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The carbon-isotopic composition of shell carbonate is shown to be in isotopic equilibrium with bicarbonate dissolved in the water. By measurement of both 13C and 18O in a series of brackish-water shells a mean growth temperature can be deduced.
The shell-forming glands of Japanese quail fed p,p'-DDT or p,p'-DDE had carbonic anhydrase activity 16 to 19 percent lower than shell glands of quail on a diet free of pesticides.
Molecular recognition based on length was found to occur between oppositely charged pairs of flexible and randomly coiled block copolymers in an aqueous milieu. Matched pairs with the same block lengths of polyanions and polycations exclusively formed even in mixtures with different block lengths. These assemblies of the charged segments with matched chain lengths then formed larger core-shell-type supramolecular assemblies with an extremely narrow size distribution due to the strict phase separation between core- and shell-forming segments.
Cluster spectroscopy, aided by ab initio theory, was used to determine the detailed structure of a complete hydration shell around an anion. Infrared spectra of size-selected O(2)-. (H(2)O)(n) (n = 1 to 4) cluster ions were obtained by photoevaporation of an argon nanomatrix. Four water molecules are required to complete the coordination shell. The simple spectrum of the tetrahydrate reveals a structure in which each water molecule is engaged in a single hydrogen bond to one of the four lobes of the pi* orbital of the superoxide, whereas the water molecules bind together in pairs. This illustrates how water networks deform upon accommodating a solute ion to create a distinct supramolecular species.
Infectious bovine rhinotracheitis virus was isolated from the soft-shelled tick (Ornithodoros coriaceus). Serological identification of the isolated viruses was confirmed by restriction endonuclease digestion of purified virus DNA. These isolations indicate that the soft-shelled tick may be a vector for infectious bovine rhinotracheitis virus. This may be the first reported isolation of mammalian herpesvirus from an arthropod vector.
Frequency distribution patterns of aerobic-facultative microflora, obtained by random selection of macrocolonies from samples of haddock fillets and shucked soft-shelled clams before and after treatment with doses of from 50,000 to 800,000 rad of Co(60) gamma rays, are presented, with analyses and interpretation. The data showed that a decided change occurred in the constitution of the microbial populations of both products: from a mixed gram-negative-gram-positive flora to a predominantly gram-positive flora immediately after irradiation. The great majority of these surviving microorganisms were micrococci, sporeforming bacilli, and certain yeasts, molds, and actinomyces. During storage at refrigeration temperatures above freezing, the microflora changed from the descendants of the more radioresistant gram-positive species to the more prolific gram-negative psychrophilic species that flourish at these low temperatures. Micrococci and gram-positive rods declined somewhat during the rise of the actively proteolytic-lipolytic pseudomonads and related species, but still remained at high enough levels to contribute significantly to the spoilage observed at different times in storage. The eventual spoilage of haddock fillets was characterized by discoloration of the cream-white tissue with water-soluble yellow, green, and red bacterial pigments; degradation of the tissue, by proteolytic and other microbial enzymes, to a watery, flaccid mass; and formation of volatile compounds that smelled putrid, rancid, and generally foul and pungent. Shucked soft-shelled clams displayed a different spoilage pattern, changing to a variegated brown-gray and forming a matted or loose gelatinous mass from which arose stale, acrid, putrid, sulfurous odors. These differences are believed to be attributable to the varied biochemical nature of the tissues involved, the Eh potential within the tissues, the time sequence of microfloral change, and the species and types of microorganisms associated with each product. Staphylococci were present in small numbers in some of the samples tested, but did not appear to be species of public health significance. Gram-negative enteric rods were also encountered, but were considered to be of doubtful public health importance. More detailed investigations, designed to study the effects of Co(60) gamma radiations on such microbes in sea foods, would be useful in assessing the problem further.
Filamentous structures designated Triton shells were obtained from virulent Mycoplasma pneumoniae by treatment with Triton X-100. Monoclonal antibodies directed against M. pneumoniae were used in conjunction with radioimmunoprecipitation and Western blotting to detect immunologically reactive polypeptides in Triton shells. The major adhesin, protein P1, was associated with these structures.
A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.
