The Unique Chemistry of Eastern Mediterranean Water Masses Selects for Distinct Microbial Communities by Depth

The waters of the Eastern Mediterranean are characterized by unique physical and chemical properties within separate water masses occupying different depths. Distinct water masses are present throughout the oceans, which drive thermohaline circulation. These water masses may contain specific microbial assemblages. The goal of this study was to examine the effect of physical and geological phenomena on the microbial community of the Eastern Mediterranean water column. Chemical measurements were combined with phospholipid fatty acid (PLFA) analysis and high-throughput 16S rRNA sequencing to characterize the microbial community in the water column at five sites. We demonstrate that the chemistry and microbial community of the water column were stratified into three distinct water masses. The salinity and nutrient concentrations vary between these water masses. Nutrient concentrations increased with depth, and salinity was highest in the intermediate water mass. Our PLFA analysis indicated different lipid classes were abundant in each water mass, suggesting that distinct groups of microbes inhabit these water masses. 16S rRNA gene sequencing confirmed the presence of distinct microbial communities in each water mass. Taxa involved in autotrophic nitrogen cycling were enriched in the intermediate water mass suggesting that microbes in this water mass may be important to the nitrogen cycle of the Eastern Mediterranean. The Eastern Mediterranean also contains numerous active hydrocarbon seeps. We sampled above the North Alex Mud Volcano, in order to test the effect of these geological features on the microbial community in the adjacent water column. The community in the waters overlaying the mud volcano was distinct from other communities collected at similar depths and was enriched in known hydrocarbon degrading taxa. Our results demonstrate that physical phenomena such stratification as well as geological phenomena such as mud volcanoes strongly affect microbial community structure in the Eastern Mediterranean water column.     

Cutting edge: precursor frequency affects the helper dependence of cytotoxic T cells.

Generation of CTL immunity often depends on the availability of CD4 T cell help. In this report, we show that CTL responses induced by cross-priming can be converted from CD4-dependent to CD4-independent by increasing the frequency of CTL precursors. In the absence of CD4 T cells, high numbers of CTL precursors were able to expand in number and become effector CTL. The ability of high frequencies of CD8 T cells to override help was not due to their ability to signal CD40 via expression of CD154. These findings suggest that when precursor frequencies are high, priming of CD8 T cell responses may not require CD4 T cell help.     

Construction of interleukin-1 alpha mutants using unequal contamination of synthetic oligonucleotides.

Proteins without readily available three-dimensional structural data present a difficult problem in the exploration of structure/function relationships. Saturation mutagenesis using contaminated oligonucleotides can identify potentially interesting regions of such a protein. This technique, in which synthesized oligonucleotides contain low-level base substitutions, allows random mutations to be placed throughout a gene sequence. Using double-stranded cassettes, a region of the human interleukin-1 alpha gene has been altered using such mutagenic oligonucleotides. However, instead of contaminating both strands of the gene sequence at the same level, each strand of the insert was contaminated at a different level. Several recombinants were sequenced and the effects of the mutations on the activity of the proteins were examined. Contaminating the two oligonucleotides at different levels produced a significantly different distribution of nucleotide changes from that seen if both strands were contaminated at the same level. The observed distribution followed the average of the distributions for each of the two contamination levels. This resulted in roughly equal frequencies of 1 to 5 nucleotide changes per clone with very few clones containing the wild-type nucleotide sequence. This helped overcome the redundancy in the genetic code, resulting in a high frequency of amino acid changes, and allowed changes at every amino acid to be sampled in a small number of mutants. This procedure can allow a gene sequence to be screened rapidly by removing most wild-type sequences from analysis while making sure that there are many amino acid changes in the resultant mutants.     

Ion exchange process for removing glucosyltransferase from crude glucoamylase

The advant of a new range of high-protein capacity cellulosic ion exchangers suitable for use on an industrial scale made it worthwhile investigating the conditions necessary to remove the contaminating enzyme glucosyltransferase from a commercial preparation of crude glucoamylase. The potential of the SP derivative, SP Indion((R)), for achieving this separation is shown. At pH 2.5 the glucosyltransferase was selectively adsorbed by the ion exchanger, and 99% of the glucoamylase was recovered in the eluate from the column. Purification of an Aspergillus culture filtrate by this method will require careful control of the ionic strength of the culture medium if it is to be used without the additional step of cation exchange to lower than pH to 2.5.     

