Amino-terminal sequence, synthesis, and membrane insertion of glycoprotein B of herpes simplex virus type 1.

Glycoprotein B (gB) was purified from cells infected with two strains (KOS and F) of herpes simplex virus type 1. Determination of amino acid sequence at the NH2 termini revealed, by comparison with amino acid sequence deduced from previously published nucleotide sequence, that gB is made with a cleavable signal sequence of 29 or 30 amino acids, depending on the virus strain. Analysis of gB translated in vitro in the presence and absence of membranes showed that gB is inserted into membranes and glycosylated cotranslationally; a large portion of the gB polypeptide made in vitro is protected from proteolysis by membranes; the large protected fragment carries N-linked carbohydrate and is probably the NH2 terminus based on locations of signals for the addition of N-linked carbohydrate; and the size of the protected fragment is 93 kilodaltons (kDa) for gB made in vitro and associated with dog pancreas membranes, whereas both 93- and 98-kDa protected fragments can be detected for gB made in vivo. These last results are consistent with a previous proposal that gB may traverse the membrane three times.     

Atrial peptide inactivation by rabbit-kidney brush-border membranes

Atriopeptin (AP) 24, containing amino acids Ser103-Tyr126 of the carboxy-terminal portion of the atrial natriuretic peptide prohormone, was degraded rapidly by rabbit kidney brush border membranes. The rate of degradation of AP24 measured by the loss of vasorelaxant activity followed a similar time course to the decrease in peptide peak area measured by high-performance liquid chromatography. Inactivation of AP24 produced peptide fragments which were separated by HPLC. The major products were purified individually and their peptide sequences determined. Results indicate that AP24 was proteolytically cleaved at three peptide bonds: Ser103-Ser104, Cys105-Phe106 and Ser123-Phe124. des-Ser103-AP24 had similar vasorelaxant activity to AP24, while AP24 cleaved at Cys105-Phe106 was inactive. Regarding the proteolytic cleavage at Ser123-Phe124, there was an accumulation of the C-terminal tripeptide, Phe-Arg-Tyr, only at the later time points of the incubation. Degradation experiments were repeated with an amino- and carboxy-terminal protected peptide, acetyl-AP24-amide. Peptide sequence analysis of the major degradation products of this peptide revealed that the critical peptide bond cleaved was Cys105-Phe106. We conclude that the Cys-Phe peptide bond renders atrial peptides highly susceptible to proteolysis by renal brush border membranes, resulting in inactivation.     

Direct Evidence for Changes in the Resistance of Legume Root Nodules to O2 Diffusion

Indirect evidence suggests that legumes can adjust rapidly theresistance of their root nodules to O₂ diffusion. Here we describeexperiments using O₂ specific micro-electrodes and dark fieldmicroscopy to study directly the operation of this diffusionbarrier. The O₂ concentration sensed by the electrode decreasedsharply in the region of the inner cortex and was less than1.0 mmol m^–3 throughout the infected tissue in nodulesof both pea (Pisum sativum) and french bean (Phaseolus vulgaris).In a number of experiments the ambient O₂ concentration wasincreased to 40% while the electrode tip was just inside theinner cortex. In 13 out of 21 cases the O₂ concentration atthis position either remained low and unchanged or increasedirreversibly to near ambient values. In the remaining casesthe O₂ concentration increased after 1 to 2.5 min and then decreasedto its former value. These results are ascribed to an increasein resistance of the barrier in response to increased O₂ fluxinto the nodule. It was shown microscopically that air spacesboth at the boundary between the infected zone and the innercortex, and within the infected zone started to disappear 3min after nodules were exposed to high ambient O₂ concentrationsand had disappeared completely after 8 min. These spaces werenot changed by exposure of the nodule for 10 min to either N₂or air. Key words: Oxygen, root nodules, air spaces     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Delayed primary reconstruction of subtotal nail bed loss using a split-thickness nail bed graft on decorticated bone

Split-thickness nail bed grafting is the accepted method of treatment for injuries involving loss of nail bed tissue. The nail bed of the great toe may be used without donor-site morbidity, and nail bed grafting may be combined with other procedures for fingertip reconstruction. A case of fingertip avulsion injury with loss of the nail plate, nail bed, and periosteum over the exposed distal phalanx of the thumb was reconstructed by a split-thickness nail bed graft placed directly on granulating decorticated bone. The length, appearance, and function of the injured dominant thumb were preserved.     

