Complete Oxidation of Propionate, Valerate, Succinate, and Other Organic Compounds by Newly Isolated Types of Marine, Anaerobic, Mesophilic, Gram-Negative, Sulfur-Reducing Eubacteria

Anaerobic enrichment cultures with either propionate, succinate, lactate, or valerate and elemental sulfur and inocula from shallow marine or deep-sea sediments were dominated by rod-shaped motile bacteria after three transfers. By application of deep-agar dilutions, five eubacterial strains were obtained in pure culture and designated Kyprop, Gyprop, Kysw2, Gylac, and Kyval. All strains were gram negative and grew by complete oxidation of the electron donors and concomitant stoichiometric reduction of elemental sulfur to hydrogen sulfide. The isolates used acetate, propionate, succinate, lactate, pyruvate, oxaloacetate, maleate, glutamate, alanine, aspartate, and yeast extract. All isolates, except strain Gylac, used citrate as an electron donor but valerate was oxidized only by strain Kyval. Fumarate and malate were degraded by all strains without an additional electron donor or acceptor. Kyprop, Gyprop, and Gylac utilized elemental sulfur as the sole inorganic electron acceptor, while Kysw2 and Kyval also utilized nitrate, dimethyl sulfoxide, or Fe(III)-citrate as an electron acceptor.     

Effects of GTP,forskolin, sodium fluoride,serotonin, dopamine,and carbachol on adenylate cyclase inTeleost retina

Adenylate cyclase activity can be stimulated in goldfish retina by forskolin, GTP, NaF, dopamine and serotonin. Pharmacological characterisation of the dopamine and serotonin responses shows them to be mediated through specific receptors. A synergistic increase in the level of C-AMP is observed following application of forskolin together with NaF, GTP, dopamine, or serotonin. Dopamine and serotonin with or without GTP produced an additive response. When NaF and GTP are both together their combined effect in elevating C-AMP levels in the presence or absence of forskolin is less than additive. These results suggest that forskolin may be interacting with a G_s protein as well as directly stimulating adenylate cyclase. Increases in the level of C-AMP observed following application of forskolin or dopamine are decreased by carbachol in a dose-dependent manner. The carbachol response is blocked by pertussis toxin and is insensitive to the phosphodiesterase inhibitor, IBMX, suggesting an involvement of a G_i protein. Carbachol attenuation of elevated C-AMP levels is inhibited by atropine while pirenzapine has little effect suggesting the presence of a M2-type receptor.     

Control of potato cyst-nematode, Globodera rostochiensis, and root-knot nematode, Meloidogyne incognita, by organosphosphorus, carbamate, benzimidazole and other compounds

Control of potato cyst-nematode, Globodera rostochiensis, was examined on potato or tomato in pots and on potato in field plots by various chemicals incorporated into the soil at planting. The most effective nematicides were organophosphorus compounds, generally of the type O,O-diethyl O-phosphoro-thioates or O,O-diethyl phosphorodithioates, carbamates and benzimidazoles. In organic soils, the more lipophilic compounds were less effective, presumably because of sorption onto soil organic matter. Foliar sprays of chemicals, including oxamyl which is known to be translocated to roots, gave poor control of G. rostochiensis. The root-knot nematode, Meloidogyne incognita, on tomato, widely used in screening for nematicidal activity, was controlled by aldicarb or phoxim incorporated into the soil at planting, but not by benomyl or thiabendazole, in contrast to the moderate effectiveness of these latter two chemicals against G. rostochiensis.     

Analysis of gammabeta, betagamma, gammagamma, betabeta multiple turns in proteins.