Exudation of amino acids by intact and damaged roots of wheat and peas

Summary Wheat and pea seedlings were grown aseptically in solution-culture and the total free amino nitrogen released by the roots was determined by a quantitative ninhydrin test. Amino nitrogen from wheat plants after 14 days growth was not detected by the test, indicating the release of less than 3 µg of amino nitrogen from a culture of 15 plants. Pea plants of the same age released from 2 to 7 µg per plant. Paper chromatograms of highly concentrated undisturbed solution-cultures revealed up to 13 amino compounds from wheat and 11 from pea. The pattern of amino acids in exudates was similar to that in crushed roots, except for an unidentified amino compound which was detected only in exuded material. The total amino nitrogen and relative proportions of several amino acids in the root exudates of sand-grown peas was influenced by several ratios of oxygen and carbon dioxide supplied to the root zone. Roots, experimentally damaged by swirling and rinsing in sand, released in 1 hour amino nitrogen of from 73 to 120 per cent of that released by normal exudation over a 2-week period. Our findings suggest that experimental and environmental root damage may be responsible for a large proportion of organic materials released by growing plant roots.Trade names are used in this publication to provide specific information. Their use does not constitute a guarantee of the products named and does not signify that they are approved by the U.S. Department of Agriculture to the exclusion of others of suitable composition.     

Acute lethality after x-irradiation of Tribolium confusum adults

Recently emerged adults of Tribolium confusum, maintained at 30° in flour-yeast medium, were exposed to X-ray doses between 8 and 75 kR at dose rates of 225 or 1000 R/min. In all cases, there was no significant mortality for about 10 days, and all deaths attributable to the irradiation occurred during the next 10 to 15 days; after the lower doses, many insects survived this critical period and then lived on for many months. The relative dose-independence of the survival-time of decedents suggested that there is a specific mode of death, i.e., an as-yet-unrecognized acute lethal syndrome. Irradiated beetles incubated at 22° exhibited about twice as great a delay in the onset of mortality, and a mortality period with about twice the duration, as those at 30°, suggesting the necessity of a sequence of metabolic events in the development of lethality. Beetles on a cornstarch diet were at least as radioresistant as beetles on flour yeast; beetles at 22° were distinctly more sensitive than those kept at 30°. Older beetles, exposed 4 or 10 months after emergence, showed progressive increases in acute radiosensitivity. Implications of these findings for radiobiological investigations on adult insects are discussed.

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Lethalite aigüe apres irradiation aux rayons X des adultes de Tribolium Confusum

Résumé La plupart des insectes utilisés dans les expériences de radiobiologie ont une longévité relativement brève à l'état adulte. De ce fait, il est difficile ou impossible de distinguer la mortalité aigüe liée à des mécanismes spécifiques d'une diminution nonspécifique de la résistance à un stress.La longévité des adultes de Tribolium rend possible de séparer ces deux catégories de réponses, elle peut aussi permettre la mise en évidence des différents modes de léthalité aigüe.Des adultes récemment formés de Tribolium confusum, maintenus à 30° dans le milieu farine-levure, ont été exposés à des doses de rayons X de 8 à 75 kR aux taux de 225 ou 1000 R/min. Dans tous les cas, il n'y a pas eu de mortalité significative pendant 10 jours, et toutes les morts attribuables à l'irradiation ont eu lieu au cours des 10 à 15 jours suivants; après les doses les plus faibles, beaucoup d'insectes ont survécu à cette période critique et ont alors vécu de nombreux mois. L'indépendance relative par rapport à la dose de la durée de la survie des descendants suggère l'existence d'un mode spécifique de mortalité, ou d'un syndrome léthal aigu.Les insectes irradiés en élevage à 22° ont présenté un délai de début de mortalité environ le double et une durée de la période de mortalité environ double de ceux enregistrés à 30°. Ceci représente un Q₁₀ d'environ 2,3, qui suggère la nécessité d'une succession de phénomènes métaboliques dans le développement de la léthalité.Des insectes sur un régime à base d'amidon de maïs étaient au moins aussi radiorésistants que ceux sur farine et levure; les insectes à 22° furent sans conteste plus sensibles que ceux élevés à 30°. Des coléoptères plus âgés, exposés 4 ou 10 mois après leur formation, ont montré des augmentations progressives dans la radiosensibilité aigüe.Ces résultats ont deux conséquences importantes pour d'autres recherches radiobiologiques sur les insectes adultes. La mise en évidence de l'existence d'un syndrome léthal spécifique et de ses délais de manifestation devrait accélérer l'analyse des facteurs intervenant dans la mort des insectes irradiés; d'autre part dans toute expérimentation radiobiologique sur des insectes adultes, les imagos doivent être d'un âge uniforme et connu.

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This work was supported by U. S. Public Health Service, NIH Grant GM-10208. During 1964–1965, the senior author held a Special Fellowship (1-F3-GM23423), U. S. Public Health Service, at the Department of Zoology, University of Cambridge; he is indebted to Professor C. F. A. Pantin, F. R. S., and other members of the Department, and especially to Dr. George Salt, F. R. S., both for extending use of their facilities and for many illuminating discussions.     

