We characterized the binding of 125I-[Nle8, Nle18, Tyr34]parathyroid hormone-(1-34) amide [125I-nlPTH-(1-34)] to renal plasma membranes prepared from chicks to determine the effects of secondary hyperparathyroid states on renal PTH receptors. This radioligand exhibited specific binding to membranes with high affinity (Kd, 2-3 X 10(-9) M). Agonists or competitive antagonists of PTH were effective in competing for binding sites labeled with 125I-nlPTH-(1-34), whereas an inactive fragment of PTH, salmon calcitonin, and bovine growth hormone did not compete with the radioligand for renal PTH receptors. Newly hatched chicks raised on control diet with adequate vitamin D and calcium or diets deficient in either vitamin D or calcium were used to study the regulation of renal PTH receptors in experimental models of secondary hyperparathyroidism. We found that both experimental diets resulted in marked hypocalcemia and progressive loss of renal cyclic AMP responsiveness to PTH in vitro. Associated with this refractoriness to the hormone was a marked reduction in PTH receptors in membranes from both vitamin D-deficient and calcium-deficient chick kidney. No change in the affinity of the PTH receptors was found. Vitamin D3, in a single dose of 250 micrograms, partially restored serum calcium of vitamin D-deficient birds toward normal by 72 h and also partly restored renal cyclic AMP responsiveness to PTH and the PTH receptor number toward control values. We conclude that renal refractoriness to PTH observed in experimentally hyperparathyroid animals models is due to a marked loss of plasma membrane receptor sites for PTH without an apparent change in the affinity of the receptors for the hormone.

Genetic transformation studies were used to determine relatedness within the family Pasteurelluceae. Among strains with <60 % relatedness to Haemophilus influenzae based on deoxyribonucleic acid hybridization, two groups were identified; one, showing competition for homospecific transformation with H. influenzae, contained Haemophilus parainfluenzae, Haemophilus parasuis, Haemophilus aphrophilus, Haemophilus paraphrophilus, Pasteurellu pneumotropica, Pasteurella multocida, and ActinobaciElus actinomycetemcomitans, and the other, showing little or no competition for homospecific transformation with H. influenme, contained Haemophilus ducreyi, Haemophilus parahaemolyticus, Actinobacillus ligniermii, Actinobacillw equuli, Actinobacillus pleuropneumoniae, and Pasteurellu ureae. Such groupings support existing studies which have used only deoxyribonucleic acid hybridization or numerical analysis. Currently there is much interest in the taxonomy of the family Pasteurellaceae, particularly the genus Haemophilus, and it is recognized that traditional genus designation based on limited phenetic traits may be inadequate (15). Despite requirements for hemin (X factor) or nicotinamide adenine dinucleotide (V factor), deoxyribonucleic acid (DNA) hy-bridization studies have suggested that Haemophilus pleuropneumoniae is more closely related to members of the genus Actinobacillus ; that Haemophilus ducreyi is a mono-specific genus unrelated to the type species Haemophilus influenzae; that avian species of Haemophilus are related to Pasteurella species; and that Haemophilus somnus, Haemo-philus agni and Histophilus ovis are related and belong to a single distinct species (4, 13, 15, 16, 25). Overall DNA sequence relationships as determined by DNA hybridization studies have become the standard parameter for determining taxonomic relationships, and the concept of the genospecies is thought to be of little value (11). Recent studies, however, hqve demonstrated regional differences within the genome for both presumed rates and mechanisms of evolution (17). We think, therefore, that taxonomic methods based on characteristics other than overall sequence relatedness will be necessary for completeness. Not long after Alexander and Leidy recognized transformation in H. influenzae in 1950, this genetic method was used in classification studies for members of the genus Haemophilus (2, 12). Since that time, much work has been done on the molecular events that accompany transformation , and several steps in transformation that are of potential taxonomic significance have been identified: (i) development of competence, (ii) recognition and uptake of transforming DNA, (iii) integration of DNA in the recipient genome, and (iv) expression of phenotype in the recipient cell (6, 9, 11, 18-20, 22). To avoid problems of expression of phenotype, we have chosen markers thought to be conserved, such as ribosomal subunits or (in this study) a subunit of the DNA-dependent ribonucleic acid polymerase (7). To avoid problems of variability in development o...

