Synthesis of biomolecules from N2, CO,and H2O by electric discharge

A model primitive gas containing a mixture of N₂, CO and water vapor over a water pool (300 mL, 37 °C) was subjected to electric discharges. The discharge vessel (7 L in volume) was equipped with a CO₂ absorber (The CO₂ being formed during the discharge), thus simulating possible absorption of CO₂ in the primitive ocean. The vessel also has a cold trap ( –15 °C), which protects the primary products against the further decomposition in the discharge phase by enabling these products to adhere to the trap. Since the partial pressures of CO and N₂ decreased at rates of 1.5–1.7 cmHg day^–1 and 0.1–0.2 cmHg day^–1, respectively, the gases were added at regular intervals. The solution was analyzed at regular intervals for HCN, HCHO and urea, and maximum concentrations of about 50, 2, and 140 mM were observed. The discharge phase was continued for 6 months. In the solution, glycine (5.6% yield based on the carbon), glycylglycine (0.64%), orotic acid (0.004%) and small amounts of the other amino acids were found.     

Prebiotic synthesis of orotic acid parallel to the biosynthetic pathway

By heating an aqueous solution of aspartic acid and urea, carbamylaspartic acid is first formed and then the molecule is cyclized to dihydroorotic acid (DHO) with loss of water. Irradiation of an aqueous solution of DHO with a tungsten lamp yields orotic acid by photo-dehydrogenation of the molecule. This pathway of orotic acid formation is quite similar to that of biosynthesis of the molecule.     

Synchronisation of cancer cell lines of human origin using methotrexate

U937 human histiocytic lymphoma cell line and SW626 ovarian carcinoma line of human origin were synchronised using very low, nontoxic concentrations (0.04-0.08 microM for 16-24 h) of methotrexate (MTX) under standard culture conditions. Satisfactory synchrony was achieved to study S phase events. Various kinetic behaviours and biological properties of the synchronised cells are considered for characterisation of the system. MTX-synchronisation was compared with that induced by aphidicolin (APC) alone and by serum deprivation and APC. In some cancer cell lines MTX appears to be the best choice for obtaining highly synchronised cell populations without cytotoxicity or physiological perturbations.     

Metabolic control analysis. An application of signal flow graphs.

In order to study particle phagocytosis and glycogenolysis simultaneously, this study was designed to develop a direct-read-out method to monitor Kupffer-cell function continuously, based on the uptake of colloidal carbon by the isolated perfused rat liver. Livers were perfused for 20 min with Krebs-Henseleit buffer saturated with O2/CO2 (19:1). Colloidal carbon (1-2 mg/ml) was added to the buffer, and absorbance of carbon was monitored continuously at 623 nm in the effluent perfusate. Since colloidal-carbon uptake was proportional to A623, rates of uptake were determined from the influent minus effluent concentration difference, the flow rate and the liver wet weight. Rates of colloidal-carbon uptake were 50-200 mg/h per g and were proportional to the concentration of carbon infused. Data from light-microscopy and cell-separation studies demonstrated that carbon was taken up exclusively by non-parenchymal cells and predominantly by Kupffer cells. Further, the amount of colloidal carbon detected histologically in non-parenchymal cells increased as the concentration of colloidal carbon in the perfusate was elevated. When Kupffer cells were activated or inhibited by treatment with endotoxin or methyl palmitate, carbon uptake was increased or decreased respectively. Taken together, these results indicate that Kupffer-cell function can be monitored continuously in a living organ. This new method was utilized to compare the time course of phagocytosis of carbon by Kupffer cells and carbohydrate output by parenchymal cells. Carbohydrate output increased rapidly by 69 +/- 9 mumol per g within 2-4 min after addition of carbon and returned to basal values within 12-16 min. However, carbon uptake by the liver did not reach maximal rates until about 15 min. Infusion of a cyclo-oxygenase inhibitor, aspirin (10 mM), caused a progressive decrease in carbohydrate output and blocked the stimulation by carbon completely. Aspirin neither altered rates of carbon uptake nor prevented stimulation of carbohydrate release by addition of N2-saturated buffer. The data from these experiments are consistent with the hypothesis that output of mediators by Kupffer cells, presumably prostaglandin D2 and E2, occurs transiently as Kupffer cells begin to phagocytose foreign particles in the intact organ, a process which continues at high rates for hours.     

Application of electrical analogues for control analysis of simple metabolic pathways.

I have used electrical analogues for calculating the Flux Control Coefficients of metabolic pathways. An analogue circuit consists of resistances that are connected in series (or parallel) with a voltage (or current) source. In constructing the analogues, each of the enzymes in the pathway is associated with a resistance whose magnitude depends on the Elasticity Coefficients of the enzymes. These circuits can be designed in a heuristic fashion directly from the configuration of the pathway, without the necessity of writing down the governing equations with the use of Summation and Connectivity Theorems. The Flux Control Coefficients of the enzymes are represented by voltages across (or currents through) the resistances and are determined by an application of Ohm's Law. Results are given for (a) a simple linear pathway without feedback or feedforward regulation, and (b) a linear pathway with feedback inhibition. The analogue circuits are also convenient for assessing the relative importance of the various enzymes in flux control, and for simplifying the structure of a given pathway.     

Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42-amino-acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age ( r = −0.05, p = 0.59), severity of cognitive impairment ( r = 0.22, p = 0.21), or duration of AD symptoms ( r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.     

Efficient transgenic plant regeneration throughAgrobacterium-mediated transformation of Chickpea (Cicer arietinum L.)

Three genotypes of chickpea ICCV-1, ICCV-6 and a Desi (local) variety were tested for plant regeneration through multiple shoot production. The embryo axis was removed from mature seeds, the root meristem and the shoot apex were discarded. These explants were cultured on medium containing MS macro salts, 4X MS micro salts, I35 vitamins, 3.0 mg/1 BAP, 0.004 mg/1 NAA, 3% (w/v) sucrose and incubated at 26⁰C. The explants were transformed withAgrobacterium tumefaciens strain LBA4404 with binary vector pBI121 containing theuidA andnptIl genes. Multiple shoots were repeatedly selected with kanamycin. The selected kanamycin resistant shoots were rooted on MS medium supplemented with 0.05 mg/1 113A. The presumptive transformants histochemically stained positive for GUS. Additionally, nptll assay confirmed the expression ofnptII in kanamycin resistant plants. Transgenic plants were transferred to soil and grown in the green house.Abbreviations BAP 6-benzylamino purine - 2,4-D 2,4dichlorophenoxy acetic acid - IAA Indole acetic acid - IBA Indole butaric acid - NAA Naphthalene acetic acid     

Direct high-level secretion into the culture medium of tuna growth hormone in biologically active form byBacillus brevis

The characteristic features of theBacillus brevis system developed by us are very high productivity of heterologous proteins and very low extracellular proteinase activity. However, the production level of eucaryotic proteins with this system was generally one or two orders of magnitude lower than that of bacterial proteins. Therefore, we have explored methods for increasing the production efficiency as to animal proteins. Signal peptide modification was found to be very effective for high-level secretion of tuna growth hormone (tGH). Modification of the signal peptide with higher basicity in the amino terminal region and higher hydrophobicity in the middle region brought about a ten-fold increase in tGH production. Further elevation of the tGH yield to 240 mg/l was achieved by using a low proteinase mutant and a stable plasmid, and by culturingB. brevis under optimal conditions with the addition of some chemicals. Thus, biologically active tGH can be efficiently produced directly in the medium with thisB. brevis system.