Multiplexed, Tethered Particle Microscopy for Studies of DNA-Enzyme Dynamics.

DNA is the carrier of genetic information and, as such, is at the center of most essential cellular processes. To regulate its physiological function, specific proteins and motor enzymes constantly change conformational states with well-controlled dynamics. Twenty-five years ago, Schafer, Gelles, Sheetz, and Landick employed the tethered particle motion (TPM) technique for the first time to study transcription by RNA polymerase at the single-molecule level. TPM has since then remained one of the simplest, most affordable, and yet incisive single-molecule techniques available. It is an in vitro technique which allows investigation of DNA-protein interactions that change the effective length of a DNA tether. In this chapter, we will describe a recent strategy to multiplex TPM which substantially increases the throughput of TPM experiments, as well as a simulation to estimate the time resolution of experiments, such as transcriptional elongation assays, in which lengthy time averaging of the signal is impossible due to continual change of the DNA tether length. These improvements allow efficient study of several DNA-protein systems, including transcriptionally active DNA-RNA polymerase I complexes and DNA-gyrase complexes.

Communication: Fragment-based Hamiltonian model of electronic charge-excitation gaps and gap closure.

Capturing key electronic properties such as charge excitation gaps within models at or above the atomic scale presents an ongoing challenge to understanding molecular, nanoscale, and condensed phase systems. One strategy is to describe the system in terms of properties of interacting material fragments, but it is unclear how to accomplish this for charge-excitation and charge-transfer phenomena. Hamiltonian models such as the Hubbard model provide formal frameworks for analyzing gap properties but are couched purely in terms of states of electrons, rather than the states of the fragments at the scale of interest. The recently introduced Fragment Hamiltonian (FH) model uses fragments in different charge states as its building blocks, enabling a uniform, quantum-mechanical treatment that captures the charge-excitation gap. These gaps are preserved in terms of inter-fragment charge-transfer hopping integrals T and on-fragment parameters U((FH)). The FH model generalizes the standard Hubbard model (a single intra-band hopping integral t and on-site repulsion U) from quantum states for electrons to quantum states for fragments. We demonstrate that even for simple two-fragment and multi-fragment systems, gap closure is enabled once T exceeds the threshold set by U((FH)), thus providing new insight into the nature of metal-insulator transitions. This result is in contrast to the standard Hubbard model for 1d rings, for which Lieb and Wu proved that gap closure was impossible, regardless of the choices for t and U.

The lupus susceptibility locus Sle3 is not sufficient to accelerate atherosclerosis in lupus-susceptible low density lipoprotein receptor-deficient mice.

Cardiovascular disease risk is increased in individuals suffering from systemic lupus erythematosus. Understanding the mechanism(s) of systemic lupus erythematosus-accelerated atherosclerosis is critical for the development of effective therapies. Our laboratory previously demonstrated that radiation chimeras of systemic lupus erythematosus-susceptible B6.Sle1.2.3 and low density lipoprotein receptor (LDLr)(-/-) mice have augmented atherosclerosis, which is associated with increased T-cell burden and activation in the lesion. The goals of this study were to further define specific immune mechanisms that mediate accelerated atherosclerosis and to determine whether the gene interval Sle3, which is linked to lupus-associated T-cell dysregulation, was sufficient to modulate atherogenesis. We transferred B6.Sle3 or C57Bl/6-derived bone marrow cells into lethally irradiated LDLr( -/-) mice (hereafter referred to as LDLr.Sle3 and LDLr.B6, respectively). Sixteen weeks after transplantation, the mice were placed on a western-type diet for 8 weeks. Our analyses revealed that LDLr.Sle3 mice had increased auto-antibody production against double-stranded DNA and cardiolipin compared with LDLr.B6 controls. We also found an increase in atherosclerosis-associated oxLDL antibodies. Antibody isotypes and serum cytokine analysis suggested that the humoral immune response in LDLr.Sle3 mice was skewed toward a Th2 phenotype. This finding is consistent with lupus-associated immune dysregulation. Additionally, LDLr.Sle3 mice had decreased serum cholesterol and triglyceride levels. However, there was no difference in lesion area or cellular composition of lesions between the two groups. These data demonstrate that, despite no change in lesion area, transfer of Sle3-associated T-cell dysregulation alone to LDLr-deficient mice is sufficient to decrease serum cholesterol and to exacerbate humoral immune responses that are frequently associated with atherosclerosis.

