Temperature-induced Alteration Research Articles

Anion and Temperature Responsive Molecular Switches Based on Trimetallic Complexes of Ru(II) and Os(II) That Demonstrate Advanced Boolean and Fuzzy Logic Functions.

Anion- and temperature-induced alteration of the photoredox behaviors of our recently reported trimetallic complexes was carried out to fabricate potential molecular switches. The triads [(phen)2Ru(d-HIm-t)M(t-HIm-d)Ru(phen)2]6+ (phen = 1,10-phenanthroline, d-HIm-t = heterotopic bipyridine-terpyridne type bridging ligand, and M = RuII and OsII) possess two acidic imidazole NH protons that upon interaction with anions give rise to considerable changes in their photophysical and electrochemical behaviors. Red-shifts of the absorption and emission maximum and a decrease in the M3+/M2+ potential occur when the triads are treated with basic anions. In fact, the triads can function as triple-channel sensors for F-, AcO-, CN-, OH-, and H2PO4- in acetonitrile and as selective probes for CN- and SCN- in water. The equilibrium constants for the receptor-anion interaction are on the order of 106 M-1, while the limit of detection was on the order of 10-9 M. Temperature plays a key role in the luminescence properties of the triads by adjusting the energy barrier between the emitting triplet metal-to-ligand charge transfer and non-emitting triplet metal-centered levels. A decrease in temperature leads to increases in the emission intensity and lifetime of the triads displaying the "on state", whereas an increase in temperature leads to a decrease in their emission characteristics and thus indicates the "off state" and that the process is fully reversible. In essence, the triads could function as potential switches on the basis of the reversible change in their spectral and redox behaviors under the influence of anion, acid, and temperature. The most important outcome of this study is mimicking several advanced Boolean and fuzzy logic functions by utilizing the spectral response of the triads upon the action of said external stimuli.

Temperature-induced alteration of the extracellular matrix in the tropical sea anemone, Exaiptasia pallida

Coral bleaching results from the loss of the endosymbiotic dinoflagellate the cells of coral. The symbionts provide photosynthetic products for nourishment of the coral, and loss of the symbiont can result in death of the coral. Many stressors can cause coral bleaching, but we are focusing on the effect of increased water temperature in this research because of rising oceanic temperatures. We are how investigating how increased temperature affects the extracellular matrix (ECM) using the tropical sea anemone, Exaiptasia pallida as our model organism, since this anemone has similar physiological characteristics and similar symbionts as coral. Research on the effects of a heat-shock (from 25°C to 30°C) in anemones embedded, sectioned, and then stained with the Russel-Movat Pentachrome stain showed that the mesoglea of the anemone, was altered after a 12 hour heat-shock compared to control anemones. To confirm these findings, staining with an additional ECM stain, Masson’s Trichrome, will be conducted. Additionally, we are investigating how alteration in the ECM because of increased temperature affects apoptosis to determine if there is a positive correlation between temperature-induced changes in the ECM and loss of cells through apoptosis. Results from these experiments will help us better understand the timing and consequences of temperature-induced breakdown of the coral-algal symbiosis.

Laser-induced alteration of Raman spectra for micron-sized solid particles

The Raman Laser Spectrometer (RLS) instrument on board of the future ESAs ExoMars mission will analyze micron-sized powder samples in a low pressure atmosphere. Such micron-sized polycrystalline solid particles might be heated by the laser during the Raman measurements. Here, we report on the temperature-induced alteration of Raman spectra from micron-sized polycrystalline solid particles by comparing Raman spectra on silicon and the rock forming minerals olivine and pyroxene taken at different laser intensities and different ambient temperatures. Our analyses indicate that laser-induced heating results in both broadening and shifting of characteristic Raman lines in the Stokes and anti-Stokes spectral regions. For elementary crystalline silicon a significant local temperature increase and relevant changes in Raman spectra have been observed in particles with median sizes below 250µm. In comparison, significantly weaker laser-induced Raman spectral changes were observed in more complex rock-forming silicate minerals; even for lower grain sizes. Laser power densities realized in the RLS ExoMars instrument should cause only low local heating effects and, thus, negligible frequency shifts of the major Raman lines in common silicate minerals such as olivine and pyroxene.

