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Attraction and feeding assays were conducted on blueberry maggot, Rhagoletis mendax Curran, to three protein baits, ammonium acetate, and sucrose. Flies fed significantly longer on concentrations of 25 and 50% SolBait than they did on any of the concentrations tested for Nu-Lure, AY50% (Mauri Yeast Australia), or a water control. The number of flies arriving at SolBait in an attraction assay was significantly higher than for Nu-Lure and a water control but was not different from AY50%. Flies fed less on aqueous solutions of 1 and 4% ammonium acetate, a known fruit fly attractant, than they did on either 0.25% ammonium acetate or water. Aqueous concentrations of 8, 16, and 32% sucrose elicited greater feeding responses from flies than either 4% sucrose or water. These findings suggest that SolBait is a superior protein bait based on attraction and feeding assays. Development of alternative baits should contain at least 8% sucrose, as a significant feeding stimulant, and some amount of ammonium acetate as an attractant. Future work should determine whether the feeding deterrence of ammonium acetate could be reduced or even eliminated in the presence of sucrose.

Aims: The aim of this study was to examine the perioperative nutritional status, body mass indices (BMI) and nutritional intakes of patients undergoing a modified D2 gastrectomy (preserving pancreas and spleen) for carcinoma to determine whether a relationship exists between the above and outcomes., Methods: Fifty consecutive patients [median age 71 years, 38 male] with gastric adenocarcinoma were studied prospectively., Results: Seven patients (14%) were obese (BMI > 30 kg/m2), 16 patients (32%) were overweight (BMI > 25 kg/m2), 21 patients (42%) were of normal weight (BMI 20-25 kg/m2), and six patients (12%) were underweight (BMI < 20 kg/m2). Operative morbidity was commoner in underweight patients (33%) when compared with overweight patients (17%, P = 0.391) and patients of normal weight (14%, P = 0.289). Fatal complications, however (two patients, 4%) were confined to overweight patients (P = 0.118). Preoperative serum albumin levels were significantly higher in overweight patients (43 g/dl) compared to underweight patients (34.5 g/dl; P = 0.003), though no correlation was found between patients' serum albumin levels and postoperative morbidity (r = -0.023, P = 0.877). Overweight patients were significantly less likely to achieve their protein requirements postoperatively than underweight patients (P = 0.037). Early enteral feeding contributed to 56% of the median energy requirements and 45% of the median protein requirements on the seventh postoperative day., Conclusion: BMI alone is a poor indicator of outcomes after modified D2 gastrectomy for carcinoma. The role of early enteral nutrition in patients undergoing gastrectomy for cancer deserves further evaluation., (Copyright 2003 Elsevier Ltd.)

Background: Although acute complications necessitating emergency hospital admission are well documented in patients with carcinoma of the colon, comparable data for patients with gastric carcinoma is thin. The aim of this study, therefore, was to examine the outcomes of patients presenting to hospital as acute admissions with emergency complications of previously undiagnosed gastric cancer., Methods: Three hundred consecutive patients with gastric adenocarcinoma were studied prospectively, and subdivided into two groups according to whether the patients were referred as acute emergencies ( n = 116) or as outpatients ( n = 184)., Results: The commonest emergency complications were: abdominal pain (57%), vomiting (41%), gastrointestinal bleeding (37%), dysphagia (26%), and a palpable mass (18%). Stages of disease were significantly more advanced in patients presenting acutely (I : II : III : IV = 7 : 11 : 27 : 71) compared with patients referred via outpatients (20 : 23 : 50 : 91, Chi(2) = 3.955; DF, 1; P = 0.047). R0 gastrectomy was significantly less likely after acute presentation (23 patients; 20%) compared with patients referred via outpatients (70 patients; 38%; Chi(2) = 11.037; DF, 1; P = 0.001). Cumulative 5-year survival for patients referred acutely was 9%, compared with 22% after outpatient referral (Chi(2) = 9.11; DF, 1; P = 0.0025). Multivariate analysis revealed two factors to be significantly and independently associated with durations of survival: stage of disease (hazard ratio [HR], 1.742; 95% confidence interval [CI], 1.493-2.034; P = 0.0001) and presentation with acute complications (HR, 1.561; 95% CI, 1.151-2.117; P = 0.004)., Conclusion: Emergency complications of gastric cancer are a significant and independent prognostic marker of poor outcome.

African trypanosomes, such as Trypanosoma brucei, are protozoan parasites of mammals that were first described over 100 hundred years ago. They have long been the subjects of biological investigation, which has yielded insights into a number of fundamental, as well as novel, cellular processes in all organisms. In the last decade or so, genetic manipulation of trypanosomes has become possible through DNA transformation, allowing yet more detailed analysis of the biology of the parasite. One facet of this is that DNA transformation has itself been used as an assay for recombination and will undoubtedly lead to further genetic approaches to examine this process. Here we describe protocols for DNA transformation of Trypanosoma brucei, including two different life cycle stages and two different strain types that are distinguished by morphological and developmental criteria. We consider the application of transformation to recombination, as well as the uses of transforming the different life cycle stages and strain types.

In a field study in Hawaii, color-marked protein-deprived and protein-fed female melon flies, Bactrocera cucurbitae Coquillett, were released within canopies of unsprayed sorghum plants (a nonhost of melon flies) outside of a border area of unsprayed or bait-sprayed sorghum plants or open space that surrounded cucumbers, a favored host of melon flies. Application of bait spray to sorghum or sugarcane surrounding host plants of melon flies is a common practice for melon fly control in Hawaii. GF-120 Fruit Fly Bait spray proved very effective in preventing protein-deprived females from alighting on cucumbers (23% of released females were observed dead on bait-sprayed sorghum; 0% were observed alive on cucumbers), but proved less effective in suppressing protein-fed females (14% of released females were observed dead on bait-sprayed sorghum; 11% were observed alive on cucumbers). No females were found dead on unsprayed sorghum. Compared with open space surrounding cucumbers, the presence of unsprayed sorghum as surrounding border area neither significantly enhanced nor significantly inhibited the ability of either type of female with respect to finding cucumbers. Greenhouse cage assays revealed that compared with droplets of water, droplets of GF-120 Fruit Fly Bait spray were highly attractive to protein-deprived females within 1 h of bait spray application to sorghum, but lost about half of their attractiveness within 5 h and all of it within 24 h under the dry greenhouse conditions used for maintaining baited-sprayed sorghum plants in these assays. Laboratory cup assays showed that bait spray droplets remained highly toxic to protein-deprived females 24 h after application, but lost nearly half of their toxicity within 4 d under laboratory exposure and nearly all of it after approximately 8 mm of rainfall. Combined findings suggest that application of GF-120 Fruit Fly Bait spray to nonhost plants for melon fly control either be made often enough to overcome loss of attractiveness of bait spray droplets to females or that bait spray be applied to nonhost plants that are themselves attractive to the females.

