First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.     

Cytoplasmic suppression of malignancy

Summary Using both normal and transformed rat liver epithelial cells to prepare cytoplasmic hybrids (cybrids) we have found evidence to support the theory that the cytoplasm from a normal cell can suppress tumorigenicity. A unique aspect of this study is that all of the cells utilized, both normal and malignantly transformed, were derived from an original cloned cell. We found that fusing cytoplasts from normal cells to malignantly transformed whole cells resulted in cybrid clones which, when injected into newborn rat pups, isogenic with those from which the cell culture was initiated, yielted tumors in 51% of the animals injected compared to 92% of the animals injected with the tumorigenic parent. Those animals that did develop tumors from the cybrid cells survived longer than those injected with cells from the tumorigenic parent. Thus, the cybrid, formed of cytoplasm from both parents, was less tumorigenic than the malignantly transformed parent cell. When reconstituted cells were prepared by fusing cytoplasts from normal cells with karyoplasts from malignantly transformed cells, a situation in which essentially all of the cytoplasm of the reconstituted cell is derived from normal cells, the tumorigenic phenotype was extinguished. This work was supported in part by United States Public Health Service grant CA12056, and grant CA09100 from the National Cancer Institute, Bethesda, MD. This work is partial fulfillment for the degree of Doctor of Philosophy for B.A.I.     

Care Needs of the Elderly Treated at a Dental School1

Increased longevity and recently intensified emphasis on gerodontology mandate that dental students'exposure to clinical care of the elderly be enhanced. The extent to which individual students can be exposed to geriatric dental care depends on the availability of elderly patients to dental school clinics and the treatment needs of these patients. The purposes of this study were (1) to determine the dental treatment needs of geriatric patients who seek care at a dental school, and (2) to ascertain if differences exist between the needs of older versus relatively young geriatric patients. Data collected on 128 elderly patients during a three month period indicate that 57% of the aged were either edentulous at examination or treatment planned to become edentulous and receive two full dentures. The remaining 43% were treatment planned to remain dentulous and receive therapies other than full upper and lower dentures. More older geriatric patients required full dentures than their younger cohorts. More young elderly required prophylaxes, scalings, root planing therapy, dental restorations, and partial dentures. Additional to their denture requirements, aged patients appear to have sufficient non-prosthetic needs to allow for meaningful gerodontic experience by students.     

Chemically synthesized galactosyl ficaprenyl diphosphate as an intermediate in the biosynthesis of the Salmonella O-antigenic polysaccharide

The membrane fraction from a mutant of Salmonella anatum deficient in UDPgalactose-4-epimerase, utilized synthetic ficaprenyl alpha-D-galactosyl diphosphate as a substrate in the biosynthesis of the O-polysaccharide portion of lipopolysaccharide which has a mannosylrhamnosylgalactose repeating sequence. The galactosyl lipid was prepared by chemical synthesis from D-galactose and ficaprenol extracted from Ficus elastica. Membrane preparations catalyzed the transfer of rhamnose from TDP-rhamnose onto membrane-bound ficaprenyl galactosyl diphosphate forming rhamnosylgalactosyl ficaprenyl diphosphate; the reaction was dependent on the prior insertion of the synthetic glycosyl-lipid into the membrane, and was proportional to incubation time up to 4 min at 29 degrees C. When both TDP-rhamnose and GDP-mannose were added, the product formed was O-polysaccharide. These results indicate that the chemically synthesized ficaprenyl galactosyl diphosphate can be an active substrate for the in vitro synthesis of the Salmonella O-polysaccharide.     

Formation and possible functions of alpha-putrescinylthymine in bacteriophage phi W-14 DNA: analysis of bacteriophage mutants with decreased levels of alpha-putrescinylthymine in their DNAs.