Intracranial angioplasty and stenting: long-term results from a single center.

BACKGROUND AND PURPOSE: Large-vessel intracranial atherosclerotic stenosis carries a proved stroke risk of 8%-22% per year with "best medical therapy." The long-term clinical neurologic and angiographic outcomes of angioplasty and/or stent placement for intracranial atherosclerosis in a consecutive series of patients are presented. METHODS: The demographics, procedural details, procedural outcome, and long-term neurologic follow-up in 60 consecutive patients with 71 lesions, undergoing a total of 84 procedures, were analyzed. RESULTS: Angioplasty alone was performed in 62 procedures; 22 procedures involved stent placement. The periprocedural stroke+death rate was 4.8%. The overall complication-free success rate was 90.5%. Restenosis occurred in 23 lesions at a mean of 4.6 months; 13 were re-treated without complication. There were 4 strokes and 4 non-neurologic deaths during 224 patient-years of follow-up. The annualized stroke rate was 1.8%, and the annualized stroke+all-cause death rate was 3.0%. CONCLUSIONS: The stroke and death rates in this consecutive series of patients with severe intracranial atherosclerotic stenosis treated with optimal endovascular therapy are considerably less than those associated with the natural history of intracranial atherosclerosis treated with maximal medical therapy. Intracranial angioplasty with conditional stent placement is technically feasible and clinically effective with a substantial reduction in long-term stroke and death.

Atomic force microscopy study of DNA deposited on poly L-ornithine-coated mica.

Analyses of individual biomolecules, like DNA, or DNA-protein complexes, via atomic force microscopy, require 'gentle' methods to immobilize DNA on surfaces, which allow the ensemble of molecules to adopt conformations dictated primarily by their physical characteristics, and which possibly permit the use of a wide selection of buffers. We show that poly-L-ornithine-coated mica is a good substrate for fast, reliable deposition of DNA for wet or dry imaging. The surface firmly secures DNA, which retains the B-form helical rise (0.34 nm bp(-1)). The conformations of DNA that result are reminiscent of three-dimensional random coils projected on to a plane. The contrast is good, especially in solution, and buffers with physiological concentrations of salt with or without divalent cations may be used. This is important for comparison of scanning probe microscopy results with those obtained by different techniques.

Nature of charge carriers in disordered molecular solids: are polarons compatible with observations?

Polaronic theories for charge transport in disordered organic solids, particularly molecularly doped polymers, have been plagued by issues of internal consistency related to the magnitude of physical parameters. We present a natural resolution of the problem by showing that, in the presence of correlated disorder, polaronic carriers with binding energies Delta approximately 50-500 meV and transfer integrals J approximately 1-20 meV are completely consistent with the magnitudes of field and temperature dependent mobilities observed.

Three-dimensional magneto-optic trap for micro-object manipulation.

A magneto-optic trap for micro-objects is described. Magnetic beads were trapped by optical tweezers while being rotated by a new integrated magnetic manipulator. Rotation was achieved with eight electromagnets with tip-pole geometry. The time orbital potential technique was used to achieve rotation of magnetic beads. Trapping in three dimensions and rotation of magnetic beads on three axes are demonstrated with forces up to 230 pN and force momenta of up to 10(-16)N m . A position-detection apparatus based on an interferometric scheme provides nanometer sensitivities in a few milliseconds.

Use of phosphosynapsin I-specific antibodies for image analysis of signal transduction in single nerve terminals.