A 6-year experience with immediate reconstruction after mastectomy for cancer

A 6-year retrospective review is presented of 185 patients who underwent immediate reconstruction of the breast at the same operation as mastectomy for carcinoma. The patients were treated at two institutions under similar protocols of patient selection, surgical technique, and postoperative care. A detailed evaluation is presented from both the oncologic and surgical points of view. The data support the conclusion that immediate reconstruction of the breast does not alter survival or cancer recurrence rates and does not interfere with the treatment of primary or secondary disease. A low incidence of significant surgical complications is also detailed. Combined with previous reports answering psychological concerns about this mode and timing of reconstruction, this review offers significant reassurance about the overall safety of immediate reconstruction. The authors therefore recommend immediate reconstruction of the breast as a safe treatment option for the woman facing mastectomy.     

Mapping of the structural gene for the herpes simplex virus type 2 counterpart of herpes simplex virus type 1 glycoprotein C and identification of a type 2 mutant which does not express this glycoprotein

The gene encoding glycoprotein F (gF) of herpes simplex virus type 2 (HSV-2) was mapped to the region of the viral genome from 0.62 to 0.64 map units. This region is colinear with, and partially homologous to, the region of the HSV-1 genome previously shown to encode gC. Mapping of the gF gene was done by insertion of HSV-2 DNA fragments into the thymidine kinase gene of an HSV-1 virus and screening of the resultant recombinant viruses for the expression of gF. In this way, DNA sequences necessary for the expression of gF in infected cells were also delimited. Because several plaque morphology mutants (syncytial mutants) of HSV-1 have previously been shown to be gC-, a syncytial mutant of HSV-2 (GP) was tested for the expression of gF. It was found to be gF-, indicating that gF is not essential for replication of HSV-2 in cell culture, just as gC is not essential for replication of HSV-1. This result also suggests that the gF- and gC- phenotypes are related in the same, as yet undefined, way to the expression of a syncytial marker. A proposal to change the name of HSV-2 gF to gC (gC-2) is discussed.     

Characterization of a herpes simplex virus type 2 75,000-molecular-weight glycoprotein antigenically related to herpes simplex virus type 1 glycoprotein C.

Evidence is presented that the herpes simplex virus type 2 glycoprotein previously designated gF is antigenically related to herpes simplex virus type 1 gC (gC-1). An antiserum prepared against type 1 virion envelope proteins immunoprecipitated gF of type 2 (gF-2), and competition experiments revealed that the anti-gC-1 component of the antiserum was responsible for the anti-gF-2 cross-reactivity. An antiserum prepared against fully denatured purified gF-2, however, and three anti-gF-2 monoclonal antibodies failed to precipitate any type 1 antigen, indicating that the extent of cross-reactivity between gC-1 and gF-2 may be limited. Several aspects of gF-2 synthesis and processing were investigated. Use of the enzymes endo-beta-N-acetylglucosaminidase H and alpha-D-N-acetylgalactosaminyl oligosaccharidase revealed that the fully processed form of gF-2 (about 75,000 [75K] apparent molecular weight) had both complex-type N-linked and O-linked oligosaccharides, whereas newly synthesized forms (67K and 69K) had only high-mannose N-linked oligosaccharides. These last two forms were both reduced in size to 54K by treatment with endo-beta-N-acetylglucosaminidase H and therefore appear to differ only in the number of N-linked chains. Neutralization tests and radioiodination experiments revealed that gF-2 is exposed on the surfaces of virions and that the 75K form of gF-2 is exposed on cell surfaces. The similarities and differences of gF-2 and gC-1 are discussed in light of recent mapping results which suggest collinearity of their respective genes.     

Modified Agglutination Test for Pasteurella tularensis

Several modifications in technique were incorporated into the standard agglutination test for Pasteurella tularensis. Reciprocal shaking of all tubes in a Kahn shaker was introduced to increase the rate of agglutination and quantity of agglutinated cell mass, making it possible to report preliminary results within 4 hr. Increased incubation time at a higher temperature was used to favor the rate of agglutination. A serum control for each serum tested was necessary to detect false positive tests. Finally, a verification procedure with 5% NaCl used as the diluent was instituted to prevent these false positive reactions.