The sterile insect technique (SIT) is used to control wild Mediterranean fruit fly introductions in California and Florida in the U.S. In the past, bait sprays containing malathion proved invaluable in treating new outbreaks or large populations before the use of SIT. Recently, a spinosad protein bait spray, GF-120, has been developed as a possible alternative to malathion, the standard insecticide in protein bait sprays. In this study, protein-deficient and protein-fed Vienna-7 (sterile, mass-reared, "male-only" strain) flies and wild males and females were evaluated to determine the effectiveness of the GF-120 protein bait containing spinosad with respect to bait attraction, feeding, and toxicology. There were no effects of diet or fly type on feeding duration in small laboratory cages. Wild flies, however, registered more feeding events than Vienna-7 males. Flies that fed longer on fresh bait died faster. Protein-deficient flies were more active and found the bait more often than protein-fed flies. Data suggest that adding protein to the diet of SIT flies may decrease their response to baits, therefore, reduce mortality, and thus, allow the concurrent use of SIT and bait sprays in a management or eradication program.

Irradiation of puparia in Mediterranean fruit fly, Ceratitis capitata (Wiedemann), sterile insect release programs can negatively affect adult fly performance. Emergence, survivorship, lure attraction, and mating competition tests were performed on irradiated and unirradiated Mediterranean fruit flies in Hawaii. Unirradiated flies of the Vienna-7 (tsl) strain had higher emergence, flight ability, and survivorship compared with irradiated flies. In general, unirradiated flies were more responsive to trimedlure, but this effect was not consistent for all strains at every age. Laboratory strains, of both unirradiated and irradiated flies, responded to trimedlure at a younger age than wild flies, which may be a result of inadvertent selection for decreased development time in laboratory-reared flies. Mating competition tests with irradiated and unirradiated flies showed no significant differences. Costs associated with the irradiation process and the development of alternative control techniques are discussed.

Thrombocytopenia is a common occurrence in moderate to severe crotaline envenomation. The exact mechanism by which rattlesnake venom leads to thrombocytopenia is unclear, but aggressive treatment with crotaline-specific antivenom often leads to resolution of this disorder. Crotalinae Polyvalent Immune Fab (CroFab(TM), Protherics Inc., Nashville, TN) (crotaline Fab) is now available for the treatment of symptomatic rattlesnake envenomation. Although recurrence of thrombocytopenia has been reported in patients after envenomation treated with crotaline Fab, cases refractory to this therapy have not been described. We report a case of severe crotaline envenomation that appears to have exhibited two separate episodes of thrombocytopenia, only one of which responded to antivenom. The second, later phase was refractory to both crotaline Fab as well as traditional Antivenin (Crotalinae) Polyvalent (Wyeth-Ayerst Pharmaceuticals, Philadelphia, PA) (ACP). By reviewing the literature regarding venom-induced thrombocytopenia, we attempt to explain this "biphasic" phenomenon and the inability of crotaline Fab to reverse this toxic effect.

Background: The aim of this study was to examine the accuracy of laparoscopy is staging patients with gastric cancer in comparison with preoperative computed tomography (CT) examination., Methods: One hundred patients out of a consecutive series of 258 patients with gastric adenocarcinoma underwent a preoperative staging CT followed by a staging laparoscopy. The strengths of the agreement between the CT stage, the laparoscopic stage, and the final histopathological stage were determined by the weighted Kappa statistic (Kw)., Results: The strengths of agreement between the CT stage and the final histopathological stage were Kw = 0.336 (95% confidence interval [CI]; 0.172-0.5; P = 0.0001) for T stage and 0.378 (95% CI; 0.226-0.53; P = 0.0001) for M stage, compared with 0.455 (95% CI; 0.301-0.609; P = 0.0001) and 0.73 (95% CI; 0.596-0.864; P = 0.0001) for the laparoscopic T and M stages, respectively. Unsuspected metastases that were not detected by CT, were found in 21 patients at laparoscopy, all of whom had T3 or T4 locally advanced tumors evident on CT., Conclusions: Preoperative laparoscopic staging of gastric cancer is indicated for potential surgical candidates with locally advanced disease in the absence of metastases on CT. The aim of this study was to examine the accuracy of laparoscopy in staging patients with gastric cancer in comparison with preoperative computed tomography (CT) examination.

The relationship between operative time, the intermediate equivalent value (IEV) and the complexity of common general surgical operations was examined. Correlation was found between the BUPA schedule values for procedures categorized as intermediate and major, but complex major vascular reconstruction and oesophagogastric resection for cancer occupied significantly more theatre time than the four intermediate equivalents allocated by the Collins or BUPA schedule. Moreover, anaesthetic preparation time for complex major surgery in the latter surgical subspecialities contributed at least one further intermediate value. Re-evaluation of the ideal IEV weighting of all surgical operations including anaesthetic input from larger similar audits would allow more accurate audits of surgeons' work-load, and also facilitate transparent intensive management of operating theatre resource.

Background: To determine the role of body mass index (BMI) in a Western population on outcomes after modified D2 gastrectomy (preserving pancreas and spleen where possible) for gastric cancer., Methods: Eighty-four consecutive patients undergoing an R0 modified D2 gastrectomy for gastric cancer were studied prospectively. Male patients with a BMI of greater than 24.7 kgm(-2) and female patients with a BMI of greater than 22.6 kgm(-2) were classified as overweight and compared with control patients with BMIs below these reference values., Results: Thirty-eight of the patients (45%) were classified as overweight. The median BMI of the overweight patients was 27.0 kgm(-2) (range, 22.7-34.7 kgm(-2); 27 males) compared with 21.2 kgm(-2) (range, 15.2-24.7 kgm(-2), 31 males) for control patients. Operative morbidity and mortality were 26% and 7.9% in overweight patients compared with 22% and 6.5% in control patients (morbidity, chi(2) = 0.240; df = 1; P = 0.624; mortality, chi(2) = 0.059; df = 1; P = 0.808). Cumulative survival at 5 years was 52% for overweight patients compared with 55% for control patients (chi(2) = 0.15; df = 1; P = 0.7002). In a multivariate analysis, the number of lymph node metastases (hazard ratio, 1.441; 95% confidence interval [CI], 1.159-1.723; P = 0.009) and splenectomy (hazard ratio, 12.111; 95% CI, 9.645-14.577; P = 0.043) were independently associated with the duration of survival., Conclusion: High BMIs were not associated with increased operative risk, and longterm outcomes were similar in the two groups after modified D2 gastrectomy.

