Typical patients with SAH (SAH group, n = 13) and 20 healthy individuals were also extracted as control groups. The degree of brain edema was scored visually as none, mild, or severe, and the CT values of the HDAs and brain parenchyma were measured. These parameters were compared among the groups. We also compared the prognosis between the pseudo-SAH(+) and pseudo-SAH(-) groups. RESULTS: On CT, pseudo-SAH was associated with severe brain edema, whereas there was mild or no edema without pseudo-SAH. The CT values of the HDAs in the pseudo-SAH(+) group were significantly lower than those of the CPA-SAH and SAH groups (P < .0001). The brain parenchyma of the pseudo-SAH(+) group had the lowest CT values among all of the groups (P < .0001). The prognosis of the pseudo-SAH(+) group was significantly poorer than that of the pseudo-SAH(-) group in terms of both clinical outcome (P = .02) and survival (P = .046). CONCLUSION: The findings of pseudo-SAH have several imaging characteristics differing from SAH and predict a poor prognosis. This provides important information that can be used for deciding treatment strategies.

Effect of time for mating and gestation length on reproductive efficiency in dogs.

The purpose of this study was to examine the effect of time for mating and gestation length on reproductive efficiency in dogs. Groups of eight, six and six beagle bitches were mated with a total of three sires on days 3, 5 and 7, respectively, after the luteinizing hormone (LH) surge. All the bitches whelped successfully. The gestation lengths (the intervals from the LH surge to the whelping) were 65.1 +/- 1.9, 65.5 +/- 1.9 and 68.0 +/- 1.8 days, respectively. This length of mating 7 days after the LH surge was significantly longer than that of mating 3 and 5 days after the LH surge (p < 0.05). The litter sizes were 5.5 +/- 2.2, 6.2 +/- 1.9 and 4.8 +/- 2.1, respectively and there was no significant difference between any of the groups. In conclusion, mating 3-7 days after a bitch's LH surge results in a good reproductive efficiency and the intervals from the LH surge to the whelping of mating 7 days after the LH surge shows a longer day significantly in comparison with mating 3 and 5 days after the LH surge.

Inhibitory effects of safe and novel SOD derivatives, galactosylated-SOD, on hepatic warm ischemia/reperfusion injury in pigs.

BACKGROUND/AIMS: Oxygen-derived free radicals such as superoxide play an important role in ischemia/reperfusion (IR) injury during and after extensive liver surgery or liver transplantation. Superoxide dismutase (SOD) has protective effects against hepatic IR injury. The effect of native SOD is, however, limited because of rapid elimination from the blood circulation and poor affinity for liver cells. It was reported by our collaborators that a SOD derivative modified with galactose (Gal-SOD) was selectively delivered well to hepatocytes by direct attachment to galactose receptors. In the present study, the efficacy of this agent for attenuating hepatic warm IR injury was investigated using the pig model. METHODOLOGY: After 45-min clamping of the hepatic artery and portal vein, pigs were divided into 3 groups according to the following treatments. Ten milliliters of normal saline in Group 1 (n=5), 10,000 units/kg of native SOD in Group 2 (n=5) and 10,000 units/kg of Gal-SOD in Group 3 (n=5) were given just prior to hepatic reperfusion. Liver function including clearance of total bile acid (TBA) and hyaluronic acid (HA) was investigated. Lipid peroxidase of the liver tissue (LPO) and histological findings were examined. In addition, survival rates of the pigs in each group were evaluated. RESULTS: The survival rates at the 7th day after the operation were 60%, 80%, 100% in Groups 1, 2 and 3, respectively. Liver function tests, clearance of TBA and HA, and LPO levels were significantly improved in Groups 3 over findings in Groups 1 and 2. Congestion of hepatic tissues and vacuolization of hepatocytes in Group 3 were less than those in Groups 1 and 2. These results suggested that oxygen-derived free radicals were scavenged by Gal-SOD and IR injury was attenuated. CONCLUSIONS: A safe and novel agent, Gal-SOD has a protective effect against hepatic warm IR injury.

Late-onset biliary leakage after laparoscopic cholecystectomy using laparoscopic coagulating shears. Report of a case.

We recently encountered a rare case of late-onset biliary leakage after laparoscopic cholecystectomy using laparoscopic coagulating shears (LCS). The patient was a 49-year-old Japanese man who had undergone a laparoscopic cholecystectomy at Hamamatsu Medical Center after a diagnosis of cholecystolithiasis associated with localized adenomyomatosis. The cystic duct and the cystic artery were closed using LCS instead of metal endoclips. The postoperative course was uneventful, and the patient was discharged on the 4th operative day. However, on the 7th day after the surgery, the patient developed severe upper abdominal pain and was readmitted to our center with the diagnosis of a late biliary leakage, which was confirmed by an endoscopic retrograde cholangiogram. We then treated the leak successfully with endoscopic nasobiliary drainage.