We have developed a semi-quantitative method for indirectly revealing variations in the concentration of second messengers (Ca(2+), cyclic AMP) in single presynaptic boutons by detecting the phosphorylation of the synapsins, excellent nerve terminal substrates for cyclic AMP- and Ca(2+)/calmodulin-dependent protein kinases. For this purpose, we employed polyclonal, antipeptide antibodies recognising exclusively synapsin I phosphorylated by Ca(2+)/calmodulin-dependent protein kinase II (at site 3) or synapsins I/II phosphorylated by either cAMP-dependent protein kinase or Ca(2+)/calmodulin-dependent protein kinase I (at site 1). Cerebellar granular neurones in culture were double-labelled with a monoclonal antibody to synapsins I/II and either of the polyclonal antibodies. Digitised images were analysed to determine the relative phosphorylation stoichiometry at each individual nerve terminal. We have found that: (i) under basal conditions, phosphorylation of site 3 was undetectable, whereas site 1 exhibited some degree of constitutive phosphorylation; (ii) depolarisation in the presence of extracellular Ca(2+) was followed by a selective and widespread increase in site 3 phosphorylation, although the relative phosphorylation stoichiometry varied among individual terminals; and (iii) phosphorylation of site 1 was increased by stimulation of cyclic AMP-dependent protein kinase but not by depolarisation and often occurred in specific nerve terminal sub-populations aligned along axon branches. In addition to shedding light on the regulation of synapsin phosphorylation in living nerve terminals, this approach permits the spatially-resolved analysis of the activation of signal transduction pathways in the presynaptic compartment, which is usually too small to be studied with other currently available techniques.

Analysis of difference in vivo effects of TCDD between c-src +/+ mice, c-src deficient, -/+ and -/- B6, 129-Src(tm 1 sor) mice and their wild-type littermates.

Toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were tested on two strains of c-src deficient B6, 129-Src(tm 1 sor) mice, c-src -/+ and c-src -/- and their matched wild-type littermates c-src +/+ mice along with another c-src +/+ mice, from the same genetic lineage, B6, 129-Fos(tm 1 Pa) mice. The most conspicuous effect of c-src deficiency on the toxicity of TCDD appears to be the reduced hepatotoxicity. TCDD-treated c-src deficient mice show only modest degrees of hepatomegaly and triglycerides accumulation as compared to treated wild-type mice.

FAK+ and PYK2/CAKbeta, two related tyrosine kinases highly expressed in the central nervous system: similarities and differences in the expression pattern.

Focal adhesion kinase (FAK) and proline-rich tyrosine kinase 2/cell adhesion kinase beta (PYK2/CAKbeta) are related, non-receptor, cytoplasmic tyrosine kinases, highly expressed in the central nervous system (CNS). In addition, FAK+ is a splice isoform of FAK containing a 3-amino acid insertion in the carboxy-terminal region. In rat hippocampal slices, FAK+ and PYK2/CAKbeta are differentially regulated by neurotransmitters and depolarization. We have studied the regional and cellular distribution of these kinases in adult rat brain and during development. Whereas PYK2/CAKbeta expression increased with postnatal age and was maximal in the adult, FAK+ levels were stable. PYK2/CAKbeta mRNAs, detected by in situ hybridization, were expressed at low levels in the embryonic brain, and became very abundant in the adult forebrain. Immunocytochemistry of the adult brain showed a widespread neuronal distribution of FAK+ and PYK2/CAKbeta immunoreactivities (ir). PYK2/CAKbeta appeared to be particularly abundant in the hippocampus. In hippocampal neurons in culture at early stages of development, FAK+ and PYK2/CAKbeta were enriched in the perikarya and growth cones. FAK+ extended to the periphery of the growth cones tips, whereas PYK2/CAKbeta appeared to be excluded from the lamellipodia. During the establishment of polarity, a proximal-distal gradient of increasing PYK2/CAKbeta-ir could be observed in the growing axon. In most older neurons, FAK+-ir was confined to the cell bodies, whereas PYK2/CAKbeta-ir was also present in the processes. In vitro and in vivo, a subpopulation of neurons displayed neurites with intense FAK+-ir. Thus, FAK+ and PYK2/CAKbeta are differentially regulated during development yet they are both abundantly expressed in the adult brain, with distinctive but overlapping distributions.

Differential toxicities of TCDD in vivo among normal, c-src knockout, geldanamycin- and quercetin-treated mice.

Although we have previously reported the result of our preliminary study on the reduced toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in vivo in male c-src deficient, -/+ B6, 129-src(tmlSor) mice as compared to those in wild-type mice, there are still two major shortcomings of the above study: (a) the low number of individuals tested, (b) in some of the comparison tests, C57BL/6J mice (i.e. inbred B6 mice) were used as wild-type control mice. Since then we increased our laboratory breeding program and thereby the availability of B6, 129-/+, -/- and true littermate wild-type +/+ individual mice. The results of critical in vivo toxicity comparison tests, involving 6-13 mice per test group, showed that there are considerable variations expressed in toxicity within each group of c-src deficient mice. Nevertheless, when a large enough number of individuals were tested two toxic effects were found to be less expressed in src-deficient mice. They were: (a) excess fatty deposits and (b) the mottled appearance of the liver which were commonly observed in TCDD-treated wild type mice, but not in c-src deficient mice. The former trend was also confirmed by both liver lipid analysis and histological examinations of the affected livers. As for the biochemical parameters, the hepatic nuclear protein binding to C/EBP (CCAAT/enhancer binding protein) response element appears to be uniformly reduced by the action of TCDD in + / + mice, but not in -/+ or -/- mice.