An in situ biotinylated DNA probe assay was evaluated as an adjunct to anti-cytomegalovirus early nuclear antigen indirect immunofluorescence and cytopathic effect on cytomegalovirus-infected monolayers in shell vial cultures. Viral infection was detected by early nuclear antigen indirect immunofluorescence at 24 h and by DNA probe assay and shell vial cytopathic effect at 5 days.
A cluster of poliovirus type 3-positive specimens cultured in shell vials led to the discovery of significant cross contamination between vials related to the type of vial cap. Simulated testing with colored media in the shell vials indicated that the most contamination occurred with the plastic snap cap and that the least occurred with the screw cap. Measures to prevent cross contamination in clinical virology laboratories are discussed.
This study compared PCR and an assay for cytomegalovirus (CMV) pp65 antigenemia (CMV-vue; INCSTAR Corp.) with a quantitative shell vial culture (QSVC) technique for the detection of CMV in serial blood specimens from 46 solid-organ transplant recipients. In a comparison based on 535 specimens tested by PCR and QSVC, CMV was detected by PCR in 41 and by QSVC in 37 of 43 recipients at risk of CMV infection. The mean number of days after transplantation of initial detection of CMV was 29.9 for PCR and 34.0 for QSVC (P = 0.01). The antigenemia assay was performed on 395 specimens, including 304 of those also tested by PCR. In these specimens, CMV was detected by the antigenemia assay, QSVC, and PCR in 30, 32, and 35 (respectively) of 38 patients at risk, with no statistically significant difference in the time to detection. Each of the assays detected CMV in similar proportions of patients with and without clinically significant CMV infection. PCR stayed positive longer after transplantation than the other assays but frequently returned to negative when more than 6 months had elapsed after transplantation. The antigenemia assay and PCR stayed positive longer after institution of antiviral therapy than QSVC. PCR can provide highly sensitive detection of CMV viremia, but a PCR assay for CMV is not yet available in kit form. The pp65 antigenemia assay and shell vial culture are quantifiable and comparable in sensitivity. Either is recommended for rapid detection of CMV in blood specimens from solid-organ transplant recipients.
Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever. We correlated the results of 205 R. conorii blood and skin cultures for 157 patients and the results of 48 detections of R. conorii in circulating endothelial cells (CEC) for 41 patients with relevant serological, clinical, and therapeutic data. R. conorii was cultured from 40% of patients and 29.8% of samples. R. conorii was detected in CEC in 50% of samples, representing 46.2% of patients. When these calculations were limited to the samples from untreated patients prior to their seroconversion to R. conorii, the sensitivity of culture was 59%, whereas it remained at 50% for detection in CEC. We also performed PCRs for the detection of R. conorii on eight shell vial supernatants from positive cultures and on 43 blood samples. Only nonfrozen supernatants from fresh cultures were positive. The methods described in this report are suitable for use in all laboratories. Our findings suggest that for samples to be suitable for culture they must be collected prior to the initiation of an antibiotic regimen, as early as possible in the course of the disease, and be inoculated onto shell vials with minimal delay, if R. conorii is to be successfully isolated. For patients who have been treated or who have a delayed diagnosis, detection of R. conorii in CEC remains helpful.
Cytomegalovirus (CMV) infectious titers and DNA levels were determined by quantitative shell vial culture and quantitative-competitive PCR with blood samples from 10 renal transplant recipients with active CMV infection. Blood samples were stored at either room temperature or 4 degrees C and were processed at intervals of 0, 6, 24, 48, and 72 h. All samples were culture and PCR positive at baseline. Whereas the sensitivity of shell vial culture progressively declined, with only 55% positive at 24 h and 10% positive at 48 h, all samples remained PCR positive at all time points. Furthermore, the infectious titer diminished by 83 to 91% by 24 h compared to that at baseline (P < 0.0001), but quantitative DNA levels did not decline over time. Storage temperature had no significant effect on either infectious titer or DNA levels.
