Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size.     

Orientation in a desert lizard (Uma notata): time-compensated compass movement and polarotaxis

Summary The diurnal escape response of fringetoed lizards (Uma notata) startled by predators demonstrates clear directional orientation not likely to depend on local landmarks in the shifting sands of their desert environment. Evidence that celestial orientation is involved in this behavior has been sought in the present experiments by testing the effects of (1) phase shifting the animal's internal clock by 6 h and (2) by training the lizards to seek shelter while exposed to natural polarization patterns. In the first case, 90° shifts in escape direction were demonstrated in outdoor tests, as expected if a time-compensated sun or sky polarized light compass is involved. In the second instance, significant bimodale-vector dependent orientation was found under an overhead polarizing light filter but this was only evident when the response data were transposed to match the zenithe-vector rotation dependent on the sun's apparent movement through the sky. This extends to reptiles the capacity to utilize overheade-vector directions as a time-compensated sky compass. The sensory site of this discrimination and the relative roles of sun and sky polarization in nature remain to be discovered.     

Mapping of brain activity in mesencephalic and diencephalic structures of toads during presentation of visual key stimuli: a computer assisted analysis of (14C)2DG autoradiographs

Summary The (¹⁴C)2DG autoradiographic technique has been employed to quantitatively map glucose utilization in the mesencephalon, the diencephalon and the cerebellum, of toads in response to configurational moving visual stimuli: (i) a 0.4 cm × 2.8 cm worm-like stripe (W) which elicited prey catching responses, (ii) a 8.4 cm × 8.4 cm square (S) that released predator avoidance responses, and (iii) a 2.8 cm × 0.4 cm antiworm-like stripe (A) which elicited no motor activity.For various brain nuclei different relationships were obtained: The optic tectum showed statistical significant higher 2DG uptake during worm-stimulation (¯X _W) than during antiworm stimulation (¯X _A), i.e.¯X _W>¯X _A. The latter visual pattern led to a 2DG utilization that was statistically significant stronger than during stimulation with a square (¯X _S), i.e.¯X _A>¯X _S. Thus, in comparison between right and left hemisphere as well as between brains the following ratios were obtained:Optic tectum:¯X _W>¯X _A>¯X _S; nucleus isthmi:¯X _W>¯X _A-¯X _s; posterodorsal lateral thalamic nucleus:¯X _S>¯X _A>¯X _W; posteroventral lateral thalamic nucleus:¯X _S>¯X _A¯X _W; posterior thalamic nucleus:¯X _W>¯X _A¯X _S; anteripr division of the lateral thalamic nucleus:¯X _W>¯X _A¯X _S; anterior thalamic nucleus:¯X _A>¯X _S>¯X _W; nucleus of Bellonci and dorsal division of the ventrolateral thalamic nucleus:¯X _W¯X _A¯X _S; cerebellum:¯X _S¯X _W>¯X _A.Abbreviations A anterior thalamic nucleus - Cb cerebellum - Hyp hypothalamus - Ist nucleus isthmi - cl. Ist contralateral Ist - La lateral thalamic nucleus, anterior division - Lpd lateral thalamic nucleus, posterodorsal division - Lpv lateral thalamic nucleus, posteroventral division - MP medial pallium - NB/VLd nucleus of Bellonci and ventrolateral thalamic nucleus, dorsal division - P posterior thalamic nucleus - PO preoptic area - Sna snapping evoking area=ventrolateral tectum - Str striatum - Tec tectum opticum     

Rapid recovery from K current inactivation on membrane hyperpolarization in molluscan neurons

Recovery from K current inactivation was studied in molluscan neurons using two-microelectrode and internal perfusion voltage clamps. Experiments were designed to study the voltage-dependent delayed outward current (IK) without contamination from other K currents. The amount of recovery from inactivation and the rate of recovery increase dramatically when the membrane potential is made more negative. The time course of recovery at the resting potential, -40 mV, is well fit by a single exponential with a time constant of 24.5 s (n = 7). At more negative voltages, the time course is best fit by the sum of two exponentials with time constants at -90 mV of 1.7 and 9.8 s (n = 7). In unclamped cells, a short hyperpolarization can cause rapid recovery from inactivation that results in a shortening of the action potential duration. We conclude that there are two inactivated states of the channel and that the time constants for recovery from both states are voltage dependent. The results are discussed in terms of the multistate model for K channel gating that was developed by R. N. Aldrich (1981, Biophys. J., 36:519-532).     