Contingency genes are common in pathogenic microbes and enable, through pre-emptive mutational events, rapid, clonal switches in phenotype that are conducive to survival and proliferation in hosts. Antigenic variation, which is a highly successful survival strategy employed by eubacterial and eukaryotic pathogens, involves large repertoires of distinct contingency genes that are expressed differentially, enabling evasion of host acquired immunity. Most, but not all, antigenic variation systems make extensive use of subtelomeres. Study of model systems has shown that subtelomeres have unusual properties, including reversible silencing of genes mediated by proteins binding to the telomere, and engagement in ectopic recombination with other subtelomeres. There is a general theory that subtelomeric location confers a capacity for gene diversification through such recombination, although experimental evidence is that there is no increased mitotic recombination at such loci and that sequence homogenisation occurs. Possible benefits of subtelomeric location for pathogen contingency systems are reversible gene silencing, which could contribute to systems for gene switching and mutually exclusive expression, and ectopic recombination, leading to gene family diversification. We examine, in several antigenic variation systems, what possible benefits apply.

The tsetse fly transmitted salivarian trypanosome, Trypanosoma congolense of the subgenus Nanomonas, is the most significant of the trypanosomes with respect to the pathology of livestock in sub-Saharan Africa. Unlike the related trypanosome Trypanosoma brucei of the subgenus Trypanozoon, the major surface molecules of the insect stages of T. congolense are poorly characterized. Here, we describe the purification and structural characterization of the glutamic acid and alanine-rich protein, one of the major surface glycoproteins of T. congolense procyclic and epimastigote forms. The glycoprotein is a glycosylphosphatidylinositol-anchored molecule with a galactosylated glycosylphosphatidylinositol anchor containing an sn-1-stearoyl-2-l-3-HPO(4)-1-(2-O-acyl)-d-myo-inositol phospholipid moiety. The 21.6-kDa polypeptide component carries two large mannose- and galactose-containing oligosaccharides linked to threonine residues via phosphodiester linkages. Mass spectrometric analyses of tryptic digests suggest that several or all of the closely related glutamic acid and alanine-rich protein genes are expressed simultaneously in a T. congolense population growing in vitro.

The trypanosome variant surface glycoprotein (VSG) is first expressed during differentiation to the infective, metacyclic population in tsetse fly salivary glands. Unlike the VSG genes expressed by bloodstream form trypanosomes, metacyclic VSGs (MVSGs) have their own promoters. The scarcity of metacyclic cells has meant that only indirect approaches have been used to study these promoters, and not even their identities have been agreed on. Here, we isolated trypanosomes by dissection from salivary glands and used an approach involving 5' rapid amplification of cDNA ends to identify the transcription start site of three MVSGs. This shows that the authentic start site is that proposed for the MVAT series of MVSGs (K. S. Kim and J. E. Donelson, J. Biol. Chem. 272:24637-24645, 1997). In the more readily accessible procyclic trypanosome stage, where MVSGs are normally silent, we used reporter gene assays and linker scanning analysis to confirm that the 67 bp upstream of the start site is a promoter. This is confirmed further by accurate initiation in a homologous in vitro transcription system. We show also that MVSG promoters become derepressed when tested outwith their endogenous, subtelomeric loci. The MVSG promoters are only loosely conserved with bloodstream VSG promoters, and our detailed analysis of the 1.63 MVSG promoter reveals that its activity depends on the start site itself and sequences 26 to 49 bp and 56 to 60 bp upstream. These are longer than those necessary for the bloodstream promoter.

Aim: The aim of this study was to compare the accuracy of spiral computed tomography (CT) performed by a specialist radiologist within a multi disciplinary team (MDT) framework with that of radiologists working outside of this framework., Materials and Methods: One hundred and ten patients [median age 70 (35-86)yr, 71m] underwent either a preoperative CT performed by the MDT specialist consultant radiologist (n=60) or a CT performed by one of 13 other consultant radiologists (n=50). The strength of the agreement between the CT stage and the histopathological stage was determined by the weighted Kappa statistic (Kw)., Results: Sensitivity for T, N and M stage were 64%, 65% and 25% for MDT specialist CT, compared with 24%, 24% and 5% for control CT. Specificity for T, N and M stage were 68%, 59% and 95% for MDT specialist CT compared with 79%, 94% and 93% for control CT. Kw for T, N and M stage were 0.314, 0.350 and 0.255 for MDT specialist CT compared with 0.088, 0.102 and -0.019 for control CT. Unsuspected metastases were found in 12 patients staged by MDT specialist CT compared with 19 patients staged by control CT (Chi2=4.366, df=1,p =0.037)., Conclusion: Improved patient selection for surgery should maximize use of limited resource.

At Mediterranean fruit fly rearing centers, genetic sexing strains such as the male-only temperature sensitive lethal (tsl) strains, are replacing many of the older bisexual strains, such as Maui-93. Three tests were performed to compare the performance of the Vienna-4 (which carries tsl) and Maui-93 strains. Using standard laboratory quality control tests, the Maui-93 strain had higher percent fliers and a lower percentage deformed and partially eclosed flies. Analyses of data from field mark-recapture tests with Jackson traps failed to find significant differences in recapture numbers between the two strains. In field cage survivorship tests, the Vienna-4 strain showed significantly lower mortality in comparison to the Maui-93 strain. These results suggest that although the quality of Vienna-4 flies is lower than Maui-93 at the time of adult emergence, it is comparable with or better than Maui-93 several days later, because weaker flies have been eliminated from the cohort. The benefits of releasing only sterile males, instead of both males and females, was not factored into the quality assessment in this study, but would be an additional benefit in using a strain that carries a tsl mutation.

OxyContin (oxycodone hydrochloride controlled release) is a long-acting preparation of oxycodone that is used as an opioid analgesic to treat chronic pain conditions. We report a patient who ingested a massive quantity of OxyContin and had altered mental status, noncardiogenic pulmonary edema, and hypoventilation that proved refractory to naloxone administration. She required mechanical ventilation for 3 days before recovering completely. The severity and length of poisoning was likely related both to the quantity and formulation of the oxycodone ingested.

African trypanosomes are unicellular parasites that use DNA recombination to evade the mammalian immune response. They do this in a process called antigenic variation, in which the parasites periodically switch the expression of VSG genes that encode distinct Variant Surface Glycoprotein coats. Recombination is used to move new VSG genes into specialised bloodstream VSG transcription sites. Genetic and molecular evidence has suggested that antigenic variation uses homologous recombination, but the detailed reaction pathways are not understood. In this study, we examine the recombination pathways used by trypanosomes to integrate transformed DNA into their genome, and show that they possess at least two pathways of homologous recombination. The primary mechanism is dependent upon RAD51, but a subsidiary pathway exists that is RAD51-independent. Both pathways contribute to antigenic variation. We show that the RAD51-independent pathway is capable of recombining DNA substrates with very short lengths of sequence homology and in some cases aberrant recombination reactions can be detected using such microhomologies.

