Functional and ultrastructural effects of nontypeable haemophilus influenzae in a hamster trachea organ culture system

Summary A hamster trachea organ culture system was utilized to evaluate quantitatively the effects of a strain of nontypeableHaemophilus influenzae (NTHI) and culture supernatants of the same strain on ciliary activity. Tracheal explants were maintained in organ culture for 96 to 144 h and ciliary activity was observed daily with an inverted microscope. Explants continuously exposed to a strain of NTHI had a progressive decline in ciliary activity which was significantly lower than uninfected controls evaluated concomitantly by 48 h of exposure and thereafter. Histologic studies revealed a progressive degeneration of mucosal cells and exfoliation of ciliated cells. Scanning electron microscopy showed little adherence of NTHI to the mucosal surface. Sterile broth cultures of NTHI and supernatants of organ cultures infected with the same NTHI strain had no adverse effect on ciliary activity. Infected tracheal explants treated with ampicillin 24, 48, or 72 h after continuous bacterial challenge had no significant decline in ciliary activity compared to controls. The lack of adherence and the histologic changes observed when hamster trachea cultures were infected with NTHI suggested a toxin might mediate the damage observed. Broth and organ culture supernatants, however, produced no damage. Therefore, further studies are needed to determine the role, if any, of a toxin in the production of damage to hamster tracheal explants by NTHI. This work was supported by a Merit Review grant from the Veterans Administration and by Grant AI-19641 from the National Institute of Allergy and Infectious Diseases.     

Oral Ultrastructure and Oral Development of the Misaligned Undulating Membrane Mutant of Tetrahymena thermophila1

The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation. The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA). The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs. Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5. The effect of the mutation involves a proliferation of excess basal bodies in the UM field. Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs. This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs. This level of regional control is not clearly evident when a single UM is present. The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development. On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena.     

Manipulus Rubiacearum III. Deppeeae,a new tribe of Rubioideae (Rubiaceae)

A new tribe,Deppeeae, in subfamily Rubioideae Verdc. of the Neotropical Rubiaceae is described. It is easily distinguished by the presence of raphides, contorted or modified alternative aestivation of the corolla, numerous small ovules, and placenta attached to the middle of the septum. Three genera are known to be members of the tribe,Deppea, Schenckia, andOmiltemia. A hypothetical phylogenetic tree for the tribe is presented.     

Reply to Claudio Cobelli and Gianna Toffolo,identifiability from parameter bounds. Structural and numerical aspects. Math. Biosci. 71:237–243 (1984)

We present an analysis of receptor mediated endocytosis which includes the following elements: ligand binding to receptors, interaction of the ligand-receptor complex with coated pits, internalization of coated pit contents, recycling of receptors, and degradation of ligand. The model accounts quantitatively for epidermal growth factor binding and clustering in coated pits at 4°C, for its internalization and degradation at 37°C, and for EGF receptor down-regulation. Steady state analysis of the model indicates that the slope and intercept of a Scatchard plot are functions of the kinetic parameters of the endocytic loop and do not necessarily reflect the affinity and number of receptors in metabolically active cells. Moreover, the model predicts that for homogeneous receptors, a Scatchard plot can be either linear or nonlinear, depending on the concentration of proteins in coated pits which interact with ligand-receptor complexes. A slight generalization of the model in which phorbol ester-receptor complexes compete with EGF-receptor complexes for the same coated pit proteins provides a quantitative explanation for the loss of the high affinity portion of the EGF Scatchard plot subsequent to preincubation with phorbol esters. This explanation leads to the prediction of a local homology between a portion of the phorbol ester receptor sequence and a portion of the EGF receptor sequence.     