The number of gamma-turns in a representative protein dataset selected from the current Protein Data Bank has increased almost seven times during the past decade. Eighty percent classic gamma-turns and 57% inverse gamma-turns are associated as multiple turns with either another y-turn or a beta-turn. We refer to these as multiple turns of the (gammabeta)1,2,3 or (betagamma)1,2,3 type, depending upon whether the gamma-turn is before or after the beta-turn along the protein chain, respectively. However, for multiple turns involving only gamma-turns, we follow the nomenclature analogous to that proposed earlier for the multiple (or double) beta-turns. Fifty-eight per cent beta-turns are associated as multiple turns with another beta-turn. We extracted multiple turns from the protein dataset and classified them on the basis of individual gamma- or beta-turn types and the number of overlapping residues. Furthermore, we evaluated the amino acid positional potentials and determined the statistically significant amino acid preferences, hydrogen bond/side-chain interaction preferences in the multiple turns and secondary structure preferences for residues immediately flanking these turns. The results of our analysis would be useful in the modeling, prediction or design of multiple turns in proteins. The amino acid sequence corresponding to the multiple turn, position in the protein chain, PDB Code/chain in which multiple turn is present and the individual turn types constituting the multiple turns are available from our website and this information would also be integrated in our Database of Structural Motifs in Proteins (http://www.cdfd.org.in/dsmp.html).     

Shape,loading, and motion in the bioengineering design,fabrication, and testing of personalized synovial joints

With continued development and improvement of tissue engineering therapies for small articular lesions, increased attention is being focused on the challenge of engineering partial or whole synovial joints. Joint-scale constructs could have applications in the treatment of large areas of articular damage or in biological arthroplasty of severely degenerate joints. This review considers the roles of shape, loading and motion in synovial joint mechanobiology and their incorporation into the design, fabrication, and testing of engineered partial or whole joints. Incidence of degeneration, degree of impairment, and efficacy of current treatments are critical factors in choosing a target for joint bioengineering. The form and function of native joints may guide the design of engineered joint-scale constructs with respect to size, shape, and maturity. Fabrication challenges for joint-scale engineering include controlling chemo-mechano-biological microenvironments to promote the development and growth of multiple tissues with integrated interfaces or lubricated surfaces into anatomical shapes, and developing joint-scale bioreactors which nurture and stimulate the tissue with loading and motion. Finally, evaluation of load-bearing and tribological properties can range from tissue to joint scale and can focus on biological structure at present or after adaptation.     

Anesthesia, analgesia, and euthanasia of invertebrates

Invertebrate animals have long played an important role in biomedical research in such fields as genetics, physiology, and development. However, with few exceptions, scientists, veterinarians, and technicians have paid little attention to the anesthesia, analgesia, and euthanasia of these diverse creatures. Indeed, some standard research procedures are routinely performed without anesthesia. Yet various chemical agents are available for the immobilization or anesthesia of invertebrates, ranging from gases or volatile liquids that can be pumped into either an anesthetic chamber (for terrestrial species) or a container of water (aquatic species), to benzocaine and other substances for fish. Many invertebrates are not difficult to immobilize or anesthetize and the procedures recommended in this article appear to be safe; however, none should be considered totally risk-free. Analgesia of invertebrates is as yet a largely unexplored field; until scientific data are available, other measures can promote the well-being of these animals in the laboratory. For euthanasia, various methods (physical or chemical or a combination of both) have been recommended for different taxa of invertebrates, but most have not been properly studied under laboratory conditions and some can be problematic in the context of research procedures and tissue harvesting. Furthermore, relevant data are scattered, sometimes available only in languages other than English, and there is no international approach for seeking and collating such information. In this article I review various methods of anesthesia, analgesia, and euthanasia for terrestrial and aquatic invertebrates, as well as areas requiring further research.     

Influence of methods of preparation on the infectivity, agglutination, activity, and ultrastructure of bloodstream trypanosomes

Contrary to the reports of others, the surface coat of Trypanosoma brucei brucei was not removed by extensive washing in the various media investigated; in fact, other than a deformed profile when washed in saline, ultrastructure was little affected by column separation and washing in any of these media. Washing in saline increased the agglutinability but reduced the activity and infectivity of the organisms. Washing in bicine-buffered saline-glucose did not impair activity or infectivity but increased agglutinability, albeit to a lesser extent than after washing in phosphate-buffered saline-glucose. The inclusion of 0.1% serum or plasma in the washing medium (phosphate-buffered saline-glucose or bicine-buffered saline-glucose) increased the activity of the T. b. brucei and did not increase agglutinability or impair infectivity. T. congolense and T. vivax were more susceptible to ionic strength changes than were T. b. brucei or T. lewisi, in that not only was their activity impaired, but they began to aggregate on standing in lower ionic strength buffers.     