Detection of enteroviruses and adenoviruses mainly in fecal specimens by rapid culture with inoculation onto cell monolayers in flat-bottom tubes by centrifugation and immunofluorescence staining with genus-specific monoclonal antibodies was compared with that by the conventional virus isolation procedure. For both conventional culture and shell vial culture human lung fibroblast cells and tertiary monkey kidney cells were used. For enterovirus detection, 979 clinical specimens (916 stool specimens, 56 cerebrospinal fluid specimens, and 7 nasopharyngeal swabs) were used. Conventional culture detected 74 enterovirus isolates. A cytopathic effect compatible with the presence of an enterovirus after 3 days of incubation occurred in 25 of the 74 (34%) specimens that eventually became positive. The detection rate for enteroviruses by rapid cell culture after 2 to 3 days of incubation was 42 of 74 (57%). The genus-specific enterovirus monoclonal antibody did not react with strains of echovirus types 22 and 23 or enterovirus type 71. Rapid cell culture for the detection of adenoviruses was performed with 567 clinical specimens (536 stool specimens, 25 cerebrospinal fluid specimens, and 6 miscellaneous specimens), in which 42 adenoviruses were found by conventional culture. Nine of the 42 (21%) adenovirus isolates were detected by conventional culture within 3 days after inoculation, whereas 21 (50%) were found by rapid cell culture within 2 to 3 days. Only two of the nine specimens found to be positive for the enteric adenovirus type 41 by conventional culture as well by a type-specific enzyme-linked immunosorbent assay (ELISA) tested positive by rapid cell culture. In conclusion, the rapid shell vial assay allows the early detection and identification of enteroviruses and adenoviruses in clinical specimens but is markedly less sensitive than the conventional isolation procedure according to the eventual results of the conventional isolation procedure. Conventional cell culture remains a prerequisite for serotyping of enteroviral isolates. On the basis of the results for adenovirus type 41, the rapid detection of adenoviruses was not considered to be useful for the detection of clinically relevant adenoviruses in fecal samples.
A blood sample from a patient who returned from Algeria with a fever inoculated on human embryonic lung fibroblasts by the shell vial cell culture technique led to the recovery of Rickettsia prowazekii. The last clinical strain was isolated 30 years ago. Shell vial cell culture is a versatile method that could replace the classic animal and/or embryonated egg inoculation.
Simultaneous coinfection of Spodoptera frugiperda cells with baculovirus recombinants containing the genes for VP6 and VP7 of bovine rotavirus strain C486 resulted in the production of spherical particles resembling smooth, double-shelled rotavirus. These particles were predominantly located in the cell culture supernatant instead of being cell associated. Pretreatment of infected-cell culture supernatants with CaCl2, prior to particle purification, was found to enhance the smooth appearance of the particles. The authenticity of the proteins making up the particles was demonstrated by their reactivity with antiserum specific for double-shelled C486 rotavirus.
The three-dimensional structure of single-shelled bluetongue virus has been determined to a resolution of 3 nm by using electron cryomicroscopy and image-processing techniques. The single-shelled virion has a diameter of 69 nm. The three-dimensional structure of the virion has icosahedral symmetry with a triangulation number of 13 in a left-handed configuration. The three-dimensional structure can be described in terms of two concentric layers of density surrounding a central core density. Two distinctive features of the outer layer are the 260 knobby capsomeres located at all the local and strict threefold axes and the aqueous channels located at all the five- and six-coordinated positions. These protrusions extend outward from an inner radius of 28 nm. They are interconnected out to a radius of 30 nm by saddle-shaped densities across the local and strict twofold axes. The aqueous channels surrounded by these capsomeres are about 8 nm wide at the outer surface and 8 nm deep. Some of these channels extend inward, penetrating the inner layer. These channels may provide pathways for transporting the metabolites and mRNA during the transcriptase activity of the particles. The inner layer is a featureless smooth bed of density except for the indentations in register with the channels of the outer layer. We propose that the 260 capsomeres in the outer layer are made up of trimers of the major protein, VP7, and that the inner layer is composed of the second major protein, VP3. The density in the central portion of the structure at a radius of less than 21 nm is likely due to the minor proteins and the genomic RNA.
Rice dwarf virus (RDV) is a double-shelled particle that contains a major capsid protein (P8), a major core protein (P3), several minor core proteins, and viral genomic double-stranded RNA. Coexpression of P8 and P3 in transgenic rice plants resulted in formation of double-shelled, virus-like particles (VLPs) similar to the authentic RDV particles. The VLPs were not detected in transgenic rice plant cells expressing P8 alone. This in vivo result suggests that P8 interacted with P3 and that these two proteins provide the structural integrity required for the formation of VLPs in rice cells independently of other structural proteins, nonstructural proteins, or viral genomic double-stranded RNAs.