Optical phonons, OH vibrations, and structural modifications of phlogopite at high temperatures: An in-situ infrared spectroscopic study

The thermal behavior of optical phonons and OH vibrations of phlogopite (a trioctahedral mica) was examined at temperatures up to 1000 K using in situ infrared spectroscopy. The results showed that with increasing temperature, O–K bands in phlogopite exhibited a relatively strong variation in frequency in a manner similar to those in muscovite. The work revealed that different types of OH bands (fundamentals and combinations) have very different thermal behavior or temperature dependence, and their absorption coefficients are commonly not constant on heating. OH combination bands that are associated with summation processes of multi-phonon interactions commonly show a decrease in their intensities on heating, but in contrast combination bands due to difference processes generally exhibit an increase. This means that temperature dependencies of their absorption coefficients need to be considered when using the Beer-Lambert law to determine or estimate OH contents or hydrogen concentrations at high temperatures. The results showed a structural anomaly associated with a discontinuity in the temperature derivative of the wavenumber of Al–O and Si–O vibrations and O–H stretching near 600 K. However, framework-related phonon modes in the FIR and MIR regions do not suggest a break of the original monoclinic structural symmetry in the investigated temperature region. The complex changes are attributed to temperature-induced alteration of local configuration involving TO4 tetrahedra and a possible change of the orientation of OH dipoles, in addition to a previously reported distortion of MO6 octahedra. Increasing temperature to 1000 K also causes partial dehydroxylation, as evidenced by the disappearance of the OH band near 3623 cm−1 and the decrease in OH band height and area of other OH bands. The study did not record the formation of H2O inside phlogopite as a result of partial dehydroxylation. The work offers new data and findings that have important implications in understanding the complex structural modifications and the behavior of phonon modes and the thermal stability of hydroxyls on approaching the dehydroxylation, as well as the way hydrogen is released from micas at high temperatures. Our data also show that phologpite becomes less transparent with increasing temperature suggesting a change of radiative properties and ability to transmit heat, which could be of interest for modeling thermal-transmission in crustal rocks.

Variations in Protein–Flavin Hydrogen Bonding in a Light, Oxygen, Voltage Domain Produce Non-Arrhenius Kinetics of Adduct Decay

Light, oxygen, voltage (LOV) domains utilize a conserved blue light-dependent mechanism to control a diverse array of effector domains in biological and engineered proteins. Variations in the kinetics and efficiency of LOV photochemistry fine-tune various aspects of the photic response. Characterization of the kinetics of a key aspect of this photochemical mechanism in EL222, a blue light responsive DNA binding protein from Erythrobacter litoralis HTCC2594, reveals unique non-Arrhenius behavior in the rate of dark-state cleavage of the photochemically generated adduct. Sequence analysis and mutagenesis studies establish that this effect stems from a Gln to Ala mutation unique to EL222 and homologous proteins from marine bacteria. Kinetic and spectroscopic analyses reveal that hydrogen bonding interactions between the FMN N1, O2, and ribityl hydroxyls and the surrounding protein regulate photocycle kinetics and stabilize the LOV active site from temperature-induced alteration in local structure. Substitution of residues interacting with the N1-O2 locus modulates adduct stability, structural flexibility, and sequestration of the active site from bulk solvent without perturbation of light-activated DNA binding. Together, these variants link non-Arrhenius behavior to specific alteration of an H-bonding network, while affording tunability of photocycle kinetics.