Intracytoplasmic injection of porcine, bovine, mouse, or human spermatozoon into porcine oocytes

We determined the incidence of activation, male pronuclear formation, and apposition of pronuclei in porcine oocytes following intracytoplasmic injection of various porcine sperm components and foreign species spermatozoa, such as that of cattle, mouse or human. The porcine oocytes were activated by injection of a spermatozoon or an isolated sperm head. In contrast, injection of either sperm tail or a trypsin- or NaOH-treated sperm head failed to induce oocyte activation. Because injection of mouse, bovine, or human spermatozoon activated porcine oocytes, the sperm-borne activation factor(s) is not strictly species-specific. Male pronuclear formation and pronuclear apposition were observed in porcine oocytes following injection of porcine, bovine, mouse or human spermatozoa. Electrical stimulation following sperm cell injection did not enhance the incidence of male pronuclear formation or pronuclear apposition compared with sperm cell injection alone (P > 0.1). Following porcine sperm injection, the microtubular aster was organized from the neck of the spermatozoon, and filled the whole cytoplasm. In contrast, following injection of bovine, mouse, or human spermatozoon, the maternal-derived microtubules were organized from the cortex to the center of the oocytes, which seems to move both pronuclei to the center of oocytes. Cleavage to the two-cell stage was observed at 19-21 hr after injection of porcine spermatozoon. However, none of the oocytes following injection of mouse, bovine, or human spermatozoa developed to the mitotic metaphase or the two-cell stage. These results suggested that the oocyte activating factor(s) is present in the perinuclear material and that it is not species-specific for the porcine oocyte. Self-organized microtubules seemed to move the pronuclei into center of oocytes when foreign species spermatozoa were injected into porcine oocytes.     

Analysis of fronto-orbital advancement for Apert, Crouzon, Pfeiffer, and Saethre-Chotzen syndromes

The purposes of this study were (1) to document outcome after primary fronto-orbital advancement for the four major eponymous craniosynostotic syndromes (Apert, Crouzon, Pfeiffer, and Saethre-Chotzen) and (2) to identify factors that might influence need for primary and secondary fronto-orbital advancement or foreheadplasty. Also tested was the hypothesis that coincident sagittal synostosis could modulate brachycephaly and affect whether a primary or secondary frontal operation was necessary. Data were collected on age and indications for initial operation, type of primary and secondary frontal procedures, and concomitant sagittal synostosis. Patients initially managed by subcranial Le Fort III were included in the study group but excluded from analysis of fronto-orbital advancement. Patients treated by monobloc advancement or Le Fort III osteotomies with frontal grafting or Anderl modification were assessed as having had primary fronto-orbital advancement. Minimum time to follow-up was 5 years. A total of 126 patients met inclusion criteria. Lateral photographs were examined to assess preoperative and postoperative sagittal position of supraorbital rims-to-globes. Frontal re-advancement was indicated if the corneal apex was anterior to the supraorbital rim. Foreheadplasty was indicated for unacceptable frontal contour and normal supraorbital rim-to-globe relationship. Primary correction for frontal retrusion was not required in 4 percent of Apert (1 of 25), 16 percent of Crouzon (7 of 44), 6 percent of Pfeiffer (2 of 31), and 19 percent of Saethre-Chotzen (5 of 26) patients. Of those infants who had a primary fronto-orbital advancement, reoperation for either supraorbital retrusion or frontal deformity was necessary in all 16 Apert patients and in 5 of 19 Crouzon (26 percent), 10 of 26 Pfeiffer (38 percent), and 13 of 20 Saethre-Chotzen (65 percent) patients (p < 0.001). Age at initial fronto-orbital advancement did not influence reoperative rate. No correlation was found between concomitant sagittal synostosis and necessity for primary or secondary frontal correction (p = 0.22). In summary, phenotypic diagnosis was determinant for outcome as defined by need for secondary fronto-orbital advancement, foreheadplasty, or both. Apert patients had the highest incidence of reoperation for frontal retrusion or forehead contour. Crouzon and Saethre-Chotzen patients were most likely to express a minor phenotype and not require fronto-orbital correction. Coincident sagittal synostosis did not influence frontal projection, as reflected in need for either primary or secondary frontal advancement.     

