Keyhole approach for repair of congenital duodenal obstruction.

BACKGROUND: We report on our experience of repair of congenital duodenal obstruction using a circumumbilical incision. The aim of this report is to describe how a Bianchi approach provides a safe and invisible alternative to transverse abdominal incision for the repair of duodenal atresia. METHODS: Between January 2005 and December 2009, we treated 13 cases with congenital duodenal obstruction using a circumumbilical incision (Group I) and 14 cases with this condition repaired using a standard transverse right upper abdominal incision (Group II). Surgical procedures included a diamond-shaped duodenoduodenostomy as originally described by Kimura and standard duodenal web excision. The circumumbilical incision utilized at our institution is a classic Bianchi procedure. The 2 groups were compared with regard to patient demographics, operative reports and postoperative outcomes. RESULTS: There were no differences in preoperative parameters such as gestational age, age at surgery, or body weight at operation between the 2 groups. The circumumbilical cohort and transverse incision cohort had similar rates of congenital anomalies (61.54% vs. 64.29%), Kimura diamond-shaped anastomosis (61.54% vs. 64.29%) with only a slight female predominance in Group I. The mean operating time in Group I was 65.0 min while mean duration of the operation in Group II was 64.64 min. The difference between groups was statistically not significant (p>0.05). The mean time to full enteral feeding for patients with an umbilical incision was significantly shorter (p<0.0001) compared to patients with a standard incision (6.92 days vs. 11.86 days). Mean postoperative hospital stay was longer for patients in Group II (19.71 days vs. 12.38 days; p<0.0001). The postoperative course was uneventful for all patients. There were no intra- or postoperative complications. CONCLUSION: We report on a first series comparing umbilical and transverse right upper abdominal incision for the treatment of congenital duodenal obstruction. Our results suggest that an umbilical incision offers all the benefits of a minimal access approach, including earlier feeding and shorter times to discharge. We consider our approach an intermediate step, with laparoscopy likely to become the "gold standard" for the treatment of congenital duodenal obstruction.

The origin of atmospheric oxygen on Earth: the innovation of oxygenic photosynthesis.

The evolution of O(2)-producing cyanobacteria that use water as terminal reductant transformed Earth's atmosphere to one suitable for the evolution of aerobic metabolism and complex life. The innovation of water oxidation freed photosynthesis to invade new environments and visibly changed the face of the Earth. We offer a new hypothesis for how this process evolved, which identifies two critical roles for carbon dioxide in the Archean period. First, we present a thermodynamic analysis showing that bicarbonate (formed by dissolution of CO(2)) is a more efficient alternative substrate than water for O(2) production by oxygenic phototrophs. This analysis clarifies the origin of the long debated "bicarbonate effect" on photosynthetic O(2) production. We propose that bicarbonate was the thermodynamically preferred reductant before water in the evolution of oxygenic photosynthesis. Second, we have examined the speciation of manganese(II) and bicarbonate in water, and find that they form Mn-bicarbonate clusters as the major species under conditions that model the chemistry of the Archean sea. These clusters have been found to be highly efficient precursors for the assembly of the tetramanganese-oxide core of the water-oxidizing enzyme during biogenesis. We show that these clusters can be oxidized at electrochemical potentials that are accessible to anoxygenic phototrophs and thus the most likely building blocks for assembly of the first O(2) evolving photoreaction center, most likely originating from green nonsulfur bacteria before the evolution of cyanobacteria.

Possible relationship of mechanisms of Mn2+ oxidation and formation of plant photosystem II manganese cluster.

The mechanisms of Mn2+ cation oxidation in alkaline, neutral and slightly acidic media were studied. In all cases, the Mn2+ oxidation resulted in the formation of the structure[see text]. The formal resemblance and differences in the Mn2O3 structure and Klein's model of the Mn cluster of PS II were noted. The necessity of the primary ligation of Mn2+ cations was discussed for both the decrease in the Mn2+ oxidation potential and the stability of the Mn2O3 structure. It was supposed that Mn2O3 is an initial block for the assembly of the inorganic core of the photosynthetic water-oxidizing complex.

Identical amino acid sequence of the aroA(G) gene products of Bacillus subtilis 168 and B. subtilis Marburg strain.

