Cloning and nucleotide sequence of the Salmonella typhimurium LT2 gnd gene and its homology with the corresponding sequence of Escherichia coli K12

Summary The complete nucleotide sequence of the Salmonella strain LT2 gnd gene for 6-phosphogluconate dehydrogenase was determined. The gene contains 1404 bases and encodes a 468 amino acid polypeptide, which is the same as for Escherichia coli K12. The DNA sequence shows 14.8% difference between the two and the amino acid sequence 3.6% difference. Changes are mostly in the third codon base and most of the amino acid changes are conservative.     

In vitro holding and PLD repair

Summary The Stationary or Plateau-Phase of commonly used rodent cell lines like the V79 are often assumed to be quiescent (non-mitotic). An analysis of cell turnover in V79 plateau-phase cultures through BrUdR-incorporation combined with FUdR-block and light exposure (S-phase cytocide) revealed such cultures to be in a state of kinetic equilibrium. Even when the state of maximal permissible density was acquired, at least 50% of the population of cells were cycling within the time for one population doubling. Attempts at holding the cells from cycling (through nutrient-depletion and serum-privation) were unsuccessful, although the turnover-rate was reduced. Our assays for X-irradiated clonogenic survivors after attempted holding combined with delayed plating (DP) showed differences in the survival curves for exponentially growing and confluent cultures. Elimination of cycling cells by S-phase cytocide removed these differences. Since a significant fraction of plateau-phase cells are not mitotically quiescent (Q), one must eliminate the proliferating (P) fraction if one wishes to examine the PLDR of the Q cells. For V79 cells, removal of the P cells eliminates the higher survival (usually interpreted as Q cell PLDR) of plateau-phase cells.     

Discrimination of jittered sonar echoes by the echolocating bat,Eptesicus fuscus: The shape of target images in echolocation

1. Behavioral experiments with jittering echoes examined acoustic images of sonar targets in the echolocating bat, Eptesicus fuscus, along the echo delay or target range axis. Echo phase, amplitude, bandwidth, and signal-to-noise ratio were manipulated to assess the underlying auditory processes for image formation. 2. Fine delay acuity is about 10 ns. Calibration and control procedures indicate that this represents temporal acuity rather than spectral discrimination. Jitter discrimination curves change in phase when the phase of one jittering echo is shifted by 180 degrees relative to the other, showing that echo phase is involved in delay estimation. At an echo detectability index of about 36 dB, fine acuity is 40 ns, which is approximately as predicted for the delay accuracy of an ideal receiver. 3. Compound performance curves for 0 degrees and 180 degrees phase conditions match the crosscorrelation function of the echoes. The locations of both 0 degrees and 180 degrees phase peaks in the performance curves shift along the time axis by an amount that matches neural amplitude-latency trading in Eptesicus, confirming a temporal basis for jitter discrimination.     

Analysis of the oligosaccharides from the roots of Arnica montana L., Artemisia absinthium L., and Artemisia dracuncula L.

The oligosaccharides extracted from the roots of Arnica montana L., Artemisia absinthium L. and Artemisia dracunculus L. have been analysed by thin-layer and gel-permeation chromatography to assess their applicability as ‘guide’ substances for pharmacological activity. Differences observed in the oligosaccharide component composition of such extracts might be more related to the vegetative stage of the plants at time of harvest than to the species themselves. In addition to a series of non-reducing oligofructosides, a series of reducing inulin-type oligosaccharides was found at the initial stages of growth, whereas in later stages of growth only non-reducing oligofructosides were present. These differences have been related to different stages of biosynthetic activity within the plants.     