Evidence for organochlorine contamination in tissues of salmonids in Lake Tahoe.

To evaluate the extent of organochlorine pollution in the Sierra Nevada ecosystem, residues of certain organochlorines in lake trout and Kokanee fish from Lake Tahoe, an alpine lake located between the Sierra Nevada Mountain Range and the Carson Range of California and Nevada, were analyzed. Multiresidue analysis in fish muscle revealed wet weight concentrations of total polychlorinated biphenyls (PCBs) in the range 18 to 430 ppb and of p,p'-DDE in the range 5 to 85 ppb in the two fish species studied. In one large lake trout sample (6.6 kg), which was studied in more detail as compared with others, residue levels of PCB (267 ppb), toxaphene (154 ppb), a chlordane mixture (78 ppb), and a DDT mixture (154 ppb) were found in muscle. Full spectra of specific PCB congeners and p,p'-DDE were obtained from fish fat tissues and their identities were confirmed using gas chromatography/mass spectrometry (GC-MS). The results of total PCB analysis indicated that residues found in fish consisted mostly of moderately (tri- to tetrachloro-) to highly (penta- to heptachloro-) chlorinated biphenyls. For all fish residues analyzed, the best match to PCB residue profiles was with Aroclor 1260 or 1262.

Sequences of cDNAs encoding calmodulin, and partial structures of calmodulin kinase, and a calcium channel of kdr-resistant and -susceptible German cockroaches, Blattella germanica.

Complementary DNA sequences of genes encoding calmodulin, partial structures of calmodulin-dependent protein kinase II (CaM-kinase II) and an L-type-like calcium channel al subunit (IVS5-IVS6-EF hand region) were identified and compared between susceptible and kdr strains of German cockroach, Blattella germanica. For this purpose, polymerase chain reactions (PCR) were used to obtain their sequences using cDNA from poly(A) + RNA isolated from their heads and thoraces. No mutation differences were found in all three sequences of calcium-regulating proteins between susceptible and strain. Northern blot analysis, however, showed reduced expressions of CaM-kinase II mRNA in two kdr strains. Western blot analysis with an antibody preparation against CaM-kinase II on protein levels confirmed the above strain difference in the titer of this enzyme. In contrast, the levels of calmodulin as well as that of an L-type-like calcium channel gene expression were not different between susceptible and kdr strains.

Use of c-Src and c-Fos knockout mice for the studies on the role of c-Src kinase signaling in the expression of toxicity of TCDD.

Previously, we reported that several of the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were not fully expressed in c-src -/- and -/+ mice (1). In the current study, we studied the basic molecular mechanism of their differential responses. First, we could show that chemical inhibition of c-src kinase could produce practically the same phenomenon of reduced toxicity of TCDD in wild-type mice cotreated with geldanamycin, a specific chemical inhibitor known to suppress c-src kinase. Second, we established that the level of the Ah receptor associated with c-src kinase (2) is indeed low in c-src deficient mice as well as geldanamycin-treated mice. We could show, at the same time, that the effect of c-src deficiency on the toxicity of TCDD is very selective; that is, despite the reduction of many of its toxic signs, the enlargement of liver, induction of cytochrome P450, and other drug-metabolizing enzymes took place normally in those c-src-deficient mice. Apparently, induction of these detoxification enzymes are independent of c-src-mediated pathway. Based on the known signaling pathways of c-src, we tested c-fos-deficient mice and found that some of the c-src-dependant toxic signs of TCDD such as thymic atrophy and decrease in adipose tissue weight were also reduced in c-fos-deficient mice, indicating that these two toxic effects are likely to be mediated through both c-src and c-fos. However, other TCDD responses noticeable in c-fos-deficient mice; downregulation of receptors for epidermal growth factor (EGF), tumor necrosis factor (TNF alpha), and retinoic acid; and up-regulation of the T3 receptor. These findings clearly show that c-fos mediates only a part of c-src signaling pathway in transducing these specific toxic actions of TCDD as mediated by c-fos.

