MARINICHLORELLA KAISTIAE GEN. ET SP. NOV. (TREBOUXIOPHYCEAE,CHLOROPHYTA) BASED ON POLYPHASIC TAXONOMY1

We describe three coccoid green algal strains belonging to a new genus and species, Marinichlorella kaistiae Z. Aslam, W. Shin, M. K. Kim, W.‐T. Im et S.‐T. Lee, in seawater samples from the South Sea of Korea. These strains were maintained at 25°C–30°C under a 12:12 light:dark (L:D) photoregime in an ASN‐III medium at a pH of 7.5. These strains were tolerant of high salinity (7.5% NaCl) (w/v) and temperature (40°C). Molecular phylogenetic analyses using 18S rRNA gene sequence data resolved these organisms to a clade separate from green coccoid algae with similar morphology. The DNA–DNA hybridization results demonstrated very low relatedness of these organisms to phylogenetically related species of the genera Chlorella and Parachlorella. The molar guanine + cytosine content (G + C mol%) of the genomic DNA of these organisms ranged from 64.7 to 69.1 mol%. Based on molecular phylogeny, DNA–DNA hybridization, and other morphological studies, we propose a new taxon, Marinichlorella kaistiae, to describe these strains and classify them in the family Chlorellaceae. The type strain is KAS007^T (= KCTC AG10303^T = IAM C‐620^T).     

Nitrogen fixation associated with a hotspring green alga

A unicellular alga which can grow in the light without a combined nitrogen source was isolated from a hot spring. The cells were almost spherical, usually 5–10 m in diameter. Absorption spectra of the watersoluble pigments and of the acetone-extracted ones revealed the existence of chlorophyll a and b and the absence of phycobilins. Thin sections examined by electron microscopy revealed an eukaryotic organization with features typical of the coccoid green algae (the Chlorococcales). Cells divided by internal cytokinesis and subsequent liberation of daughter cells from the parental wall, in a way similar to Chlorella. The alga reduced acetylene to ethylene and incorporated ¹⁵N₂ into cell protoplasm when incubated in a low oxygen atmosphere. Nitrogenase activity was light-dependent, microaerophilic and thermophilic. Although the association of symbiotic nitrogen fixing prokaryotes with the cells may still be possible, any such organisms have not so far been detected.Abbreviations Used DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - Chl chlorophyll - MBM modified Bristol medium - TLC thin layer chromatography     

Refixation of xylem sap CO2 in Populus deltoides

Vascular plants have respiring tissues which are perfused by the transpiration stream, allowing solubilization of respiratory CO₂ in the xylem sap. The transpiration stream could provide a conduit for the internal delivery of respiratory CO₂ to leaves. Trees have large amounts of respiring tissues in the root systems and stems, and may have elevated levels of CO₂ in the xylem sap which could be delivered to and refixed by the leaves. Xylem sap from the shoots of three Populus deltoides trees had mean dissolved inorganic carbon concentrations (CO₂+H₂CO₃+HCO^?₃) ranging from 0. 5 to 0. 9 mM. When excised leaves were allowed to transpire 1 mM[¹⁴C]NaHCO₃, 99. 6% of the label was fixed in the light. Seventy-seven percent of the label was fixed in major veins and the remainder was fixed in the minor veins. Autoradiography confirmed that label was confined to the vasculature. In the dark, approximately 80% of the transpired label escaped the leaf, the remainder was fixed in the major veins, slightly elevating dark respiration measurements. This indicates that the vascular tissue in P. deltoides leaves is supplied with a carbon source distinct from the atmospheric source fixed by interveinal lamina. However, the contribution of CO₂ delivered to the leaves in the transpiration stream and fixed in the veins was only 0. 5% of atmospheric CO₂ uptake. In the light 90% of the label was found in sugar, starch and protein, a pattern similar to that found for atmospheric uptake of[¹⁴C]CO₂. Compared with leaves labelled in the light, leaves labelled in the dark had more label in organic acid, amino acid and protein and less label in sugar and starch. After a 5-s pulse the majority of the label fed to petioles in both the light and the dark was found in malate. The majority of the label was found in malate at 120 s in the dark; only 2% of the label was found in phosphorylated compounds at 120 s. The proportion of label found in phosphorylated compounds increased from 17% at 5 s to 80% at 120 s in the light. This suggests that CO₂ delivered to leaves in the light via the transpiration stream is fixed in the veins, a small portion through dark fixation into malate, the remainder by C-3 photosynthesis.     

Helicobacter pylori: A Fickle Germ

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.     