Ammonium ions prevent methylation of uridine to ribothymidine inAzotobacter vinelandii tRNA

It was shown that tRNA fromAzotobacter vinelandii grown in the presence of ammonium chloride lacks ribothymidine while that grown in the absence of the ammonium salt contains this modified nucleoside. [³²P]-Labelled tRNA from this organism grown in a medium containing the ammonium salt was digested with RNase T1 and the pseudouridinecontaining tetranucleotide, common to all tRNAs was isolated and analysed for the nucleoside replacing the ribothymidine. It was found to be uridine. Cells previously labelled with [³²P]-phosphate in the ammonium salt medium were washed and incubated in the ammonium saltfree medium to test whether ribothymidine would be formed upon removal of the ammonium ions. Methylation of the uridine did not take place.     

Enzyme-linked immunosorbent assay for secalonic acid D

A competitive enzyme-linked immunosorbent assay was developed for the xanthone dimer secalonic acid D. The immunogen and enzyme marker were prepared by direct reaction of secalonic acid D with bovine serum albumin and horseradish peroxidase, respectively. The resultant conjugates were characterized by UV/VIS spectra and thin layer chromatography. The hapten:protein ratios in the conjugates were estimated by difference UV/VIS spectra and by fluorescent techniques. Immunization procedures were conducted utilizing New Zealand rabbits over a period of 12 weeks. The competitive enzyme-linked immunosorbent assay on microtiter plates showed that secalonic acid D was detectable within a range of 250–25 000 ng/assay.     

Dye coupling in the organ of Corti

Summary Dye-coupling in an in vitro preparation of the supporting cells of the guinea-pig organ of Corti was evaluated by use of the fluorescent dyes, Lucifer Yellow, fluorescein and 6 carboxyfluorescein. Despite the presence of good electrical coupling in Hensen cells (coupling ratios >0.6) the spread of Lucifer yellow was inconsistent. Hensen cells are very susceptible to photoinactivation, i.e., cell injury upon illumination of intracellular dye; and this in conjunction with Lucifer Yellow's charge and K+-induced precipitability may account for its variability of spread. Fluorescein and 6 carboxyfluorescein, on the other hand, spread more readily and to a greater extent than Lucifer Yellow, often spreading to cell types other than those of Hensen. Dye spread is rapid, occurring within a few minutes. These results suggest that molecules of metabolic importance also may be shared by the supporting cells of the organ of Corti.     

Fine structure of the malaria parasite Plasmodium falciparum in human hepatocytes in vitro

Summary Recent advances in the ability to culture the hepatic forms of mammalian malaria parasites, particularly of the important human pathogen Plasmodium falciparum have provided novel opportunities to study the ultrastrucural organisation of the parasite in its natural host cell the human hepatocyte. In this electron-microscopic and immunofluorescence study we have found the morphology of both parasite and host cell to be well preserved. The exoerythrocytic forms, which may be found at densities of up to 100/cm², grow at rates comparable to that in vivo in the chimpanzee. In the multiplying 5- and 7-day schizogonic forms the ultrastructural organisation of the parasite bears striking resemblances to other mammalian parasites, e.g., the secretory activity and distribution of the peripheral vacuole system, but also homology with avian parasites, e.g., in nuclear and nucleolar structure and mitochondrial form. The latter homologies support earlier suggestions of the close phylogenetic relationship of P. falciparum with the avian parasites. Evidence is also presented showing the persistence of the cytoskeleton of the invasive sporozoite within the cytoplasm of the ensuing rapidly growing vegetative parasites.     

Stability of tissue culture medium pH as a function of autoclaving,time, and cultured plant material

Autoclaving is a standard procedure for sterilizing nutrient media for plant tissue cultures. Most tissue cultures are grown at pH 5.2 to 5.8 with pH adjustments being made prior to autoclaving. This paper reports that there are significant differences between initial pH levels and pH levels following autoclaving, particularly in the pH range of 5.7 to 8.5. This effect is noted with and without agar. In addition, we report that with time the pH of the medium drifts into the acid range. When Cucumis callus was added to the medium, the pH was changed significantly within 48 hours. The amount and direction (increase or decrease of pH) was significantly correlated with the original pH. This suggests that researchers should be wary of the true pH situation in their medium. In addition, in publications authors should specify whether their medium pH value was determined before or after autoclaving.