TGF beta 2 and TGF beta 3 are potent survival factors for midbrain dopaminergic neurons.

The vertebrate ventral midbrain contains 3-4 x 10(4) dopaminergic neurons that influence motor activity, emotional behavior, and cognition. Recently, glial cell line-derived neurotrophic factor (GDNF) was shown to be a potent survival factor for these dopaminergic neurons in culture. However, many midbrain dopaminergic neurons project to targets that do not express GDNF. We report here that transforming growth factors (TGFs) TGF beta 2 and TGF beta 3, which are distantly related to GDNF, also prevent the death of cultured rat embryonic midbrain dopaminergic neurons at picomolar concentrations. Furthermore, we find that TGF beta 2, TGF beta 3, and GDNF are expressed sequentially as local and target-derived trophic factors and that subpopulations of dopaminergic neurons projecting to distinct targets have access to only one of these factors. These findings are consistent with the idea that GDNF, TGF beta 2, and TGF beta 3 are physiological survival factors for developing midbrain dopaminergic neurons and may have applications as therapeutics for Parkinson's disease, a neurodegenerative disorder of dopaminergic neurons.

Persephin, a novel neurotrophic factor related to GDNF and neurturin.

A novel neurotrophic factor named Persephin that is approximately 40% identical to glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) has been identified using degenerate PCR. Persephin, like GDNF and NTN, promotes the survival of ventral midbrain dopaminergic neurons in culture and prevents their degeneration after 6-hydroxydopamine treatment in vivo. Persephin also supports the survival of motor neurons in culture and in vivo after sciatic nerve axotomy and, like GDNF, promotes ureteric bud branching. However, in contrast to GDNF and NTN, persephin does not support any of the peripheral neurons that were examined. Fibroblasts transfected with Ret and one of the coreceptors GFRalpha-1 or GFRalpha-2 do not respond to persephin, suggesting that persephin utilizes additional, or different, receptor components than GDNF and NTN.

Calcium-dependent Ret activation by GDNF and neurturin.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) define a new family of neurotrophic factors that play crucial roles in survival and differentiation of various neurons. Recent studies demonstrated that GDNF and NTN use a multicomponent receptor system in which glycosyl-phosphatidylinositol (GPI)-linked cell surface proteins and Ret receptor tyrosine kinase function as the ligand-binding and signalling components, respectively. In the present study, we investigated the role of Ca2+ ions for biochemical and biological activities of Ret because Ret has a unique structure of the extracellular domain with the cadherin-like motif. The results demonstrated that Ca2+ ions might be required for the complex formation of Ret and GDNF or NTN that induces Ret oligomerization and autophosphorylation. Full morphological differentiation of neuroblastoma cells by these neurotrophic factors was also Ca2+-dependent. These findings thus suggested that, in addition to GPI-linked cell surface proteins, Ca2+ ions are components of the signal transducing complex formed by Ret and GDNF protein family.

Feedback control of sex determination by dosage compensation revealed through Caenorhabditis elegans sdc-3 mutations.

In Caenorhabditis elegans, sex determination and dosage compensation are coordinately controlled through a group of genes that respond to the primary sex determination signal. Here we describe a new gene, sdc-3, that also controls these processes. In contrast to previously described genes, the sex determination and dosage compensation activities of sdc-3 are separately mutable, indicating that they function independently. Paradoxically, the sdc-3 null phenotype fails to reveal the role of sdc-3 in sex determination: sdc-3 null mutations that lack both activities disrupt dosage compensation but cause no overt sexual transformation. We demonstrate that the dosage compensation defect of sdc-3 null alleles suppresses their sex determination defect. This self-suppression phenomenon provides a striking example of how a disruption in dosage compensation can affect sexual fate. We propose that the suppression occurs via a feedback mechanism that acts at an early regulatory step in the sex determination pathway to promote proper sexual identity.

Signal sequence of human preproparathyroid hormone is inactive in yeast.

