Plasmid-encoded protein: the principal factor in the "metabolic burden" associated with recombinant bacteria

Experimental elucidation of the metabolic load placed on bacteria by the expression of foreign protein is presented. The host/vector system is Escherichia coli RR1/pBR329 (amp(r), cam(r), and let(r)). Plasmid content results, which indicate that the plasmid copy number monotonically increases with decreasing growth rate, are consistent with the literature on ColE1-like plasmids. More significantly, we have experimentally quantified the reduction in growth rate brought about by the expression of chloramphenicol-acetyl-transferase (CAT) and beta-lactamase. Results indicate a nearly linear decrease in growth rate with increasing foreign protein content. Also, the change in growth rate due to foreign protein expression depends on the growth rate of the cells. The observed linear relationship is media independent and, to our knowledge, previously undocumented. Furthermore, the induction of CAT, mediated by the presence of chloramphenicol, is shown to occur only at low growth rates, which further increases the metabolic load.Results are vdelineated with the aid of a structured kinetic model representing the metabolism of recombinant E. coli. In this article, several previous hypotheses and model predictions are justified and validated. This work provides an important step in the development of comprehensive, methabolically-structured, kinetic models capable of prediciting optimal conditions for maximizing product yield.     

Isolation and characterization of noradrenalin storage granules of bovine adrenal medulla

A method is described for the preparation of (1) the heavy population of bovine adrenal chromaffin granules ($\text{S}{}_{\mathrm{<mtext>H</mtext>}}\text{(}\text{average sedimentation coefficient}\text{) = 12 400}\text{S}$ in 0.25 M sucrose) essentially free from contamination with mitochondria and other organelles, and (2) a subpopulation of this heavy population which is highly enriched in noradrenalin ($\text{?95\%}$ of the total catecholamine is noradrenalin). The method is based on isopycnic gradient centrifugation using a self-generating gradient of polyvinylpyrrolidone-coated colloidal silica particles (Percoll) in 0.5 M sucrose medium.The isolated population of noradrenalin granules appeared highly electron dense in transmission electron microscopy and revealed a rather narrow size distribution. The specific content of amine and adenine nucleotides (with reference to total granule protein) was markedly higher than for the total population of heavy chromaffin granules. The molar ratio of amines to adenine nucleotides was, however, lower in the noradrenalin granules, i.e. 4.8 vs. 11.9.     

Chironomid communities as water quality indicators

Recent mathematical indices summarizing biological communities of indicators are recapitulated. Improvements of these indices based on weighting according to width of trophic ranges of each species are suggested. Their principle deficiencies, however, are pointed out.
Revised lists of characteristic profundal as well as littoral and sublittoral chironomids in Nearctic and Palearctic lakes show that at least 15 characteristic chironomid species communities can be delineated, 6 in each of the oligotrophic and the eutrophic ranges and 3 in the mesotrophic range. It is proposed that these communities be lettered consecutively in the Greek alphabet from α (alpha) to o (omikron). A key to the 15 divisions based on the species associations in the profundal zone of harmonic lakes is put forward. There is very good correlation between the 15 divisions and the ratios of average total phosphorus to mean lake depth and average chlorophyll a to mean lake depth.
The ratio of chironomids to oligochaetes and the distribution patterns of single species have proven useful in pin-pointing localized areas of pollution. The primary mechanism governing the distribution of chironomid communities in oligotrophic and mesotrophic lakes appears to be the availability of food materials rather than the annual hypolimnetic oxygen concentration. In eutrophic lakes the relationships between organic matter accumulation and oxygen levels are so interdependent as to be inseparable.     