Ultrastructure of the endosymbionts of the whitefly,Bemisia tabaci andTrialeurodes vaporariorum

Summary The ultrastructure of the mycetocytes and mycetome micro-organisms of the sweetpotato whitefly,Bemisia tabaci Genn. andTrialeurodes vaporariorum West are described. InB. tabaci, two morphologically distinct types of micro-organisms were observed in mycetocytes. The predominant type lacked a distinct cell wall, was pleomorphic in shape with a surrounding vacuole. The second type was a coccoid organism, with inner and outer cell membranes. The coccoid organism was often found in groups of varying number within vacuoles, and in many cases appeared to be undergoing degradation. InT. vaporariorum mycetocytes, pleomorphic and coccoid organisms were found, although the coccoid micro-organism inT. vaporariorum, had a thicker cell wall than the coccoid micro-organism inB. tabaci.Abbreviations C coccoid micro-organism - P pleomorphic micro-organism     

Characterization of Subsurface Bacteria Associated with Two Shallow Aquifers in Oklahoma

The bacterial microflora of two shallow aquifers (saturated subsurface zones) in Oklahoma was characterized by direct observation with light and electron microscopy, by plating, and by examination of colony morphology and distribution. Isolated bacterial strains were also examined. Total cell counts varied only slightly (2.9 × 10⁶ to 9.8 × 10⁶ g [dry wt]⁻¹) from sample to sample, whereas colony counts varied widely (6.3 × 10² to 6.5 × 10⁶ CFU g [dry wt]⁻¹). Colony counts on nutritionally rich media were lower than on low-nutrient media, especially in samples from the saturated zone. The variety of colony types growing on nutritionally rich media decreased with increasing depth and saturation. Colony counts of anaerobic bacteria also decreased with depth but were at least 100-fold lower than aerobic counts on most media. Cell morphologies of bacteria grown aerobically on plates included short rods, cocci, and actinomycete-like forms. Direct light microscopic observation of sediments revealed short, rod-shaped, and coccoid bacterial cells; endospores, actinomycete spores, and eucaryotic forms were not observed by light microscopy. Electron microscopic observation of bacteria released from the samples revealed that 85 to 90% of them were coccoid, gram-positive, Arthrobacter-like organisms, some of which were dividing or contained completed division septa; other types of gram-positive and gram-negative bacteria were present in lower numbers. Isolated bacterial strains were able to grow on both nutritionally rich and low-nutrient media. A higher proportion of gram-negative organisms was isolated than gram-positive organisms. Most of the isolates were capable of storing polyphosphate, poly-β-hydroxybutyrate, or polysaccharide. The results of this study suggest that the microbial population of these two shallow aquifers is dominated by aerobic, nutritionally versatile bacteria that can subsist on low concentrations of organic compounds without forming specialized resting cells. Other types of microorganisms, such as facultatively anaerobic bacteria and microeucaryotes, may also be present, but they represent only a small fraction of the microflora.     

Temperature-Dependent Genome Degradation in the Coccoid Form of Campylobacter jejuni

Campylobacter jejuni undergoes a dramatic morphological transformation from a corkscrew-shaped rod to a coccoid form in response to unfavorable conditions. It has been speculated that the coccoid plays an important role in the survival and dissemination of C. jejuni but questions still remain regarding the viability of coccoid cells. Characterization of the genome of coccoid cells found that newly formed coccoid cells (i.e., 1–3 days) had a SmaI-digestion profile identical to that of spiral-shaped cells; however, there was a progressive degradation of the DNA with continued incubation at 37°C. Concomitant with genome degradation was the detection of DNA in supernatants of coccoid cells. In contrast, cells incubated at 4°C retained a spiral shape and their SmaI-digestion profile for 8 weeks and released little DNA into the medium. Thus, low temperature inhibited both coccoid formation and genome degradation. Collectively, these data support the theory that the coccoid form of C. jejuni is a manifestation of cellular degradation and spiral-shaped cells, or possibly coccoid cells formed at low temperature, are the most probable candidates for a viable but nonculturable form of this pathogen.     

PHOTOSYNTHETIC PIGMENTS OF PSEUDOSCOURFIELDIA MARINA AND SELECT GREEN FLAGELLATES AND COCCOID ULTRAPHYTOPLANKTON: IMPLICATIONS FOR THE SYSTEMATICS OF THE MICROMONADOPHYCEAE (CHLOROPHYTA)1

Photosynthetic pigments of the green flagellate Pseudoscourfieldia marina (Throndsen) Manton (Micromonadophyceae) are similar to those of the coccoid Pycnococcus provasolii Guillard; prasinoxanthin is the predominant carotenoid. Other organisms that possess prasinoxanthin also possess additional pigments not found in either P. marina or P. provasolii. Uriolide, a xanthophyll previously described from the coccoid done URI 266G, was also found in Mantoniella squamata (Manton et Parke) Desikachary, Micromonas pusilla Manton et Parke and Mamiella gilva (Parks et Rayns) Moestrup, all flagellate members of the Mamiellales, and the coccoid clone IV E5G. Other unidentified carotenoids were also present in M. squamata, M. pusilla, and M. gilva. These results suggest that P. marina and the coccoid organisms URI 266G and IV E5G may be related to the Mamiellales, and that P. provasolii may be more closely related to P. marina than to M. squamata, M. pusilla, and M. gilva.     

