An autonomously propagating luciferase-encoding vector for Dictyostelium discoideum

We have constructed a luc reporter vector for Dictyostelium discoideum using a 626-bp fragment from the nuclear-associated plasmid Ddp2. The ori from Ddp2 is localized within this fragment and was used to provide an autonomous replication sequence for the reporter vector. This reporter vector was stably retained in D. discoideum AX3K cells without alteration. The vector molecule was also found to exist in relatively low copy number compared to other Dictyostelium vectors in the transformed cells. We demonstrated the utility of this vector as a reporter vector with glycogen synthase promoter/luc fusions of varying sizes.     

Spotlight on phytochrome nomenclature

These recommendations for genes encoding phytochromes were developed independently by Quail et al., but are broadly consistent with the Commission's guidelines. Their original article, kindly provided in advance of publication, appeared as a Letter to the Editor inPlant Cell (6:468–471, 1994) and is published with permission of the American Society of Plant Physiologists.     

HPLC and NMR methods for the quantitation of the (R)-enantiomer in (−)-(S)-timolol maleate drug raw materials

HPLC and ¹H-NMR methods for the quantitation of the (R)-enantiomer in (?)-(S)-timolol maleate were developed and validated. The HPLC method requires a 25 cm × 4.6 mm 5 μm Chiracel OD-H (cellulose tris-3,5-dimethylphenylcarbamate) column, a mobile phase of 0.2% (v/v) diethylamine and 4% (v/v) isopropanol in hexane at a flow rate of 1 ml/min and UV detection at 297 nm. A system suitability test was devised to verify the separation of the (R)- and (S)-enantiomers of timolol from other drug-related impurities. The NMR method requires the use of a high-field NMR spectrometer (>360 MHz) and a chiral solvating agent, (?)-(R)-2,2,2-trifluoro-1-(9-anthrylethanol) (R-TFAE). The limits of quantitation were 0.05% and 0.2% (m/m) for HPLC and NMR, respectively. The methods were applied to the determination of the (R)-enantiomer in eight lots of raw material. The results for the two methods were in very good agreement, with results ranging from 0.1 to 4.1% (m/m) by HPLC and none detected to 4.3% (m/m) by NMR. The USP method for specific rotation was found to be unsuitable for detecting the presence of low levels of the (R)-enantiomer in (?)-(S)-timolol maleate. © 1994 Wiley-Liss, Inc.     

Determination of l-α-acetylmethadol, l-α-noracetylmethadol and l-α-dinoracetylmethadol in plasma by gas chromatography—mass spectrometry

A method is described for the simultaneous determination of l-α-acetylmethadol (LAAM) and its N-demethylated metabolites, l-α-noracetylmethadol (norLAAM) and l-α-dinoracetylmethadol (dinorLAAM), in plasma by gas chromatography—chemical ionization mass spectrometry. Deuterated internal standards for each analyte serve as carriers and control for recovery during sample purification on a solid-phase extraction column (C₁₈), and subsequent separation and analysis on a DB-17 capillary column. With this method, we have determined levels of LAAM, norLAAM, and dinorLAAM in small volumes of plasma (100 μl). The limit of quantitation for all analytes was approximately 1.0 ng/g plasma and the limit of detection was approximately 0.5 ng/g plasma. An experimental application is also described where these analytes are quantitated in plasma obtained from rats before, during, and after chronic administration of LAAM-HCl. Since this technique affords a selective and sensitive means of detection of LAAM and its active, N-demethylated metabolites in small samples of blood, it may enable patient compliance to be more easily assessed by allowing samples to be collected by a simple finger-prick technique.     

A Neurofilament-Associated Kinase Phosphorylates Only a Subset of Sites in the Tail of Chicken Midsize Neurofilament Protein

Although neurofilaments are among the most highly phosphorylated proteins extant, relatively little is known about the kinases involved in their phosphorylation. The majority of the phosphates present on the two higher-molecular-mass neurofilament subunits are added to multiply repeated sequence motifs in the tail. We have examined the specificity of a neurofilament-associated kinase (NFAK) partially purified from chicken spinal cord that selectively phosphorylates the middle-molecular-mass neurofilament subunit, NF-M. Two-dimensional phosphopeptide mapping of ³²P-labeled NF-M shows that, in vitro, NFAK phosphorylates a subset of peptides phosphorylated in vivo in cultured neurons. The absence of a complete complement of labeled phosphopeptides following in vitro phosphorylation, compared with phosphorylation in vivo, is not due to a lack of availability of phosphorylation sites because the same maps are obtained when enzymatically dephosphorylated NF-M is used as an in vitro substrate. Phosphopeptide maps from in vitro-phosphorylated NF-M and those from a recombinant fusion protein containing only a segment of the tail piece of chicken NF-M reveal identical labeled peptides. The fusion protein lacks a segment containing 17 KXX(S/T)P putative phosphorylation sites contained in the tail of chicken NF-M but contains a segment that includes four KSPs and a KSD site also present in the intact tail. These results suggest (a) that NFAK mediates the phosphorylation of some, but not all, potential phosphorylation sites within the tail of NF-M and (b) that multiple kinases are necessary for complete phosphorylation of the NF-M tail.     

