Leishmania mexicana: proteinase activities and megasomes in axenically cultivated amastigote-like forms

Proteinase activities and megasomes were examined in axenically cultivated amastigote-like forms, freshly isolated lesion amastigotes, and promastigotes. Megasomes were absent in promastigotes and present in both amastigote stages, but they seemed to be less numerous and more homogeneous in cultured amastigote-like forms. Contrasting with the poor detection of proteinase activities in promastigote lysates, both types of amastigotes shared multiple proteinases, which were classified in two groups: (a) 60 to > 100 kDa, o-phenanthroline-sensitive activities; and (b) 23- to 40-kDa cysteine proteinases, of which those resolving as 35- to 40-kDa bands in gelatin gels were more clearly visualized in lysates of cultured amastigote-like forms. Incubation of both kinds of amastigotes with 0.25 to 1.0 microM of either Z-Phe-AlaCHN2 or Z-Tyr-AlaCHN2 selectively inactivated cysteine proteinases, but not the 35- to 40-kDa activities, which, again, were detected with higher intensity in cultured amastigote-like forms. The expression of the 35- to 40-kDa proteinases progressively increased when promastigotes were allowed to transform into amastigote-like forms or when lesion amastigotes were incubated at 34 degrees C for different time periods prior to exposure to Z-Phe-AlaCHN2; activities comparable to those of amastigote-like forms were attained within 24 to 48 hr. The activities resistant to Z-Phe-AlaCHN2 in vivo were fully inhibited by E-64 or Z-Phe-AlaCHN2 during gelatin digestion, suggesting that the 35- to 40-kDa proteinases were mainly inactive before cell lysis. The presence of cycloheximide (at 10, 50, and 100 micrograms/ml) during the pulse with Z-Phe-AlaCHN2 abolished the 35- to 40-kDa activities of lesion amastigotes and significantly reduced gelatin digestion by the similar enzymes of cultured amastigote-like forms. In the latter, the 35- to 40-kDa proteinases were no more detected when cycloheximide was given 60 min prior to Z-Phe-AlaCHN2. The results indicate higher rates of synthesis of the 35- to 40-kDa enzymes, and the existence of a more representative pool of inactive enzyme precursors, in cultured amastigote-like forms.     

Cross-sectional assessment of ELISA reactivity in leprosy patients, contacts, and normal population using the semisynthetic antigen natural disaccharide octyl bovine serum albumin (ND-O-BSA) in Cebu, The Philippines

An indirect enzyme-linked immunosorbent assay (ELISA) using natural disaccharide octyl bovine serum albumin (ND-O-BSA) as antigen was used in testing leprosy patients, contacts and a normal population in Cebu, The Philippines, from 1985 to 1989. A total of 1413 persons were studied. The results suggested that ELISA reactivity and the bacterial index (BI) correlate in a general way. In multibacillary (MB) leprosy, positivity ranges from 54.2% to 92.3% among patients with a BI of < 2+ to > 4+ on the Ridley scale, with an overall average of 84.5%. Paucibacillary (PB) leprosy patients have a low degree of reactivity, with only 15.0% ELISA positive. The test is more efficient in detecting MB than PB leprosy. The contacts of MB leprosy showed 6.5% positivity; contacts of PB leprosy, 7.0% positivity. The normal population showed 1.7% positive ELISA or 17 per thousand population, which is very much less than that of the household contacts. However, because the normal population is a much larger population than the household contact population in a community, more new leprosy cases would emanate from it. Leprosy workers are concerned about the transmission of the disease to household contacts. However, for the reason stated above, we should be more concerned with the silent spread of the disease to the normal population in the community.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of unphosphorylated, newly synthesized I kappa B beta in persistent activation of NF-kappa B

Stimulation with inducers that cause persistent activation of NF-kappa B results in the degradation of the NF-kappa B inhibitors, I kappa B alpha and I kappa B beta. Despite the rapid resynthesis and accumulation of I kappa B alpha, NF-kappa B remains induced under these conditions. We now report that I kappa B beta is also resynthesized in stimulated cells and appears as an unphosphorylated protein. The unphosphorylated I kappa B beta forms a stable complex with NF-kappa B in the cytosol; however, this binding fails to mask the nuclear localization signal and DNA binding domain on NF-kappa B, and the I kappa B beta-NF-kappa B complex enters the nucleus. It appears therefore that during prolonged stimulation, I kappa B beta functions as a chaperone for NF-kappa B by protecting it from I kappa B alpha and allowing it to be transported to the nucleus.     