Suppression of C/EBPalpha and induction of C/EBPbeta by 2,3,7,8-tetrachlorodibenzo-p-dioxin in mouse adipose tissue and liver.

We examined the effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on two transcription factors, CAAT/enhancer binding protein-alpha (C/EBPalpha) and beta (C/EBPbeta), involved in the coordination of gene expression in adipose and liver. A single dose of TCDD (100 microg/kg) to male C57BL mice resulted in a time- and dose-dependent decrease in the level of C/EBPalpha mRNA in adipose tissue and liver, and a reciprocal increase in C/EBPbeta mRNA. Gel shift analysis using hepatic nuclear extracts from control and TCDD-treated mice and an oligonucleotide containing a C/EBP recognition element revealed a time-dependent change in DNA-protein complexes formed. Bands corresponding to C/EBPalpha, as determined by supershift analysis, diminished in TCDD-treated animals over a 7-day time period, whereas two new bands corresponding to C/EBPbeta, not present in control extracts, were increased significantly in treated samples. TCDD induced C/EBPbeta mRNA in wild-type mouse hepatoma cells, but not in aryl hydrocarbon receptor (AhR) nuclear translocator-deficient hepatoma cells. Induction in wild-type hepatoma cells was antagonized effectively by a molar excess of alpha-naphthoflavone. These results showed that TCDD caused rapid, reciprocal changes in C/EBPalpha and C/EBPbeta mRNAs and DNA binding in the adipose and liver of male C57BL mice and induced C/EBPbeta in hepatoma cells in an AhR-dependent manner. C/EBPs play vital roles in the coordination of energy homeostasis, and their alteration by TCDD may provide insight into the mechanism by which TCDD perturbs energy storage and utilization in vivo.

Continuous fentanyl infusion: use in severe cancer pain.

OBJECTIVE: To describe the use of a continuous fentanyl infusion in an adult cancer patient. CASE SUMMARY: A 66-year-old white woman diagnosed with metastatic pancreatic carcinoma required hospital admission for pain control after receiving five different chemotherapy regimens. Morphine 2 mg/h i.v. was initiated and the dosage was titrated upward to a total of 6613 mg/d by hospital day 16. As hospital supplies of opioids became depleted over a holiday weekend, therapy was changed to a continuous infusion of hydromorphone 70 mg/h on hospital day 17, then changed to a continuous fentanyl infusion beginning with a dosage of 500 micrograms/h. The fentanyl dosage was titrated to 4250 micrograms/h by hospital day 20. She died comfortably on hospital day 22 while receiving this dosage. DISCUSSION: Continuous infusions of opioids, particularly morphine and hydromorphone, are frequently used for control of cancer pain and are safe and effective when administered by this route. Transdermal fentanyl has been shown to effectively manage chronic cancer pain, and use of continuous subcutaneous fentanyl has been reported. However, reports of continuous intravenous fentanyl infusion in the cancer pain literature are limited. Our patient achieved good pain control with a continuous infusion of fentanyl 4250 micrograms/h. CONCLUSIONS: Continuous fentanyl infusion should be considered for the treatment of cancer pain in patients requiring high doses who become refractory to other opioids, when other opioids cause intolerable adverse effects, when patients have a true morphine allergy, or when high-dose requirements threaten to deplete existing stock of alternate opioids.

Nanoscopic structure of DNA condensed for gene delivery.

Scanning force microscopy was used to examine DNA condensates prepared with varying stoichiometries of lipospermine or polyethylenimine in physiological solution. For the first time, individual DNA strands were clearly visualized in incomplete condensates without drying. Using lipospermine at sub-saturating concentrations, discrete nuclei of condensation were observed often surrounded by folded loops of DNA. Similar packing of DNA loops occurred for polyethylenimine-induced condensation. Increasing the amount of the condensing agent led to the progressive coalescence or aggregation of initial condensation nuclei through folding rather than winding the DNA. At over-saturating charge ratios of the cationic lipid or polymer to DNA, condensates had sizes smaller than or equal to those measured previously in electron micrographs. Polyethylenimine condensates were more compact than lipospermine condensates and both produced more homogeneously compacted plasmids when used in a 2-4-fold charge excess. The size and morphology of the condensates may affect their efficiency in transfection.

