Influence of single amino acids on the development of hamster one-cell embryos in vitro

One-cell hamster embryos placed in culture have always shown a complete block to development at the two-cell stage. In a preliminary study using a chemically defined culture medium containing 20 amino acids (HECM-1), many one-cell embryos were able to escape the "two-cell block" and develop to the four-cell stage. Use of a simpler formulation containing only the amino acids hypotaurine and glutamine revealed marked inhibitory and stimulatory effects of adding the other amino acids. In the first experiment, 19 amino acids were separately examined for effects on one-cell embryo development. Six amino acids (phenylalanine, valine, isoleucine, tyrosine, tryptophan, and arginine) inhibited embryo development (reduced mean cell number; MCN), and three others (glycine, cystine, and lysine) stimulated development (increased MCN), compared with basic medium containing only glutamine and hypotaurine (low control). When the responses with the six inhibitory amino acids were totalled, only 3 of 185 (2%) one-cell embryos reached the six-or seven-cell stage compared to a total of 15 of 76 (20%) embryos that developed to these stages using the three stimulatory amino acids. When tested together in a second experiment, the six inhibitory amino acids significantly reduced the MCN, from 4.28 +/- 0.44 (low control) to 3.71 +/- 0.55. In this group, 17 of 117 (15%) of one-cell embryos reached more than four-cell and only 4 of 117 (3%) reached six- or 7-cell stages, compared with 39 of 117 (33%) and 12 of 117 (10%), respectively, for the basal medium group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dimensions of face in Asia in the perspective of geography and prehistory

Data from 24 craniofacial dimensions were compiled for samples representing all the human populations of the world. These were converted into C scores and used to construct Euclidean Distance dendrograms. The populations of the world are best depicted as 8 major regional clusters representing: Africa, Amerind, Asia-Mainland, Australo-Melanesia, Eskimo-Siberia, Europe, India, and Jomon-Pacific. The mainland Asian cluster divided into northern and southern components. The Neolithic in both areas is less clearly associated with the northern or southern components and may represent the form of their undifferentiated precursors. This needs to be tested by the addition of further specimens and an adequate sample from South China. Mongols are peripheral members of the Asia-Mainland cluster and should not be thought of as typifying the region as a whole. The use of the term "Mongoloid" is inappropriate in both theory and practice.     

Estimating fermentative amino acid catabolism in the small intestine of growing pigs

Fermentative catabolism (FAAC) of dietary and endogenous amino acids (AA) in the small intestine contributes to loss of AA available for protein synthesis and body maintenance functions in pigs. A continuous isotope infusion study was performed to determine whole body urea flux, urea recycling and FAAC in the small intestine of ileal-cannulated growing pigs fed a control diet (CON, 18.6% CP; n=6), a high fibre diet with 12% added pectin (HF, 17.7% CP; n=4) or a low-protein diet (LP, 13.4% CP; n=6). ¹⁵N-ammonium chloride and ¹³C-urea were infused intragastrically and intravenously, respectively, for 4 days. Recovery of ammonia at the distal ileum was increased by feeding additional fibre when compared with the CON (P<0.05) but was not affected by dietary protein (0.24, 0.39 and 0.14 mmol nitrogen/kg BW/day for CON, HF and LP, respectively; P<0.05). Lowering protein intake reduced urea flux (25.3, 25.7 and 10.3 mmol nitrogen/kg BW/day; P<0.01), urinary urea excretion (14.4, 15.0 and 6.2 mmol N/kg BW/day; P<0.001) and urea recycling (12.1, 11.3 and 3.23 mmol nitrogen/kg BW/day; P<0.01) compared with CON. There was a rapid reduction in ¹⁵N-ammonia enrichment in digesta along the small intestine suggesting rapid absorption of ammonia before the distal ileum and lack of uniformity of enrichment in the digesta ammonia pool. A two-pool model was developed to determine possible value ranges for nitrogen flux in the small intestine assuming rapid absorption of ammonia. Maximum estimated FAAC based on this model was significantly lower when dietary protein content was decreased (32.9, 33.4 and 17.4 mmol nitrogen/kg BW/day; P<0.001). There was no impact of dietary fibre on estimates of small intestine nitrogen flux (P>0.05) compared with CON. The two-pool model developed in the present study allows for estimation of FAAC but still has limitations. Quantifying FAAC in the small intestine of pigs, as well as other non-ruminants and humans, offers a number of challenges but warrants further investigation.     

