A theoretical approach to the large-scale evolution of multicellularity and cell differentiation

In contrast to Darwinian evolution in which organisms have been selected by the instantaneous judgment of advantage or disadvantage for a mutated gene, the large-scale evolution of multicellular organisms by drastic changes in their genomes to produce new genes is theoretically formulated on the basis of the new concept of ‘biological activity’. The ‘biological activity’ of an organism is a macroscopic quantity determined by its whole genome and the environment, consisting of three terms; the energy acquired from the outside, the energy stored in the form of bio-molecules, and the systematization of multicellularity as well as of organizing genes and their products. The acquired energy minus stored energy is lost as heat, and the entropy production by the heat must compensate for the entropy reduction owing to the systematization in the organism. Under the boundary determined by this thermodynamic law, the organisms, which experienced gene duplication to produce new genes for multicellularity and cell differentiation, first decline to be minor members in a population by the increase in the energy to be stored and by the advanced systematization of cell differentiation. If the acquired energy is raised by the cooperative action of newly differentiated cells with the pre-existing types of cells, however, the ‘biological activity’ of this new style of organism can be recovered. The new style of organism generated through this evolutionary process does not necessarily expel the old style of organism to extinction but can coexist by choosing different material and energy resources. Moreover, this theory of large-scale evolution not only explains the punctuated mode of evolution indicated by paleontology but also reproduces the divergence of body plans observed in Triploblastica and Tracheophyta.     

The theory of organism and the culturalist foundation of biology

Summary After the disappearance of organism was diagnosed, the discussion about the role of a theory of organism in biology is characterised by a significant contradiction. On the one hand, the importance of a theory of organism is stated. Particularly developmental biology demands organism-centred approaches as a basis for conceptual integration. On the other hand, several modern biological disciplines such as genetics and molecular biology simply don’t need a theory of organism for their work. Consequently, the determination of the status of the organism and its relevance for biology at all is an unsolved problem. In order to clarify the methodological status of the organism in biology we start with the reconstruction of three important propositions. A life oriented approach and a hierarchy concept - which both are from a neo-Darwinian origin - are confronted with a structuralist approach of organism, that can be characterised as a non-Darwinist approach. Our own attempt for the solution of the organism problem applies the tools of culturalist methodology. In accordance to this pragmatic approach, the term organism is introduced as a concept of notion. A constructional morphological case study exemplifies the applicability of this concept. From the culturalist point of view a methodological foundation of biology can be achieved, that provides a consistent basis for a comprehensive integration of biological knowledge.     

Biocalorimetry- supported analysis of fermentation processes

The close relation between metabolic activity and heat release means that calorimetry can be successfully applied for on-line monitoring of biological processes. Since the use of available calorimeters in biotechnology is difficult because of technical limitations, a new sensitive heat-flux calorimeter working as a laboratory fermenter was developed and tested for different aerobic and anaerobic fermentations with Saccharomyces cerevisiae and Zymommonas mobilis. The aim of the experiments was to demonstrate the abilities of the method for biotechnological purposes. Fermentations as well as the corresponding heat, substrate and product analyses were reproducible. During experiments the heat signal was used as a sensitive and fast indicator for the response of the organisms to changing conditions. One topic was the monitoring of diauxic growth phenomena during batch fermentations, which may affect process productivity. S. cerevisiae was used as the test organism and a protease-excreting Bacillus licheniformis strain as an industrial production system. Other experiments focused on heat measurements in continuous culture under substrate-limiting conditions in order to analyse bacterial nutrient requirements. Again, Z. mobilis was used as the test organism. Ammonium, phosphate, magnesium, biotin and panthothenate, as important substrate compounds, were varied. The results indicate that these nutrients are required in lower amounts for growth than formerly suggested. Thus, a combination of heat measurements and other methods may rapidly improve our knowledge of nutrient requirements even for a well-known microorganism like Z. mobilis. *** DIRECT SUPPORT *** AG903062 00004     

Measurement of heat evolution and correlation with oxygen consumption during microbial growth. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 3, Pages 269-281 (1968)

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent on the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.     