Adsorption and Thermoresponsive Behavior of Poly(N-isopropylacrylamide-co-N,N‘-cystaminebisacrylamide) Thin Films on Gold

We describe the synthesis of thermoresponsive polymers made from N-isopropylacrylamide and varying amounts of a thiol-containing co-monomer, N,N'-cystaminebisacrylamide (P(NIPAm-co-CBAm)). Infrared spectroscopy revealed a backbone similar to that seen with pure PNIPAm. UV-vis spectroscopy showed that P(NIPAm-co-CBAm) undergoes a thermoresponsive phase transition around 32 degrees C in aqueous solution. The presence of the thiol groups enabled the polymer to adsorb onto gold surfaces. Following adsorption onto a gold surface, X-ray photoelectron spectroscopy showed a carbon/gold atomic ratio of 0.93 for a sample without CBAm and a ratio of 1.61 for a P(NIPAm-co-CBAm) sample with 0.20% CBAm. Quartz crystal microbalance (QCM) analysis showed increases in the mass of polymer adsorbed when the CBAm content in the polymer increased. The thermoresponsive behavior of the thin films on gold was investigated with contact angle and dissipative QCM analysis. Contact angles were measured for polymer films at both 25 and 60 degrees C. The largest temperature-induced alteration in the contact angle was seen with the 1.00% CBAm sample. Similarly, QCM-D results showed a significantly greater change in frequency and dissipation following a temperature change when CBAm was present than in pure NIPAm polymers.

Temperature-dependent modifications in the surface composition of Finemet: A ToF-SIMS study

Time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed as an in situ tool to study the temperature-induced alteration of the surface composition of amorphous Finemet, Fe 73Si 15.8B 7.2Cu 1Nb 3. Temperature was changed reversibly by cooling from room temperature to 118 K and warming back to room temperature. As a general result, the ion intensities and, consequently, the surface concentrations of the alloy constituents were found to vary non-monotonously. Therefore segregation processes were in operation the extent of which was element-specific. Most importantly, while cycling the temperature hysteresis behaviour was observed with concentration of Fe developing just opposite to that of the alloying elements. Accordingly, on cooling the alloy, the surface enrichment with B, Si, Nb, Cu attained first a maximum in the range of 248–193 K before the segregation changed the trend to establish appreciable depletion of these elements at 118 K (as compared to room temperature). By contrast, the surface iron content developed inversely and decreased first to a minimum at ∼223 K before reaching enrichment at 118 K. During warming, a maximum segregation of boron and silicon was observed at about 223 K – similar as on cooling – so that this temperature can be considered characteristic of the segregation process. Dissociative adsorption of water from the residual atmosphere occurring at low temperatures was responsible for the formation of surface hydroxides of iron, silicon and niobium; an enhanced adsorption of molecular water was observed at temperatures below 153 K. The temperature-dependent segregation and adsorption–desorption processes were found to be largely reversible, so that the surface composition of Finemet was practically restored after finishing the cooling–warming cycle. The processes and factors governing the non-monotonous temperature dependence of the surface segregation in the amorphous alloy are discussed within the frame of segregation theory and the influence of temperature-induced tensile stress on segregation.

Temperature-induced alteration of cuticular lipids are not required for transition phenomenon in ticks

The linkage between a phase change in cuticular lipids and accelerated water loss is defined as critical transition temperature (CTT), a characteristic in ticks reputed for implying habitat preference and serving as a cue to regulate water levels during feeding. The CTT is based on activation energy (Ea ) describing the effect of temperature (T) change on the rate of water loss (k) by the Arrhenius equation, k = A exp — Ea/RT. Typical CTT expression in American dog tick, Dermacentor variabilis, and lone star tick, Amblyomma americanum, occurs at 35–40 °C in the adult and 30–35°C in the larva (used for their lack of spiracles). Removing cuticular lipids from ticks produced higher evaporative losses, but this did not ablate or modify CTT or corresponding values of Ea . What changed was frequency (steric) factor A, simply as an integrated measure of increased water loss from the delipidizing treatment. This first analysis of the actual animal, rather than a cuticular lipid extract, demonstrates the failure of Ea to respond to artificially increased water loss rates, allowing for the role of cuticular lipids in tick water-proofing to be separated from CTT and the Ea .