We demonstrate, by gene deletion analysis, that Mre11 has a critical role in maintaining genomic integrity in Trypanosoma brucei. mre11(-/-) null mutant strains exhibited retarded growth but no delay or disruption of cell cycle progression. They showed also a weak hyporecombination phenotype and the accumulation of gross chromosomal rearrangements, which did not involve sequence translocation, telomere loss, or formation of new telomeres. The trypanosome mre11(-/-) strains were hypersensitive to phleomycin, a mutagen causing DNA double strand breaks (DSBs) but, in contrast to mre11(-/-) null mutants in other organisms and T. brucei rad51(-/-) null mutants, displayed no hypersensitivity to methyl methanesulfonate, which causes point mutations and DSBs. Mre11 therefore is important for the repair of chromosomal damage and DSBs in trypanosomes, although in this organism the intersection of repair pathways appears to differ from that in other organisms. Mre11 inactivation appears not to affect VSG gene switching during antigenic variation of a laboratory strain, which is perhaps surprising given the importance of homologous recombination during this process.

Trypanosome antigenic variation, involving differential expression of variant surface glycoprotein (VSG) genes, has a strong association with telomeres and with DNA recombination. All expressed VSGs are telomeric, and differential activation involves recombination into the telomeric environment or silencing/activation of subtelomeric promoters. A number of pathogen contingency gene systems associated with immune evasion involve telomeric loci, which has prompted speculation that chromosome ends provide conditions conducive for the operation of rapid gene switching mechanisms. Ku is a protein associated with eukaryotic telomeres that is directly involved in DNA recombination and in gene silencing. We have tested the hypothesis that Ku in trypanosomes is centrally involved in differential VSG expression. We show, via the generation of null mutants, that trypanosome Ku is closely involved in telomere length maintenance, more so for a transcriptionally active than an inactive telomere, but exhibits no detectable influence on DNA double strand break repair. The absence of Ku and the consequent great shortening of telomeres had no detectable influence either on the rate of VSG switching or on the silencing of the telomeric promoters of the VSG subset that is expressed in the tsetse fly.

Background: The best reported long-term survival following surgery for gastric cancer is from centers performing radical D2 gastrectomy. Yet comparative studies from European centers report higher rates of postoperative complications following D2 gastrectomy than after the less radical D1 gastrectomy, without any benefit in survival. We aimed to compare the outcome after modified D2 gastrectomy (preserving spleen and pancreas where possible), performed by specialist surgeons, with that after conventional D1 gastrectomy performed by general surgeons for gastric cancer in a large United Kingdom cancer unit., Methods: Two groups of patients were studied: a historical control group of 245 consecutive patients with gastric cancer, of whom 50 underwent a potentially curative D1 resection (median age, 69 years; 35 males) was compared with 200 consecutive patients, 72 of whom underwent a potentially curative D2 resection (median age, 71 years; 47 males)., Results: Among the 122 patients judged to have curable cancers, patients who underwent a D2 gastrectomy had lower operative mortality (8.3% vs 12%; chi(2) = 0.48; P = 0.50) and experienced fewer complications (28% vs 36%; chi(2) = 0.93; P = 0.35) than patients who underwent a D1 gastrectomy. Cumulative survival at 5 years was 56% after D2 resections, compared with 11% after D1 resections ( P < 0.00001). In a multivariate analysis, only the stage of disease (stage I, hazard ratio [HR], 0.068; P = 0.0001; stage II, HR, 0.165; P = 0.001; stage III, HR, 0.428; P = 0.053) and the level of lymphadenectomy (HR, 0.383; P = 0.00001) were independently associated with the duration of survival., Conclusion: Modified D2 gastrectomy without pancreatico-splenectomy, performed by specialist surgeons, can improve survival after R0 resections without increasing operative morbidity and mortality, when compared with D1 gastrectomy performed by general surgeons.

African trypanosomes can spend a long time in the blood of their mammalian host, where they are exposed to the immune system and are thought to take advantage of it to modulate their own numbers. Their major immunogenic protein is the variant surface glycoprotein (VSG), the gene for which must be in one of the 20--40 specialized telomeric expression sites in order to be transcribed. Trypanosomes escape antibody-mediated destruction through periodic changes of the expressed VSG gene from a repertoire of approximately 1000. How do trypanosomes exclusively express only one VSG and how do they switch between them?

The aim of this study was to assess the volume of work generated by one consultant (out of a surgical unit of seven) managing all the upper gastrointestinal malignancy in a district general hospital serving a population of 480,000. A 3-year period was prospectively audited and the volume of out-patient and in-patient workload assessed with particular reference to resource management and levels of surgical staffing. Oesophagogastric cancer accounted for a mean of 61 new cases per year, representing 5.3% of new patient referrals. Assuming that a complex major operation for an oesophagogastric cancer equates to four intermediate equivalent values (IEVs), then this translated to a mean operative workload of 186 IEVs per year, representing 16.7% of the total elective operative workload of 1140 IEVs per year. Thus, all the oesophagogastric cancer was managed by a single firm as a speciality in a district general hospital over this 3-year period, though a relatively small proportion of new patients with oesophagogastric cancer translated into a significantly greater burden on the resources of consultant manpower and operating theatre time.

We have characterised the organisation of genes encoding the glutamate and alanine rich protein (GARP) surface coat of the procyclic and epimastigote stages of Trypanosoma congolense in the tsetse fly. The GARP genes are arranged at two, possibly physically linked, loci, one of which exhibits allelic variation. One locus contains a single GARP gene, whilst both alleles of the other have a large tandem array of polycistronically transcribed GARP genes. Sequence analysis has revealed that there are very few coding differences between different GARP genes. A sequence related to the Trypanosoma brucei expression site associated gene 4 (encoding a transmembrane protein with a cytoplasmic adenylate cyclase domain) has been identified within a region at the downstream flank of one locus. There is no evidence that, within the single trypanosome, GARP genes are as diverse as the procyclin genes that encode a corresponding coat in T. brucei.