Thermogenic effects of a "cafeteria" diet on the rat during its reproductive cycle

The heat production and oxygen consumption of intact virgin, pregnant, lactating and postlactating rats has been investigated both in groups fed a "cafeteria" diet as well as in controls. A third group of rats fed the cafeteria diet after parturition has been investigated. Pregnant rats fed a "cafeteria" diet increased their weight faster than controls. During lactation no increases in weight were observed, and in postlactation both "cafeteria" groups attained the same values higher than controls. The ingestion of a "cafeteria" diet resulted in higher heat production in all groups except lactating rats, which--in addition--showed higher heat outputs than all the other groups when the actual data were corrected by metabolic weight according to the surface law. The high lactation heat production (and oxygen consumption) can be a consequence of increased metabolic activity in the rat organism, devoted to milk production. It can be concluded that during lactation the dam energy output through the milk must absorb any increases in energy input due to the more densely-packed energy in the "cafeteria" diet, and this did not result in increased heat production.     

PCR-based gene synthesis as an efficient approach for expression of the A+T-rich malaria genome

The A+T-rich genome of the human malaria parasite Plasmodium falciparum encodes genes of biological importance that cannot be expressed efficiently in heterologous eukaryotic systems, owing to an extremely biased codon usage and the presence of numerous cryptic polyadenylation sites. In this work we have optimized an assembly polymerase chain reaction (PCR) method for the fast and extremely accurate synthesis of a 2.1 kb Plasmodium falciparum gene (pfsub-1) encoding a subtilisin-like protease. A total of 104 oligonucleotides, designed with the aid of dedicated computer software, were assembled in a single-step PCR. The assembly was then further amplified by PCR to produce a synthetic gene which has been cloned and successfully expressed in both Pichia pastoris and recombinant baculovirus-infected High Five(TM) cells. We believe this strategy to be of special interest as it is simple, accessible and has no limitation with respect to the size of the gene to be synthesized. Used as a systematic approach for the malarial genome or any other A + T-rich organism, the method allows the rapid synthesis of a nucleotide sequence optimized for expression in the system of choice and production of sufficiently large amounts of biological material for complete molecular and structural characterization.     

Cell shape: taking the heat

Preservation of cell architecture under physically stressful conditions is a basic requirement for many biological processes and is critical for mechanosensory systems built to translate subtle changes in cell shape into changes in organism behaviour. A new study reveals how an extracellular protein--Spam--helps mechanosensory organs in the fruit fly to withstand the effects of the water loss that accompanies heat shock.     

Measurement of heat evolution and correlation with oxygen consumption during microbial growth

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent On the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.     

Seasonal changes in the body and muscle heat resistance of the green toad

A study was made of the dynamics of seasonal changes in heat resistance of the organism and muscles of the green toad taken from micropopulation living in the North-Eastern suburbs of Tashkent. The heat resistance of the organism was determined by the time of onset of the irreversible heat shock caused by the immersion of animals into water at 39 +/- 0.1 degrees C. The heat resistance of muscles (m. gastrocnemius) was determined by the time of the loss of response to electrical stimulus during heating in the Ringer solution (41 +/- 0.1 degrees). It has been shown that the organism as a whole is much more dependent on seasonal variations of environmental temperature as compared with muscle tissue. Of much importance for the changes in the heat resistance of muscle tissue are cyclic hormonal changes in the organism associated with the process of reproduction and preparation to it.     

Iron uptake processes in Mycobacterium vaccae R877R, a mycobacterium lacking mycobactin

The production of exochelins (MV) was established in Mycobacterium vaccae R877R under iron-deficient conditions in concentrations about five times greater than in Mycobacterium smegmatis. M. vaccae does not produce mycobactin nor is salicylic acid secreted into the medium. A simple method is described using 55Fe-labelled culture filtrates for assessing exochelin production and which would be applicable to other mycobacteria. One of the exochelins produced (MV3) is part of an active iron uptake system and another (MV1) is responsible for a passive uptake system. MV3 exochelin has similar chromatographic properties and biological activity to the major exochelin produced by M. smegmatis: iron uptake from MV3 exochelin was inhibited by dinitrophenol, NaN3 and HgCl2, and was judged to be an active transport process. This process was not inhibited by equimolar amounts of ferri-salicylate or ferri-citrate both of which could be used separately as sources of iron for the organism. Uptake from these latter sources was insensitive to metabolic inhibitors and uncouplers. The multiplicity of pathways for iron uptake in a single organism is discussed.     