Molecular cloning of novel temperature-induced expressed sequence tags related to apoptosis in spermatogenic cells of mouse

Objective: Spermatogenesis needs the relative cool environment of the scrotum and Cryptorchidism could cause human male infertility .It is most likely due to the cease of spermatogenesis and induced apoptosis of spermatogenic cells when the testes were exposed to the mild hyperthermia environment of abdomen. In this study, we have established a unilateral operative cryptorchid mouse model and used the method of suppression subtractive hybridization (SSH) to screen temperature-induced genes related to apoptosis in Spermatogenic Cells and to investigate the mechanism of spermatogenesis arrest in experimental cryptorchidism. Design: Scrotal testis was removed surgically into abdomen to established unilateral operative cryptorchid mouse model. On day 3 after surgery, SSH was performed between scrotal testis (‘driver’) and cryptorchid testis (‘tester’) to isolate expressed sequence tags (ESTs) which represent genes overexpressed in cryptorchid testis. Novel ESTs will be used to clone full-length cDNA in the future. Materials and Methods: Male C57BL/6 mice at 8–10 wk of age were anesthetized and a small incision was made in the abdomen. The testes on right side were displaced into the abdomen and the inguinal canals on right side were suturing to prevent testes decent. On day 3 after surgery, three mice were killed and testes were removed. mRNA from cryptorchid testes and scrotal testes were extracted and pooled each. SSH was performed between scrotal testes (‘driver’) and cryptorchid testes (‘tester’) using PCR-SelectTM cDNA Substraction Kit (Clontech) according to the manufacturer’s recommendations. The substracted library cDNA was cloned into pGEM-T vector (Promega) and total 168 individual recombinant clones were picked. After screening by reversed northern blots, total 54 recombinant clones were sequenced to analyze insert sequences. Results: 4 novel ESTs were isolated. Homology searches revealed that among them 3 ESTs were unknown cDNA and one EST was identical to a hypothetical protein whose function had not been reported. Conclusion: Experimental unilateral cryptorchidism provide us suitable model for studying temperature-induced alteration of gene expression in the process of hypospermatogenesis and spermatogenic cell apoptosis, because the environmental temperature was the only difference between cryptorchid and scrotal testes in experimental cryptorchidism mice. By using SSH technique, we have identified 4 novel ESTs overexpressed in cryptorchid testis. In further study, we can begin from these ESTs to clone full-length cDNA represent novel genes which may play important roles in temperature-induced germ cell apoptosis.

Cellular mechanisms underlying temperature-induced bleaching in the tropical sea anemone Aiptasia pulchella.

Temperature-induced bleaching in symbiotic cnidarians is a result of the detachment and loss of host cells containing symbiotic algae. We tested the hypothesis that host cell detachment is evoked through a membrane thermotropic event causing an increase in intracellular calcium concentration, [Ca(2+)](i), which could then cause collapse of the cytoskeleton and perturb cell adhesion. Electron paramagnetic resonance measurements of plasma membranes from the tropical sea anemone Aiptasia pulchella and the Hawaiian coral Pocillopora damicornis labeled with 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) revealed no membrane thermotropic event. In addition, intracellular imaging using Fura-2AM as well as labeling anemones with (45)Ca revealed no significant change in [Ca(2+)](i). However, bleaching could be evoked at ambient temperature with 25 mmol l(-1) caffeine without affecting [Ca(2+)](i). [Ca(2+)](i) could be altered with ionomycin in isolated host cells, but ionomycin could not induce bleaching in A. pulchella. As caffeine can affect levels of intracellular protein phosphorylation, the ability of other agents that alter intracellular levels of protein phosphorylation to evoke bleaching was investigated. The protein phosphatase inhibitor vanadate could induce bleaching in A. pulchella. Two-dimensional gels of (32)P-labeled proteins from cold-shocked, caffeine-treated and control anemones show that both temperature shock and caffeine alter the array of phosphorylated host soluble proteins. We conclude that cnidarian bleaching is linked to a temperature-induced alteration in protein phosphorylation.

Temperature-induced alteration of the polytene X chromosome structure in male larvae of the strain In(1)BM2 (reinverted) of Drosophila melanogaster.