Proline Metabolism in Leishmania donovani Promastigotes*

Oxygen uptake of Leishmania donovani culture promastigotes was stimulated by L-proline and to a lesser extent by L-glutamate and L-arginine. L-proline reversed partially KCN-induced inhibition of respiration and completely, inhibition caused by malonate. Labeled proline, glutamate, alanine, and arginine were detected by thin layer chromatography in the free amino acid pool from cells incubated with L-proline-¹⁴C. Labeled tricarboxylic acid cycle intermediates, α-ketoglutarate, succinate, fumarate, malate, and oxaloacetate, also were found by this method in extracts from organisms incubated with L-proline-¹⁴C which contained also pyruvate. Cells incubated with malic acid-¹⁴C contained labeled alanine, glutamate, and arginine. Labeled L-proline was not found in promastigotes incubated with D-glucose-¹⁴C, although arginine, glutamate, and alanine were detected in extracts from these organisms. Indirect evidence for the presence of a NADP-dependent malic enzyme was obtained by Ochoa's method. All results suggest the presence of a proline-glutamate interconversion pathway in L. donovani promastigote culture forms.     

Coccoid Helicobacter pylori Not Culturable In Vitro Reverts in Mice

An experimental rodent model was used to demonstrate the viability of the coccoid form of Helicobacter pylori. Concentrated suspensions were prepared for the two different morphologies: at 2 days incubation for the bacillary forms and at 20 days incubation for the “dormant” forms. The strains used for incubation were two fresh isolates from humans with duodenal ulceration, and two collection strains. Five hundred microliters of culture (OD₅₅₀ = 5 Mc Farland) of Helicobacter pylori with bacillary (2-5×10⁹ CFU/ml) and coccoid (0 CFU/ml) morphology were inoculated intragastrically in BALB/c mice. The gastric mucosa of the mice was colonized by Helicobacter pylori with the administration of fresh bacillary and coccoid cultures and not with the established cultures. Helicobacter pylori was isolated at 1 week after inoculation with the administration of fresh bacillary cultures, while fresh coccoid Helicobacter pylori was recovered in mice stomachs after 2 weeks of inoculation. After colonization, histopathologic changes occurred after 1 month from inoculation; all colonized mice showed a systemic antibody response to Helicobacter pylori. These results support the thesis of the viability of coccoid Helicobacter pylori non-culturable in vitro and confirm that concentrated bacterial suspensions are able to colonize and to produce gastric alterations in this suitable animal model.     

Biosynthesis of indole-3-acetic acid in protoplasts,chloroplasts and a cytoplasmic fraction from barley (Hordeum vulgare L.)

Protoplast preparations from barley (Hordeum vulgare L.) enzymatically converted [5-³H]tryptophan to [³H]indole-3-acetic acid (IAA). Both a chloroplast and a crude cytoplasmic fraction, isolated from protoplasts that had previously been fed [5-³H]tryptophan, contained [³H]IAA. Chloroplast and cytoplasmic preparations, isolated from protoplasts and thereafter incubated with [5-³H]tryptophan, also synthesized [³H]IAA, although, in both instances the pool size was less than 50% of that detected in the in-vivo feeds. There were no significant differences in the amounts of [³H]IAA that accumulated in protoplast and chloroplast preparations incubated in light and darkness.Abbreviations HPLC high-performance liquid chromatography - IAA indole-3-acetic acid - RC radiocounting     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Detection and Capturing of <Superscript>14</Superscript>C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads

