An indirect immunofluorescence assay using a cell culture-derived antigen for detection of antibodies to the agent of human granulocytic ehrlichiosis.

An indirect immunofluorescence assay for the detection of human antibodies to the agent of human granulocytic ehrlichiosis (HGE) was developed and standardized. Antigen was prepared from a human promyelocytic leukemia cell line (HL-60) infected with a tick-derived isolate of the HGE agent (USG3). Suitable antigen presentation and preservation of cellular morphology were obtained when infected cells were applied and cultured on the slide, excess medium was removed, and cells were fixed with acetone. Use of a buffer containing bovine serum albumin and goat serum reduced background fluorescence, and use of an immunoglobulin G (gamma-specific) conjugate reduced nonspecific binding. The assay readily detected specific antibody from HGE patients and did not detect antibody from healthy individuals. No significant reactivity was noted in sera from patients with high titers of antibodies to other rickettsial species. We were able to identify antibodies reactive to USG3 antigen in samples from areas where HGE is endemic that had tested negative to other rickettsial agents. Animal sera reactive against Ehrlichia equi or Ehrlichia phagocytophila bound to the HGE antigen, indicating that the assay may be useful for veterinary use. Comparability between two different laboratories was assessed by using coded human sera exchanged between laboratories. Results from the two laboratories were similar, indicating that the assay can be easily integrated into use for routine testing for HGE. The assay was then compared to an assay using horse neutrophils infected with ehrlichiae. The two assays gave comparable results, indicating that the cell culture-derived antigen can be used for testing samples that have been previously tested with E. equi as an antigen. The new assay offers several advantages over other immunofluorescence methods that use animal-derived antigen and is suitable for use in testing for human antibodies to the HGE agent.     

New stand magnifiers do not meet rated levels of performance

A new range of stand magnifiers has been released by the COIL company in the United Kingdom. Examination of these magnifiers reveals that they fail to deliver the rated magnifications labelled prominently on the appliances, as a result of the manufacturer's conformance with the requirements of the German DIN standard and the use of back vertex power (F'_v) rather than equivalent dioptric power (F_m) of the magnifier. In this study we provide information on the optometric parameters of these new stand magnifiers that will assist the more accurate specification of improvements in vision expected from their use.     

Effects of food deprivation on cocaine base smoking in rhesus monkeys

Studies have shown that both food deprivation and response cost have important influences on the magnitude of self-administration of a wide variety of psychoactive drugs. In an attempt to extend these findings to the smoked route of drug self-administration, the effects of food allotment and fixed-ratio (FR) value were evaluated in four male rhesus monkeys trained to smoke cocaine base. In the first phase of the experiment, monkeys were trained to self-administer smoked cocaine base under a chained progressive-ratio (PR), fixed-ratio (FR) schedule during daily experimental sessions. Monkeys were required to make 20 lever-press responses and then five inhalations on a smoking spout to obtain the first smoke delivery. The lever ratio then increased to 60, 140, 300, 620, 1260, 2540, and 4940 for each successive smoke delivery. The initial lever ratio value was reset to 20 at the beginning of each daily session. The body weights of three monkeys were determined under free-feeding conditions. Monkeys were then restricted to 100 g food and, when body weights had stabilized, the daily food allotment was increased to 150 g, approximately 210 g, or greater than 400 g (satiation). As the daily food allotment and body weight increased, the mean number of smoke deliveries decreased in two of three monkeys. In the second phase of the experiment, three monkeys were maintained under either food-satiated or food-restricted conditions. Body weights were maintained at approximately 90% of their free-feeding weights under food-restricted conditions. The cost of the drug (lever FR value) was constant within each experimental session, but was increased after 3 consecutive days of stable responding. Fixed-ratio values were increased from 128 to 256, 512, 1024, and 2048. Monkeys were required to complete the lever FR value and then to make five inhalations on the smoking spout to gain access to 1.0 mg/kg per delivery cocaine base. The mean number of smoke deliveries increased at FR 256, 512, and 1024 when monkeys were food-restricted as opposed to food-satiated. Correspondingly, the mean number of responses increased under food-restricted conditions. Responding continued to increase over a wider range of FR values, and the peak number of responses was higher under food-restricted, as opposed to food-satiated conditions. These results, using the smoking route of administration, are consistent with the hypothesis that food deprivation increases the self-administration of reinforcing drugs.     

Immunological characterization of dehydroepiandrosterone sulfotransferase from human liver and adrenal.