Fluorescent, photoaffinity, and biotinyl analogs of oxytocin

This study reports the solid phase synthesis and biological activities of two oxytocin analogs, [1-desamino, 4-lysine,7-(L-3,4,-dehydroproline)]oxytocin and [1-desamino, 4-threonine,7-(L-3,4-dehydroproline),8-lysine]oxytocin, and several fluorescent, photoaffinity, or biotinylated derivatives of these analogs and of oxytocin. The activities (in IU/mg) of the lysine-containing parent compounds, respectively, were as follows: uterus (without Mg++) 4.8 and 54; uterus (with Mg++) 19 and 440; milk ejection 65 and 414. The above analogs were coupled through the chemically reactive epsilon-amino group of lysine in position 4 or 8 or, in the case of oxytocin, through the N-terminal amino group of fluoresceine, photoaffinity, or biotinyl ligands. Fluoresceine coupled in position 1 of oxytocin gave an analog of low to moderate uterine (3.8 without Mg+ and 1.9 with Mg++) and milk ejection (7.9) activities. Analogs with biotin or fluoresceine coupled to lysine in position 4 had moderate uterine (11 and 23 without Mg++; 38 and 11 with Mg++) and milk ejection (33 and 13) activities. Analogs with fluoresceine, photoaffinity, or biotinyl labels coupled to lysine in position 8 retained good uterine (106, 62, and 147 without Mg++; 79, 78, and 509 with Mg++) and milk ejection (101, 181, and 247) activities and represent potentially useful experimental tools for studying hormone-receptor interactions and for receptor localization and isolation.     

Formation, conversion, and utilization of isorhodopsin, rhodopsin, and porphyropsin by rod photoreceptors in the Xenopus retina

The visual pigment content of rod photoreceptors in Xenopus larvae was reduced greater than 90% through a combination of vitamin A-deficient diet and constant light. Thereafter, a dose of either all-trans-retinol or 9-cis-retinal was injected intramuscularly, leading to the formation of a rhodopsin (lambdamax 504 nm) or isorhodopsin (lambdamax 487-493 nm) pigment, respectively. Electrophysiological measurements were made of the threshold and spectral sensitivity of the aspartate-isolated PIII (photoreceptoral) component of the electroretinogram. These measures established that either rhodopsin or isorhodopsin subserved visual transduction with the same efficiency as the 519 nm porphyropsin pigment encountered normally. When animals with rhodopsin or isorhodopsin were kept in darkness or placed on a cyclical lighting regimen for 8 days, retinal densitometry showed that either pigment was being converted to porphyropsin; significantly more porphyropsin was formed as a result of cyclical lighting than after complete darkness.     

Influence of Cadmium, Zinc, and Lead on Growth, Trap Formation, and Collagenase Activity of Nematode-Trapping Fungi

Growth and morphogenesis of seven species of nematode-trapping fungi and the activity of a collagenase produced by Arthrobotrys amerospora were measured in the presence of various concentrations of divalent cadmium, zinc, or lead. In general, growth varied with species and was dependent on the metal present and the concentration at which it was tested. Cadmium was found to exhibit the greatest toxicity followed by zinc and lead, respectively. In most cases, inhibition of growth was directly correlated with a decreased capacity to form traps. However, in a few cases, trap formation was inhibited either more or less than was growth. The activity of the collagenase was less sensitive than was growth or trap formation to heavy-metal inhibition.     

Synthesis, biological activity, and tritiation of L-3, 4-dehydroproline-containing peptides

A series of analogs of thyroliberin (TRH) ([L-delta3Pro3]-TRH, [D-delta3Pro3]-TRH, [L-3-MeHis2, L-delta3Pro3]-TRH) in which proline was replaced by L- or D-3, 4-dehydroproline was synthesized. The analogs exhibited approximately the same biological activity as the corresponding proline-containing peptides. These analogs and others in which 3, 4-dehydroproline is present at the NH2-terminal, COOH-terminal or internal positions in the peptide were successfully reduced with deuterium or tritium to provide the 3, 4-2H2-proline or 3,4-3H2-proline analogs, respectively, with near theoretical values of substitution. A novel procedure for the chemical resolution of DL-3, 4-dehydroproline is also described.     