Development of broad spectrum antibodies to heat shock protein 70s as biomarkers for detection of multiple stress by pollutants and environmental factors.

To test the hypothesis that broad spectrum antibodies may be developed as biomarkers useful in detecting the consequence of combined environmental stresses in a wide variety of tissues and organisms, a stretch of 16 amino acids, TVPAYFNDSQRQATKDA, a well-conserved portion of heat shock 70 proteins, was identified, against which specific antibodies could be designed. This stretch of peptide was synthetically prepared and used as a hapten for antibody preparation by coupling to keyhole lympet hemocyanin, injecting into a rabbit, collecting its blood, and purifying an IgG-rich fraction. The resulting polyclonal antibody was found to react with many heatshock protein (HSP) 70s in every species tested so far, including two species of fish and one amphibian, two arthropod, and one plant species. To relate the reactivity of this antibody preparation to heat shock proteins known to be induced by environmental stress, a Western blot assay method was used to study several organisms under unstressed or stressed conditions. Invariably, heat treatment caused a rise in the titer or HSP70 and/or glucose-regulated proteins. In addition, in some species chemical stresses were also found to be manifested in the form of an increased titer of these proteins.

Altered in vivo toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in C-SRC deficient mice.

Administration of a single i.p. dose of 115 microg/kg of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to homozygous and heterozygous c-src deficient mice (i.e. c-src -/- and -/+ mice) and their wild-type littermates (c-src +/+ mice) induced differential toxic responses. In c-src +/+ mice, there were clear-cut signs of the toxicity of TCDD, such as the loss of weight in the body, thymus and adipose tissue, whereas in c-src -/+ mice these effects were modest and were not statistically significant. Yet, hepatomegaly, a characteristic effect of TCDD, took place in all three strains of mice. Histological examination of liver samples from control mice and from mice treated with TCDD for 10 days showed that there are qualitative differences in the expression of the effects of TCDD between control and treated mice as well as between c-src -/+ and +/+ mice. In the case of c-src +/+ mice, the predominant lesions were lipid accumulation, glycogen depletion, edema formation and necrosis, as shown by the presence of large areas of ballooning degeneration, and cellular influx of fluid. These changes were demonstrated only marginally in c-src -/+ mice. The predominant effect in -/+ mice was edema formation. At a high dose of TCDD (345 microg/kg), all of the +/+ mice died within 34 days, whereas none of the c-src -/+ mice died. Together these results clearly indicate that some of the toxic effects of TCDD are not fully expressed in c-src deficient mice.

Cloning and sequencing of the para-type sodium channel gene from susceptible and kdr-resistant German cockroaches (Blattella germanica) and house fly (Musca domestica).

Using reverse transcription polymerase chain reactions (RT-PCR), the DNA sequence for the main membrane-spanning region (IS3 through IVS6) of the gene encoding the alpha-subunit of the para sodium channel of the German cockroach, Blattella germanica, has been determined. The overall structure of the open reading frame region of this B. germanica gene is very similar to that of the para gene of Drosophila melanogaster, and that of the partially sequenced para gene of Musca domestica. On the other hand, it is distinctly different from that of the DSC gene (Drosophila sodium channel). As a result of a side-by-side comparison of the para gene sequences of the susceptible CSMA strain and the kdr resistant VT strain of B. germanica, one mutation (TTG to TTC) at the approximate center of the IIS6 membrane-spanning segment was found to result in an amino acid change from L to F. While the functional meaning of this mutation for the operation of the para sodium channel remains to be studied, this region is very highly conserved among all sodium channels identified so far, and is one of the most hydrophobic areas of the entire alpha-subunit. For comparison, we have studied the same region of the para sodium channel of both kdr and susceptible SBO strain of the housefly, Musca domestica. We found the homologous type of mutation, CTT to TTT, resulting in the same amino acid alteration (L to F) at this site. However, in the case of houseflies both kdr and susceptible strains contained both L and F versions of the protein. The ratio of TTT to CTT was significantly higher in the kdr strain of M. domestica than in the three susceptible strains examined.