The DNA synthesized in the nonpermissive host by the noncomplementing mutants am36 and am42 of bacteriophage phi W-14 contains about half the wild-type level of alpha-putrescinylthymine (putThy) and a correspondingly greater level of thymine. The mechanisms whereby thymine nucleotides are excluded from replicating DNA are functional in both mutants because neither of them incorporates exogenous thymidine into DNA. It is proposed that (i) in wild-type phi W-14, the conversion of hydroxymethyluracil to putThy at the polynucleotide level is sequence specific, but that to thymine is nonspecific; and (ii) in the mutants, the sequence-specific recognition is impaired so that more thymine and less putThy are formed. The thymine-rich DNA can be packaged into phage particles. In the case of am42, the phage particles are morphologically indistinguishable from and have essentially the same polypeptide composition as wild-type particles. However, the DNA molecules they contain are about 11% shorter than those in wild-type phage, am42rev4, a revertant of am42, contains DNA with about 70% of the normal level of putThy; these molecules are about 3% shorter than wild-type DNA. The properties of am42 and am42rev4 are consistent with the suggestion that putThy facilitates the very tight packing of phi W-14 DNA (Scraba et al., Virology 124:152-160, 1983). It also appears that the putThy content of phi W-14 DNA can be reduced by no more than 30% without adversely affecting the production of viable progeny; for example, the burst size of am42rev4 is about 25% of that of the wild type.     

Cultivation and Survival Studies of Neisseria gonorrhoeae in a Human Diploid Cell Strain

Neisseria gonorrhoeae was cultivated in a human diploid cell strain (WI-38). Eighty percent of the cultures contained viable gonococci for at least 4 m at 36°C, as evidenced by subculture to brain heart infusion broth. Monthly subcultures of bacteria could be made to fresh WI-38 cultures for at least 11 monthly passages with a 69% survival rate. The identity of gonococci was confirmed by morphology, gram staining, oxidase testing and fermentation reactions. Viability in brain heart infusion broth, minimum essential medium (Eagle), and WI-38 spent fluid was of much shorter duration. The organisms grown in WI-38 cultures appeared to orient largely in the vicinity of the WI-38 cells as well as within the cytoplasm of the cells.     

Gallium-67 Scans in the Diagnosis of Pyelonephritis

Gallium-67 (⁶⁷Ga) citrate was administered intravenously (50 microcuries per kg of body weight) to patients in whom acute and chronic urinary tract infections were suspected. Scanning was done, using both the Anger-type scintillation camera and the rectilinear scanner, 24 to 78 hours after injection of the isotope.The preliminary results imply that ⁶⁷Ga renal uptake is present in patients with pyelonephritis whether overt or silent, as well as in patients with uretero-sigmoidostomies. However, ⁶⁷Ga renal uptake is not present in patients with radiographic evidence of chronic pyelonephritis without active infection and in patients without renal disease.     

Properties of adenovirus messenger ribonucleic Acid synthesized in vitro

Adenovirus deoxyribonucleic acid (DNA) was used as template for the in vitro synthesis of viral-specific ribonucleic acid (RNA). When the kinetics of the reaction were compared by using native and heat-denatured DNA templates, the latter synthesized RNA at a slower rate. The fate of the DNA after acting as template and physical characteritstics of the RNA product were studied. The DNA template, according to its sedimentation rate, was not significantly degraded by the Micrococcus lysodeicticus RNA polymerase. The products of the RNA polymerase reaction had the following properties. (i) Hybridization experiments revealed a high degree of complementarity (50 to 70%) for its homologous DNA. (ii) A very low complementarity (6 to 7%) was found for its heterologous DNA. (iii) The sedimentation rate of the synthetic RNA in a sucrose gradient was 5 to 10S when native DNA was used as the template. When heat-denatured DNA was used, the resulting RNA product, free of the template, sedimented at a rate of 3 to 16S. A rapidly sedimenting (>30S) DNA-RNA complex resulted when denatured DNA was the template. The DNA moiety of the complex was sensitive to 125 μg of deoxyribonuclease per ml. The RNA of the complex, however, was fully refractory to 50 μg of ribonuclease per ml. When the adenovirus DNA was sonically treated and then used as template, the RNA product sedimented at 3 to 9S. The heat-denatured sonically treated DNA template yielded a DNA-RNA complex that also sedimented at an unusually fast rate (>18S).     

CAMBIAL DIVISIONS IN PSEUDOTSUGA MENZIESH

Mitotic activity in the vascular cambium was determined from nine samples from a single internode in each of four Douglas-fir (Pseudotsuga menziesii [Mirb.] Franco) trees. Counts of mitotic nuclei and populations of potentially dividing cells in each sample were used to determine the average mitotic index per core. The sampling error was determined for the average mitotic index per core and the internode as a whole. Significant differences were found between the mitotic indexes of samples within the internodes of three of the trees; however, no differences were observed in the rate of division among trees. A significant correlation was established between the number of cells in the cambial zone and the average mitoses per core per sample.