The zonal ultracentrifuge was used for separation of Treponema pallidum from large volumes of rabbit testicular syphiloma extracts by continuous-flow centrifugation in a cesium chloride density gradient. The gradient was linear with radius from a density of 1.05 to 1.36 g/ml. Operating speeds were 15,000 rev/min for the continuous-flow phase and 25,000 rev/min for a 30-min banding period. A total of 9 x 109 (24.3%) treponemes were recovered from the original extract. Of the treponemes recovered, 88% formed a band at a density of 1.170 to 1.211 g/ml. Within the limits of present methods of assay, these fractions were relatively free from testicular particulates and protein when compared with treponemes recovered after differential centrifugation. Observations of the isolated fractions by dark-field and electron microscopy indicated a lack of gross morphological damage to T. pallidum. Their antigenic characteristics were also retained, as evidenced by their ability to react with syphilitic sera in the indirect fluorescent-antibody procedure. At present, Treponema pallidum can be propagated only in vivo. Therefore, harvesting of the organism must involve its extraction from the tissue in which it is grown, usually rabbit testes. It is obvious that large quantities of spirochetes free from host contaminants are required for use in analytical studies and to produce effective vaccines. The zonal centrifuge has proven useful for separating particles ranging from whole cells to large protein molecules (1, 2, 4-6). Since the organism falls within this particle size, these techniques should be applicable to separating T. pallidum from tissue debris. This paper describes the application of continuous-flow gradient centrifugation to the separation of T. pallidum from tissue debris. MATERIALS AND METHODS Rabbit testes infected with T. pallidum, Nichol's virulent strain, were finely minced and agitated in 0.85% sodium chloride (50 ml/testis) on a rotator at 190 cycles/min for 30 min. The supernantant fluid was centrifuged at 300 x g for 10 min to remove

The three type strains and a reference strain of Mobiluncus examined in this study had a gram-positive type of cell walls. All four strains were susceptible to vancomycin and resistant to colistin, and the overall susceptibility patterns with other antimicrobial agents were consistent with those of gram-positive microorganisms. Strains of Mobiluncus mulieris were lysed by 3% potassium hydroxide, and strains of Mobiluncus curtisii were not. However, all of the strains examined had intermediate levels of Limulus amebocyte-lysate reactivity that was not lipopolysaccharide associated. Lipopolysaccharide was not detected when whole-cell lysates were digested with proteinase K, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and silver stained. In addition, 2-keto-3-deoxyoctulosonic acid and heptose were not detected by gas chromatography of 0-methyloxime acetate derivatives of whole-cell carbohydrates. Saponification and gas chromatography of whole-cell fatty acids showed that none of the four strains examined had detectable levels of hydroxylated fatty acids. Hydroxylated fatty acids or aldehyde fatty acids were not detected in acid hydrolysates of crude cell membrane preparations. (1,4,17,27,30,35,37,39,41,45,48), although the taxonomic position of members of this genus is still unresolved (15,43).The genus Mobiluncus has been tentatively assigned to the family Bacteroidaceae (43), which includes obligately anaerobic straight, curved, or helical gram-negative and gram-positive rod-shaped bacteria (18). The Gram reactions of strains of Mobiluncus are typically gram negative or variable (15, 43), although electron micrographs show that structurally these organisms have gram-positive cell walls (41, 43) and lack an outer membrane. Likewise, anaerobic curved rod-shaped organisms belonging to the genera Butyrivibrio, Lachnospira, and Acetivibrio (B), which are also members of the family Bacteruidaceae, stain gram negative but are structurally gram positive. Among anaerobic curved rods consistent with Mobiluncus, fatty acid aldehydes (presumably from plasmologens) have been detected, while hydroxlated fatty acids (found predominately in gram-negative bacteria) have not been found (39). Mobiluncus species are resistant to some antimicrobial agents which traditionally inhibit gram-negative microorganisms and are susceptible to some antimicrobial agents which inhibit gram-positive microorganisms (1,8,15,42,43,44). Limulus amebocyte lysate (LAL) reactivity, an indicator of gram-negative endotoxin, has also been reported (29).In this study, we compared characteristics of the type * Corresponding author.strains and a reference strain of Mobiluncus since previous reports have included organisms of undetermined taxonomic position which resemble members of this genus. We examined four strains of Mobiluncus for the following characteristics which are indicative of cell wall type: cell lysis, lipopolysaccharide (LPS), endotoxin reactivity, fatty acid content, antimicrobial agent susceptibility, and carbohyd...

Thirty isolates of Haemophilus ducreyi collected in Thailand in 1984 were characterized by plasmid content. Three novel plasmids with estimated molecular masses of 1.8, 2.6, and 2.8 MDa were observed in 29 isolates, in addition to the 3.2-, 5.7-, and 7.0-MDa beta-lactamase and 4.4-MDa sulfonamide resistance plasmids. At least three of the seven plasmids were observed in each of the 29 isolates. The number and diversity of plasmids observed in these isolates of H. ducreyi distinguish them from strains previously described.

No abstract

However, except for the Reiter organism, similar studies of cultured spirochetes in sera from syphilitic humans have not been reported, although several of these strains have been described as variants of Treponema pallidum. Therefore, the Reiter, English Reiter, Noguchi, Nichols, and Kazan-2 strains (purportedly T. pallidum) and the FM, N-39, and MRB strains of

No abstract