The potential of a polyphasic PCR-dGGE approach in evaluating microbial diversity of natural whey cultures for water-buffalo Mozzarella cheese production: bias of culture-dependent and culture-independent analyses

A polyphasic PCR-DGGE approach was used to describe the microbial population occurring in natural whey cultures (NWCs) for water-buffalo Mozzarella cheese production. Total microbial community was assessed without cultivation by analyzing DNA directly extracted from the original samples of NWC. In addition, DNA extracted from bulks of cells formed by harvesting colonies from the serial dilution agar plates of a variety of culture media was used to profile the "cultivable" community. The 16S rDNA V3 region was amplified using DNA from NWC as well as DNA from bulks as templates and the amplicons were separated by DGGE. The microbial entities occurring in NWCs were identified by partial 16S rDNA sequencing of DGGE bands: four lactic acid bacteria (LAB) closest relative of Streptococcus thermophilus, Lactococcus lactis, Lactobacillus delbrueckii and Lactobacillus crispatus were revealed by the analysis of DNA directly extracted from NWC while two other LAB, Lactobacillus fermentum and Enterococcus faecalis, were identified by analyzing DNA from the cultivable community. The developed PCR-DGGE analysis of the "cultivable" community showed good potential in evaluating microbial diversity of a dairy environment: it usefully highlighted the bias introduced by selective amplification when compared to the analysis of the total community from NWC and allowed suitability of media and growth conditions to be evaluated. Moreover, it could be used to complete the culture independent study of microbial diversity to give information on concentration ratios among species occurring in a particular environment and can be proposed for rapid identification of dominant microorganisms in alternative to traditional tools.     

Analysis of energetic amino acid metabolism in Acyrthosiphon pisum: A multidimensional approach to amino acid metabolism in aphids

Aphids are highly specialized insects that feed on the phloem-sap of plants, the amino acid composition of which is very unbalanced. Amino acid metabolism is thus crucial in aphids, and we describe a novel investigation method based on the use of ¹⁴C-labeled amino acids added in an artificial diet. A metabolism cage for aphids was constructed, allowing for the collection and analysis of the radioactivity incorporated into the aphid body, expired as CO₂, and rejected in the honeydew and exuviae. This method was applied to the study of the metabolism of eight energetic amino acids (aspartate, glutamate, glutamine, glycine, serine, alanine, proline, and threonine) in the pea aphid, Acyrthosiphon pisum. All these amino acids except threonine were subject to substantial catabolism as measured by high ¹⁴CO₂ production. The highest turnover was displayed by aspartate, with 60% of its carbons expired as CO₂. For the first time in an aphid, we directly demonstrated the synthesis of three essential amino acids (threonine, isoleucine, and lysine) from carbons of common amino acids. The synthesis of these three compounds was only observed from amino acids that were previously converted into glutamate. This conversion was important for aspartate, and lower for alanine and proline. To explain the quantitative results of interconversion between amino acids, we propose a compartmentation model with the intervention of bacterial endosymbiotes for the synthesis of essential amino acids and with glutamate as the only amino acid supplied by the insect to the symbiotes. Moreover, proline exhibited partial conversion into arginine, and it is suggested that proline is probably indirectly involved in excretory nitrogen metabolism. © 1995 Wiley-Liss, Inc.     

Leprosy in the early medieval Lauchheim community

Leprosy was a well-recognized and dreaded disease in medieval Europe (5th-15th century AD). It is reported to have reached Germany with the Roman invasion. A much larger fraction than previously assumed appears to have been affected by leprosy in the medieval period. This article estimates the frequency (i.e., the prevalence at death) of leprosy among adult people buried in the Lauchheim early medieval cemetery. Seven different dichotomous osteological lesions indicative of leprosy are analyzed, and it is possible to score at least one of these conditions on 110 adult skeletons (aged 15 or more). The scores were transformed to a statistic--lambda (lambda)--indicating the likelihood that the person to whom the skeleton belonged suffered from leprosy. The analyses indicate that 16% (95% confidence interval: 9-23%) of adult people in Lauchheim died with osteological signs of leprosy. Leprosy was significantly more prevalent among men than women. The lambda statistic indicates that people who died with signs of leprosy did not differ in the distribution of age at death from those who did not have such signs. Some of the leprosy-related lesions had a statistically significant nonrandom dispersal on the cemetery; but there is no clear pattern to this and the significant results could be easily attributed to a type-1 error in the statistical analysis.     