Whole genome sequencing of meticillin-resistant Staphylococcus aureus.

BACKGROUND: Staphylococcus aureus is one of the major causes of community-acquired and hospital-acquired infections. It produces numerous toxins including superantigens that cause unique disease entities such as toxic-shock syndrome and staphylococcal scarlet fever, and has acquired resistance to practically all antibiotics. Whole genome analysis is a necessary step towards future development of countermeasures against this organism. METHODS: Whole genome sequences of two related S aureus strains (N315 and Mu50) were determined by shot-gun random sequencing. N315 is a meticillin-resistant S aureus (MRSA) strain isolated in 1982, and Mu50 is an MRSA strain with vancomycin resistance isolated in 1997. The open reading frames were identified by use of GAMBLER and GLIMMER programs, and annotation of each was done with a BLAST homology search, motif analysis, and protein localisation prediction. FINDINGS: The Staphylococcus genome was composed of a complex mixture of genes, many of which seem to have been acquired by lateral gene transfer. Most of the antibiotic resistance genes were carried either by plasmids or by mobile genetic elements including a unique resistance island. Three classes of new pathogenicity islands were identified in the genome: a toxic-shock-syndrome toxin island family, exotoxin islands, and enterotoxin islands. In the latter two pathogenicity islands, clusters of exotoxin and enterotoxin genes were found closely linked with other gene clusters encoding putative pathogenic factors. The analysis also identified 70 candidates for new virulence factors. INTERPRETATION: The remarkable ability of S aureus to acquire useful genes from various organisms was revealed through the observation of genome complexity and evidence of lateral gene transfer. Repeated duplication of genes encoding superantigens explains why S aureus is capable of infecting humans of diverse genetic backgrounds, eliciting severe immune reactions. Investigation of many newly identified gene products, including the 70 putative virulence factors, will greatly improve our understanding of the biology of staphylococci and the processes of infectious diseases caused by S aureus.

Selective importation of RNA into isolated mitochondria from Leishmania tarentolae.

All mitochondrial tRNAs in kinetoplastid protozoa are encoded in the nucleus and imported from the cytosol. Incubation of two in vitro-transcribed tRNAs, tRNA(Ile)(UAU) and tRNA(Gln)(CUG), with isolated mitochondria from Leishmania tarentolae, in the absence of any added cytosolic fraction, resulted in a protease-sensitive, ATP-dependent importation, as measured by nuclease protection. Evidence that nuclease protection represents importation was obtained by the finding that Bacillus subtilis pre-tRNA(Asp) was protected from nuclease digestion and was also cleaved by an intramitochondrial RNase P-like activity to produce the mature tRNA. The presence of a membrane potential is not required for in vitro importation. A variety of small synthetic RNAs were also found to be efficiently imported in vitro. The data suggest that there is a structural requirement for importation of RNAs greater than approximately 17 nt, and that smaller RNAs are apparently nonspecifically imported. The signals for importation of folded RNAs have not been determined, but the specificity of the process was illustrated by the higher saturation level of importation of the mainly mitochondria-localized tRNA(Ile) as compared to the level of importation of the mainly cytosol-localized tRNA(Gln). Furthermore, exchanging the D-arm between the tRNA(Ile) and the tRNA(Gln) resulted in a reversal of the in vitro importation behavior and this could also be interpreted in terms of tertiary structure specificity.

RseB binding to the periplasmic domain of RseA modulates the RseA:sigmaE interaction in the cytoplasm and the availability of sigmaE.RNA polymerase.

The Escherichia coli sigmaE regulon has evolved to sense the presence of misfolded proteins in the bacterial envelope. Expression of periplasmic chaperones and folding catalysts is under the control of sigmaE RNA polymerase. The N-terminal domain of RseA sequesters sigmaE in the cytoplasmic membrane, preventing its association with core RNA polymerase. The C-terminal domain of RseA interacts with RseB, a periplasmic protein. The relative concentration of sigmaE:RseA:RseB is 2:5:1 and this ratio remains unaltered upon heat shock induction of the sigmaE regulon. Purification from crude cellular extracts yields cytoplasmic, soluble sigmaE RNA polymerase as well as membrane sequestered sigmaE.RseA and sigmaE.RseA.RseB. RseB binding to the C-terminal domain of RseA increases the affinity of RseA for sigmaE by 2- to 3-fold (Kd 50-100 nM). RseB binds also to the misfolded aggregates of MalE31, a variant of maltose binding protein that forms inclusion bodies in the periplasm. We discuss a model whereby the RseB-RiseA interaction represents a measure for misfolded polypeptides in the bacterial envelope, modulating the assembly of sigmaE RNA polymerase and the cellular heat shock response.