In vitro characterization of FlgB, FlgC, FlgF, FlgG, and FliE, flagellar basal body proteins of Salmonella

The bacterial flagellar basal body is a rotary motor. It spans the cytoplasmic and outer membranes and drives rapid rotation of a long helical filament in the cell exterior. The flagellar rod at its central axis is a drive shaft that transmits torque through the hook to the filament to propel the bacterial locomotion. To study the structure of the rod in detail, we have established purification procedures for Salmonella rod proteins, FlgB, FlgC, FlgF, FlgG, and also for FliE, a rod adapter protein, from an Escherichia coli expression system. While FlgF was highly soluble, FlgB, FlgC, FlgG and FliE tended to self or cross-aggregate into fibrils in solutions at neutral pH or below, at high ionic strength, or at high protein concentration. These aggregates were characterized to be beta-amyloid fibrils, unrelated to the rod structure formed in vivo. Under non-aggregative conditions, no protein-protein interactions were detected between any pairs of these five proteins, suggesting that their spontaneous, template-free polymerization is strongly suppressed. Limited proteolyses showed that FlgF and FlgG have natively unfolded N and C-terminal regions of about 100 residues in total just as flagellin does, whereas FlgB, FlgC and FliE, which are little over 100 residues long, are unfolded in their entire peptide chains. These results together with other data indicate that all of the ten flagellar axial proteins share structural characteristics and folding dynamics in relation to the mechanism of their self-assembly into the flagellar axial structure.     

Separation of optical isomers of scopolamine, cocaine, homatropine, and atropine

Different drug stereoisomers can have different physiological and therapeutic effects. Difficulties in separating optical isomers often make it impractical to market stereochemically pure products or to monitor isomeric contamination. This is not thought to be a problem with drugs isolated from biological sources (the alkaloids, for example). However, small amounts of isomeric impurities also exist in many biological systems. More importantly the isolation and purification process can cause partial or complete racimization in some cases. Great care must be taken in the handling of some drugs and an efficient, sensitive means to monitor racimization is important. Liquid chromatographic separation on a chiral beta-cyclodextrin bonded phase can be an effective technique in many cases. Its use in separating optical isomers of dl-scopolamine, dl-hyoscyamine, dl-homatropine, and dl-cocaine is discussed.     

8-Nitroxanthine,a product of myeloperoxidase,peroxynitrite, and activated human neutrophils,enhances generation of superoxide by xanthine oxidase

In biology, chiral recognition usually implies the ability of a protein, such as an enzyme or a drug receptor, to distinguish between the two enantiomeric forms of a chiral substrate or drug. Both diastereoisomerism and specific contacts between enzyme/receptor and substrate/drug are necessary. The minimum requirement is for four contact points including four nonplanar atoms (or groups of atoms) in both probe and target. The molecular models described by Easson and Stedman and by Ogston require three binding sites in a plane. A modified model with three binding sites in three dimensions is described. Under certain circumstances this model allows binding of both enantiomeric forms of a substrate or a drug. Enantiomer superposition of two enantiomers at an active site occurs in some specific cases (e.g., phenylalanine ammonia-lyase, isocitrate dehydrogenase) and is likely in others. The nature of enantiomer binding to racemase enzymes is discussed.     

Lack of renal effects of DOCA, ACTH, spironolactone, and angiotensin II in Squalus acanthias

Angiotensin II was infused intravenously in spiny dogfish sharks (Squalus acanthias). There were no significant effects on arterial blood pressure, glomerular filtration rate, urine flow, or Na excretion either in comparison with pre- and postinfusion values or in comparison with values measured in a control group of fish given elasmobranch saline intravenously. In other dogfish, glomerular filtration rate, urine flow, and Na and K excretory rates were measured for 3 days following implantation of desoxycorticosterone (DOCA), adrenocorticotropin (ACTH), or spironolactone; a control group was given no drug. There were no significant differences between these four groups of fish with respect to any of the measured parameters. These results suggest that the dogfish kidney is not a target organ for several substances known to affect renal function, either directly or indirectly, in other animals.     