The biosynthesis of human preproparathyroid hormone (hpreproPTH) and the processing to mature parathyroid hormone (hPTH) was investigated in yeast. Cells were transformed with a plasmid that carried a fusion gene made of the yeast pyruvate kinase promoter, complementary DNA (cDNA) encoding a slightly modified form of hpreproPTH and the transcription termination signal from yeast triosephosphate-isomerase. In transformed yeast cells we identified a protein that was recognized by a PTH antiserum and, on gel electrophoresis, comigrated with hpreproPTH marker. The amino-terminal sequence of the protein was consistent with that of hpreproPTH, indicating that the hormone precursor is not processed. It was localized inside the cell, when analyzed in pulse-chase experiments by trypsin accessibility in intact and lysed spheroplasts. In contrast, when mRNA from these yeast cells and from human parathyroid tissue was translated into preproPTH in a reticulocyte lysate supplemented with canine pancreatic microsomes, the preproPTHs from both mRNAs were transported and cleaved with identical efficiencies. We conclude that hpreproPTH is synthesized in yeast but not recognized and processed like a precursor of a secreted protein by the yeast secretory apparatus.

Interleukin-1beta-induced promatrilysin expression is mediated by NFkappaB-regulated synthesis of interleukin-6 in the prostate carcinoma cell line, LNCaP.

Previously, our laboratory showed that interleukin-1beta (IL-1beta) secreted by lipopolysaccharide-activated monocytes induces promatrilysin expression in the prostate carcinoma cell line, LNCaP. We now demonstrate that IL-1beta-induced promatrilysin expression is mediated by an indirect mechanism that requires nuclear factor Kappa B (NFkappaB)-dependent synthesis of IL-6. Inhibition of protein synthesis with cycloheximide blocked IL-1beta-mediated induction of matrilysin mRNA suggesting that synthesis of one or more additional factors is required for IL-1beta-induced promatrilysin protein expression. Blockage of NFkappaB transactivation activity abrogated IL-1beta-induced promatrilysin expression to baseline levels suggesting that NFkappaB transactivation activity is necessary. Inhibition of IL-6 activity attenuated IL-1beta-induced promatrilysin, but not NFkappaB transactivation activity indicating that IL-6 acts downstream of NFkappaB in potentiation of IL-1beta-mediated promatrilysin expression. Inhibition of protein synthesis with cycloheximide did not alter IL-6-induced induction of matrilysin mRNA indicating that, contrary to the mechanism by which IL-1beta regulates promatrilysin expression, IL-6-mediated matrilysin mRNA expression does not require new protein synthesis. Transient transfection with dominant negative STAT3 inhibited IL-1beta- and IL-6-induced promatrilysin. These data provide evidence that NFkappaB-mediated IL-6 synthesis is required for IL-1beta-induced promatrilysin expression, and IL-6 signaling through STAT3 plays a role in IL-1beta-induced promatrilysin expression.

A DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis can be maintained as an extrachromosomal element.

A 4.05-kb DNA fragment containing the ADE2 gene from Schwanniomyces occidentalis was cloned into the pUC19 vector. When an ade2 strain of Sc. occidentalis was transformed with this plasmid, pADE-2 was found to integrate into the host chromosome and was also present in a variety of extrachromosomal species. These extrachromosomal elements were present in multiple copies, varied in molecular mass and were composed of polymerized forms of pADE-2. A fragment containing the ADE2 gene was used to transform a Sc. occidentalis ade2 mutant, as either a linear or circularized molecule. The linear form integrated into the host genome, whereas the circularized form was found as a stably maintained extrachromosomal element with no evidence of integration or detectable loss of the Ade+ phenotype upon subculturing of transformed yeast under nonselective conditions for 60 generations. The ratio of the number of extrachromosomal ADE2 genes to genomic ADE2 ranged from 3.8 to 6.6.

Characterization of a beta-tubulin gene and a beta-tubulin gene products of Brugia pahangi.