Large scale expression and purification of recombinant RNA in Escherichia coli

Stable, folded RNA are involved in many key cellular processes and can be used as tools for biological, pharmacological and/or molecular design studies. However, their widespread use has been somewhat limited by their fragile nature and by the difficulties associated with their production on a large scale, which were limited to in vitro methods. This work reviews the novel techniques recently developed that allow efficient expression of recombinant RNA in vivo in Escherichia coli. Based on the extensive data available on the genetic and metabolic mechanisms of this model organism, conditions for optimal production can be derived. Combined with a large repertoire of RNA motifs which can be assembled by recombinant DNA techniques, this opens the way to the modular design of RNA molecules with novel properties.     

Physiological consequences of intermittent exercise during compensable and uncompensable heat stress.

Time-weighted averaging is a traditional method used in heat stress analyses to approximate, in terms of a single continuous level of heat production, the rate of heat production from complex intermittent exercise patterns. Physiological responses during intermittent and continuous exercise were studied in four subjects exposed to heat stress in which evaporation was either free or severely restricted. Intermittent work consisted of repeated 10-min exercise-rest patterns. Continuous work was at the time-weighted average of intermittent exercise: 3.3 mets. When heat stress was uncompensable, intermittent work induced more physiological strain than continuous work: endurance time was 14 min less (P less than 0.05); core temperature at 60 min was 0.40 degrees C higher (P less than 0.05); and, after 30 min of exposure, the rate of core temperature rise was 33% greater. The difference in the rate of heat storage was not satisfactorily explained by a discrepancy in the average rate of heat production or in the calculated rate of surface heat loss. Alternatively, the results may be partially explained by interruptions in the usual rate of heat transport via the cutaneous circulation. These interruptions may be caused by nonthermal factors associated with postural and work load transitions. Although the mechanisms are not totally understood, it is clear that application of the time-weighted averaging method can lead to erroneous overprediction of endurance time and should be applied with discretion.     

Accumulation of polyhydroxyalkanoates by Microlunatus phosphovorus under various growth conditions

Microlunatus phosphovorus is an activated-sludge bacterium with high levels of phosphorus-accumulating activity and phosphate uptake and release activities. Thus, it is an interesting model organism to study biological phosphorus removal. However, there are no studies demonstrating the polyhydroxyalkanoate (PHA) storage capability of M. phosphovorus, which is surprising for a polyphosphate-accumulating organism. This study investigates in detail the PHA storage behavior of M. phosphovorus under different growth conditions and using different carbon sources. Pure culture studies in batch-growth systems were conducted in shake-flasks and in a bioreactor, using chemically defined growth media with glucose as the sole carbon source. A batch-growth system with anaerobic–aerobic cycles and varying concentrations of glucose or acetate as the sole carbon source, similar to enhanced biological phosphorus removal processes, was also employed. The results of this study demonstrate for the first time that M. phosphovorus produces significant amounts of PHAs under various growth conditions and with different carbon sources. When the PHA productions of all cultivations were compared, poly(3-hydroxybutyrate) (PHB), the major PHA polymer, was produced at about 20–30% of the cellular dry weight. The highest PHB production was observed as 1,421 mg/l in batch-growth systems with anaerobic–aerobic cycles and at 4 g/l initial glucose concentration. In light of these key results regarding the growth physiology and PHA-production capability of M. phosphovorus, it can be concluded that this organism could be a good candidate for microbial PHA production because of its advantages of easy growth, high biomass and PHB yield on substrate and no significant production of fermentative byproducts.     

Elevated serine catabolism is associated with the heat shock response in Escherichia coli.

The biochemical events associated with the heat shock response are not well understood in any organism, nor have the signals that initiate the induction of heat shock protein synthesis been identified. In this work, we demonstrate that the rate of serine catabolism of Escherichia coli cells grown in glucose minimal medium supplemented with serine is elevated three- to sevenfold when the growth temperature is shifted from 37 to 44 degrees C. Elevations in growth temperature and mutations or treatments that lead to elevated basal rates of serine catabolism at 37 degrees C result in the excretion into the culture medium of acetate derived from exogenous serine. Increases in the basal level of serine catabolism at 37 degrees C do not per se induce a heat shock response but are associated with abnormalities in the pattern of induction of heat shock polypeptides following a temperature shift. We postulate that the events responsible for or resulting from the elevation in serine catabolism associated with a shift-up in temperature modulate the induction of 3 of the 17 heat shock polypeptides identified in E. coli. These observations suggest that heat shock diverts serine away from the production of glycine and C1 units, which are required for initiation of protein synthesis and for nucleotide biosynthesis, and towards acetyl coenzyme A and acetate.     

