Physiological characterization of strain DCB-1, a unique dehalogenating sulfidogenic bacterium

Strain DCB-1 is an obligately anaerobic bacterium which carries out the reductive dehalogenation of halobenzoates and was previously known to grow only on pyruvate plus 20% ruminal fluid. When various electron acceptors were supplied, thiosulfate and sulfite were found to stimulate growth. Sulfide was produced from thiosulfate. Cytochrome c and desulfoviridin were detected. The mol% G+C was 49 (at the thermal denaturation temperature). Of 55 carbon sources tested, only pyruvate supported growth as the sole carbon source in mineral medium. Lactate, acetate, L- and D-malate, glycerol, and L- and D-arabinose stimulated growth when supplemented with 10% ruminal fluid and 20 mM thiosulfate. In mineral medium, pyruvate was converted to acetate and lactate, with small amounts of succinate and fumarate accumulating transiently. During growth with thiosulfate, all of these products accumulated transiently. Addition of excess hydrogen to pyruvate-grown cultures resulted in diversion of carbon to formate, lactate, and butyrate, which caused a decrease in cell yield. We conclude that strain DCB-1 is a new type of sulfidogenic bacterium.     

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Expression of herpes simplex virus type 1 DNA polymerase in Saccharomyces cerevisiae and detection of virus-specific enzyme activity in cell-free lysates.

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.     

Cytokinin Secretion by Frankia sp. HFP ArI3 in Defined Medium

Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N⁶- (Δ²-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography-mass spectrometry, at levels of approximately 1 ng/ml culture medium.     

Isolation and characterization of a cDNA that encodes the peptide core of the secretory granule proteoglycan of human promyelocytic leukemia HL-60 cells

A cDNA that encodes the peptide core of the secretory granule proteoglycan of the human promyelocytic leukemic cell line, HL-60, has been isolated and analyzed. When human genomic DNA was digested and probed under conditions of low stringency with a rat cDNA that encodes a Mr = 18,600 serine/glycine-rich proteoglycan peptide core in L2 yolk sac tumor cells (Bourdon, M. A., Oldberg, A., Pierschbacher, M., and Ruoslahti, E. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1321-1325) and basophilic leukemia-1 cells (Avraham, S., Stevens, R. L., Gartner, M. C., Austen, K. F., Lalley, P. A., and Weis, J. H. (1988) J. Biol. Chem. 263, 7292-7296), a number of DNA fragments were identified. A HL-60 cell-derived cDNA library was therefore screened under conditions of low stringency with the rat probe to identify and isolate a human homologue of this rat proteoglycan peptide core. Analysis of the resulting human cDNA clones indicated that the proteoglycan peptide core that is expressed in HL-60 cells is Mr = 17,600 and contains an 18-amino acid glycosaminoglycan attachment region that consists primarily of alternating serin and glycine. Northern blot analysis of total RNA probed with the human cDNA revealed that the major message for this proteoglycan peptide core in HL-60 cells is approximately 1.3 kilobase pairs in size. When a Southern blot of digested human genomic DNA was probed with the human cDNA, three bands of approximately 6, 9, and 12 kilobase pairs were detected. However, when the Southern blot was probed with the XmnI----3' fragment of this human cDNA, one prominent band was detected, indicating that a single gene encodes this protein in the human. Analysis of the DNA from human/mouse and human/hamster somatic cell hybrids probed with the human cDNA demonstrated that the gene that encodes this molecule resides on human chromosome 10. Because the proteoglycans that are present in the secretory granules of different types of rat and mouse mast cells possess small peptide cores that are rich in serine and glycine, we propose that this HL-60 cell-3 derived cDNA encodes the peptide core of the proteoglycan that is expressed in the secretory granules of this human promyelocytic cell.     

Effect of a sprint training protocol on growth rate, conversion efficiency, food consumption and body composition of rainbow trout, Salmo gairdneri Richardson

Rainbow trout were sprint-trained (30 s duration) once or twice on alternate days for a period of 6 weeks. Swim speed for the first 10 s of a training bout averaged 11.4 bls for group 2 (trained once) and 10.2 bl s ⁻¹ for group 3 (trained twice). Food consumption, growth rate and conversion efficiency were measured over 2-week periods. Food consumption was 31-38% less for the trained groups than for the control group (group 1). The growth rates of control and trained fish increased gradually over the training period. The growth rate of trained fish was always significantly less (48-81%) than that of control fish. Although conversion efficiency was significantly less for group 3 at the beginning of training, no other significant differences in conversion efficiency were recorded. Maintenance rations were high in the initial period for all groups, but were lower than the initial values in the second and third periods. While condition factor was significantly lower for the trained groups, there were no differences in percent tissue protein, lipid, or moisture.     

Responsiveness of genetically epilepsy-prone rats to intracerebroventricular morphine-induced convulsions

The sensitivity to intracerebroventricular morphine-induced convulsions was determined in members of the severe seizure (GEPR-9) and moderate seizure (GEPR-3) colonies of genetically epilepsy-prone rats as well as in non-epileptic control rats. GEPR-9s were more sensitive to morphine-induced wet-dog shakes, rearing with bilateral forelimb clonus and generalized clonus than controls of GEPR-3s. GEPR-3s were less sensitive to morphine-induced wet-dog shakes and rearing with bilateral forelimb clonus than controls. Both high and extremely low doses of morphine in GEPR-9s elicited tonic extensor convulsions resembling the characteristic sound-induced convulsion of GEPR-9s. The results suggest that opiotergic systems may contribute to the pathophysiology of the seizure-prone condition in GEPR-9s. Further, differences in responsiveness of opiotergic systems in GEPR-3s and GEPR-9s may partially account for differences in seizure severity in the characteristic sound-induced seizures of these two types of GEPRs.     

Computer-designed prostheses for orbitocranial reconstruction

Three-dimensional imaging is an adjunct to preoperative evaluation and surgical management in some patients with complex anatomic defects of various etiologies. Deformities defined by conventional computerized tomography can be viewed as accurate three-dimensional images calculated from the original scan. The images are viewed on a high-resolution video monitor and can be photographed for a permanent record. A computer-controlled milling device can use these data to fabricate prostheses. The prostheses aid reconstructive surgery through use as an alloplastic implant, as a template to fashion autogenous bone grafts, or as a model for tissue removal. We have utilized three-dimensional imaging in combination with computer-assisted prosthesis manufacture in six patients with complex orbitocranial deformities. Four patients have undergone reconstructive surgery with satisfactory results and no complications thus far. The use of computer-designed prostheses adds a new aspect to orbitocranial reconstructive surgery that facilitates increased accuracy in the correction of anatomic defects.     

Transverse alternating field electrophoresis (TAFE)

The rapid development of electrophoretic technology during the past five years has given us a stream of improvements in the area of pulsed-field separation techniques. This progression has culminated in the conception of TAFE, a simple, high resolution technique providing straight lane geometry and separation of fragments up to 9 million base pairs in length. Pulsed-field techniques will unquestionably play a major role in the forthcoming analysis of the human genome by facilitating restriction mapping and cloning of large fragments. We have no doubt that TAFE will allow scientists to do this type of genetic analysis, faster and better than ever before.