A genomic clone containing a beta-tubulin gene from the parasitic nematode Brugia pahangi was isolated. This gene was sequenced to determine its size, structural organization, and corresponding primary amino acid sequence. The coding sequence of the beta-tubulin gene spans 3.8 kb, is organized into 9 exons and expresses an mNRA of 1.8 kb which codes for a protein of 448 amino acids. The predicted beta-tubulin amino acid sequence is 89%, 94%, 90% and 88% identical to the chicken beta 2, and the Caenorhabditis elegans ben-1, tub-1 and mec-7 gene products, respectively. Southern hybridization analyses demonstrated that there is only one copy of this gene isotype but that other distinct beta-tubulin genes may exist in the Brugia pahangi genome. A nematode specific antipeptide rabbit antiserum raised against the predicted amino acid sequence of the extreme carboxy-terminal region of the B. pahangi beta-tubulin was used to identify beta-tubulin isoforms in adult nematodes and microfilariae. Isoforms detected by this nematode-specific antipeptide antiserum were identical in both adult worms and microfilariae and did not differ from the isoform patterns detected by a monoclonal antibody recognizing a conserved beta-tubulin epitope. This suggests that this carboxy-terminal peptide is highly represented in the beta-tubulin isoforms of B. pahangi.

Expression of cloned beta-tubulin genes of Haemonchus contortus in Escherichia coli: interaction of recombinant beta-tubulin with native tubulin and mebendazole.

Two distinct beta-tubulin cDNA isotypes (beta 8-9 and beta 12-16) from Haemonchus contortus were expressed for the first time in Escherichia coli and characterised by their specific mebendazole (MBZ) binding and polymerization properties. Beta-tubulin was expressed without translational fusion to an E. coli sequence under the regulation of the tryptophan promoter in the pTrp2 vector. Beta-tubulin was produced in large amounts in insoluble 'inclusion bodies'. The inclusion bodies were purified and solubilised and the beta-tubulin renatured by treatment with urea followed by dilution with alkaline buffer and a shift to physiological pH. The yield was more than 10 mg of beta-tubulin per litre of cell culture. The recombinant tubulin produced was recognized in Western blot by specific anti-beta-tubulin antibodies. Tritiated MBZ binding to the recombinant H. contortus beta-tubulin was measured in the presence or absence of whole, tubulin-free or tubulin-rich extracts of H. contortus. Some [3H]MBZ high-affinity binding (HB) to 'pure' (no other eukaryotic protein present) beta 8-9 or beta 12-16 was observed. Enhanced high-affinity binding was observed when recombinant beta 8-9 or beta 12-16 were mixed and pre-incubated with whole supernatants or tubulin-enriched extracts from H. contortus. The enhancement was more than additive. Beta 12-16 bound more MBZ and caused a greater enhancement than beta 8-9. Mixing recombinant beta 8-9 or beta 12-16 with whole supernatants or tubulin-enriched fractions from H. contortus promoter polymerization at 37 degrees C. Use of 35S-labelled protein showed that the polymer contained recombinant tubulin. Western blot using specific anti-alpha-tubulin monoclonal antibodies showed that the polymer contained alpha-tubulin. Similarly the recombinant nematode beta-tubulin co-polymerized with tubulin from chicken brain. Our data suggest that the recombinant beta-tubulin can interact and copolymerize with parasite or chicken tubulin. Furthermore the interaction of recombinant nematode beta-tubulin with native tubulin and/or microtubule associated proteins (MAPs) resulted in the formation of high-affinity MBZ-binding sites. However, interaction of recombinant beta-tubulin with microtubule proteins from chicken brain did not result in the formation of high-affinity MBZ-binding sites.

Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus.

Phosphofructokinase (PFK), the key regulatory enzyme in glycolysis, has been cloned from the pathogenic parasitic nematode Haemonchus contortus by functional complementation in Escherichia coli. An E. coli strain deleted for both PFK loci (strain DF1020) was transformed with plasmid DNA from a lambda ZAP II H. contortus cDNA library. Two out of 3 x 10(7) transformants were able to grow on minimal medium with mannitol as the sole carbon source. A plasmid, pPFK, containing a 2.7-kb insert, was isolated from one of these transformants and conferred on DF1020 the ability to grow on mannitol (the PFK phenotype). The complemented cells contain PFK enzyme activity, absent in the E. coli mutant, at levels considerably higher than in wild type E. coli. Sequence analysis of the 2.7-kb insert shows an open reading frame that predicts a 789-amino acid protein that has approximately 70% similarity to mammalian PFKs. The amino acid sequence around asp182, thought to be the catalytic site, is completely conserved from nematodes to mammals.

Cloning of a cDNA encoding phosphoenolpyruvate carboxykinase from Haemonchus contortus.