Carbon cycling in the hypersaline microbial mats from Chiprana Lake, Spain is primarily dependent on phototrophic microorganisms with the ability to fix CO₂ into organics that can be further utilized by aerobic as well as anaerobic heterotrophic bacteria. Here, mat pieces were incubated in seawater amended with ¹⁴C sodium bicarbonate and the incorporation of the radiocarbon in the small subunit ribosomal RNA (SSU rRNA) of mat organisms was followed using scintillation counter and autoradiography. Different domains of SSU rRNA were separated from the total RNA by means of streptavidin-coated magnetic beads and biotin-labeled oligonucleotide probes. The ¹⁴C label was detected in isolated RNA by both scintillation counter and autoradiography, however the latter technique was less sensitive. Using scintillation counter, the radiolabel incorporation increased with time with a maximum rate of 0.18 Bq ng⁻¹ detected after 25 days. The bacterial SSU rRNA could be captured using the magnetic beads, however the hybridization efficiency was around 20%. The captured RNA was radioactively labeled, which could be mainly due to the fixation of radiocarbon by phototrophic organisms. In conclusion, the incubation of microbial mats in the presence of radiolabeled bicarbonate leads to the incorporation of the ¹⁴C label into RNA molecules through photosynthesis and this label can be detected using scintillation counter. The used approach could be useful in studying the fate of fixed carbon and its uptake by other microorganisms in complex microbial mats, particularly when species-specific probes are used and the hybridization efficiency and RNA yield are further optimized.     

Urease Activity and Urea Gene Sequencing of Coccoid Forms of H. pylori Induced by Different Factors

Helicobacter pylori exists in two morphologic forms: spiral shaped and coccoid. The nonculturable coccoid forms were believed to be the morphologic manifestations of cell death for a long time. However, recent studies indicate the viability of such forms. This form of H. pylori is now suspected to play a role in the transmission of the bacteria and is partly responsible for relapse of infection after antimicrobial treatment. Urease activity of H. pylori is an important maintenance factor. Determination of urease activity and possible mutations in the DNA sequences of coccoid bacteria will hence contribute to the understanding of pathogenesis of infections, which these forms might be responsible for. In this study, our aim was to analyze the urease activity and investigate the urease gene sequences of coccoid H. pylori forms induced by different factors with respect to the spiral form. For this purpose, the urease activities of H. pylori NCTC 11637 standard strain and two clinical isolates were examined before and after transformation of the cells to coccoid forms by different methods such as exposure to amoxicillin, aerobiosis, cold starvation, and aging. The effects of these conditions on the urease gene were examined by the amplification of 411-bp ureA gene and 115-bp ureB gene regions by PCR technique and sequencing of the ureA gene. The urease activities of coccoid cells were found to be lower than those of the spiral form. ureA and ureB gene regions were amplified in all coccoid cells by PCR. Inducing the change to coccoid form by different methods was found to have no effect on the nucleotide sequence of the ureA gene. These results show that the urease gene region of coccoid H. pylori is highly protected under various mild environmental conditions.     

Clearance rates of sessile rotifers: in vitro determinations

We measured laboratory clearance rates of 10 rotifer and one unidentified bryozoan species from 3 different lakes using ³²P labeled algae (Chlamydomonas) or yeast (Rhodotorula). Clearance rates for all rotifers fed yeast ranged from < 2.0 to > 260 µl · animal^–1 · h^–1 depending on species. The in vitro clearance rates of two sessile rotifers (Ptygura crystallina and P. pilula) were not significantly different from previously measured in situ rates (Wallace and Starkweather 1983). Clearance rates for 5 rotifers fed algae ranged from < 5.0 to > 90.0 µl · animal^–1 · h^–1. Ptygura beauchampi, P. crystallina, P. pilula, Floscularia conifera, and F. melicerta ingested both cell types but their clearance rates varied substantially among species and between cell types. There was a substantial time-dependent loss of 32P from formalin-fixed animals (Sinantherina socialis) awaiting processing. This loss stabilized at approximately 20 hours and was estimated to be about 40% of the initial ingested label. Clearance rates for the bryozoan fed yeast or algae were highly variable, ranging from < 1.0 to > 3 000 µl · animal^–1 · h^–1.     

Effect of natural zooplankton feeding on the tissue and skeletal growth of the scleractinian coral<Emphasis Type="Italic"> Stylophora pistillata</Emphasis>

Laboratory experiments were designed to estimate the ingestion rates of the scleractinian coral Stylophora pistillata under varying prey concentrations and feeding regimes and to assess the effect of feeding on the tissue and skeletal growth. Six sets of corals were incubated under two light (80 and 300 µmol photons m^–2 s^–1) and three feeding levels (none, fed twice, and fed six times per week) using freshly collected zooplankton. Results showed that the number of prey ingested was proportional to prey density, and no saturation of feeding capability was reached. Capture rates varied between 0.5 and 8 prey items 200 polyp^–1 h^–1. Corals starved for several days ingested more plankton than did fed corals. Fed colonies exhibited significantly higher levels of protein, chlorophyll a, and chlorophyll c₂ per unit surface area than starved colonies. Feeding had a strong effect on tissue growth, increasing it by two to eight times. Calcification rates were also 30% higher in fed than in starved corals. Even moderate levels of feeding enhanced both tissue and skeletal growth, although the processes involved in this enhancement remain to be determined.     