Dehydroepiandrosterone sulfotransferase (DHEA-ST), a steroid sulfotransferase (ST), has recently been purified from human liver cytosol and partially characterized. DHEA-ST has a subunit molecular mass of 35 kDa and is responsible for the majority of the sulfation of steroids and bile acids in the liver. For these studies, polyclonal antibodies to human liver DHEA-ST were raised in rabbits. The anti-human liver DHEA-ST antibodies were used to characterize the immunoreactivity of DHEA-ST in human liver and to study the relationship of human adrenal DHEA-ST to the liver form of the enzyme. Immunoblot analysis of several different human liver cytosol samples with the rabbit anti-human liver DHEA-ST antiserum detected only a single 35-kDa protein in each liver. Anti-human liver DHEA-ST antibodies also did not react with either form of phenol sulfotransferase (PST), P-PST or M-PST, present in human liver cytosol. DHEA-ST activity was purified from the 100,000 x g supernatant fraction of human adrenal tissue by DEAE-Sepharose CL-6B chromatography and 3',5'-diphosphoadenosine-agarose affinity chromatography. Human adrenal DHEA-ST was shown to have a molecular mass of 35 kDa, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunoblot analysis of human adrenal cytosol revealed that the anti-human liver DHEA-ST antibodies reacted specifically with the 35-kDa subunit of DHEA-ST. The apparent Km values for DHEA and 3'-phosphodenosine-5'-phosphosulfate obtained with human adrenal DHEA-ST were 1.0 microM and 1.6 microM, respectively. Adrenal DHEA-ST demonstrated the same pattern of reactivity towards different steroid substrates as did human liver DHEA-ST, and neither form of DHEA-ST was found to sulfate cortisol. The results of this study suggest that DHEA-ST is the major steroid ST present in human liver and adrenal tissue and that the physical, biochemical, and kinetic properties of adrenal DHEA-ST are similar if not identical to those of the liver form of the enzyme.     

Escape responses following elimination of the giant interneuron pathway in the cockroach, Periplaneta americana

The entire set of giant interneurons (GIs) in the nerve cord of the cockroach, Periplaneta americana, was ablated using either electrolytic or surgical techniques. All animals with these lesions were capable of turning and running away from standard wind puffs. However, all animals responded much less frequently to standard wind stimuli following lesion, and the latency of their responses was significantly increased. These results are discussed in terms of a GI role in extremely short latency escape responses, and the idea that non-GI pathways, perhaps associated with head sensory structures, need to be considered in the normal control of escape in the cockroach.     

Pheochromocytoma and paraganglioma: comparison of MR imaging with CT and I-131 MIBG scintigraphy

To ascertain the magnetic resonance (MR) imaging characteristics of pheochromocytomas and paragangliomas and to compare MR with computed tomography (CT) and iodine-131 metaiodobenzylguanidine (I-131 MIBG), 19 patients (18 with pheochromocytomas, one with a paraganglioma) were studied. The 18 patients with pheochromocytomas had had positive findings with I-131 MIBG scintigraphy. Abdominal pheochromocytomas were generally hypointense compared with normal liver on T1-weighted MR images and extremely hyperintense on T2-weighted MR images. MR imaging was preferable to CT in the evaluation of primary pheochromocytomas due to superior tissue characterization, particularly in the patient with hypertension and borderline catecholamine levels. For patients with recurrent or metastatic disease, the data suggest that I-131 MIBG scintigraphy is the examination of choice.     

Chaperonin-mediated assembly of wild-type and mutant subunits of human propionyl-CoA carboxylase expressed in Escherichia coli

We developed a bacterial expression system for the human alpha and beta cDNAs of propionyl-CoA carboxylase (PCC). These cDNAs (less the putative mitochondrial matrix targeting presequences) were co-expressed in Escherichia coli on one plasmid vector with each cDNA having its own IPTG-inducible promoter. Only negligible amounts of active PCC were measured despite the presence of both alpha and beta subunits as indicated by Western blot analysis and the almost complete biotinylation of the alpha subunit. Co-expression of this plasmid with a second plasmid vector over-expressing the E. coli chaperonin proteins, groES and groEL, resulted in a several hundred-fold increase in PCC specific activity, to a level comparable with that found in crude human liver extracts. PCC was partially purified on monomeric avidin affinity resin and the presence of both alpha and beta subunits was demonstrated, thereby confirming the assembly of both subunits into an active enzyme. Deficiency of either alpha PCC or beta PCC results in propionic acidemia, an autosomal recessive disorder. We used this expression system to characterize one missense mutation previously described in five Japanese alleles, namely C1283T (Thr428lle) in beta PCC. This mutation, when expressed in E.coli under the same conditions as that of wild-type PCC, had null activity, despite the presence of assembled alpha PCC and beta PCC subunits. This bacterial expression system can be useful for analysis of either alpha PCC or beta PCC mutations. Our findings indicated that the groES and groEL chaperonin proteins were essential for folding and assembly of the human PCC heteromeric subunits.