Biochemical and metabolic data have led to the conclusion that the enzyme phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) contributes to a critical point of divergence in energy conservation pathways between mammals and nematodes. To facilitate the determination of the molecular basis for host vs parasite differences in PEPCK, we have cloned a cDNA encoding this enzyme from a parasitic nematode of ruminants, Haemonchus contortus. H. contortus PEPCK was cloned by functional complementation of a PEPCK-, malic enzyme- strain of Escherichia coli (E1786) using an egg stage H. contortus cDNA library in lambda ZAPII. Selection was for growth on malate as the sole carbon source (malate+ phenotype). We isolated a plasmid, pPEPCK, which reproducibly confers a malate+ phenotype in E1786. The sequence of the 2.0-kb EcoRI insert of pPEPCK predicts a 612-amino acid protein which shows about 74% similarity to Drosophila melanogaster and chicken PEPCK. Extracts of E1786[pPEPCK], but not E1786, contain IDP- or GDP-dependent PEPCK enzyme activity. Sequence analysis revealed that the open reading frame (ORF) in pPEPCK lacked a 5' initiation codon and was probably expressed as an in-frame fusion protein with beta-galactosidase. A strategy combining library screening with PCR analysis of positive clones led to the identification of a clone encoding 6 additional NH2-terminal amino acids, including a Met, which, by comparison with known PEPCK amino acid sequences, is likely to be the translation initiation site.

Three beta-tubulin cDNAs from the parasitic nematode Haemonchus contortus.

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of beta-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian beta-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of beta-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three beta-tubulin cDNAs from this organism, beta 12-16, beta 12-164, and beta 8-9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes. beta 8-9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode beta-tubulins with mammalian beta-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to beta 8-9 and beta 12-16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to beta 8-9.

Haemonchus contortus: the role of two beta-tubulin gene subfamilies in the resistance to benzimidazole anthelmintics.

The role of beta-tubulin genes in benzimidazole (BZ) resistance was investigated using one susceptible (S) and two resistant (Rt and Rc) strains of Haemonchus contortus. The Rt strain was isolated from the field on the basis of thiabendazole resistance. The Rc strain was derived from the S strain by treatment with cambendazole. cDNAs, derived from the S strain, encoding two isoforms of beta-tubulin (beta 12-16 and beta 8-9), alpha-tubulin and phosphofructokinase (Pfk) were used as probes for Southern hybridization analysis of genomic DNA digested by restriction enzymes. Genomic DNA was isolated from a pool of worms or single worms. The restriction-enzyme fragment length polymorphism (RFLP) differences among these strains depended on the enzyme and the probe used. When digested with Stu I or Hpa I, and probed under stringent conditions with beta 8-9 or beta 12-16, fewer fragments were seen in the Rt and Rc strains than in the S strain. Different hybridizing fragments were found in different individuals. The frequency of individuals bearing certain fragments hybridizing to beta 12-16 or beta 8-9 in the susceptible population was reduced significantly in the resistant populations. Some differences in RFLP between these strains were observed when probed with alpha-tubulin or Pfk, but the changes were not consistent with fragments being lost from the resistant strains as observed for beta-tubulin probes. These changes in RFLP pattern correlate with changes in the binding profiles of BZs and isoelectric isoform patterns reported previously for these strains. The data confirm that reduced heterogeneity within the population is associated with BZ resistance. Our results show that both the beta 8-9 and the beta 12-16 subfamilies of beta-tubulin are affected to a similar extent by this reduction in heterogeneity in a resistant population.

Mechanism-based screening: discovery of the next generation of anthelmintics depends upon more basic research.

The therapeutic arsenal for the control of helminth infections contains only a few chemical classes. The development and spread of resistance has eroded the utility of most currently available anthelmintics, at least for some indications, and is a constant threat to further reduce the options for treatment. Discovery and development of novel anthelmintic templates is strategically necessary to preserve the economic and health advantages now gained through chemotherapy. As the costs of development escalate, the question of how best to discover new drugs becomes paramount. Although random screening in infected animals led to the discovery of all currently available anthelmintics, cost constraints and a perception of diminishing returns require new approaches. Taking a cue from drug discovery programmes for human illnesses, we suggest that mechanism-based screening will provide the next generation of anthelmintic molecules. Critical to success in this venture will be the exploitation of the Caenorhabditis elegans genome through bioinformatics and genetic technologies. The greatest obstacle to success in this endeavour is the paucity of information available about the molecular physiology of helminths, making the choice of a discovery target a risky proposition.