Extraction of hemicellulose from ryegrass straw for the production of glucose isomerase and use of the resulting straw residue for animal feed

The hemicellulose fraction of ryegrass straw was extracted with NaOH and used for the production of glucose isomerase by Streptomyces flavogriseus. The level of hemicellulose extracted increased proportionately with increasing NaOH concentration up to about 4%, then the rate of increase slowed down. Hemicellulose extraction was facilitated by the combined application of heat and NaOH. Approximately 15% hemicellulose (12% as pentosan) could be obtained by treating straw with 4% NaOH for either 3 hr at 90°C or 24 hr at room temperature. The highest level (3.04 units/ml culture) of intracellular glucose isomerase was obtained when the organism was grown at 30°C for two days on 2% straw hemicellulose. The organism also produced a high yield of glucose isomerase on xylose or xylan. The NaOH-treated straw residue, after removal of hemicellulose, had approximately 75% higher digestibility and 20% higher feed efficiency for weanling meadow voles than untreated straw. Thus, the residue could be used as animal feed. A process for the production of glucose isomerase and animal feed from ryegrass straw was also proposed.     

Microcalorimetric investigation of metabolism in rat hepatocytes cultured on microplates and in cell suspensions

In the present work, heat production rate in rat hepatocytes has been measured by use of thermopile heat conduction calorimeters. Both hepatocytes cultured in monolayers on microplates and hepatocytes in suspensions were used for microcalorimetric measurements. The highest heat production rate was found in newly cultured cells; thereafter, a gradual decrease was noted. After 1 day of culture, metabolic activity had reached a steady state that lasted about 4 days. A cell-density dependence of heat production was found, both in cell suspensions and in cultured hepatocytes on microplates. Higher cell concentration in the calorimeter ampoule was accompanied by decreasing heat production per cell. The heat output recorded for hepatocytes cultured on microplates (25 X 10(3) cells) was found to be 0.327 +/- 0.13 nW per cell after 24-48 h. Addition of sodium azide and sodium fluoride to tissue culture medium reduced heat production rate in cultured hepatocytes by 60 and 20%, respectively. Recording of heat production with the present calorimetric technique is relatively simple and fast, and offers the possibility to perform measurements in small samples of cultured hepatocytes on microplates, thus allowing long-term as well as repeated measurements on the same cell population.     

Analysis of aflatoxin-B1-degrading microbes by use of a combined toxicity-profiling method

To monitor cytotoxic and genotoxic effects of aflatoxin, a luminescent assay employing Aliivibrio fischeri as a test organism and a colorimetric assay based on the SOS-Chromotest were adapted to our needs. The aim of this method-developing work was to be able to select - from a collection of environmental isolates - microbes that degrade aflatoxin without production of harmful intermediates and by-products, in a fast and cost-effective way. By the combination of the two modified assays, microbes that met these criteria have been successfully selected. Among thirty-three isolates, the strain Rhodococcus rhodochrous NI2 proved to be the best aflatoxin-B1-degrading microbe, with the weakest harmful biological effects throughout aflatoxin-B1-degradation. Our findings underline the necessity to employ bio-tests in biodegradation assays, as cytotoxicity and/or genotoxicity may occur even after substantial degradation of the toxins.     

Biological role of melatonin during summer season related heat stress in livestock

Heat stress can cause a significant financial burden to livestock producers by decreasing all productive functions in livestock. The major strategies associated with relieving heat stress in livestock are through use of sheds, fans, or evaporative cooling. Such practices are not possible where the animals are reared in a semi-intensive system. This necessitates developing other strategies to counteract the adverse effects of heat stress. A new strategy involving the feeding of melatonin (MEL) has been evaluated by a few researchers. Melatonin has hypothermic and antioxidant effects and may counter the detrimental effect of heat stress on livestock production. The aim of the paper is to review evidence for and against the use of MEL as an anti-heat stress agent. The early suggestion of a functional antagonism between the pineal and the adrenal gland became additionally reinforced by experimental and clinical findings indicating that MEL may be able to protect the organism against heat stress-induced damages. Melatonin effectively protects against heat stress, by a variety of mechanisms. As animals in tropical countries are exposed to heat stress during much of the year, MEL with its potential beneficial effects may be useful as an anti-heat stress agent to prevent the loss of production.