A DNA fragment containing the aroA(G) gene of Bacillus subtilis 168, encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase-chorismate mutase, was cloned and sequenced. The N-terminus of the protein encoded by aroA(G) showed homology with chorismate mutase encoded by aroH of B. subtilis and with the chorismate mutase parts of proteins encoded by the pheA and tyrA genes of Escherichia coli. The C-terminus of the aroA(G) product has sequence similarity with 3-deoxy-D-manno-octulosonate 8-phosphate synthase of E. coli. It was shown that the proteins encoded by the aroA(G) gene of B. subtilis 168 and the aroA gene of B. subtilis ATCC 6051 Marburg strain are identical, so the observed differences in DAHP synthase activity from these two strains must result from other changes.

Photoreduction of silicomolybdate in chloroplasts by agents accelerating the deactivation reactions of the water-oxidizing system.

Uncouplers of photosynthetic phosphorylation, CCCP, TTFB and PCP, inhibited light-induced O2 evolution in the Hill reaction with SiMo (I50 approximately 20, 3 and 45 microM, respectively), but only insignificantly diminished SiMo photoreduction by pea chloroplasts. The same properties were exhibited by the ADRY agent ANT2p. CCCP, TTFB and PCP are oxidizable compounds with redox potentials of +1.17, +1.18 and +1.09 V (pH 6.0), as determined by cyclic voltammetry. Similarly to NH2OH, the tested uncouplers can apparently serve as electron donors for photosystem II.

Densovirus of Aedes aegypti as an expression vector in mosquito cells.

We have constructed an infectious DNA clone containing the genome of Aedes aegypti densovirus (AeDNV) in a bacterial plasmid. When this clone was transfected into Aedes albopictus C6/36 mosquito cells, the AeDNV genome rescued from the plasmid and replicated as the wild-type virus. To investigate the cloned virus as an expression vector, the reporter gene encoding beta-galactosidase (beta-gal) was inserted into four large open reading frames (ORF) observed in the AeDNV genome. When these recombinant constructs were transfected into Aedes albopictus C6/36 cells, the beta-gal was expressed efficiently from the right ORF (encoding capsid proteins, Vps) and the mid ORF (encoding putative nonstructural protein 2). A low level of expression was found from the left ORF (encoding nonstructural protein 1, NS1), and no expression was detected from the ORF observed on the minus strand of the AeDNV genome. The expression from the right, mid, and left ORFs can be trans-activated with NS1. A putative nuclear targeting sequence observed in the N-terminus of the AeDNV Vps is presumed to be responsible for transport of the chimeric beta-gal into nucleus. The recombinant AeDNV genomes (carrying the beta-gal gene) supplied with the AeDNV capsid proteins can be packaged into infectious transducing particles. Our results indicate that the genome of AeDNV can serve as a vector for delivery and expression of foreign genes in mosquito cells with subsequent targeting of the product to the desired cell compartment.

Crystal structure of the holotoxin from Shigella dysenteriae at 2.5 A resolution.

Shigella dysenteriae is the pathogen responsible for the severe form of dysentery in humans. It produces Shiga toxin, the prototype of a family of closely related bacterial protein toxins. We have determined the structure of the holotoxin, an AB5 hexamer, by X-ray crystallography. The five B subunits form a pentameric ring, encircling a helix at the carboxy terminus of the A subunit. The A subunit interacts with the B pentamer via this C-terminal helix and a four-stranded mixed beta-sheet. The fold of the rest of the A subunit is similar to that of the A chain of the plant toxin ricin; both are N-glycosidases. However, the active site in the bacterial holotoxin is blocked by a segment of polypeptide chain. These residues of the A subunit would be released as part of the activation mechanism of the toxin.

Characterization of comE, a late competence operon of Bacillus subtilis required for the binding and uptake of transforming DNA.

The binding and transport of DNA by competent Bacillus subtilis requires the assembly of a specialized apparatus. We present here the characterization of comE, an operon under competence control that is required for both DNA binding to the competent cell surface, and for uptake. comE contains three open reading frames (ORF1-3) read in the forward direction, preceded by a long untranslated leader sequence and an apparent E sigma A promoter. A minor promoter also is responsible for transcription of ORF2 and -3. A transcript containing a single ORF is produced in the reverse direction. The reverse ORF overlaps ORF1 and the untranslated comE leader. The comE transcript is present at a very low level during growth and at an elevated level in stationary-phase cells. Conversely, the reverse transcript is present during exponential growth and disappears during stationary phase. The reverse ORF resembles prokaryotic and eukaryotic pyrroline-5'-carboxylate reductases, while ORF2 is similar to several dCMP deaminases. ORF1 and ORF3 are predicted to be integral membrane proteins. The latter is specifically required for DNA uptake but not for binding.