Antigenic variation is an immune evasion strategy used by African trypanosomes, in which the parasites periodically switch the expression of VSG genes that encode their protective variant surface glycoprotein coat. Two main routes exist for VSG switching: changing the transcriptional status between an active and an inactive copy of the site of VSG expression, called the bloodstream VSG expression site, or recombination reactions that move silent VSGs or VSG copies into the actively transcribed expression site. Nothing is known about the proteins that control and catalyze these switching reactions. This study describes the cloning of a trypanosome gene encoding RAD51, an enzyme involved in DNA break repair and genetic exchange, and analysis of the role of the enzyme in antigenic variation. Trypanosomes genetically inactivated in the RAD51 gene were shown to be viable, and had phenotypes consistent with lacking functional expression of an enzyme of homologous recombination. The mutants had an impaired ability to undergo VSG switching, and it appeared that both recombinational and transcriptional switching reactions were down-regulated, indicating that RAD51 either catalyzes or regulates antigenic variation. Switching events were still detectable, however, so it appears that trypanosome factors other than RAD51 can also provide for antigenic variation.

African trypanosomes first express the variant surface glycoprotein (VSG) at the metacyclic stage in the tsetse fly vector, in preparation for transfer into the mammal. Metacyclic (M)VSGs comprise a specific VSG repertoire subset and their expression is regulated differently from that of bloodstream VSGs, involving exclusively transcriptional regulation during the life cycle. To identify basic structural and functional features that may be common to MVSG telomeric transcription units, we have characterized the anatomy and transcription of the telomere containing the ILTat 1.61 MVSG gene. This telomere contains pseudogenes of the ESAG1 and ESAG9 families found in bloodstream VSG transcription units. The 1.61 MVSG occupies a monocistronic transcription unit and is transcriptionally controlled through the life cycle. The 1.61, and also the 1.22, MVSG transcription initiation site sequences resemble eukaryotic initiator elements. Sequence comparison reveals that four out of five characterized MVSG expression sites have a conserved region 2.0-4.7 kb long upstream of the MVSG. In some cases, this region contains not only the transcription initiation site that we have observed to be active in fly-transmitted trypanosomes but also, upstream, another sequence, described elsewhere as a 'putative promoter' for the MVAT set of M/VSGs (Nagoshi YL, Alarcon CM, Donelson JE. A monocistronic transcript for a trypanosome variant surface glycoprotein, Mol Biochem Parasitol 1995;72:33-45). In fly-transmitted trypanosomes, the latter element is transcriptionally silent. Our analysis of the structure of MVSG telomeres suggests that metacyclic expression sites arose from bloodstream expression sites.

Transformation of the free-living nematode Caenorhabditis elegans with promoter/reporter gene constructs is a very powerful technique to examine and dissect gene regulatory mechanisms. No such transformation system is available for parasitic nematode species. We have exploited C. elegans as a heterologous transformation system to examine activity and specificity of parasitic nematode gene promoters. Using three different parasite promoter/lac Z reporter constructs strict tissue-specific expression is observed. Upstream sequences of the Haemonchus contortus gut pepsinogen gene pep-1 and cysteine protease gene AC-2 direct expression exclusively in gut cells, while promoter sequence of the Ostertagia circumcincta cuticular collagen gene colost-1 directs hypodermal-specific expression. Mutation analysis indicates that AC-2 promoter function is dependent on a GATA-like motif close to the translation start site, similar to our findings with the C. elegans cpr-1 cysteine protease gene. While the spatial expression of these parasite promoters in C. elegans correlates with their expression in the parasite, the exact timing of expression does not. This suggests that regulatory mechanisms influencing the timing of expression may have evolved more rapidly than those controlling spatial expression of structural genes.

A number of mechanisms have been described by which African trypanosomes undergo the genetic switches that differentially activate their variant surface glycoprotein genes (VSGs) and bring about antigenic variation. These mechanisms have been observed mainly in trypanosome lines adapted, by rapid syringe passaging, to laboratory conditions. Such "monomorphic" lines, which routinely yield only the proliferative bloodstream form and do not develop through their life cycle, have VSG switch rates up to 4 or 5 orders of magnitude lower than those of nonadapted lines. We have proposed that nonadapted, or pleomorphic, trypanosomes normally have an active VSG switch mechanism, involving gene duplication, that is depressed, or from which a component is absent, in monomorphic lines. We have characterized 88 trypanosome clones from the first two relapse peaks of a single rabbit infection with pleomorphic trypanosomes and shown that they represent 11 different variable antigen types (VATs). The pattern of appearance in the first relapse peak was generally reproducible in three more rabbit infections. Nine of these VATs had activated VSGs by gene duplication, the tenth possibly also had done so, and only one had activated a VSG by the transcriptional switch mechanism that predominates in monomorphic lines. At least 10 of the donor genes have telomeric silent copies, and many reside on minichromosomes. It appears that trypanosome antigenic variation is dominated by one, relatively highly active, mechanism rather than by the plethora of pathways described before.

We have identified a gene encoding a new member of the Caenorhabditis elegans GATA transcription factor family, elt-3. The predicted ELT-3 polypeptide contains a single GATA-type zinc finger (C-X2-C-X17-C-X2-C) along with a conserved adjacent basic region. elt-3 mRNA is present in all stages of C. elegans development but is most abundant in embryos. Reporter gene analysis and antibody staining show that elt-3 is first expressed in the dorsal and ventral hypodermal cells, and in hypodermal cells of the head and tail, immediately after the final embryonic cell division that gives rise to these cells. No expression is seen in the lateral hypodermal (seam) cells. elt-3 expression is maintained at a constant level in the epidermis until the 2(1/2)-fold stage of development, after which reporter gene expression declines to a low level and endogenous protein can no longer be detected by specific antibody. A second phase of elt-3 expression in cells immediately anterior and posterior to the gut begins in pretzel-stage embryos. elt-1 and lin-26 are two genes known to be important in specification and maintenance of hypodermal cell fates. We have found that elt-1 is required for the formation of most, but not all, elt-3-expressing cells. In contrast, lin-26 function does not appear necessary for elt-3 expression. Finally, we have characterised the candidate homologue of elt-3 in the nematode Caenorhabditis briggsae. Many features of the elt-3 genomic and transcript structure are conserved between the two species, suggesting that elt-3 is likely to perform an evolutionarily significant function during development., (Copyright 1999 Academic Press.)

In the mammalian bloodstream, African trypanosomes express the variant surface glycoprotein (VSG), continual switching of which allows evasion of the host immune response. Bloodstream VSG genes are transcribed from polycistronic bloodstream expression sites with promoters which are located 45-60 kb upstream. These promoters are not exclusively stage-regulated, being active in the insect midgut stage where VSG is not expressed. However, the metacyclic VSG (M-VSG) genes, a small subset activated when VSG synthesis begins in the metacyclic stage in the tsetse fly salivary glands, are transcriptionally activated specifically in that stage from promoters <3 kb upstream. Using deletion mapping and transient transfection, we show that the entire 1.22 M-VSG gene promoter region (171 bp) is required for full activity in metacyclic-derived trypanosomes. However, a subsidiary, bloodstream stage-specific activity is present in its 5' half which directs transcription initiation very close to the initiation site used in metacyclic-derived trypanosomes. Our results imply that the M-VSG gene promoter is longer and more complex than other VSG gene promoters.

