Folate-dependent enzymes in cultured Chinese hamster ovary cells: evidence for mutant forms of folylpolyglutamate synthetase

A Chinese hamster ovary auxotroph requiring glycine + adenosine + thymidine (CHO AUXB1) was shown by us previously to lack several folylpolyglutamate synthetase (FPGS) type activities. Two revertants of AUXB1 (one spontaneous and one Pt(S0₄)₂ induced) have been isolated and found to contain altered forms of this enzyme. The revertant enzymes are more sensitive to heat inactivation (37 °C, pH 7.4 or 9.0) than the parent CHO enzyme. Increased sensitivity of revertant FPGS is observed irrespective of whether one assays the specific catalysis of radioactive tetrahydropteroyldi- or tetraglutamate synthesis. ATP and MgCl₂ protect both revertant and parent CHO FPGS against rapid heat denaturation at pH 9.0, but not at pH 7.4. A genetically related auxotroph (CHO AUXB3) contains one-fifth the parent amount of FPGS. AUXB3 FPGS shows a normal sensitivity to 37 °C heat inactivation, but it has an altered substrate saturation and specificity pattern when assayed for tetrahydropteroyldi[U-¹⁴C]glutamate synthesis. Also, unlike the FPGS from parent CHO and a genetically unrelated mutant requiring only glycine (CHO AUXB2), the AUXB3 enzyme specifically lacks tetrahydropteroyltetra[U-¹⁴C]glutamate synthetase activity. These findings and polyethylene glycol fusion data with AUXB2 indicate that AUXB1 and AUXB3 each carry a mutation in the structural gene for a CHO FPGS that catalyzes tetrahydropteroyldi- as well as tetraglutamate formation. The altered form of FPGS in AUXB3 is responsible for its glycine + adenosine auxotrophy under standard culture conditions.     

Aerobic photolysis of alkylcobalamins: quantum yields and light-action spectra

Quantum yields (φ) for the aerobic photolysis of 5′-deoxyadenosylcobalamin (dAB₁₂), methylcobalamin (MeB₁₂), propylcobalamin (PrB₁₂), and ethylcobalamin (EtB₁₂) were determined as a function of the irradiation wavelength. φ Determinations were made for both the base-on and base-off forms of each compound (except base-off dAB₁₂) at incident wavelengths from 250 nm to 570 nm. As a rule, the φs were high (0.1–0.5) and they varied significantly with respect to the irradiation wavelength. In general, each alkylcobalamin at pH 7.0 displayed a quantum yield spectrum distinct from its base-off form at pH 1.0. Across most of the spectrum, the φs of the base-off form were appreciably smaller than the base-on φs of the same compound. An exception to this generality was MeB₁₂ for which the φs at pH 1.0 were about the same as, or slightly greater above 450 nm than those at pH 7.0. At pH 7.0 and in the visible region the trend of the φs was dAB₁₂ < MeB₁₂ < PrB₁₂ < EtB₁₂. Under neutral conditions each compound showed a broad quantum yield peak in the 450–470 nm region.From the quantum yield and absorption spectra, photolysis spectra were calculated for 5.0 × 10^?5m solutions of each compound. The light-action spectra accurately give the relative rates/μ Einstein that these solutions photolyze at each wavelength. Thus, for example, MeB₁₂ photolyzed faster at pH 7.0 versus pH 1.0 in 510 nm light, but it photolyzed slower at pH 7.0 versus pH 1.0 in 450 nm light. Solutions of each compound photolyzed faster in the ultraviolet region as opposed to the visible (e.g., 310 nm versus 510 nm).Our findings show that the previously reported photolysis rates estimated by others with tungsten lamps provide no valid information about the intrinsic photolability of various alkyl-cobalt bonds. This also applies to the relative white-light photolysis rates reported for the base-on versus the base-off form of MeB₁₂. All such relative rates are artifacts which represent only the extent of overlap between the true action spectrum and the light emission spectrum of an incandescent lamp.     

Synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives of ribavirin

The synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives (13 and 15, respectively) of the antiviral agent ribavirin are described. Direct glycosylation of 2',3'-O-isopropylideneribavirin with either tetra-O-acetyl-alpha-D-glucopyranosyl bromide (4) or tetra-O-acetyl-alpha-D-galactopyranosyl bromide (8) under Koenigs-Knorr conditions (i.e., silver carbonate, silver perchlorate, and Drierite in dichloromethane) followed by O-deacetylation of the reaction product gave the corresponding ortho esters. However, treatment of 2',3'-di-O-acetyl-5'-O-tritylribavirin (11) with 4 under the Bredereck modification of the Koenigs-Knorr reaction (i.e., silver perchlorate and Drierite in nitromethane) and subsequent deacetylation furnished the desired 1-(5-O-beta-D-glucopyranosyl-beta-D-ribofuranosyl)-1,2,4-triazole-3-carb oxamide (13). Similarly, reaction of 11 with 8 in the presence of AgClO4, and deprotection of the condensation product, gave 5'-O-beta-D-galactopyranosylribavirin (15). The beta-anomeric configuration of the D-glucosyl and D-galactosyl groups of 13 and 15 was assigned by 1H-n.m.r. studies.     