The pharmacology of FMRFamide-related neuropeptides in nematodes: new opportunities for rational anthelmintic discovery?

The chemotherapeutic control of helminth parasites is compromised by the limited number of classes of anthelmintic drugs. Discovery of novel anthelmintics is impeded by the lack of novel screening technologies that overcome the difficulties inherent in screens based on whole organism toxicity. The development and implementation of mechanism-based screens for new anthelmintics offers great promise for the revitalization of antiparasitic drug discovery. However, mechanism-based screens must be based on a thorough understanding of the proteins or processes that offer the best chance for selective chemotherapeutic intervention. Basic research on the characterization of nematode FMRFamide-related peptides (FaRPs) has revealed that these peptides are ubiquitously distributed in helminths. Chemical identification of a number of nematode FaRPs has been achieved, and these peptides have potent and profound effects on the nematode neuromuscular system. Physiological processes mediated by nematode FaRPs (and other helminth neuropeptides) offer potential targets for the discovery of novel anthelmintics.

Feasibility of biolistic gene therapy in burns.

Skin is an especially attractive target for genetic manipulation because it is readily accessible and easily monitored for both the presence and the expression of inserted genes. This study was designed to assess the feasibility of particle mediated gene transfer to burned skin and to compare the transfection efficiency, anatomic distribution, and duration of transgene expression achievable in normal versus burned skin. Two days following scald injury of varying depths in 60 degrees C water (10 s: superficial partial; 20 s: deep partial; 40 s: full thickness) reporter gene (beta-galactosidase) constructs were delivered using a gene gun at various helium pressures (200-600 psi) to normal and burned skin. A time course study was performed to examine the kinetics of transgene expression. Animals received a superficial partial thickness burn and were sacrificed 12 h, 1, 3, 5, 7, 14, or 21 days after gene transfer. India Ink injection and immunohistochemistry were used to assess the depth of the scald injury. Transfection efficiency was measured in skin homogenates 24 h after gene transfer by morphometric and chemoluminescent assays. We found that the extent of tissue damage was directly related to the duration of heat source exposure. Reporter gene activity was significantly higher in superficial partial thickness burns compared to normal controls and gradually declined with increasing tissue injury. No activity was seen in the full thickness burn group. Beta-galactosidase activity reached a maximum level 12 h after gene transfer in both normal and superficial partial thickness burned skin with no levels seen after 5 days post-transfection. These findings indicate that particle-mediated gene transfer in thermally injured skin is feasible and may provide a means of introducing biologic agents into injured tissue capable of enhancing bacterial clearance and improving wound healing.

Hemodynamic correlates of outcome in patients undergoing orthotopic liver transplantation. Evidence for early postoperative myocardial depression.

OBJECTIVE: To describe the hemodynamic and oxygen transport patterns in survivors and nonsurvivors following liver transplantation (LT) and to assess their relationship to organ failure and mortality. DESIGN: Retrospective cohort. SETTING: Surgical ICU in a tertiary care university teaching hospital. PATIENTS: Consecutive series of 113 adults undergoing LT between 1984 and 1992. Patients were excluded if they died intraoperatively (n = 2), required retransplantation (n = 8), or their records were incomplete (n = 7). MEASUREMENTS AND MAIN RESULTS: Preoperative severity of illness was assessed by the acute physiology and chronic health evaluation (APACHE) II scoring system. Hemodynamic and oxygen transport variables were recorded immediately preoperatively and sequentially every 12 h during the first 2 postoperative days. Organ failures (pulmonary, renal, cardiovascular, hepatic, and central nervous system) were assessed for patients in the postoperative period. Patients were grouped as survivors (n = 82) or nonsurvivors (n = 14) with a mortality rate of 15%. Preoperative APACHE II scores were significantly lower in survivors compared with nonsurvivors (7 +/- 0 vs 11 +/- 2; p = 0.029). Both preoperatively and postoperatively, survivors sustained a relatively higher mean arterial pressure, stroke volume index, left ventricular stroke work index, cardiac index, and oxygen delivery as compared with nonsurvivors (p < 0.01). The postoperative decline in systemic blood flow that was seen in both groups was particularly prominent in nonsurvivors during the first 12 h following LT (p < 0.03). Nonsurvivors sustained an approximately fivefold increase in the rate of organ failure (p < 0.0001); all patients (n = 6) with 4 or more organ failures died. CONCLUSION: Nonsurvivors of LT have less cardiac reserve pretransplant; postoperatively, they demonstrate early myocardial depression and subsequently lower levels of cardiac index and oxygen delivery. Patients who develop these hemodynamic patterns are more prone to organ failure and death.