African trypanosomes evade the mammalian host immune response by antigenic variation, the continual switching of their variant surface glycoprotein (VSG) coat. VSG is first expressed at the metacyclic stage in the tsetse fly as a preadaptation to life in the mammalian bloodstream. In the metacyclic stage, a specific subset (<28; 1 to 2%) of VSG genes, located at the telomeres of the largest trypanosome chromosomes, are activated by a system very different from that used for bloodstream VSG genes. Previously we showed that a metacyclic VSG (M-VSG) gene promoter was subject to life cycle stage-specific control of transcription initiation, a situation unique in Kinetoplastida, where all other genes are regulated, at least partly, posttranscriptionally (S. V. Graham and J. D. Barry, Mol. Cell. Biol. 15:5945-5956, 1985). However, while nuclear run-on analysis had shown that the ILTat 1.22 M-VSG gene promoter was transcriptionally silent in bloodstream trypanosomes, it was highly active when tested in bloodstream-form transient transfection. Reasoning that chromosomal context may contribute to repression of M-VSG gene expression, here we have integrated the 1.22 promoter, linked to a chloramphenicol acetyltransferase (CAT) reporter gene, back into its endogenous telomere or into a chromosomal internal position, the nontranscribed spacer region of ribosomal DNA, in both bloodstream and procyclic trypanosomes. Northern blot analysis and CAT activity assays show that in the bloodstream, the promoter is transcriptionally inactive at the telomere but highly active at the chromosome-internal position. In contrast, it is inactive in both locations in procyclic trypanosomes. Both promoter sequence and chromosomal location are implicated in life cycle stage-specific transcriptional regulation of M-VSG gene expression.

As the metacyclic trypanosome stage develops in the tsetse fly salivary glands, it initiates expression of variant surface glycoproteins (VSGs) and does so by each cell activating, at random, one from a small subset of metacyclic VSG (M-VSG) genes. Whereas differential activation of individual VSG genes in the bloodstream occurs as a function of time, to evade waves of antibody, it is believed that the aim in the metacyclic stage is simultaneously to generate population diversity. M-VSG genes are activated in their telomeric loci and belong to monocistronic transcription units, unlike all other known trypanosome protein-coding genes, which appear to be transcribed polycistronically. The promoters of these metacyclic expression sites (M-ESs) have the unique property, in this organism, of being switched on and off in a life-cycle stage specific pattern. We have found that the 1.22 M-ES promoter is regulated according to life cycle stage, differential control being exerted through different elements of the promoter and under the influence of its genomic locus. We have characterized in detail the telomeres containing the 1.22 and 1.61 M-ESs. Upstream of the M-ES is a possibly haploid, non-transcribed region with some degenerate sequences homologous with expression site associated genes (ESAGs) that occur in bloodstream VSG expression sites. Further upstream (respectively, 22 and 13 kb upstream of the 1.22 and 1.61 VSG genes) are alpha-amanitin sensitive transcription units that may be polycistrons and are transcribed in all examined life cycle stages. They contain a number of genes. The differences between metacyclic and bloodstream ESs may have important consequences for life cycle regulation, genetic stability, phenotype complexity and adaptability of the metacyclic stage as it infects different host species.

Approximately 53,000 serologic tests and viral isolation studies were performed on 1,700 nonhuman primate specimens for evidence of past and/or current viral infection. Information, other than the requested test, generally was not provided with the specimen. This lack of information does not permit any attempt at interpretation of results. Requested testing included a large number of diverse viral agents in approximately 40 primate species. The resulting data are in keeping with those of previous studies and offer an insight into the needs of colony management, as well as some general information on the overall frequency of infection with the indicated viruses. Inasmuch as the results represent testing of single specimens, they are not to be construed as "diagnostic," and simply indicate past infection as represented by the presence of antibody in the test animal. Viral isolation results are listed, and the number of positive results versus the number of animals tested emphasizes the limitations of the procedure. Investigations such as these continue to assist in the maintenance of healthy nonhuman primate colonies. This information also supports continued use of nonhuman primates for research in human viral infections and may be helpful in terms of animal selection for use in xenotransplants.

The large number of genes involved in antigenic variation in African trypanosomes has been the focus of a wide literature that describes an almost bewildering array of mechanisms for their differential activation. To the outsider searching for an underlying strategy for antigenic variation, this can appear as a rather disordered and confusing picture. Here, David Barry argues that an understanding of which mechanisms are significant, which ones are primarily inconsequential and which ones perhaps even arise from overdependence on laboratory models, might be achieved by turning attention to trypanosomes that have not undergone adaptation in laboratory conditions. Application of such an approach has led to a proposal for a main mechanism for antigenic variation.

The Caenorhabditis elegans cuticle collagens are encoded by a multigene family of between 50 and 100 members and are the major component of the nematode cuticular exoskeleton. They are synthesized in the hypodermis prior to secretion and incorporation into the cuticle and exhibit complex patterns of spatial and temporal expression. We have investigated the cis regulatory requirements for tissue- and stage-specific expression of the cuticle collagen gene dpy-7 and have identified a compact regulatory element which is sufficient to specify hypodermal cell reporter gene expression. This element appears to be a true tissue-specific promoter element, since it encompasses the dpy-7 transcription initiation sites and functions in an orientation-dependent manner. We have also shown, by interspecies transformation experiments, that the dpy-7 cis regulatory elements are functionally conserved between C. elegans and C. briggsae, and comparative sequence analysis supports the importance of the regulatory sequence that we have identified by reporter gene analysis. All of our data suggest that the spatial expression of the dpy-7 cuticle collagen gene is established essentially by a small tissue-specific promoter element and does not require upstream activator or repressor elements. In addition, we have found the DPY-7 polypeptide is very highly conserved between the two species and that the C. briggsae polypeptide can function appropriately within the C. elegans cuticle. This finding suggests a remarkably high level of conservation of individual cuticle components, and their interactions, between these two nematode species.