Problems associated with the study of cellular oscillations

Data from the article by Brodsky et al. (2000) have been examined in order to confirm that the fluctuations they report are not random but have a rhythmic basis, thereby verifying their conclusions regarding the existence of periodicity in protein synthesis. Reasons are outlined why oscillations should be expected in all cellular biochemical studies. Associated practical problems are briefly discussed together with comments on the validity and value of less rigorous methods of analysis. The subject of aliasing is raised as justification for doubting the validity of much published data, especially where periodicity has not been suspected. The existence of oscillations indicates the need for a thorough re-evaluation of our understanding of cell biochemistry.     

Several pseudomonads,associated with the cultivated mushrooms Agaricus bisporus or Pleurotus sp., are hemolytic

Pseudomonas tolaasii, causing brown blotch disease on cultivated mushrooms, and yielding a white line precipitate towards P. “reactans”, has been shown to induce lysis of erythrocytes. Some Finnish strains isolated from diseased mushroom fruit bodies, although harboring the typical features of P. tolaasii, proved to be distinct, and have been allocated to a nov. sp. P. costantinii. We examined in these study whether all brown blotch causing agents were hemolytic. The induction of erythrocytes lysis seemed to be a rather common feature of mushroom associated-pseudomonads, especially for strains involved in the production of a white-line-in agar.     

Embryonic integument and "molts" in Manduca sexta (Insecta,Lepidoptera)

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed.     

Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10–18 areas, and increased in only 1–3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.     

The effects of sensory stimulation on the free amino acid pool of the developing brain

Developing chicks were subjected to three different paradigmata of sensory stimulation, and the effects on the free amino acid concentrations in the blood and in various brain regions were monitored. The free amino acid pool of individual brain regions was found to be affected in a treatment-and age-specific manner. The increased neuronal activity resulting from sensory stimulation seems to affect intrinsic factors involved in the regulation of the free amino acid pool, most likely via modulations of the rate of metabolization of individual amino acids and/or of the rate of synthesis and degradation of individual proteins in certain brain regions.Special Issue dedicated to Prof. Holger Hydén.     

Formation of polyol-fatty acid esters by lipases in reverse micellar media

The synthesis of polyol-fatty acid esters has strong implications in such industries as foods, cosmetics, and polymers. We have investigated these esterification reactions employing the polyols ethylene glycol, 2-monoglyceride, and sugars and their dervatives with the biocatalyst lipase in water/AOT/isooctane reverse micellar media. For the first reaction, 50-60% conversion was achieved and product selectivity toward the monoester over the diester shown possible by employing lipase from Rhizopus delemar. A simple kinetic model based on the formation of acyl-enzyme intermediate accurately predicted the effect of polyol concentration but not the effect of fatty acid or water concentration probably due to the model exclusion of paritioning effects. The success of this reaction in reverse micellar media is due greatly to its capacity to solubilize large quantities of glycol despite the media's overall hydrophobicity. The second reaction, investigated for its potential for production of "mixed" glycerides, also achieved about 50% conversion but had only a small portion of triglyceride in its product distribution. Also, isomerization of the 2-monoglyceride to 1-monoglyceride, followed by hydrolysis of the latter, unfortunately occurred to a significant extent. Attempts at esterification with hexoses and their derivatives such as glucose and mannitol produced no convesion.     

Changes of amino acid gradients in brain tissues induced by microwave irradiation and other means

Focused microwave irradiation to the head (FMI) has been used extensively by neurochemists for rapid inactivation of enzymatic activity in brain tissues and the preservation, for in vitro analysis, of in vivo substrate concentrations. Periodically the suitability of this technique for regional studies has been questioned. Evidence has now been obtained, on the basis of altered concentration gradients for GABA and taurine from the Substantia Nigra (SN) to an Adjacent Dorsal Area (ADJ), that FMI not only inactivates enzymes, but also facilitates rapid diffusion of small molecules from areas of high concentrations to adjacent areas of lower concentration. To a lasser extent, the implantation of plastic injection cannulas also decreased these concentration gradients. These results offer clear evidence that FMI is ill suited and unreliable for studies designed to map and compare the in vivo regional concentrations of diffusible organic molecules (such as amino acids) in brain tissues. Any invasive technique that compromises membrane barriers is likely to produce smaller similar effects.     

Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast

Summary Fluorescence of 1-anilinonaphthalene-8-sulfonate in yeast membranes appears to be caused predominantly by binding to lipids (ANS_proteinANS_lipid120) as indicated by the fluorescence lifetime, degree of polarization, and excitation spectra. It was insensitive to short-circuiting the membrane potential. Fluorescence intensity increased as cells (especially after pretreatment with energy donors such as glucose) were exposed to some amino acids, in particular, aspartic and glutamic acids. The character of fluorescence shifted to that of protein-bound ANS, suggesting an exposure of new protein sites accessible to the probe. This shift could be prevented by inhibitors of energy transduction as well as of transport. TheK _1/2 of the shift was at 2.5mm aspartic acid.     

Reevaluation of dominance hierarchy in bonobos (Pan paniscus)

While dominance relationships have been widely studied in chimpanzees, in bonobos, dominance style and linearity of hierarchy are still under debate. In fact, some authors stated that bonobo hierarchy is nonlinear/ill-defined, while others claimed that it is fairly linear. In this paper, we test the hypothesis that a shift in group composition determines changes in linearity of hierarchy. To test this hypothesis, we collected data on one of the largest captive groups in the world, in the Apenheul Primate Park (The Netherlands). We investigated the linearity of the hierarchy in two different periods, with a shifting group composition. We used the corrected Landau's index and David's scores to estimate which animals were most dominant. The major overall result of our study is that hierarchy is fairly nonlinear in this group: during the first study period (eight adults), the hierarchy was nonlinear, whereas during the second one (six adults), it failed to reach statistical linearity. We argue that the reduction of the number of adults is the principal factor affecting linearity. We also found that dominance interactions were evenly distributed across sex classes in both study periods. Furthermore, no correlation was observed between age/body weight and rank. As for the overall dominance relationship between males and females, our results suggest that there is no exclusive female dominance in the Apenheul group. The dominance style of bonobos may be loose and differentially expressed in diverse groups or in the same group, along with shifting conditions.     

Protein restriction and branched‐chain amino acid restriction promote geroprotective shifts in metabolism

The proportion of humans suffering from age‐related diseases is increasing around the world, and creative solutions are needed to promote healthy longevity. Recent work has clearly shown that a calorie is not just a calorie—and that low protein diets are associated with reduced mortality in humans and promote metabolic health and extended lifespan in rodents. Many of the benefits of protein restriction on metabolism and aging are the result of decreased consumption of the three branched‐chain amino acids (BCAAs), leucine, isoleucine, and valine. Here, we discuss the emerging evidence that BCAAs are critical modulators of healthy metabolism and longevity in rodents and humans, as well as the physiological and molecular mechanisms that may drive the benefits of BCAA restriction. Our results illustrate that protein quality—the specific composition of dietary protein—may be a previously unappreciated driver of metabolic dysfunction and that reducing dietary BCAAs may be a promising new approach to delay and prevent diseases of aging.     

Aromatic amino acid metabolism during organogenesis in rice callus cultures

The activity during root and shoot initiation of key enzymes involved in aromatic amino acid metabolism was examined in rice ( Oryza sativa L. cv. Bala) callus cultures. Increased activities of the enzymes quinate:NAD⁺ oxidoreductase (EC 1.1.1.24), shikimate kinase (EC 2.7.1.71), chorismate mutase (EC 5.4.99.5), anthranilate synthase (EC 4.1.3.27) and tryptophan synthetase (EC 4.2.1.20) were noticed in organ-forming callus compared to proliferating callus of rice, especially prior to the visible manifestation of form. These results suggest a correlation between organogenesis and the aromatic amino acid pathway.     