Purification and crystallization of Shiga toxin from Shigella dysenteriae.

The protein toxin produced by Shigella dysenteriae consists of one enzymatically active A subunit of 293 amino acid residues and five B subunits of 69 amino acid residues that are involved with cell attachment. The holotoxin has been purified by blue Sepharose and chromatofocusing column chromatography. Two crystal forms of purified holotoxin have been grown by vapor diffusion. One grows as fine needles, hexagonal in cross-section, which do not diffract well enough to characterize crystallographically. The second grows as thin plates that diffract to at least 3 A resolution. Their space group is P2(1)2(1)2(1) with unit cell dimensions of a = 132.0 A, b = 146.0 A and c = 82.5 A. The asymmetric unit of the crystals is likely to contain two AB5 units.

Sex pheromones of male yellowfin Baikal sculpin (Cottocomephorus grewingki): Isolation and chemical studies.

Three components of the male yellowfin Baikal sculpin pheromonal signal have been isolated from urine by diethyl ether extraction, thinayer chromatography (TLC), and high-performance liquid chromatography (HPLC). Using mass spectrometry, we have identified two of them as testosterone (T) and 11ß-hydroxytestosterone (11HT). These steroids are synthesized in testes during full spermatogenesis, and they are excreted in male urine with milt. The third component is not a steroid. It is more likely to be a polyene alcohol (farnesol). 2Z,6E-Farnesol possesses behavioral activity.

Nucleotide sequence and genomic organization of Aedes densonucleosis virus.

Over 99% of the genome of Aedes densonucleosis virus was determined. Two types of the viral DNA were found that differ only in four nucleotides (nt) in the 5' noncoding part and whose sizes are 4009 nt (more copious) and 4012 nt, respectively. Both 146 nt at the 3' end and 164 nt at the 5' end could assume a similar T-shaped structure; but unlike the adeno-associated virus, Aedes DNA has a unique primary DNA sequence at each terminus. However, the crossarms of these structures are built of the same sequences. An imperfect direct repeat of 34 nt was observed in the 5' noncoding part. The plus strand has three large open reading frames (ORF): a left ORF, a right ORF, and a mid ORF (within the left ORF). The left ORF codes for the nonstructural protein NS-1 (97.5K) featured by an NTP-binding domain, and the right ORF encodes the both capsid proteins, the smaller of which (39K) is supposed to be derived from the larger one (40.5K) by proteolytic cleavage. There is also an ORF in the minus strand. The putative polypeptide coded by this ORF is extremely hydrophobic.

Hormonal sensitivity of adenylate cyclase incorporated in proteoliposomes.

Proteoliposomes containing adenylate cyclase (AC) from rabbit heart ventricles were obtained using a novel reconstitution procedure from solubilized state. The enzyme preparation can be stimulated by 5'guanylylimidodiphosphate (GppNHp) and NaF, but not by isoproterenol. Hormonal responsiveness of AC is restored by either isoproterenol trapped by the proteoliposomes during the reconstitution or pretreatment of proteoliposomes with alamethicin. GTP in the presence of alamethicin decreases the affinity of beta-adrenoceptors to the agonist, thus confirming that the properties of reconstituted AC system do not differ from the native one. It is demonstrated that the degree of AC activation by isoproterenol depends strongly on the beta-adrenoceptors content in the proteoliposomes, which in turn can be changed artificially in the process of reconstitution. The described reconstitution technique might be a useful tool for investigating the role of component stoichiometry in the functioning of hormone-regulated AC-system.

Toxic and protective effects of antioxidants in biomembranes.

Natural and synthetic antioxidants (AOs) are widely used as stabilizers of biomembranes against lipid peroxidation (LPO). Natural AOs (tocopherols, ubiquinols) containing hydrocarbon "tails" do not disturb the membrane lipid bilayer. Synthetic AOs devoid of hydrocarbon radicals may perturb the lipid bilayer. It was shown that AOs devoid of hydrocarbon tails (butylated hydroxytoluene, 2,2,5,7,8-pentamethyl-6-hydroxychromane) exerted toxic effects on erythrocyte membranes (induced hemolysis), on sarcoplasmic reticulum membranes (inhibited Ca2+-transport) and on platelet membranes (initiated Ca2+-dependent aggregation) in vitro. These AOs are the substrates of cytochrome P-450, and underwent oxidative hydroxylation. This suggests that they have short half-life times in biomembranes and in the organism. Antioxidants with hydrocarbon tails, are hydroxylated at very low rates and are slowly excreted. Antioxidants devoid of hydrocarbon tails, are 10-20 fold more potent LPO inhibitors than the corresponding AOs with hydrocarbon tails. The strategy of AOs application for long and short-term stabilization of biomembranes against LPO in vivo is discussed.