EFFECTS OF LIGHT ON PHOTOSYNTHESIS,GRAZING, AND POPULATION DYNAMICS OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA PISCICIDA (DINOPHYCEAE)1

In studying how environmental factors control the population dynamics of Pfiesteria piscicida Steidinger et Burkholder, we examined the influence of light regime on kleptoplastidic photosynthesis, growth, and grazing. Prey (Rhodomonas sp.)‐saturated growth rate of P. piscicida increased (0.67 ± 0.03 d^?1 to 0.91 ± 0.11 d^?1) with light intensity varying from 0 to 200 μmol photons·m^?2·s^?1. No significant effect was observed on grazing, excluding the possibility that light enhanced P. piscicida growth through stimulating grazing. Light‐grown P. piscicida exhibited a higher gross growth efficiency (0.78 ± 0.10) than P. piscicida incubated in the dark (0.32 ± 0.16), and photosynthetic inhibitors significantly decreased growth of recently fed populations. These results demonstrate a role of kleptoplastidic photosynthesis in enhancing growth in P. piscicida. However, when the prey alga R. sp. was depleted, light's stimulating effect on P. piscicida growth diminished quickly, coinciding with rapid disappearance of Rhodomonas‐derived pigments and RUBISCO from P. piscicida cells. Furthermore, the effect of light on growth was reversed after extended starvation, and starved light‐grown P. piscicida declined at a rate significantly greater than dark‐incubated cultures. The observed difference in rates of decline appeared to be attributable to light‐dependent cannibalism. Using a 5‐chloromethylfluorescein diacetate staining technique, cannibalistic grazing was observed after 7 days of starvation, at a rate four times greater under illumination than in the dark. The results from this study suggest that kleptoplastidy enhances growth of P. piscicida only in the presence of algal prey. When prey is absent, P. piscicida populations may become vulnerable to light‐stimulated cannibalism.     

Silver tolerance and accumulation in yeasts

Summary Debaryomyces hansenii (NCYC 459 and strain 75-21),Candida albicans (3153A),Saccharomyces cerevisiae (X2180-1B),Rhodotorula rubra (NCYC 797) andAureobasidium pullulans (IMI 45533 and ATCC 42371) were grown on solid medium supplemented with varying concentrations of AgNO₃. Although Ag⁺ is highly toxic towards yeasts, growth on solid media was still possible at Ag concentrations of 1–2 mM. Further subculture on higher Ag concentrations (up to 5 mM) resulted in elevated tolerance. The extent of Ag tolerance depended on whether Ag-containing plates were exposed to light prior to inoculation since light-mediated reduction of Ag⁺ to Ag⁰ resulted in the production of a less toxic silver species. Experimental organisms exhibited blackening of colonies and the surrounding agar during growth on AgNO₃-containing medium especially at the highest Ag concentrations tested. All organisms accumulated Ag from the medium; electron microscopy revealed that silver was deposited as electron-dense granules in and around cell walls and in the external medium. X-ray microprobe analysis indicated that these granules were metallic Ag⁰ although AgCl was also present in some organisms. Volatile and non-volatile reducing compounds were produced by several test organisms which presumably effected Ag⁺ reduction to Ag⁰.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Effects of ionophores on vitellogenin uptake by Hyalophora oocytes

Oocytes of Hyalophora cecropia that were incubated in vitro with [³⁵S]vitellogenin incorporated label within 10 min into an intermediate-density compartment identified by sucrose density gradient centrifugation. During a subsequent 20-min chase this presumptive endosomal label was transferred to a compartment with the higher density of protein yolk spheres. When vitellogenin uptake was inhibited by 10 μM nigericin or monensin, or 50 μM carbonyl cyanide m-cholorophenylhydrazone, a somewhat larger and more focused peak of label accumulated in the endosome region of the gradient, and the transfer of this label to the yolk spheres was blocked. Valinomycin, at concentrations as high as 100 μM, did not inhibit uptake or processing, even though successful insertion into the oocyte membrane could be demonstrated by the effects of this ionophore on the membrane potential and K⁺ permeability of the follicle. Inhibition of processing by nigericin and monensin is consistent with a model of endocytosis in which the ionophores prevent acidification of the endosomes by promoting H⁺-K⁺ exchange with the cytoplasm. Several alternative possibilities were ruled out by physiological analyses entailing the measurement of cytoplasmic pH and membrane potentials.