Energy-dispersive X-ray analysis of the extracellular cadmium sulfide crystallites of Klebsiella aerogenes

Klebsiella aerogenes forms electron-dense partieles on the cell surface in response to the presence of cadmium ions in the growth medium. These particles ranged from 20 to 200 nm in size, and quantitative energy dispersive X-ray analysis established that they comprise cadmium and sulfur in a 1:1 ratio. This observation leads to the conclusion that the particles are cadmium sulfide crystallites. A combination of atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and acid-labile sulfide analysis revealed that the total intracellular and bound extracellular cadmium:sulfur ratio is also 1:1, which suggests that the bulk of the cadmium is fixed as extracellular cadmium sulfide. The tolerance of K. acrogenes to cadmium ions and the formation of the cadmium sulfide crystallites were dependent on the buffer composition of the growth medium. The addition of cadmium ions to phosphate-buffered media resulted in cadmium phosphate precipitates that remove the potentially toxic cadmium ions from the growth medium. Electrondense particles formed on the surfaces of bacteria grown under these conditions were a combination of cadmium sulfide and cadmium phosphates. The specific bacterial growth rate in the exponential phase of batch cultures was not affected by up to 2mM cadmium in Tricine-buffered medium, but formation of cadmium sulfide crystallites was maximal during the stationary phase of batch culture. Cadmium tolerance was much lower (10 to 150 M) in growth media buffered with Tris, Bistris propane, Bes, Tes, or Hepes. These results illustrate the importance of considering medium composition when comparing levels of bacterial cadmium tolerance.Abbreviations EDXA Energy dispersive X-ray analysis - AAS Atomic absorption spectroscopy - TEM Transmission electron microscopy - SEM Scanning electron microscopy - ICP-MS Inductively coupled plasma mass spectrometry - ALSA Acid-labile sulfide analysis     

Purification and some properties of β-mannanase from Bacillus sp.

-Mannanase produced by Bacillus sp. W-2, isolated from decayed commercial konjak cake, was purified from the culture supernatant by (NH₄)₂ SO₄ precipitation, adsorption to konjak gel, and column chromatography with DEAE-cellulose, Sephadex G-100 and Sephacryl S-200. Its molecular size was estimated by SDS-PAGE as 40 kDa, and by gel filtration as 36 kDa. The enzyme was most active at pH 7 and 70°C and was stable for at least 1 h between pH 5 and 10 and below 60°C. Its activity was completely inhibited by Hg²⁺. The enzyme hydrolysed galactomannan better than glucomannan and mainly produced mannose and mannobiose.The authors are with the Department of Bioproductive Science, Faculty of Agriculture, Utsunomiya University. Utsunomiya, Tochigi 321, Japan     

Adaptations and responses of Dasymutilla occidentalis (Hymenoptera: Mutillidae) to predators

The mutillid wasp Dasymutilla occidentalis possesses several adaptations and exhibits a number of responses wich appear to be of defensive value: a long mobile sting with powerful venom; a strong, rounded and slippery cuticle; an ability to run very rapidly and evasively; an aposematic warning coloration pattern; and the ability to respond to an attack by making stridulatory sounds and by releasing a chemical secretion or both. The effectiveness of these defenses is supported by tests utilizing various vertebrate and arthropod predators. The raison d'être of the multiple lines of defense possessed by D. occidentalis and the relative value of each line of defense are discussed. It is postulated that aposematic coloration, audible stridulation, and a volatile defensive exudate all function primarily as part of an early warning system enabling a predator to recognize this wasp-with its very algogenic venom-as unpalatable and potentially dangerous.