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.     

Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells

Polymorphonuclear leukocytes (PMN) traverse an endothelial cell (EC) barrier by crawling between neighboring EC. Whether EC regulate the integrity of their intercellular adhesive and junctional contacts in response to chemotaxing PMN is unresolved. EC respond to the binding of soluble mediators such as histamine by increasing their cytosolic free calcium concentration ([Ca++]i) (Rotrosen, D., and J.I. Gallin. 1986. J. Cell Biol. 103:2379-2387) and undergoing shape changes (Majno, G., S. M. Shea, and M. Leventhal. 1969. J. Cell Biol. 42:617-672). Substances such as leukotriene C4 (LTC4) and thrombin, which increased the permeability of EC monolayers to ions, as measured by the electrical resistance of the monolayers, transiently increased EC [Ca++]i. To determine whether chemotaxing PMN cause similar changes in EC [Ca++]i, human umbilical vein endothelial cells (HUVEC) maintained as monolayers were loaded with fura-2. [Ca++]i was measured in single EC during PMN adhesion to and migration across these monolayers. PMN-EC adhesion and transendothelial PMN migration in response to formyl-methionyl-leucyl-phenylalanine (fMLP) as well as to interleukin 1 (IL-1) treated EC induced a transient increase in EC [Ca++]i which temporally corresponded with the time course of PMN-EC interactions. When EC [Ca++]i was clamped at resting levels with a cell permeant calcium buffer, PMN migration across EC monolayers and PMN induced changes in EC monolayer permeability were inhibited. However, clamping of EC [Ca++]i did not inhibit PMN-EC adhesion. These studies provide evidence that EC respond to stimulated PMN by increasing their [Ca++]i and that this increase in [Ca++]i causes an increase in EC monolayer permeability. Such [Ca++]i increases are required for PMN transit across an EC barrier. We suggest EC [Ca++]i regulates transendothelial migration of PMN by participating in a signal cascade which stimulates EC to open their intercellular junctions to allow transendothelial passage of leukocytes.     

Attempted Glass Formation in Pure KHSO4

Recent attempts to vitrify KHSO₄ have been fruitless, bringing into question the glass-forming ability of this, the only hitherto known glass-forming sulfate. Since KHSO₄ is easily converted to the pyrosulfate at temperatures above its melting point, it is likely that previously reported KHSO₄ glass samples were actually mixtures of the bisulfate and the pyrosulfate, with the pyrosulfate aiding vitrification.     

Short-chain phorbol ester constituents of croton oil

Five phorbol diesters, three 4-deoxy-4α-phorbol diesters, two phorbol monoesters, and one 4-deoxy-4α-phorbol monoester were isolated from a commercial sample of croton oil and characterized spectroscopically. Their purification was achieved using combinations of droplet countercurrent chromatography, low-pressure column chromatography over phase-bonded silica gel, and preparative thin layer chromatography. All of these isolates were shown to possess short-chain ester functionalities, with 12-O-tiglylphorbol-13-isobutyrate, 12-O-(2-methyl)butyrylphorbol-13-isobutyrate, 12-O-(2-methyl)butyrylphorbol-13-acetate, 12-O-tiglyl-4-deoxy-4α-phorbol-13-isobutyrate, 12-O-tiglyl-4-deoxy-4α-phorbol-13-acetate, 12-O-(2-methyl)butyryl-4-deoxy-4α-phorbol-13-acetate, phorbol-12-tiglate and 4-deoxy-4α-phorbol-13-acetate being new compounds. A contribution from the Program for Collaborative Research in the Pharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago.     

An investigation of the viral pathogenesis of Kikuchi-Fujimoto disease. Lack of evidence for Epstein-Barr virus or human herpesvirus type 6 as the causative agents