Accuracy of pulse oximetry to estimate HbO2 fraction of total Hb during exercise

The accuracy of two pulse oximeters (Ohmeda 3700 and Biox IIa) was evaluated during cycle ergometer incremental exercise in 10 healthy subjects. The exercise protocol began at 30 W with the power output being increased 15 W.min-1 until volitional fatigue. Ear and finger probe pulse oximetry measurements of available hemoglobin (%Spo2) were compared with arterial oxyhemoglobin fraction of total hemoglobin (%HbO2) measured directly from arterial blood samples using a CO-oximeter. To provide a wide range of %HbO2 values, four subjects exercised under hypoxic conditions [inspired partial pressure of O2 (PIo2) = 107 Torr], while the remaining six subjects exercised under normoxic conditions (PIo2 = 150 Torr). Because carboxyhemoglobin (HbCO) or methemoglobin (MetHb) is not measured by pulse oximeters, %HbO2 was corrected for HbCO and MetHb and expressed as percent arterial O2 saturation of available Hb (%Sao2). Small and insignificant differences (P greater than 0.05) existed between SpO2 (all 3 instruments) and %SaO2 at the lowest work rate and the highest power output achieved. Regression analyses of %SpO2 vs. %SaO2 produced correlation coefficients of r = 0.82 [standard error of the estimate [(SEE) = 1.79], r = 0.89 (SEE = 1.48), and r = 0.93 (SEE = 1.14) for the Biox IIa, Ohmeda 3700 (ear), and the Ohmeda 3700 (finger) pulse oximeters, respectively. We conclude that pulse oximetry, within the above limits of accuracy, is useful in estimating %SaO2 during exercise in healthy subjects.     

The precise radioimmunoassay of adenosine: minimization of sample collection artifacts and immunocrossreactivity.

Anti-adenosine antibodies were produced in rabbits immunized with N6-carboxymethyladenosine conjugated to methyl albumin. 125I-N6-Aminobenzyladenosine was synthesized and used as a high-specific-activity, high-affinity ligand. A radioimmunoassay (RIA) was developed that can detect 6.25 nM (312.5 fmol) of underivatized adenosine and cross-reacts less than 0.02% with adenine nucleotides and guanosine and not at all with 1 mM inosine. The sensitivity of the RIA can be increased to a detection limit of 0.125 nM (6.25 fmol) by derivitizing samples with benzyl bromide to form N6-benzyladenosine. The assay was adapted to an automated RIA procedure. Assay precision was increased by: (i) inhibiting slight adenosine deaminase activity present in anti-sera; (ii) treating buffers and albumin used in the RIA with charcoal to remove contaminating adenosine; and (iii) correcting for a small but variable component of immunoreactivity not attributable to adenosine. A second antibody prepared with a 2',3'-disuccinyladenosine-albumin conjugate was also found to detect some non-adenosine-mediated immunoreactivity in plasma samples. Immunointerference in human plasma was eliminated in samples treated with ZnSO4/Ba(OH)2 or partially purified over C18 Sep Paks to remove nucleotides and assayed after sample benzylation or succinylation. Human blood was mixed with a novel "stop" solution that was optimized to inhibit adenosine formation from AMP by greater than 99% and to inhibit adenosine uptake into red cells and degradation by greater than 94%. Human plasma/stop solution was assayed by RIA and HPLC with equivalent results.     

Application of an immune-tolerizing procedure to generate monoclonal antibodies specific to an alternate protein isoform of bovine growth hormone.

An immune-tolerizing protocol was employed to generate monoclonal antibodies to a variant protein isoform of bovine growth hormone arising from alternative pre-mRNA processing. Variant bovine growth hormone used for immunization was obtained by expression in bacteria and electroelution of the protein from preparative sodium dodecyl sulfate-polyacrylamide gels. Balb/c mice were first immunized with wild-type bovine growth hormone in the presence of the cytotoxic drug cyclophosphamide, thereby tolerizing the mouse to common epitopes shared among the two proteins. Subsequently, the mice were immunized with variant bovine growth hormone to produce antibodies specific to variant epitopes. Comparisons of fusions resulting from standard and tolerizing immunization protocols resulted in a significantly enhanced production of variant bovine growth hormone-specific antibodies as a result of the immunotolerizing protocol. The specificity of the antibodies to the variant growth hormone was substantiated by differential enzyme-linked immunosorbent assay and Western blot. Nearly all hybridomas positive for variant growth hormone were negative for wild-type growth hormone. Finally, the antibodies were used to demonstrate intracytoplasmic staining of COS I cells transiently transfected with a variant growth hormone-producing plasmid. Given the power of the polymerase chain reaction to conveniently clone alternatively processed mRNA species, followed by expression in bacteria to provide antigen, the immunotolerizing protocol provides a convenient general method for producing antibodies specific to desired protein isoforms.