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Zusammenfassung Die Mutillide Wespe Dasymutilla occidentalis besitzt verschiedene Anpassungen und zeigt eine Anzahl von Reaktionen, die für die Verteidigung von Wert sind: ein langer, beweglicher Stachel mit starkem Gift, eine starke, runde und glatte Kutikula, die Möglichkeit, sehr schnell und ausweichend zu laufen sowie ein aposematisches Warn-Farben-Muster. Desweiteren ist sie fähig, einem Angriff mit einem knisternden Ton entgegenzutreten sowie ein chemisches Sekret abzugeben oder auch beides. Im Labor durchgeführte Zusammenstöße zwischen vertebraten und invertebraten Räubern und D. occidentalis beweisen klar den Wert der ganzen Anzahl von Verteidigungsmechanismen für die Wespe.Diese Zusammenstöße liefern auch einen Einblick in die raison d'être der vielseitigen Verteidigung. die stärkere Kutikula und ihre Glätte, Haupteigenschaften zum überleben gegen Räuber, funktionieren gleichzeitig mit dem Stich zum Schutz gegen Vertebraten und mit schneller Fluchtmöglichkeit zum Schutz gegen die meisten Invertebraten. Das akustische Geräusch scheint eine Hilfsverteidigung zu sein wenigstens gegen einige Spinnen und vermutlich auch gegen einige Vertebraten. Die chemische Sekretion, die hauptsächlich aus 4-methyl-3-heptanon besteht, scheint möglicherweise direkten Verteidigungswert gegen einige Eidechsen zu haben und funktioniert höchstwahrscheinlich hauptsächlich im Zusammenhang mit der roten und schwarzen aposematischen Färbung und dem knisternden Ton als vielseitiges Warnsystem, das fähig ist, durch Signalisieren allen potentiellen vertebraten Räubern mitzuteilen, daß dieses Insekt ungenießbar ist.

     

An in situ assay for induced diphtheria-toxin-resistant mutants of diploid human fibroblasts

The sensitivity of diploid human fibroblasts to the cytotoxic effects of diphtheria toxin (DT) depended on the cell growth status. Exponentially growing cells treated with 10^?3-1 lethal flocculating units (LF) of DT/ml for 4 days survived with a frequency of 4 × 10^?4. However, the DT-resistant phenotype of colonies isolated under these conditions was not stable. When the growth of the cells had been arrested by confluence or deprivation of serum growth factors prior to treatment with DT (4 days, 10^?3-0.6 LF/ml), the survival decreased to 2 × 10^?6 and the resistance of isolated colonies was stable. An in situ assay for induced DT-resistant mutants was developed in order to avoid problems associated with the possible reduced viability of the mutants relative to that of wild-type cells. A reproducible and linear dose response was obtained for the induction of DT-resistant mutants by ethylnitrosourea. The mutants were induced with high frequency by this compound (e.g., 10^?3 mutants/viable cell at a 37% survival dose); complete expression of the mutant phenotype occurred after 6 generations of growth under nonselective conditions. Isolated mutant colonies showed stable resistance to DT and were cross-resistant to Pseudomonas aeruginosa exotoxin A.     

Identifying human cells capable of metabolizing various classes of carcinogens

Human cells that appear capable of metabolizing various classes of carcinogens have been identified using one of two methods: metabolism of tritiated benzo(a)pyrene to aqueous-acetone soluble forms or inhibition of cellular DNA synthesis. Each of the assay systems was optimized and the results on 15 human epithelial cell lines were compared. One or more cell lines were found to activate each of four classes of carcinogens examined: polycyclic hydrocarbons, aromatic amines, heterocyclic hydrocarbons, and nitrosamines. Cells that appeared capable of metabolizing polycyclic hydrocarbons or aromatic amines by these methods were also found to produce metabolites which were cytotoxic to cocultivated human xeroderma pigmentosum fibroblasts after a 48-hr exposure to the carcinogen.     