In Drosophila melanogaster, the polytene X chromosome of male third instar larva appears twice as wide as an unpaired female X chromosome or an autosome. This characteristic morphology of the male X chromosome is correlated with the increased rate of transcription of the sex-linked genes, which ensures gene dosage compensation. In male third instar larvae of the strain In(1)BM2 (reinverted), polytene nuclei manifest unusually puffy X chromosomes at 18 +/- 1 degrees C. Such 'puffy X' chromosomes are pompons, that is, despite the increased width of the chromosome, transcription remains at the wild-type level. This characteristic is a caveat to the invariable correlation between polytene chromosome puffs and transcription, and suggests that the mutant X chromosomes arise due to perturbation of a pathway that controls the structure but not the transcription of the polytene X chromosome. In this report we present evidence that the pompons of In(1)BM2 (reinverted) arise due to spiralization of the male X chromosome, which results in condensing of the chromosome. This unusual structural alteration can be induced only in male larvae of this strain, at the third instar larval stage, through temperature shifts from 24 +/- 1 degrees C to 18 +/- 1 degrees C and during recovery from cold shock. Furthermore, extract from male adult, pupae and third instar larvae can induce chromosome condensation in wild-type larvae in vitro. This new evidence not only explains the absence of correlation between chromosome width and transcription of the pompons of In(1)BM2 (reinverted), but also suggests that the chromosomal rearrangement perturbs a pathway that regulates the condensation of chromosomes.

Temperature-induced alteration to root longevity in Lolium perenne

The production and mortality of roots is an important factor determining nutrient and carbon fluxes in terrestrial ecosystems. However, the effect of temperature on the longevity of roots is not understood. In this study the impact of changes in temperature on the longevity of Lolium perenne roots was determined.

H-NMR study of temperature-induced structure alteration at the active site of horse heart cytochrome c.

The molecular structure of the active site of horse heart met-cyano cytochrome c, as a function of temperature, has been investigated using 1H-NMR. A temperature dependence study of the NMR spectra revealed that one heme methyl proton resonance exhibits anti-Curie behavior, i.e., the hyperfine shift increases with increasing temperature. Analyses of the average heme methyl proton hyperfine shift and the proximal His imidazole proton resonances indicated that the iron-His bonding interaction in this protein is essentially independent of temperature. Since such an anomalous temperature dependence of the heme methyl proton resonance disappears in met-cyano complex of a heme peptide prepared by enzymatic degradation of the protein [Smith, M. and McLendon, G. (1981) J. Am. Chem. Soc. 103, 4912-4921], the anti-Curie behavior observed for the heme methyl proton resonance in met-cyano cytochrome c is attributed to a rotational displacement of the heme about the iron-His bond relative to the protein moiety due to a temperature-dependent conformational alteration of the heme-protein linkage. Such rotational mobility of heme at the active site of a protein may be responsible for the anomalous temperature dependence of heme methyl proton hyperfine shifts reported for many c-type ferri cytochromes.

Temperature-induced alteration of inositolphosphorylceramides in the putative glycosylated lipid precursors of Tetrahymena mimbres glycosylphosphatidylinositol-anchored proteins

Tetrahymena species contain relatively prominent glycosylphosphatidylinositol (GPI)-anchored proteins as well as their putative precursor phosphatidylinositol (PI) glycans. We have characterized the lipid components of the two principal T. mimbres PI glycans. Following their purification by preparative TLC, the PI glycans were hydrolysed in methanolic HCl or NaOH, and resulting lipids were analysed by chromatography and mass spectrometry. The two PI glycans contained nearly identical lipid moieties having long-chain bases with N-linked fatty acids. The predominant long-chain base, 3-O-methylsphinganine, was first assumed to be O-methylated as an artifact of hydrolysis, but subsequently, on the basis of control experiments, it was shown to be naturally occurring. PI glycans from cells grown at 28 degrees C contained primarily palmitic acid (79%) and some stearic acid (11%), whereas the principal PI glycan from 38 degrees C-grown T. mimbres contained 65% stearic acid. In 15 degrees C-grown cells stearic acid accounted for only 2% of ceramide-bound fatty acids and was almost totally replaced by palmitic acid (95%). The distributions of fatty acids bound to T. mimbres GPI-anchored proteins [Ko, Hung and Thompson (1995) Biochem. J. 307, 115-121] were similar but not identical to those of the PI glycans described here. Temperature-induced specification of the lipid components of mature T. mimbres GPI-anchored proteins appears to be established both at the level of PI-glycan synthesis and the level of PI-glycan utilization for protein attachment.