Allelic variation at the frizzled locus of Drosophila

The epidermis of Drosophila has a tissue polarity that is manifested by a parallel array of polarized structures (primarily hairs and bristles). The production of normal tissue polarity requires the function of the frizzled (fz) locus. We have isolated a large number of alleles at this locus and have phenotypically characterized more than 25 of them. We have found extensive allelic variation that a previous study failed to detect. Most of the alleles fall into a hypomorphic to amorphic series. Two alleles, however, have unusual properties. These alleles, which in general are moderately strong alleles, fail to produce a rough eye phenotype that is characteristic of all the other moderate or strong fz alleles. Thus, these two alleles are tissue specific in effect. Furthermore, these two alleles also have a neomorphic or antimorphic effect on hair polarity in one region of the wing.     

Evidence that insulin and guanosine triphosphate regulate dephosphorylation of the beta-subunit of the insulin receptor in sarcolemma membranes isolated from skeletal muscle.

When sarcolemma membranes isolated from rat skeletal muscle were incubated with [gamma-32P]ATP, a membrane protein of apparent Mr 95,000 was rapidly phosphorylated, with the 32P content reaching a maximum within 2 s. On the basis of immunoprecipitation with anti-insulin-receptor antiserum, phosphoamino acid analysis and Mr, this protein probably represents the beta-subunit of the insulin receptor. Similarly, on incubation of the membrane with adenosine 5'-[gamma-[35S]thio] triphosphate the 95 kDa protein was thiophosphorylated, indicating thiophosphorylation of the beta-subunit of the insulin receptor on the basis of immunoprecipitation studies. The effect of insulin on the phosphorylation of this protein in the membrane was studied. Insulin induced a 20% decrease in the 32P labelling of the protein when the membranes were phosphorylated for 10 s. This insulin effect was dose-dependent, with half-maximal effect obtained at 2-3 nM-insulin. Addition of GTP, but not GDP or guanosine 5'-[beta, gamma-imido]triphosphate, enhanced the effect to 35% inhibition, with half-maximal effect of GTP obtained at 0.5 microM. GTP had no effect on the phosphorylation of the protein in the absence of insulin. Analysis of this insulin effect showed that insulin increased the rate of dephosphorylation of the 95 kDa protein in the membrane. In contrast, insulin had no effect on thiophosphorylation of the 95 kDa membrane protein after incubation with adenosine 5'-[gamma-[35S]thio]triphosphate. Since thiophosphorylated proteins are less sensitive to phosphatase action, these investigations suggest that insulin stimulated a protein phosphatase activity in a GTP-dependent manner. The possibility that GTP-regulatory proteins are involved in the action of insulin on the phosphorylation of the insulin receptor and other membrane proteins is discussed.     

A study of phi X-174 DNA torus and lambda DNA torus tertiary structure and the implications for DNA self-assembly

Hydrated torus shaped complexes were examined by transmission electron microscopy in both spermidine-condensed linear and nicked circular phi X-174 DNA and lambda DNA preparations. Freeze-etch replicas of both these torus samples, produced with very low Pt metal deposition levels (9APt/C), were found to have circumferentially wound single DNA double helix size surface fibers in the range of 30A width. Measurements of torus inner and outer circumference as well as ring thickness were performed. Observed differences in the torus dimension distributions from circular phi X-174 DNA and linear phi X-174 DNA may be related to the different topological constraints on DNA folding in these two samples (1). On the basis of annulus thickness measurements phi X-174 DNA toruses, in contrast to lambda DNA toruses, were observed to fall into two classes identified as being formed from monomer DNA condensation and multimer DNA condensation. All of the torus substructure and population dimensions observed here are consistent with the continuous circumferential DNA winding model of torus organization proposed by Marx and Reynolds (1) to explain the micrococcal nuclease cleavage properties of the toruses. End-on view measurements of the torus thickness were made from micrographs obtained by extensive tilting of the object replica. These direct measurements confirmed quaternary structure interpretations made from simple strand packing models. We compared the measured torus properties in this linear DNA size series (5386-48000 bp). With increasing DNA length the pattern of DNA strand self-assembly was found to be more varied producing lambda DNA toruses of varying shape. The relevance of our study to the problem of lambda bacteriophage DNA head packaging was discussed.     

The acquired immune deficiency syndrome and epidemic of infection with human immunodeficiency virus: costs of care and prevention in an inner London district.

The epidemic of the acquired immune deficiency syndrome (AIDS) and infection with human immunodeficiency virus (HIV) necessitates early planning of services and allocation of resources. The use of hospital resources by patients with AIDS and the planned additional costs of clinical and preventive services for the epidemic of infection with HIV were calculated for an inner London health district that has treated 18% of the cases in the United Kingdom. Patients with AIDS required on average 50 days of inpatient hospital care each at an estimated current average lifetime cost of pounds 6800. These costs, however, underestimated the additional capital and revenue costs of planned new preventive and treatment services, estimated as being pounds 388,000 revenue and pounds 472,000 capital for 1986-7. It is important to invest now in preventive services throughout the United Kingdom to reduce the future social and financial costs of AIDS.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.