Super-motifs and evolution of tandem leucine-rich repeats within the small proteoglycans--biglycan, decorin, lumican, fibromodulin, PRELP, keratocan, osteoadherin, epiphycan, and osteoglycin

Leucine-rich repeats (LRRs) with 20-30 amino acids in unit length are present in many proteins from prokaryotes to eukaryotes. The LRR-containing proteins include a family of nine small proteoglycans, forming three distinct subfamilies: class I contains biglycan/PG-I and decorin/PG-II; class II: lumican, fibromodulin, PRELP, keratocan, and osteoadherin; and class III: epiphycan/PG-Lb and osteoglycin or osteoinductive factor. Comparative sequence analysis of the 34 available protein sequences reveals that these proteoglycans have two types of LRRs, which we call S and T. The type S LRR is 21 residues long and has the consensus sequence of xxaPzxLPxxLxxLxLxxNxI. The type T LRR has 26 residues; its consensus sequence is zzxxaxxxxFxxaxxLxxLxLxxNxL. In both "x" indicates variable residue; "z" is frequently a gap; "a" is Val, Leu, or Ile; and I is Ile or Leu. These type S and TLRRs are ordered into two super-motifs--STT with about 73 residues in classes I and II and ST with about 47 residues in class III. The 12 LRRs in the small proteoglycans of I and II are best represented as (STT)4; the seven LRRs of class III as (ST)T(ST)2. Our analyses indicate that classes I/II and III evolved along different paths after the establishment of the precursor ST, and classes I and II also diverged after the establishment of the precursor (STT)4.     

Interactions between toxic (As, Cd, Hg and Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements in the tissues of cattle from NW Spain

Since the toxicity of one metal or metalloid can be dramatically modulated by the interaction with other toxic or essential metals, studies addressing the chemical interactions between trace elements are increasingly important. In this study correlations between the main toxic (As, Cd, Hg and Pb) and nutritional essential (Ca, Co, Cr, Cu, Fe, Mn, Mo, Ni, Se, Zn) elements were evaluated in the tissues (liver, kidney and muscle) of 120 cattle from NW Spain, using Spearman rank correlation analysis based on analytical data obtained by ICP-AES. Although accumulation of toxic elements in cattle in this study is very low and trace essential metals are generally within the adequate ranges, there were significant associations between toxic and essential metals. Cd was positively correlated with most of the essential metals in the kidney, and with Ca, Co and Zn in the liver. Pb was significantly correlated with Co and Cu in the liver. A large number of significant associations between essential metals were found in the different tissues, these correlations being very strong between Ca, Cu, Fe, Mn, Mo and Zn in the kidney. Co was moderately correlated with most of the essential metals in the liver. In general, interactions between trace elements in this study were similar to those found in polluted areas or in experimental studies in animals receiving diets containing high levels of toxic metals or inadequate levels of nutritional essential elements. These interactions probably indicate that mineral balance in the body is regulated by important homeostatic mechanisms in which toxic elements compete with the essential metals, even at low levels of metal exposure. The knowledge of these correlations may be essential to understand the kinetic interactions of metals and their implications in the trace metal metabolism.     

Biofuels, facts, fantasy, and feasibility

It is frequently claimed that green algae are intrinsically more productive, often by orders of magnitude, than higher plants commonly grown as crops for food. There is no firm evidence for this belief. On the contrary, there is much experience which shows that algae are not more but less productive. Under optimal conditions, all green organisms photosynthesize at the same rate in low light and, whilst commonly cultivated ‘sun’ species show some differences in rate in full light, these do not translate into widely different rates of accumulation of biomass. Accordingly, irrespective of crop, one acre of land, pond or bioreactor, can annually yield about enough biomass to fuel one motor vehicle or meet the calorific requirement of several people. This amount of biomass is not sufficient to make other than a very small contribution to our present road transport requirements and yet contributes significantly to global food shortages and rising prices. Reliable evidence also suggests that, if all of the inputs are taken into account, the net energy gain of liquid biofuels, derived either from algae or terrestrial crops, is either very modest or non-existent and will therefore bring about little or no sparing of carbon dioxide emissions.