Isolation of a temperature-sensitive variant Chinese hamster ovary cell line with a morphologically altered endocytic recycling compartment.

We have enriched a mutagenized population of Chinese hamster ovary (CHO) cells for those defective in endocytosis by selection for survival to treatment with transferrin (Tf)-ricin and Tf-diphtheria toxin conjugates. Surviving cells were screened with a fluorescently labeled Tf uptake assay to identify cells with morphologically aberrant endocytic phenotypes. One of the cell lines identified, B104-5, has a striking temperature-induced alteration in the morphology of its endocytic receptor recycling compartment. In parental cells the tightly clustered endocytic recycling compartment is located near the Golgi complex. In the mutant cells, following incubation at 40 degrees C, this compartment appears fragmented and widely dispersed. Surprisingly, this alteration in the morphology of the recycling compartment has no effect on the kinetics of Tf internationalization and recycling. The wild-type endocytic compartment is closely aligned with the microtubule-organizing center and the Golgi apparatus, and like the Golgi, its clustered appearance is dependent upon intact microtubules. Although the disruption of the B104-5 receptor recycling compartment morphology can be phenocopied in wild-type cells by microtubule depolymerizing drugs, the microtubule cytoskeleton in B104-5 cells appears normal in immunofluorescent staining. B104-5 cells, unlike the parental cells, do not proliferate at 40 degrees C. The mutation in B104-5 cells is recessive, as fusion with wild-type cells results in a reversion of the B104-5 phenotype. The finding that the morphology of the recycling compartment in CHO cells can be altered without affecting recycling of endocytosed Tf is consistent with the variety of recycling compartment morphologies observed among different cell lines. An interpretation of this result is that the lesion in B104-5 cells is in a gene that is involved in determining the endocytic compartment morphologies observed in different cell lines.

Phase Transitions in Liposomes Formed from the Polar Lipids of Mitochondria from Chilling-Sensitive Plants

The thermal response of mitochondrial polar lipids from a variety of chilling-sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15 degrees C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16 degrees C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15 degrees C for cucumber (Cucumus sativus L.) fruit, at 14 degrees C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15 degrees C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature-induced alteration in the structure of cell membranes.

Phytoplankton Distribution in Three Thermally Distinct Reactor Cooling Reservoirs

Phytoplankton community structure was studied in relation to physicochemical characteristics of three South Carolina reservoirs in close proximity and of similar age and bottom type. Thermal alteration, resulting from the input of cooling water from a nuclear reactor, was substantially different in each reservoir. This provided an opportunity to compare water temperature effects separated from season. Water temperature (when examined independently in statistical models) appeared to be less important than other environmental variables in determining phytoplankton community structure. Pond C, a reservoir receiving intensely heated effluent (> 20/sup 0/C ..delta..T), displayed low species diversity (Shannon-Weaver H < 2.0), except in winter. Unexpectedly, more than 50% of the total phytoplankton density in this reservoir was consistently comprised of green algae (principally Chlamydomonas sp.) despite water temperatures that averaged 47.3/sup 0/C in summer. Par Pond, having a maximum ..delta..T of 5/sup 0/C, displayed no temperature-induced alteration of phytoplankton community structure.

Role of a membranous sialyltransferase complex in the synthesis of surface polymers containing polysialic acid in Escherichia coli. Temperature-induced alteration in the assembly process.