Protective effect of nafamostat mesilate on injury of porcine hepatocytes by human plasma.

Nafamostat mesilate (NM), a protease inhibitor, possesses a cytoprotective effect and inhibits the activation of complement. The present study investigated whether NM has any protective effect against injury of porcine hepatocytes by human plasma in a bioartificial liver support system. Porcine hepatocytes were harvested and seeded at a density of 2 x 10(5) cells on a 35-mm collagen-coated plate in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal calf serum. Twenty-four hours later, the medium was replaced with human plasma with three concentrations of NM between 3.8 x 10(-5) and 3.8 x 10(-4) M and then cultured for 6 h. The viability of porcine hepatocytes, lactate dehydrogenase (LDH) levels, lidocaine clearance, porcine albumin production, and changes in complement (C3) levels were measured. The viability of porcine hepatocytes in human plasma decreased significantly to 37.7 +/- 11.4% of that in DMEM. NM improved the viability of the hepatocytes, lowered the levels of LDH, and increased lidocaine clearance and albumin production in a concentration-dependent manner. The concentrations of C3, the marker of xenogeneic reactions, did not change significantly, indicating that no hyperacute xenogeneic reaction occurred in our series. Together, our results suggested that NM exerts favorable effects on porcine hepatocytes in human plasma through direct effect such as prevention of protease activity in the plasma membrane of porcine hepatocytes rather than inhibition of complement-dependent immunoreactions.

A distinct segment of the sigma 32 polypeptide is involved in DnaK-mediated negative control of the heat shock response in Escherichia coli.

Induction of heat shock proteins in Escherichia coli is caused by a transient increase in the cellular level of sigma 32 (the rpoH gene product), a protein required for transcription of heat shock genes. Both increased synthesis and stabilization of sigma 32 contribute to the increase in sigma 32. We previously showed that heat-induced translation of sigma 32-beta-galactosidase fusion protein encoded by an rpoH-lacZ gene fusion was mediated by an mRNA secondary structure formed between two 5'-proximal segments (A and B) of rpoH coding sequence spanning some 200 nt. We now report that a portion of the sigma 32 polypeptide that corresponds to further downstream (designated region C) is involved in the DnaK-mediated negative control resulting in the shutoff of heat-induced synthesis and degradation of fusion protein. Gene fusions carrying the 5' half (433 nt) or more of the rpoH coding sequence exhibited normal shutoff of synthesis, and the fusion proteins produced were very unstable, like authentic sigma 32; both the shutoff of synthesis and the instability of protein were markedly affected by the dnaK and dnaJ mutations. In contrast, gene fusions carrying < or = 364 nt (lacking region C) and a fusion carrying most of the rpoH sequence but with a frameshift mutation specifically affecting region C exhibited little or no shutoff and produced stable proteins. These results indicate that a distinct segment of sigma 32 plays a critical role in the negative feedback control of sigma 32. The control may be exerted during or after completion of sigma 32 synthesis mediated by interaction between nascent or mature sigma 32 and DnaK/DnaJ proteins.

Heat induction of sigma 32 synthesis mediated by mRNA secondary structure: a primary step of the heat shock response in Escherichia coli.

Induction of heat shock proteins following transfer of E. coli cells from 30 degrees C to 42 degrees C depends on rapid accumulation of sigma 32, a minor sigma factor specifically required for transcription of heat shock genes. The synthesis of sigma 32 is induced by enhancing translation of its mRNA transcribed from the rpoH (htpR) gene. We previously showed that the translational control of rpoH-lacZ gene fusion is mediated by two cis-acting rpoH coding regions presumably involving mRNA secondary structure. To further examine this model, we constructed and analyzed a set of gene fusions carrying base substitution(s) or internal deletions within rpoH, including constitutive mutations predicted to destroy the mRNA secondary structure and compensatory second-site mutations that may restore the secondary structure. The results demonstrate that base pairings between the translation initiation region of some 20 nucleotides and part of the internal complementary sequences are critical for maintaining repression during steady-state growth and for modulating heat-induced synthesis of sigma 32-beta-galactosidase fusion protein upon temperature upshift. Furthermore, some of the compensatory mutations resulted in super-repressed (non-inducible) phenotypes, suggesting that the heat induction depends on a specific nucleotide sequence(s) as well as the mRNA secondary structure within the 5'-proximal regulatory segment of rpoH coding region.