Heparin induces Ca2+ release from the terminal cisterns of skeletal muscle sarcoplasmic reticulum.

Using a Ca2+-selective electrode and the chlorotetracycline fluorescence technique, the effects of heparin on Ca2+ transport in the sarcoplasmic reticulum (SR) of skeletal muscles in the absence of oxalate were investigated. It was shown that heparin (0.5-10 micrograms/ml) causes a rapid release of 40-50 nmol Ca2+/mg protein from the terminal cistern SR vesicles bound to 130-150 nmol/mg protein of Ca2+ in the presence of ATP. However, heparin has practically no effect on the longitudinal cistern fraction of SR. The effects of heparin can be prevented by ruthenium red. No influence of heparin is observed in the case of the Ca2+-induced release of Ca2+ from the terminal cisterns. When the Ca2+ release is induced by heparin, no Ca2+-induced release of Ca2+ takes place.

A comparative study on the in vitro translation products of individual RNAs from two-, three-, and four-component strains of barley stripe mosaic virus.

Passaging through plants of the three-component barley stripe mosaic virus (BSMV) strain Norwich (Norwich III) yielded a two-component isolate (Norwich II). A study was made of the sets of polypeptides translated in a rabbit reticulocyte and wheat embryo cell-free systems from individual RNAs of (1) the natural three-component strain Norwich III, (2) a two-component isolate derived from the former (Norwich II), and (3) an isolate intermediate between the three- and two-component ones, Norwich;. Translation of RNA 1 from all three variants of the Norwich strain in vitro yields a single major product with a molecular weight (Mr) of 120,000 (p120). RNA 2 from Norwich III in vitro produces two polypeptides: the viral coat protein (p23) and, in certain ionic conditions, a polypeptide of 25,000 M(r) (p25). RNA 3 from Norwich III is translated into a protein with Mr of 75,000 (p75). Conversion of Norwich III into Norwich II is accompanied by the changes in the coding specificity of RNA 2; beside the coat protein and p25, a protein of 85,000 M(r) (p85) is formed upon its translation-a feature characteristic of RNA 2 of the naturally occurring bipartite BSMV strains (Russian, type). With the Norwich(i) variety, which is marked by a significantly reduced portion of RNA 3 in the total virion RNA preparation, RNA 2 in addition to the coat protein, p25, and p85 directs the synthesis of another product with M(r) of about 78,000. Thus, in conversion of the three-component BSMV into a two-component one, the loss of RNA 3 is concomitant with the actuation in RNA 2 of a sequence coding for p85. RNAs 1-3 of the quadripartite Argentina Mild (AM) BSMV strain code in vitro for the same polypeptides as RNAs 1-3 of Norwich III. AM RNA 4 is translated as a monocistronic template into a polypeptide with Mr of 55,000 (p55). Amino acid sequences of p85, p75, and p55 are shown to overlap among these polypeptides but not to appreciably overlap with p120 sequences. Data presented here allowed a tentative structure to be proposed for genome of two-, three-, and four-component BSMV strains.

Nucleotide sequence analysis of the cloned salmon preproinsulin cDNA.

A cDNA library was constructed using polyadenylated RNA from salmon (Oncorhynchus keta) Brockmann bodies, plasmid vector pBR322, and in vitro recombinant DNA techniques. Insulin-related clones were identified with a cDNA probe generated from the same RNA and enriched for insulin sequences. Two recombinants were shown to contain the nucleotide sequence of the entire coding region and parts of the 5' and 3' untranslated regions. The salmon preproinsulin mRNA is about 760 nucleotides long, 315 of which code for the protein, while about 190 and 200 nucleotides belong to the 5' and 3' flanking regions, respectively. Comparison of the nucleotide sequences of salmon insulin mRNA with those from other species reveals that sequence conservation is limited to the regions coding for the B and A peptides and two segments of the signal peptide. The C-peptide region exhibits no significant sequence homology with the C-peptides of other vertebrates. The 5' and 3' untranslated regions of the salmon preproinsulin mRNA are homologous only with the anglerfish mRNA, whereas there is no evident homology with those of birds and mammals. In addition to establishing the sequence of the preproinsulin mRNA, cloned salmon insulin cDNA provides a specific probe for the analysis and isolation of genomic DNA fragments containing insulin genes.