Pulmonary gas exchange kinetics during exercise: physiological inferences of model order and parameters

1. 1. The ventilatory and pulmonary gas exchange responses during moderate exercise can be appropriately modelled with first-order dynamics.

2. 2. A delay term, reflecting tissue-to-lung transit time, is needed for accurate characterization, however.

3. 3. The O₂ uptake time constant ( reflects the enzymatically controlled tissue O₂ utilization.

4. 4. is appreciably longer than , consequent to the tissue CO₂ capacitance.

5. 5. As typically longer than , transient errors in alveolar and arterial blood gas tensions are predicted: small for P_CO2 but much larger for P_O2.

6. 6. At work rates above the lactate threshold, a slow and delayed component of V̇_O2 induces an additional V̇ component (“excess” V̇_O2), leading to more rapid fatigue.

7. 7. The ventilatory compensation for the metabolic acidemia at these work rates is slow, with compensation being poor for rapid-incremental exercise.

8. 8. A justifiable control model of the coupling of ventilation to metabolism must cohere with these demonstrable physiological characteristics.

The stimulatory effect of chronic lithium treatment on basal thyrotropin secretion in rats: In vivo antagonism by methylparaben

Chronic treatment of rats with lithium chloride was examined in order to determine its effect on hypothalamic monoamine and metabolite content, basal thyrotropin (TSH) secretion and thyroid function. The hypothalamic concentrations of noradrenaline (NA), dopamine (DA) and its metabolites, dihydroxyphenylacetic acid. (DOPAC) and homovanillic acid (HVA) in the lithium treated rats remained unaltered when compared to control levels. NA turnover and the NA metabolite, 3-methoxy-4-hydroxyphenylglycol (total MHPG), were significantly lower (p<0.01), whereas both serotonin (5-HT) and its metabolite, 5-hydroxyindole-3-acetic acid (5-HIAA), were significantly higher (p<0.01 and p<0.02, respectively) in the lithium treated rat hypothalami than in controls. Chronic lithium treatment significantly elevated basal TSH levels (p<0.05). This effect was antagonized by methylp-hydroxybenzoate (methylparaben, p<0.01), which did not itself affect basal TSH levels. Free serum T₃ and T₄ levels were not significantly affected by chronic lithium treatment, although T₄ tended to be slightly lower than control levels. The monoamine changes observed in the hypothalamus of lithium treated rats did not appear to account for the elevated TSH levels observed in these rats since NA activity which is generally regarded as stimulatory was decreased and 5-HT which has an inhibitory effect on TSH secretion, was increased. The elevated TSH levels may have been due to a reduced negative feedback inhibition of TSH release by the mildly reduced circulating T₄ levels caused by chronic lithium treatment. A further possibility is that the pituitary cGMP (and hence TSH) response to TRH may have been enhanced by chronic lithium treatment and methylparaben may have antagonized this effect.     

Deletion analysis of the essentiality of penicillin-binding proteins 1A, 2B and 2X of Streptococcus pneumoniae

Abstract An internal fragment from each of the penicillinebinding protein (PBP) 1A, 2B and 2X genes of Streptococcus pneumoniae , which included the region encoding the active-site serine residue, was replaced by a fragment encoding spectinomycin resistance. The resulting constructs were tested for their ability to transform S. pneumoniae strain R6 to spectinomycin resistance. Spectinomycin-resistant transformants could not be obtained using either the inactivated PBP 2X or 2B genes, suggesting that deletion of either of these genes was a lethal event, but they were readily obtained using the inactivated PBP 1A gene. Analysis using the polymerase chain reaction confirmed that the latter transformants had replaced their chromosomal copy of the PBP 1A gene with the inactivated copy of the gene. Deletion of the PBP 1A gene was therefore tolerated under laboratory conditions and appeared to have little effect on growth or susceptibility to benzylpenicillin.     

Planktivory in benthic nototheniid fish in McMurdo Sound,Antarctica

Synopsis Four species of nototheniid fish were sampled from below the sea ice near Cape Armitage, McMurdo Sound:Pagothenia borchgrevinki from just below the ice 1.5 km offshore,Trematomus bernacchii, Trematomus hansoni andTrematomus centronotus from off the bottom in about 20 m of water near the shore. Scale worms and isopods were conspicuous non-planktonic prey, and present in the three benthic fish species. The planktonic pteropod molluscLimacina helicina was numerically common in all four species of fish. The planktonic hyperiid amphipodHyperiella dilatata was also found in all fish species. WhereasP. borchgrevinki is planktivorous in accord with its pelagic habit, theTrematomus spp. clearly also feed on plankton from the water column.T. hansoni is particularly planktivorous, taking smaller copepods thanP. borchgrevinki.