metabolism of prostaglandins of the A-type

, originally introduced as an inadvertent contaminant in solutions used for evaluating the stability of prostaglandins, proved to lead to the rapid disappearance of the cyclopentenone unit of PGA₂ (as monitored by circular dichroic spectroscopy). The cyclopentenone unit is converted, in various metabolites, to a 9-keto, 9α or 9β-hydroxy group lacking the ring unsaturation. The major EtoAc-soluble 9-hydroxy metabolite (Compound-I) was shown to be 9α, 15α-dihydroxy-2,3,4,5-tetranor-13- -prostenoic acid. Similar tetranor 9-hydroxy metabolites with one additional degree of unsaturation, and with a 9β-hydroxy group, also occur but these have not been fully characterized. Only two of the wide range of 9-keto metabolites are fully characterized by mass spectral (MS) data: 9,15-oxo-2,3,4,5-tetranorprostanoic acid and 9,15-oxo-2,3,4,5-tetranor-13- -prostenoic acid. The water soluble metabolites have not been characterized further.The fully characterized metabolites together with MS data from mixtures of minor metabolites indicate that can perform the following transformations: β-oxidation, dehydrogenation at C-15, reduction of the enone carbon-carbon double bonds (both Δ^10,11 and Δ^13,14), reduction of the 9-ketone, and possibly migration of the cyclopentyl double bond (Δ^10,11 → Δ^11,12). metabolizes 15-epimeric PGA₂ equally readily with the production of similar products. PGA₁ affords less 9-keto metabolites with compound I constituting 33% of the product by HPLC analysis. displays some enantioselectivity, PGA₂ and 15-epi-PGA₂ are each metabolized more rapidly than their enantiomers. Other prostaglandins appear to be less readily metabolized.     

Enkephalin and substance P effects related to trigeminal pain

Iontophoretic applications of enkephalin (20-150 nA) reduced the spontaneous firing frequency of nociceptive neurons in the trigeminal nucleus caudalis of decerebrated cats. The response evoked by noxious stimulation (tooth pulp) was gradually inhibited during the 1st minute of application of the opioid and generally remained depressed for 5 min after the current was turned off. These effects of enkephalin were blocked by intravenously or iontophoretically administered naloxone. Nonnociceptive neurons or nociceptive neurons responding to nonnoxious inputs were less frequently inhibited by enkephalin. When tested on nonnociceptive cells, similar applications of substance P usually had little effect. Nociceptive neurons, however, were strongly excited by substance P. This action was not constant and was interrupted by periods of inactivation. Both types of peptide action were similar in temporal aspects. The results suggest a functional interrelationship between enkephalin and substance P in a trigeminal system mediating nociception.     

Temperature-sensitive deoxyribonucleic acid replication in a dnaC mutant of Bacillus subtilis.

A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenical, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment.     

Egg size and form as taxonomic criteria in Diphyllobothrium (Cestoda, Pseudophyllidea)

The size and form (length, width, and length: width ratio) of eggs of Diphyllobothrium dendriticum, D. ditremum and D. latum vary considerably among individual worms within each species. The size of eggs varies with host species and a decrease in egg size with increasing intensity of infestation is indicated. The egg size of D. latum increases during the first 10-12 days of egg production. For single worm infections in golden hamsters the mean egg length and width of D. ditremum are significantly smaller than the corresponding means of D. dendriticum and D. latum, while D. latum has significantly wider eggs than D. dendriticum. As taxonomic characteristics, egg size and form may contribute to species delimitation at the population level. For identification at the individual level the best possible accuracy is about 80%. This accuracy is considerably reduced when variation in host species and intensities of infestations are introduced. Scanning electron microscope studies did not reveal any differences among eggs of the three species.     

The morphogenesis of dimorphic pollen and anthers in Tripogandra amplexicaulis. Light microscopy and growth analysis

A characteristic of the genus Tripogandra is the presence of two dissimilar whorls of anthers. In T. amplexicaulis the short filamented anthers (S anthers) develop fertile, spheroidal pollen, the long filamented ones (L anthers) develop elongated spores, which degenerate prior to anthesis. Meiosis is regular in both types but occurs approximately two days later in the L anthers. Developmental traits of the L spores are a continuous, uniaxial extension and a sigmoid course of growth in volume accompanied by progressive vacuolarization and growth of starch grains and usually no division. The number of spores in the L anthers amounts to only 60 % of that in the S anthers, but the values for anther fresh weight and pollen sac volume of the former eventually exceed those of the latter and the relative growth rate in these variables is consistently higher in the L anthers. The relative rate in dry weight, however, is similar for both anthers and the absolute rate is clearly lower in the L anthers. These growth characteristics are reflected in a greater vacuolarization of both spores and the amoeboid tapetum in the L anthers. Cytological features of male sterile plants with hypotrophy of the tapetum are related to the sterility syndrome of the L anthers and a common hypothesis for the deviating features is presented.