Membrane-associated sialyltransferase complexes of Escherichia coli K-235 catalyze the synthesis of sialyl polymers which remain associated with the cell envelope. Sialyl monophosphorylundecaprenol is an intermediate in the formation of these unique surface structures, and fluidity of the lipid phase is required for the proper function of the enzyme complex (Troy, F.A., Vijay, I.K., and Tesche, N. (1975) J. Biol. Chem. 250, 156-163, 164-170). In membranes containing an increased unsaturated fatty acid content of the phospholipids, obtained by growing cells at 15 degrees C, synthesis of polysialic acid was uncoupled from synthesis of the sialyl lipid-linked intermediate. Using reconstruction experiments, the importance of the role of an endogenous acceptor in polymer formation was suggested by the unexpected finding that polysialic acid synthesis could be reactivated in inactive membranes by the addition of an exogenous acceptor which contained sialic acid. Concomitant with polymer synthesis was a rapid loss of labeled sialic acid from the lipid phase. The activated sialic acid was shown to be transferred directly to the exogenous acceptor. These results establish: 1) that the temperature-induced alteration in polymer synthesis resulted from the inability of cells grown at 15 degrees C to either synthesize or assemble a functional endogenous acceptor and not from a defect in the synthesis of the sialyltransferase; 2) the intermediate precursor role of lipid-soluble sialic acid in sialyl polymer synthesis; and 3) that the exogenous acceptor served directly as an "acceptor" and not as a catalytic "effector" which stimulated an inactive membrane-enzyme complex. These results are in accord with the possibility that the low temperature-induced derangement in polymer formation is a consequence of the altered lipid structure resulting from the greater unsaturated fatty acid content in the membrane phospholipids. U-14C-labeled exogenous acceptor was isolated from the culture filtrate of cells grown at 37 degrees C and purified to homogeneity by preparative polyacrylamide gel electrophoresis. The pure acceptor was characterized structurally as a homopolymer of sialic acid with a degree of polymerization of approximately 12. Potassium borohydride reduction of the acceptor prior to complete hydrolysis with neuraminidase established that the polymer possessed a free reducing terminus of sialic acid. Subsequent structural studies showed that these oligomers of sialic acid appeared in the culture filtrate as a result of acid-catalyzed hydrolysis from membrane-associated polysialic acids of about 150 to 200 sialyl residues. Marked diminution of several membrane proteins was observed for cells grown at 15 degrees C. The possible relationship of these alterations to the upward shift in unsaturated lipids and to the loss of a functional endogenous acceptor is currently under study.

Host cell growth in the presence of the thermosensitive drug resistance factor, Rts1.

We have confirmed and extended the observation of Terawaki et al. that the R factor, Rts1, alters the growth of its host at 42 C. In all media tested there was a period during which total cell numbers increased linearly, while viable counts remained constant. During this period the rate of precursor incorporation per cell particle into deoxyribonucleic acid, ribonucleic acid, and protein declined steadily. These patterns were a consequence of the accumulation of increasing numbers of cells which had lost colony-forming ability. A temperature shiftdown experiment showed that the colony formers could, after a lag, go on to divide normally, whereas most of the noncolony formers could not undergo even a limited number of divisions after shiftdown. The number of normal divisions which occurred after shiftup of Rts1 cells to 42 C was medium dependent. In rich medium there were, on the average, two or three doublings; in glucose medium, one; and in glycerol medium, only a fraction of a doubling. Even in glucose medium, however, no increase in viable counts was observed during growth at 42 C if the cells were first starved for glucose for 1 h at 42 C. A temperature shiftdown from 42 C to 27 C during glucose starvation reversed the effect of starvation at 42 C alone. These results are consistent with the hypothesis that the thermosensitive Rts1 component(s) responsible for the host effects is present at permissive temperature, but can undergo a reversible temperature-induced alteration which then interferes with some essential host function. The detrimental effects of this R factor on its host were also reflected in a heightened sensitivity to kanamycin and actinomycin D at 42 C. Electron microscope observations revealed changes in the appearance of the cell membrane. Membranous invaginations were noted at discrete sites in the cell.