Use of an adjustable tourniquet to reverse cyanosis in the newborn pig.

BACKGROUND: Previous surgical models of cyanosis have been permanent. Because normal oxygenation was not restored in these models, it is unclear whether the metabolic changes produced by prolonged exposure to hypoxemia are irreversible. We therefore designed an experimental model of cyanosis that is reversible. METHODS: The left atrial appendage was anastomosed directly to the main pulmonary artery in 8 piglets, aged 2 to 4 weeks. RESULTS: The oxygen saturation fell from 95.3% +/- 0.8% to 72.4% +/- 3.9% (p < 0.001). A tourniquet was placed around the anastomosis to produce incremental changes in the level of cyanosis. Complete tourniquet occlusion resulted in obliteration of the right to left shunt, with return of systemic oxygen saturation to baseline levels. Systemic, left atrial, and pulmonary pressures did not change during the study. CONCLUSIONS: In this acute preparation, stable hemodynamic conditions were maintained despite substantial variations in systemic levels of oxygenation. Most important, this model allows reversal of cyanosis with the return of normal oxygenation. Application of this experimental design in a chronic model may help to determine whether the metabolic effects of prolonged hypoxemia are potentially reversible.

The nervous systems of helminths as targets for drugs.

Processes that critically differentiate parasitic helminths and their hosts are obvious candidates for chemotherapeutic intervention. The recognition that neurobiology distinguishes helminths from their vertebrate hosts is due in part to the fact that several efficacious anthelmintics, derived generally from empirical screening, have been found to act selectively on the neuromuscular system of these parasites. In addition, basic physiological and pharmacological research has revealed considerable differences in the ways in which helminths and their hosts transmit information in the nervous system and respond to it in innervated tissues. Unfortunately, most of these differences have yet to be exploited in chemotherapy. The topics for this review include an analysis of mechanistic aspects of the pharmacology of anthelmintics that act on neuromuscular systems and a consideration of the prospects for discovery of novel drugs that act on this system.

Cloning and characterization of cDNAs encoding beta-tubulin from Dirofilaria immitis and Onchocerca volvulus.

Beta-Tubulin is the target for the benzimidazole anthelmintics. Unfortunately, none of these drugs is clinically useful against adult filariae. However, beta-tubulin has been shown to be a target for antibody-based toxicity to Brugia pahangi. We cloned and characterized cDNAs encoding beta-tubulin from 2 filariae, Dirofilaria immitis and Onchocerca volvulus, to explore possible explanations for benzimidazole insensitivity among adult filariae and the likelihood that epitopes of beta-tubulin could be used as antigens for a broad-spectrum filarial vaccine. The proteins predicted by these cDNAs were almost identical to the beta-tubulin previously reported from B. pahangi but were less similar to a beta-tubulin cDNA from Onchocerca gibsoni. We cloned the genomic locus for the O. volvulus beta-tubulin cDNA and compared its organization to the reported genomic loci for beta-tubulin in B. pahangi and O. gibsoni. The comparison reinforces the conclusion that the published O. gibsoni gene is in a different family, possibly the beta2 family previously described in B. pahangi. The substitution of tyr for phe at position 200 of beta-tubulin is associated with benzimidazole resistance. All 4 filarial beta-tubulins are predicted to encode phe at this position, suggesting that filarial beta-tubulin is not inherently insensitive to the benzimidazoles. A monoclonal antibody that recognizes the COOH terminus of B. pahangi beta-tubulin is lethal to this parasite in culture. The COOH terminal region is the most variable among the different isotypes of beta-tubulin and distinguishes mammalian from nematode tubulins. This region is highly conserved in 3 of the filarial beta-tubulins.