A study of TMV ts mutant Ni2519. III. Location of the reconstitution initiation sites on Ni2519 RNA.

It was suggested in an accompanying paper [Taliansky et al. (1982b), Virology, 117, 000-000] that two reconstitution initiation sites (RIS I and RIS II) were functionally active in RNA of the temperature-sensitive (ts) TMV mutant Ni2519 upon reassembly at a nonpermissive temperature; initiation of reassembly that started at two sites on the Ni2519 RNA molecule resulted in defective (ribonuclease-sensitive) virus particle (DVP) formation. RIS(s) was(were) defined as a segment(s) of Ni2519 RNA protected from the action of ribonuclease T1 by the coat protein within an incomplete nucleoprotein complex (I-NPC) formed upon limited TMV reassembly at nonpermissive (33 degrees and permissive (24 degrees) temperatures. Tl-resistant oligonucleotides protected within I-NPC were finger-printed, sequenced, and assigned to specific regions on the Ni2519 RNA molecule. It was shown that only RIS I was operative on Ni2519 RNA at 24 degrees as well as on A14 (a temperature-resistant strain, the wild type for Ni2519) RNA at both 24 and 33 degrees ; RIS I corresponded to the so-called Oa (origin of assembly) of a common TMV strain previously studied and was located at a distance of about 800 nucleotides from the 3'-end of the RNA molecule. In contrast, two RISs were revealed on Ni2519 RNA assembled at 33 degrees ; of the two RISs operative in this case the first one is identified as RIS I, while the second (RIS II) is located within 300-520 nucleotides from the 3'-end, i.e., within the coat protein gene. The latter corresponds to the so-called SERF (specifically encapsidated RNA fragment) of the RNA of common TMV.

Homology in the 3'-terminal regions of different barley stripe mosaic virus RNA species.

Preparations of [3H]DNA complementary to the individual species of barley stripe mosaic virus (BSMV) RNA (cDNA) about 1000 nucleotides long were obtained using avian myeloblastosis virus reverse transcriptase and oligo(dT)10 primer. These cDNAs were used in kinetic hybridization experiments to study the sequence homology between the 3'-terminal regions (not including 150-200 nucleotides adjacent to the 3'-end) of individual BSMV RNA species. These regions in RNA 2 and 3 are highly homologous and have no sequences in common with the respective region in RNA 1.

Aminoacylation of barley stripe mosaic virus RNA: polyadenylate-containing RNA has a 3'-terminal tyrosine-accepting structure.

Barley stripe mosaic virus (BSMV) RNA which was previously reported to contain poly(A) sequences (Agranovsky et al., 1978) can be specifically esterified with tyrosine in vitro in the presence of an aminoacyl-tRNA synthetase fraction from wheat embryos. All the three RNA components of the BSMV strain with a three-component genome (Norwich) and both RNA components of a two-component strain (Russian) can be tyrosylated. The poly(A)-containing (bound to oligo(dT)-cellulose) and poly(A)-deficient (not bound to oligo(dT)-cellulose) fractions of BSMV RNA display a similar amino acid-accepting ability. The nucleotide sequence which accepts tyrosine is coupled with the intact genomic polyadenylated BSMV RNA. The viral RNA isolated after sucrose density gradient centrifugation under drastic denaturing conditions retains its aminoacylating activity, which suggests that this activity is not due to the presence in a BSMV RNA preparation of a tyrosine tRNA associated with BSMV RNA. Inhibition of aminoacylation of the 3'-oxidized (treated with sodium metaperiodate) BSMV RNA suggests that the tyrosine-accepting structure is localized at the 3' terminus of BSMV RNA molecules. It is shown that segments of different lengths obtained upon random fragmentation can be tyrosylated. The 3'-terminal (tyrosine-accepting) poly(A)+ segments can be isolated. The shortest segments of viral RNA capable of being aminoacylated [i.e., containing both tRNA-like structure and poly(A)] consists of approximately 150-200 nucleotides. The analysis of the oligonucleotides derived from individual BSMV RNA components labeled with (32)P at the 3' end revealed two types of 3'-terminal sequences different from poly(A). It is suggested that a poly(A) sequence is intercalated between a 3'-terminal tyrosine-accepting structure and the 5'-terminal portion of poly(A)+ BSMV RNA.