Central Mechanisms of Pheromone Information Processing

An advantage of using pheromones in olfactory studies is thatthey are chemical signals for which receptor neurons are evolvedand thus elicite biologically relevant odour-information tobe processed in the brain. In many vertebrate and insect species,the olfactory system is separated into a ‘main’and an ‘accessory’ division, the latter mediatingpheromone information. In moths, the pheromone information isfirst processed in the brain in a large and sexually dimorphicstructure, the macroglomerular complex (MGC) of the antennallobe (AL). Also in vertebrates the pheromone information isprocessed in specific or modified glomerular complexes. Oneprinciple question is whether individual olfactory glomeruliare functional units, processing specific information concerningboth the chemical quality and spatiotemporal features of thestimulus, like the pheromone plume. Indeed it has been shownthat the axons of different pheromone-selective receptor neuronsproject into different MGC-glomeruli. Intracellular recordingsfrom the AL projection (output) neurons also show that informationabout single components of the pheromone blend is preservedin some output pathways, whereas other output neurons respondin a unique fashion to the blend. The information about interspecificsignals, which interrupts pheromone attraction, is processedin a specific MGC-glomerulus and is to a large extent kept separatedfrom the pheromone information throughout the AL. Many of theoutput neurons accurately encode changes in the temporal characteristicsof the stimulus. Chem. Senses 21: 269–275, 1996.     

Origin of the main class of repetitive DNA within selected Pennisetum species

In an attempt to identify relationships among genomes of the allotetraploid Pennisetum purpureum Schumach and closely related Pennisetum species with which it can be successfully hybridized, repetitive DNA sequences were examined. Digestion with KpnI revealed two highly repetitive fragments of 140 by and 160 bp. The possibility that these sequences could be used as genome markers was investigated. Average sequences were determined for the 140 by and 160 by KpnI families from P. purpureum and P. squamulatum Fresen. Average sequences (based upon four or five repeats) were determined for the P. glaucum (L.) R. Br. 140 by KpnI family and the diploid P. hohenackeri Hochst. ex Steud. 160 bp KpnI family. The average sequences of the 160 by KpnI families in P. purpureum and P. squamulatum differ by only nine bases. The 140 by KpnI families of the three related species, P. purpureum, P. squamulantum, and P. glaucum are nearly identical, and thus likely represent a recent divergence from a common progenitor or a common genome. Each repetitive sequence may contain internal duplications, which probably diverged following amplification of the original sequence. The 140 by KpnI repeat probably evolved from the 160 by KpnI repeat since the missing 18 by segment is part of the internal duplication that is otherwise conserved in the subrepeats. Tandemly arrayed repetitive sequences in plants are likely to be composed of subrepeats which have been duplicated and amplified.Florida Aqricultural Experiment Station series #R-02758     

Cortical hypometabolism in injured brain: New correlations with the noradrenergic and serotonergic systems and with behavioral deficits

Changes with time after injury in behavioral deficits, as determined by the Morris swim test, and the in vivo specific binding of HEAT, a selective ₁-adrenoreceptor ligand, were compared with the time-course of development of cortical hypometabolism in rats with focal freezing lesions. In our trauma model, cortical hypometabolism was detectable in the lesioned hemisphere at 4 hr, became maximal (50% of normal) at 3 days and diminished towards normal on days 5 and 10 post-injury. Progressive impairment of acquisition of the Morris water maze task was demonstrated up to day 3 post-lesion with improvement thereafter. On day 3 the latency to reach criterion was 60% longer in lesioned animals than in corresponding sham-operated ones. An increase in the volume of distribution of HEAT, limited to cortical areas of the lesioned hemisphere, was demonstrable at 4 hr post-lesion and reached its maximum on day 3 (200% of normal) with subsequent return toward normal on days 5 and 10. Several types of drugs were shown previously to modify the cortical hypometabolism associated with cerebral injury. The present data indicate that the same drugs also modify the in vivo binding of HEAT and the behavioral deficits induced by brain lesions. Ibuprofen, a non-steroidal anti-inflammatory drug, p-chlorophenylalanine, an inhibitor of serotonin synthesis, ketanserin, a specific 5HT₂-receptor antagonist, and prazosin, an ₁-adrenergic receptor blocker all normalized the in vivo binding of HEAT in the cortical areas of the lesioned hemisphere. All groups of animals treated with these drugs also showed subtle, but statistically highly significant improvements in latency to locate the platform in the Morris water maze. Taken together these results show good correlation between behavioral deficits, changes in ₁-noradrenergic receptor binding and cortical hypometabolism in injured brain. This supports the hypothesis that post-injury cortical hypometabolism is a reflection of cortical functional depression in which both the serotonergic and noradrenergic neurotransmitter systems play a role, compatible with their inhibitory effects in the cortex and their postulated involvement in cortical information processing.Special issue dedicated to Dr. Leon S. Wolfe.