The major surface antigen of insect stage (procyclic and epimastigote form) Trypanosoma brucei is termed procyclin or procyclic acidic repetitive protein (PARP). Procyclin/PARP is not expressed in bloodstream form parasites, which are coated instead with the variant surface glycoprotein (VSG). Although procyclin/PARP protein is not present and the mRNA is barely detectable, procyclin/PARP genes are transcribed at this life cycle stage. We examined the mechanism for down-regulation of procyclin/PARP expression in bloodstream trypanosomes by using protein synthesis inhibitors to effect accumulation of procyclin/PARP transcripts. We show that the accumulation is not due to increased transcription of procyclin/PARP genes. Further, transcripts which accumulate under these conditions are of mature size, polyadenylated and polysome-associated indicating that normally, in bloodstream trypanosomes, down-regulation of procyclin/PARP expression is exerted either during transcript processing or at the level of mRNA stability. A comparison of the inhibitor-induced accumulation of procyclin/PARP transcripts in bloodstream forms of monomorphic and pleomorphic cell lines of trypanosome stock EATRO 795 shows that accumulation occurs with similar kinetics in both cell lines.

The nematode cuticle is a multifunctional structure whose roles include exoskeleton and barrier between the animal and its environment. It is an extracellular matrix which consists predominantly of small collagen-like proteins. For those species studied, these cuticular collagens are encoded by a multigene family. In the free living nematode Caenorhabditis elegans, this family has approximately 100 members. Our data indicate a gene family of similar size in the parasitic nematode Ostertagia circumcincta. We have characterised a pair of tandemly duplicated collagen genes from O. circumcincta, colost-1 and colost-2, which we believe to be the direct homologues of col-12 and col-13, a tandemly duplicated pair previously identified in C. elegans. The interspecies comparison of these homologues indicates regions of extreme conservation. We conclude that the gene duplication event that resulted in the creation of col-12 and col-13 in C. elegans is most likely the same duplication that generated colost-1 and colost-2 in O. circumcincta, and thus this particular gene duplication precedes the divergence of the two species. These two nematode species are deeply diverged, O. circumcincta belonging to the order Strongylata and C. elegans to Rhabditata. The ability to identify direct homologues of individual cuticular collagen genes between deeply diverged species provides a powerful method for determining regions of structural importance in these small collagens. Characteristics that are conserved between homologues in divergent species, but not conserved with other members of the multigene family within one species, must relate to the specific function of that particular cuticular collagen.

The nematode Caenorhabditis elegans is contained within a multifunctional exoskeleton, the cuticle, that contains a large number of distinct collagens. As the nematode proceeds from the egg through four larval stages to the adult, transition between larval stages is marked by synthesis of a new cuticle and subsequent moulting of the old one. This is a cyclically repeated developmental event, frequently described as the moulting cycle. We have examined the temporal expression of a group of six genes encoding distinct cuticular collagens. As expected, mRNA abundance for each of the six genes tested is found to oscillate, peaking once during each larval stage. Unexpectedly, the periods of abundance for each gene do not coincide, different genes being expressed at different times relative to one another within the moulting cycle. We detect a programme of temporally distinct waves of collagen gene expression, the precise pattern of which is repeated during each of the four larval stages. This multiphasic pattern of oscillating cuticular collagen gene expression indicates an unexpected complexity of temporal control during the nematode moulting cycle and has implications for collagen trimerization and cuticle synthesis.

Antigenic variation in African trypanosomes proceeds by switching between different variant surface glycoprotein (VSG) molecules, whose extensive epitope differences enable evasion of antibody responses. Each trypanosome has approximately 1000 basic copy VSG genes inside chromosomes and a subset located at telomeres. Switching usually involves different individual basic copy genes being duplicated, as an expression linked copy, into a transcriptionally active site. In a few cases expression linked copies with a number of point mutations have been observed, leading to the suggestion that point mutagenesis provides another mechanism of antigenic variation. The most extensive example is a VSG gene that is normally activated in the metacyclic population in the tsetse fly, but the point mutations were detected in expression linked copies generated during bloodstream infection, after prolonged growth and selection. It was suggested that particularly telomeric or metacyclic VSG genes might undergo point mutagenesis during expression linked copy formation. To test this we have cloned 3 trypanosomes very soon after they had generated, during mouse infection, expression linked copies of the metacyclic VSG gene ILTat 1.22 and have detected only a single point mutation which is present in one expression linked copy, but not the corresponding basic copy, gene. This mutation does not prevent binding of a neutralizing antibody. Extensive VSG gene point mutagenesis may be a consequence merely of prolonged growth and extensive selection. There is not a single reported case of a point mutated VSG presenting a completely new set of exposed epitopes, suggesting point mutagenesis is unlikely to be an authentic mechanism for antigenic variation.

African trypanosomes are extracellular protozoan parasites that are transmitted from one mammalian host to the next by tsetse flies. Bloodstream forms express variant surface glycoprotein (VSG); the tsetse fly (procyclic) forms express instead the procyclic acidic repetitive protein (PARP). PARP mRNA is abundant in procyclic forms and almost undetectable in blood-stream forms. Post-transcriptional mechanisms are mainly responsible for PARP mRNA regulation but results of nuclear run-on experiments suggested that transcription might also be regulated. We measured the activity of genomically-integrated PARP, VSG and rRNA promoters in permanently-transformed blood-stream and procyclic form trypanosomes, using reporter gene constructs that showed no post-transcriptional regulation. When the constructs were integrated in the rRNA non-transcribed spacer, the ribosomal RNA and VSG promoters were not developmentally regulated, but integration at the PARP locus reduced rRNA promoter activity in bloodstream forms. PARP promoter activity was 5-fold down-regulated in bloodstream forms when integrated at either site. Regulation was probably at the level of transcriptional initiation, but elongation through plasmid vector sequences was also reduced.

The major surface antigen of procyclic and epimastigote forms of Trypanosoma congolense in the tsetse fly is GARP (glutamic acid/alanine-rich protein), which is thought to be the analogue of procyclin/PARP in Trypanosoma brucei. We have studied two T.congolense GARP loci (the 4.3 and 4.4 loci) whose transcription is alpha-amanitin sensitive. Whilst a transcriptional gap 5' of the first GARP gene in the cloned region of the 4.4 locus could not be detected, such a gap was present in the 5' flank of the first GARP gene in the 4.3 locus. We have located a GARP transcription start site and, using reporter gene constructs containing a putative GARP promoter region in transient transfection studies, we have demonstrated promoter activity for the test region in T.congolense. There are species-specific differences in sequences regulating expression of the two major surface antigens, GARP and procyclin/PARP: the GARP promoter is inactive in T.brucei while the procyclin/PARP promoter is inactive in T.congolense. We have defined the splice acceptor site for the 4.3 GARP gene by sequencing and by 5' RT-PCR and demonstrated microheterogeneity in GARP polyadenylation by 3' RT-PCR. It appears that some GARP and procyclin/PARP RNA processing signals, although similar, are also species-specific.