Effect of DNA repair on the cytotoxicity and mutagenicity of polycyclic hydrocarbon derivatives in normal and xeroderma pigmentosum human fibroblasts

The cytotoxicity of the “K-region” epoxides as well as several other reactive metabolites or chemical derivatives of polycyclic hydrocarbons was compared in normally-repairing human diploid skin fibroblasts and in fibroblasts from a classical xeroderma pigmentosum (XP) patient (XP2BE) whose cells have been shown to carry out excision repair of damage induced in DNA by ultraviolet (UV) radiation at a rate approx. 20% that of normal cells. Each compound tested exhibited a 2- to 3-fold greater cytotoxicity in this XP strain than in the normal strain. To determine whether this difference in survival reflected a difference in the capacity of the strains to repair DNA damage caused by such hydrocarbon derivatives, we compared the cytotoxic effect of several “K-region” epoxides in two additional XP strains, each with a different capacity for repair of UV damage. The ration of the slopes of the survival curves for each of the XP strains to that of the normal strain, following exposure to each epoxide, was very similar to that which we had previously determined for their respective UV curves, suggesting that human cells repair damage induced in DNA by exposure to hydrocarbon derivatives with the same system used for UV-induced lesions.To determine whether the deficiency in rate of excision repair in this classical XP strain (XP2BE) causes such cells to be abnormally susceptible to mutations induced by “K-region” epoxides of polycyclic hydrocarbons, we compared them with normal cells for the frequency of induced mutations to 8-azaguanine resistance. The XP cells were two to three times more susceptible to mutations induced by the “K-region” epoxide of benzo(a)pyrene (BP), 7,12-dimethylbenz(a)anthracene (DMBA), and dibenz(a,h)anthracene (DBA). Evidence also was obtained that cells from an XP variant patient are abnormally susceptible to mutations induced by hydrocarbon epoxides and, as is the case following exposure to UV, are abnormally slow in converting low molecular weight DNA, synthesized from a template following exposure to hydrocarbon epoxides, into large-size DNA.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Neuropeptides in spinal cord injury: Comparative experimental models

The possible role of endogenous opioids in the pathophysiology of spinal cord injury was evaluated utilizing a variety of experimental models and species. In the cat, we have shown that β-endorphin-like immunoreactivity was increased in plasma following traumatic spinal injury; such injury was associated with a decrease in spinal cord blood flow (SCBF) which was reversed by the opiate receptor antagonist naloxone. Naloxone treatment also significantly improved functional neurological recovery after severe injury. Thyrotropin-releasing hormone (TRH), possibly through its “anti-endorphin” actions, was even more effective than naloxone in improving functional recovery in the cat. In a rat model, utilizing a similar trauma method, TRH proved superior to naloxone in improving SCBF after injury. In addition, naloxone at high doses attenuated the hindlimb paralysis produced by temporary aortic occlusion in the rabbit. The high doses of naloxone required to improve neurological function after spinal injury suggest that naloxone's actions, if opiate receptor mediated, may be mediated by non-μ receptors. Dynorphin, an endogenous opioid with a high affinity for the κ receptor, produced hindlimb paralysis following intrathecal administration in rats. Taken together, these findings suggest that endogenous opioids, possibly acting at κ receptors in the spinal cord, may serve as pathophysiological factors in spinal cord injury.     

Correspondence of homologies in amino acid sequence and tertiary structure of protein molecules

According to the method developed previously (Kubota, Y., Takahashi, S., Nishikawa, K. and Ooi, T. (1981) J. Theor, Biol. 91, 347-361), homology among proteins may be estimated quantitatively. We extended the method to investigate the relationship of an amino acid sequence to its teritary structure and identify homologous segments which have homologous native conformations in proteins. First, we selected proper indices for the computation of correlation coefficients from 32 properties inherent to amino acids, such as hydrophobicity. The arithmetic average of correlation coefficients using six indices gave rise to a good correlation for the CD- and EF-hand regions (Ca2+ binding sites) in carp parvalbumin, but poor ones for other segments. We then applied the method to homologous proteins, the three-dimensional structures of which are known: horse hemoglobin alpha-chain and beta-chain; cytochrome c and c2; serine proteases, chymotrypsinogen and elastase; alpha-lytic protease and protease A from prokaryotic organisms. The results show that the sequence homology estimated by the present method has a good correspondence to the homology in three-dimensional structures and therefore the method is promising for the identification of important sites in sequences which have similar native conformations. For an example of the application of the method, two sequences of human interferon, one from fibroblast and the other from leukocyte, are compared, suggesting functional sites in the molecule.