Branched-chain amino acid metabolism controls membrane phospholipid structure in Staphylococcus aureus

Branched-chain amino acids (primarily isoleucine) are important regulators of virulence and are converted to precursor molecules used to initiate fatty acid synthesis in Staphylococcus aureus. Defining how bacteria control their membrane phospholipid composition is key to understanding their adaptation to different environments. Here, we used mass tracing experiments to show that extracellular isoleucine is preferentially metabolized by the branched-chain ketoacid dehydrogenase complex, in contrast to valine, which is not efficiently converted to isobutyryl-CoA. This selectivity creates a ratio of anteiso:iso C₅-CoAs that matches the anteiso:iso ratio in membrane phospholipids, indicating indiscriminate utilization of these precursors by the initiation condensing enzyme FabH. Lipidomics analysis showed that removal of isoleucine and leucine from the medium led to the replacement of phospholipid molecular species containing anteiso/iso 17- and 19-carbon fatty acids with 18- and 20-carbon straight-chain fatty acids. This compositional change is driven by an increase in the acetyl-CoA:C₅-CoA ratio, enhancing the utilization of acetyl-CoA by FabH. The acyl carrier protein (ACP) pool normally consists of odd carbon acyl-ACP intermediates, but when branched-chain amino acids are absent from the environment, there was a large increase in even carbon acyl-ACP pathway intermediates. The high substrate selectivity of PlsC ensures that, in the presence or the absence of extracellular Ile/Leu, the 2-position is occupied by a branched-chain 15-carbon fatty acid. These metabolomic measurements show how the metabolism of isoleucine and leucine, rather than the selectivity of FabH, control the structure of membrane phospholipids.     

Provocation study of persons with perceived electrical hypersensitivity and controls using magnetic field exposure and recording of electrophysiological characteristics

The aim of the present study was to investigate possible neurophysiological effects of intermittent 15 sec on/off cycle, 60 Hz, 10 μT magnetic field exposure on patients with perceived “electromagnetic hypersensitivity” (EHS), and control subjects during rest and performance of a mental arithmetic task. Twenty participants (15 female, 5 male, 31–60 years old, mean 45.8 ± 0.7 years) were invited from the group of EHS patients. Twenty volunteers (15 female, 5 male, 31–59 years old, mean 45.0 ± 0.7 years?) served as a control group. The test protocol consisted of a set of examinations: EEG, visual evoked potentials, electrodermal activity, ECG, and blood pressure. The total duration of the test was 40 min, divided into two 10 min rest periods and two 10 min periods of mathematical performance. Magnetic field and sham exposures were presented randomly during these periods, resulting in four different conditions: Field‐Rest, Sham‐Rest, Field‐Math, and Sham‐Math. The data showed significant main effects of the Group factor (EHS vs. control subjects) on heart rate (F_1,80 = 20.6; P < 0.01), heart rate spectrum ratio (F_1,80 = 9.5; P = 0.02), and electrodermal activity (F_1,76 = 4.2; P = 0.04), whereas EEG characteristics did not differ between groups. The Condition factor (mathematical task vs. relaxed) showed main effects for heart rate (F_1,80 = 14.8; P < 0.01), heart rate spectrum ratio (F_1,80 = 7.8; P = 0.06), electrodermal activity (F_1,76 = 56.8; P < 0.01), and alpha and theta spectral bands of EEG. Magnetic field exposure did not affect autonomous system or electroencephalographic variables of either group. These data do not indicate that EHS patients or control are affected by low‐level 60 Hz magnetic field exposure. However, persons reporting EHS differed from the control subjects in baseline values of investigated physiological characteristics. Perhaps EHS patients have a rather distinctive physiological predisposition to sensitivity to physical and psychosocial environmental stressors. Bioelectromagnetics 22:457–462, 2001. © 2001 Wiley‐Liss, Inc.     

Brief communication: co-detection of Bartonella quintana and Yersinia pestis in an 11th-15th burial site in Bondy, France

Historical and anthropological data suggest that skeletons excavated from an 11th to 15th century mass grave in Bondy, France, may be those of victims of the Great Plague. Using high-throughput real-time PCR investigation of the dental pulp collected from 14 teeth from five such skeletons, we detected Bartonella quintana DNA in three individuals and Yersinia pestis DNA in two individuals. DNA from five other deadly pathogens was not found. Suicide PCR genotyping confirmed Y. pestis DNA belonging to the Orientalis biotype. One individual had co-infection. These data suggest a plague epidemic in a population already infected by the body louse-transmitted B. quintana or a body louse-driven transmission of the plague that drove a medieval epidemic in inland Europe.