The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex

The role of the epithelial adhesion molecule uvomorulin in the formation of the epithelial junctional complex in the Madin-Darby canine kidney (MDCK) cell line was investigated. Experiments were carried out to determine whether specific inhibition of uvomorulin function would interfere selectively with the formation, stability, or function of the apical zonula adherens (ZA) and zonula occludens (ZO), or whether it would interfere with all forms of intercellular contact including the desmosomes. The effects of blocking antibodies and Fab fragments to uvomorulin on the formation of the junctional complex was examined with a Ca2+ switch assay for de novo junction assembly. The formation of the ZO, the ZA, and the desmosomes was assayed by fluorescence staining with an antibody to the tight junction-specific protein ZO-1, with rhodamine-phalloidin for ZA-associated actin filaments, and with an anti-desmoplakin antibody, respectively. Under different conditions and times of antibody treatment the extent of inhibition of the formation of each of the junctional elements was very similar. The ability of the cells to eventually overcome the inhibitory effect of the antibodies and form junctions correlated with the reappearance of uvomorulin at the regions of cell-cell contact. Therefore uvomorulin seems to mediate an early adhesion event between epithelial cells that is a prerequisite for the assembly of all elements of the junctional complex. In contrast, the transepithelial electrical resistance of confluent, well-established monolayers of MDCK cells grown on filters was not greatly affected by treatment with the various antibodies or Fab fragments. A small transient decrease in resistance observed with the polyclonal alpha-uvomorulin IgG may be due to a more subtle modulation of the junctional complex.     

A conserved region in the sea urchin U1 snRNA promoter interacts with a developmentally regulated factor.

The expression of the sea urchin L. variegatus U1 snRNA gene is temporally regulated during embryogenesis. Using a microinjection assay we show that a region between 203 and 345 nts 5' of the gene is required for expression. There are four conserved regions between two sea urchin species in the 345 nts 5' to the U1 gene. One region, located at about -300, binds a protein factor which is present in blastula but not gastrula nuclei. Three other potential protein binding sites within the first 200 nts 5' to the gene have been identified using a mobility shift assay and/or DNase I footprinting. Two of these regions bind factors which are not developmentally regulated and one binds a factor which is developmentally regulated. It is likely that the factor which binds at -300 is involved in expression and developmental regulation of the sea urchin U1 snRNA gene.     

Comparative efficiencies of bispecific F(ab′γ)2 and chimeric mouse/human IgG antibodies in recruiting cellular effectors for cytotoxicity via Fcγ receptors

Summary The three forms of Fc receptor carried by monocytes (FcRI, II) and natural killer (NK) cells (FcRIII) are all capable of mediating cell lysis. Here we compare the use of F(ab)₂ bispecific antibodies, specifically targetting individual FcR, and chimeric IgG mouse/human antibodies which are capable of targetting all FcR, for their ability to mediate target cell destruction. The derivatives are prepared by linking hinge sulphydryl residues via tandem thioether bonds, using a bismaleimide crosslinker: Fab from an anti-FcR mAb linked to Fab from a common anti-target mAb (BsAb), or Fab from the common anti-target mouse antibody linked to human Fc (FabFc or bisFabFc). All the derivatives targetting chick red blood cells gave efficient lysis, although different effector cell donors yielded differences in both the lytic levels achieved and the comparative efficiencies of derivatives. In contrast, significant lysis of the guinea pig lymphoblastic leukaemia, L₂C, regularly resulted only via the anti-FcRIII BsAb and the chimeric derivatives. These results suggest that the chimeric, Fc-containing derivatives mediate tumour cell lysis principally through FcRIII on NK cells. This is in contrast to the situation with the chick red blood cells where the chimeric derivatives appear capable of lysing erythrocytes by utilizing either monocytes or NK cells, because significant (50%) lysis occurred with effector cell populations magnetically depleted through either FcRII or FcRIII. A major difference between these two types of antibody derivative was their ability to function in the presence of high concentrations of normal human Fc. The lysis mediated by BsAb reactive with FcRI or II was unaffected by the presence of human Fc at 2.5 mg/ml (a concentration comparable with that yielded by IgG in plasma) whereas the BsAb recognizing FcRIII and all the Fc-containing derivatives were completely inhibited.This work has been supported by Tenovus, the Cancer Research Campaign, the Leukaemia Research Fund, Italfarmaco, Milano, Italy and the Imperial Cancer Research Fund     

Application of good laboratory practice (GLP) to culture collections of microbial and cell cultures

Although the principles and the necessity for good laboratory practice (GLP) guidelines to confirm the credibility, integrity, and quality of non-clinical laboratory studies have been known for more than a decade, culture collection activities are not subject to them. Because of recent advances in biotechnology, culture collections face increased demands not only for quality cultures but also current information. When applied in culture collections, GLP guidelines prove to be an excellent management tool as well as a cost-effective system of providing authentic and reliable microbial and cell cultures and associated data.