Interplay of two cis-acting mRNA regions in translational control of sigma 32 synthesis during the heat shock response of Escherichia coli.

When Escherichia coli cells are transferred from 30 degrees C to 42 degrees C, transcription from specific promoters recognized by RNA polymerase containing sigma 32 (the rpoH gene product) is transiently activated, resulting in induction of heat shock proteins. Transcription from heat shock promoters is activated by an increased cellular concentration of sigma 32 due to enhanced synthesis and stabilization. We have constructed and examined the expression of mutant derivatives (deletions and base substitutions) of rpoH-lacZ gene fusion. Synthesis of a sigma 32-beta-galactosidase fusion protein was found to be regulated at the translational level involving two distinct 5'-proximal rpoH coding regions. A small region immediately downstream of the initiation codon is required for potentially high-level expression, whereas a much larger internal region is required for thermal regulation--namely, repression at low temperature or nonstress conditions. The two mRNA regions act as positive and negative cis elements, respectively, in controlling rpoH translation. We propose that an interplay between these RNA regions involving secondary structure formation is important in regulating translation initiation and that transient disruption of secondary structure represents a primary step of the heat shock response.

Regulation of the heat shock response in E coli: involvement of positive and negative cis-acting elements in translation control of sigma 32 synthesis.

When cells of E coli are transferred from 30 to 42 degrees C, the cellular level of sigma 32 (rpoH gene product) increases transiently, resulting in increased transcription of a set of heat shock genes. Both increased synthesis and increased stability of sigma 32 contribute to transient accumulation of sigma 32. Evidence suggests that synthesis of sigma 32 is enhanced primarily at the level of rpoH translation. We have constructed and examined the expression of deletion derivatives of rpoH-lacZ gene fusion at 30 degrees C and after shift to 42 degrees C. It was revealed that two cis-acting sequences within the rpoH coding region are involved in thermal regulation of fusion protein synthesis. One region immediately downstream of the initiation codon is required for high level expression, whereas the other internal region is involved in repression at low temperature. Thus, these regions act as positive and negative cis-elements in thermal regulation of rpoH translation. The rpoH mRNA secondary structure model suggesting an interplay between the two regions has been proposed to account for the temperature-induced sigma 32 synthesis as a primary cellular response to the heat shock stress.

The organization and evolution of transfer RNA genes in Mycoplasma capricolum.

The genes for presumably all the tRNA species in Mycoplasma capricolum, a derivative of Gram-positive eubacteria, have been cloned and sequenced. There are 30 genes encoding 29 tRNA species. This number is the smallest in all the known genetic systems except for mitochondria. The sequences of 9 tRNA genes of them have been previously reported (1-3). Twenty-two genes are organized in 5 clusters consisting of nine, five, four and two genes (2 sets), respectively. The other eight genes exist as a single transcription unit. All the tRNAs are encoded each by a single gene, except for the occurrence of two tRNA(Lys)(TTT) genes. The arrangement of tRNA genes in the 9-gene cluster, the 5-gene cluster, the 4-gene cluster and one of the 2-gene clusters reveals extensive similarity with a part of the 21-tRNA gene cluster and/or the 16-tRNA gene cluster in Bacillus subtilis, respectively. The results suggest that the present M. capricolum tRNA genes have evolved from large tRNA gene clusters in the ancestral Gram-positive bacterial genome common to M. capricolum and B. subtilis, by discarding genes for redundant as well as non-obligate tRNAs, so that all the codons may be translated by as small a number of tRNAs as possible.

[Analysis of GM2 and anti-GM2 antibody in normal pregnancy].

Oncofetal antigen immunogenic (OFA-I), an antigen common to the fetal brain and some malignant tumors (derived from ectoderm), is immunogenic in man, and anti-OFA-I antibody appears in the sera of not only malignant patients but also pregnant women. Biochemical analysis revealed that OFA-I is ganglioside GM2 and GD2. In the present study, GM2 and anti-GM2 antibody were investigated as to the kinetics in pregnant sera and the tissue localization in placenta and decidua. The results obtained were as follows: 1. The concentration of GM2 was 76.7 pmol/ml (mean) in pregnant sera and 10.8 pmol/ml (mean) in cord sera. GM2 was not measurable in the sera of non-pregnant women. 2. GM2 was detected by thin layer chromatogram-immunostaining methods in the gangliosides extracted from decidua and term placenta. GM2 was, however, undetected from villi at an early stage. 3. In immunohistological analysis for GM2, positive findings were observed in decidua and in villous stroma of term placenta. In contrast, no distinct positive findings were observed in trophoblast at any stage. 4. Anti-GM2 antibody appeared in pregnant sera at an early stage and was detectable up to the 5th day postpartum.