Histiocytic necrotizing lymphadenitis of Kikuchi and Fujimoto is a well-defined clinicopathologic entity of unknown cause. Both the Epstein-Barr virus (EBV) and human herpesvirus type 6 (HHV-6) have been suggested as potential etiologic agents. Twenty cases of Kikuchi-Fujimoto disease were studied for the presence of EBV DNA and HHV-6 DNA by the polymerase chain reaction (PCR), and in situ hybridization in the case of EBV. Cellular DNA from sections of formalin-fixed, paraffin-embedded lymph node tissue was amplified using the PCR technique and oligonucleotide primers to the EBV BamH1 W, lymphocyte-determined membrane antigen, or the EBNA-1 region. These studies were performed in three separate laboratories. In addition, 12 cases were examined by in situ hybridization, eight of which had shown at least one positive PCR signal for EBV. The presence of HHV-6 was assessed by PCR using primers to part of the pZVH14 sequence. Biopsy specimens from eight patients (40%) showed a strong positive signal for EBV in at least one laboratory, while an additional three specimens (15%) showed a weaker positive signal. Five cases studied showed rare positive cells by in situ hybridization, and one case had scattered positive cells. All samples lacked HHV-6 genomic templates. These findings indicate that HHV-6 does not play a role in the pathogenesis of Kikuchi-Fujimoto disease and do not implicate EBV as a causal agent for Kikuchi-Fujimoto disease, since EBV was detected in only a fraction of cases with a low number of positive cells detected by in situ hybridization. Further, some discrepancies were identified in the positive results for EBV in samples studied by multiple laboratories. These results indicate that inconsistent results by PCR may occur with very low levels of viral genomes and that different laboratories perform DNA amplification at different efficiencies. Alternatively, laboratory contamination may give rise to false-positive results. Therefore, a positive result for EBV should be interpreted with caution and should be confirmed by repeated study (PCR) or by independent methodology (in situ hybridization).     

Growth advantage of human leiomyoma cells compared to normal smooth-muscle cells due to enhanced sensitivity toward insulin-like growth factor I

Human uterine leiomyomas exhibit increased IGF-I binding compared to myometrium, while both tissues show IGF-I gene expression. In this study we have examined the functional importance of these findings by testing the presence of IGF-I in 15 leiomyoma biopsies and in 18 myometrium biopsies and the capacity of smooth-muscle cells cultured from these tissues to react to IGF-I. The mean IGF-I peptide concentration in leiomyomas was 3 times higher than in myometrium. This resulted from increased IGF-I uptake in leiomyomas rather than from increased synthesis, as these tissues contain higher concentrations of type-I IGF receptors, as detected by immunohistochemistry, and equal levels of IGF-I mRNA. Blocking IGF-I transport with cytochalasin-B and with the type-I IGF receptor blocking antibody alpha IR3 in cultured cells induced decreased immunostaining intensity for IGF-I in most myometrium and leiomyoma cultures, indicating that the detected IGF-I is internalized. Depending on the culture conditions, IGF-I administration yielded increased survival or a higher proliferation rate in leiomyoma cultures than in myometrium cultures, indicating the increased importance of exogenous IGF-I for the growth of transformed smooth-muscle cells. We conclude that the increased concentrations of type-I IGF receptors in leiomyoma compared to myometrial smooth-muscle cells are functional with respect to the enhanced internalization of IGF-I and that they provide these tumor cells with a growth advantage compared to their normal counterparts.     

Critical test evaluation (1977-1992) of drug efficacy against endoparasites featuring benzimidazole-resistant small strongyles (population S) in Shetland ponies

Several compounds (n = 13 single or combinations; most at therapeutic dosages) were evaluated between 1977 and 1992 in critical tests (n = 91) against benzimidazole (BZ) resistant small strongyles (Population S) and several other species of internal parasites in Shetland ponies, mostly under 1 year old. The closed breeding herd, from which the test ponies were selected, had been treated every 8 weeks with cambendazole (CBZ) for 4 years (1974-1978) and oxibendazole (OBZ) for 14 years (1978-1992). Published field test data (1974-1992) on older ponies in the herd showed BZ resistance of small strongyles. Average efficacies in the present critical tests against small strongyles for OBZ (n = 59 animals) were high in early years (95% or higher), but gradually declined to a low of 1% in 1991. Side-resistance of small strongyles was evident in critical tests (n = 1-6/single drug or combination) for several other BZs and a pro-BZ; ivermectin and piperazine were highly active, but pyrantel pamoate exhibited weak activity. BZ resistance was evident for six small strongyle species (Cyathostomum catinatum, Cyathostomum coronatum, Cylicocylus nassatus, Cylicostephanus calicatus, Cylicostephanus goldi, and Cylicostephanus longibursatus). Activity on bots, ascarids, large strongyles, and pinworms was essentially as expected, indicating no drug resistance.