In antigenic variation in African trypanosomes, switching of the variant surface glycoprotein (VSG) allows evasion of the mammalian host immune response. Trypanosomes first express the VSG in the tsetse fly vector, at the metacyclic stage, in preparation for transfer into the mammal. In this life cycle stage, a small, specific subset (1 to 2%) of VSGs are activated, and we have shown previously that the system of activation and expression of metacyclic VSG (M-VSG) genes is very different from that used for bloodstream VSG genes (S.V. Graham, K.R. Matthews, P.G. Shiels, and J.D. Barry, Parasitology 101:361-367, 1990). Now we show that unlike other trypanosome genes including bloodstream VSG genes, M-VSG genes are expressed from promoters subject to exclusively transcriptional regulation in a life cycle stage-dependent manner. We have located an M-VSG gene promoter, and we demonstrate that it is specifically up-regulated at the metacyclic stage. This is the first demonstration of gene expression being regulated entirely at the level of transcription among the Kinetoplastida; all other protein-coding genes examined in these organisms are, at least partly, under posttranscriptional control. The distinctive mode of expression of M-VSG genes may be due to a stochastic mechanism for metacyclic VSG activation.

Thirty human and nonhuman primate sera tested at the Centers for Disease Control by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody assay (IFA), and Western blotting were retested at the Virus Reference Laboratory, Inc. by the dot-immunobinding assay (DIA). The Ebola-Reston strain of virus received from the Centers for Disease Control was prepared into a suitable DIA antigen as described for other antigens. All six Western blotting-positive sera were also positive by DIA, as were the five ELISA-positive sera. Testing by IFA, the original test of choice, indicated an additional four seropositives, all negative by the other test systems. Of 288 randomly selected macaque sera, 19 were also found to be Ebola-Reston virus-positive by DIA.

Expression of eukaryotic nuclear encoded tRNA genes requires two transcription factors, TFIIIB and TFIIIC. To determine if the highly evolutionarily diverged parasitic protozoan Trypanosoma brucei possesses any analogous factors, trypanosome nuclear extracts were prepared. Using these extracts in gel-retardation assays on trypanosome tRNA genes we detected three specific protein-DNA complexes, a highly retarded species and a less retarded pair of complexes. Introduction of mutations into the B box of a lysyl tRNA gene greatly reduced formation of all three complexes. Footprinting studies indicated that all complexes protected the B box of the tRNA gene from cleavage. The most highly retarded complex protected both the A and B boxes from DNasel cleavage. The less retarded pair of complexes showed footprints on the B box alone and the lowest B box specific complex is shown to be resistant to the polyanion heparin. All three complexes are demonstrated to induce bends in the DNA on binding.

Procyclic and epimastigote forms of Trypanosoma congolense express an immunodominant glutamic acid/alanine-rich protein (GARP) that covers the parasite surface. Although GARP shows no sequence similarity to procyclins from T. brucei, the general characteristics of the two sets of surface glycoproteins suggest that they have analogous functions, in much the same way that variant surface glycoproteins with unrelated primary sequences fulfil the same function in bloodstream form trypanosomes. Since T. brucei and T. congolense do not follow the same pathway through the tsetse fly, one possible function of procyclins might be to direct parasites to the correct compartments. As a first step towards testing this hypothesis, we have produced stably transformed procyclic forms of T. brucei in which the GARP coding region has been integrated into a procyclin expression site. GARP can be detected on the surface of these transgenic trypanosomes, uniformly distributed within the endogenous procyclin coat, but there are differences in post-translational modification when it is expressed in T. brucei rather than in T. congolense. The fact that GARP is readily accessible to antibodies which were raised against a bacterial fusion protein led us to examine its potential as a selectable surface marker for transfection. We have established a rapid and simple procedure for isolating stable transformants that provides an alternative to conventional methods of selection for antibiotic resistance.

The p34CDC2 protein kinase is a key component in the regulation of the eukaryotic cell cycle. We have isolated from the protozoan parasite Leishmania mexicana mexicana a CDC2-related kinase gene (Lmmcrk1) encoding a 34-kDa protein kinase (lmmCRK1) which has 56% amino acid identity with the human CDC2 and contains a PCTAIR motif in place of the highly conserved PSTAIR box. lmmCRK1 was detected in all life cycle stages at comparable levels, yet its histone H1 kinase activity was detected in only the promastigote form, indicating that its activity is stage-regulated at a post-translational level. lmmCRK1 did not bind p13suc1 beads and Lmmcrk1 was unable to complement a fission yeast temperature-sensitive cdc2 mutant. These data suggest that Lmmcrk1 is unlikely to be the functional L. mexicana cdc2 homologue. A distinct histone H1 kinase activity that binds p13suc1 beads (SBCRK) was also detected, with activity that correlated with the division status of the developmental forms of the parasite, being present in the dividing stages of the parasite and absent in nondividing metacyclic forms. SBCRK is a candidate for the functional CDC2 homologue, but it does not react with an anti-PSTAIR monoclonal antibody on Western blots when eluted from p13suc1 beads, indicating a divergent PSTAIR box. These data suggest that a family of CDC2-related protein kinases are present in Leishmania. Some share sequence and biochemical properties with CDC2, but significant differences also exist, possibly reflecting the evolutionary distance between Leishmania and higher eukaryotes.

Five monoclonal antibodies (mAb) were raised that bound to the surface of procyclic stage Trypanosoma congolense with high intensity in immunofluorescence. Immunoblot analysis of trypanosome lysates using 3 of these mAb revealed a diffuse SDS-PAGE band of 36-40 kDa. The purified antigen did not react with Coomassie Blue or silver stains, but did stain blue with Stains-all, indicating acidity. For the one mAb tested, the epitope was periodate-sensitive and therefore probably glycan. Although this antigen shares properties with procyclin/PARP, which forms a surface coat on procyclic Trypanosoma brucei, a search in T. congolense for homologues of a procyclin/PARP gene revealed only non-coding sequence of partial similarity. Using a differential screen, a procyclic stage T. congolense cDNA clone was isolated that encoded a putative 256-amino acid protein containing 2 peptides chemically sequenced independently by Beecroft et al. [36]. The protein, termed glutamate and alanine-rich protein (GARP), has potential hydrophobic leader and tail sequences (the latter with potential for replacement by a glycosyl phosphoinositol anchor) and no potential N-linked glycosylation sites. It has no significant sequence homology with known proteins. Antibodies against a translational fusion of GARP bound specifically in Western blots to a band very similar to that detected by the mAb and also to the purified antigen. Immunogold electron microscopy revealed a dense packing of the antigen on the cell surface. It appears that procyclic T. brucei and T. congolense have major surface proteins with structural analogy, but with no sequence homology.