Allelic polymorphism of human FcγRIIA-H/R131 receptor in American tegumentary leishmaniasis.

FcγRIIA binding to IgG subclasses with different levels of affinity is influenced by the polymorphism in the gene that encodes this receptor. The substitution of arginine (R) for histidine (H) in the 131 position defines three allelic patterns, H/H, R/R, and H/R, resulting in FcγRIIA-H/H131 affinity for IgG2 and higher affinity for IgG3 subclasses. Studies have shown the importance of genetic host factors in leishmaniasis and participation of FcγRs on the macrophage infection by amastigote forms and in the immune response to Leishmania sp. We analysed the influence of allelic diversity patterns of the receptor FcγRIIA on American tegumentary leishmaniasis (ATL). FcγRIIA-H/R131 polymorphism was determined by PCR followed by an allele-specific enzymatic digestion in 88 individuals with ATL and 98 healthy volunteer blood donors (control group). The genotypic and allelic distributions of FcγRIIA-H/R131 were similar among the studied groups as well in mild and severe clinical forms of ATL. Our results suggest no association between this allelic polymorphism and susceptibility or resistance to ATL, neither influencing the development of different clinical forms of this illness.

Yeast clathrin has a distinctive light chain that is important for cell growth.

The structure and physiologic role of clathrin light chain has been explored by purification of the protein from Saccharomyces cerevisiae, molecular cloning of the gene, and disruption of the chromosomal locus. The single light chain protein from yeast shares many physical properties with the mammalian light chains, in spite of considerable sequence divergence. Within the limited amino acid sequence identity between yeast and mammalian light chains (18% overall), three regions are notable. The carboxy termini of yeast light chain and mammalian light chain LCb are 39% homologous. Yeast light chain contains an amino-terminal region 45% homologous to a domain that is completely conserved among mammalian light chains. Lastly, a possible homolog of the tissue-specific insert of LCb is detected in the yeast gene. Disruption of the yeast gene (CLC1) leads to a slow-growth phenotype similar to that seen in strains that lack clathrin heavy chain. However, light chain gene deletion is not lethal to a strain that cannot sustain a heavy chain gene disruption. Light chain-deficient strains frequently give rise to variants that grow more rapidly but do not express an immunologically related light chain species. These properties suggest that clathrin light chain serves an important role in cell growth that can be compensated in light chain deficient cells.

Risk factors and characteristics of ocular complications, and efficacy of autologous serum tears after haematopoietic progenitor cell transplantation.

The objective of the study was to evaluate the frequency and clinical characteristics of ocular complications and their risk factors, as well as autologous serum tears (AST) for the treatment of dry eye in these patients. Data from the files of 124 patients who had undergone allogeneic haematopoietic progenitor cell transplantation (HPCT) were evaluated. In addition, 33 HPCT patients were examined and their data were compared with controls. Analysis of tears and AST was performed. Dry eye manifestation occurred in 32% of patients and was positively correlated with age over 27 years (P = 0.05), peripheral blood progenitor cell transplant (P = 0.002), chronic graft-versus-host disease (P = 0.0027), and chronic or acute myeloid leukaemia (P = 0.001). Dry mouth and Schirmer test < 5 mm were predictive factors for dry eye in HPCT patients (P = 0.002 and odds ratio 3.9 and P = 0.007, odds ratio = 5.9, respectively). Microbiological analysis revealed that six of 11 AST samples were contaminated after 30 days of use. The present study supports the role of potential risk factors for ocular complications and key elements to detect alterations in the tear film from HPCT patients. In addition, AST contamination must be considered after longer periods of use.

Erythrocyte sodium-lithium countertransport and proliferative diabetic retinopathy.

PURPOSE: To investigate whether elevated erythrocyte Na+/Li+ countertransport (Na+/Li+ CT) activity is present in patients with proliferative diabetic retinopathy (PDR). METHODS: The rate of Na+/Li+ CT activity assayed in 21 patients with type 1 diabetes mellitus (DM) presenting PDR was compared with 10 patients with nonproliferative retinopathy (NPDR) and with 11 patients with normal fundi. Twelve normal volunteers with no family history of hypertension were used as a control group. The albumin excretion rate was determined by nephelometry, and the glomerular filtration rate was measured by the plasma clearance of eidetic acid labeled with chromium-51. RESULTS: Patients with PDR showed higher diastolic blood pressure levels (mean +/- SD) compared with those with NPDR or normal fundi (95 +/- 13 versus 90 +/- 09 and 82 +/- 19 mm Hg, P = 0.02, respectively). The albumin excretion rate was higher [geometric mean (range)], and the glomerular filtration rate was lower (mean +/- SD) in patients with PDR than in those with NPDR or normal fundi [333 (2 to 5140) versus 32 (5.9 to 2200) and 6 (1.5 to 306) microg/min, P = 0.01, and 63 +/- 33 versus 99 +/- 37 and 93 +/- 43 ml/min, P = 0.02, respectively]. The mean Na+/Li+ CT in patients with PDR was significantly higher than in patients with NPDR or normal fundi and control group (0.46 +/-0.20 versus 0.32 +/- 0.12, 0.32 +/- 11, and 0.21 +/- 0.07 mM/L red blood cells (RBC)/h, respectively, P = 0.0001). In a multiple logistic regression analysis, with PDR as the dependent variable, Na+/Li+ CT (odds ratio [OR]: 4.7, confidence interval [CI]: 1.2-17.6, P = 0.02), diastolic blood pressure (OR, 3.4; CI, 1.3 to 9.6; P = 0.018), and glomerular filtration rate (OR, 5.1; CI, 1.6-17.7; P = 0.007) were the only variables that were maintained in the equation, indicating that they were the main determinants of PDR. CONCLUSIONS: Patients with type 1 DM and proliferative retinopathy have elevated erythrocyte Na+/Li+ CT.

Androgens and dry eye in Sjögren's syndrome.

Sjögren's syndrome is an extremely complex and currently incurable autoimmune disorder, which occurs primarily in females, and is associated with lacrimal gland inflammation, meibomian gland dysfunction, and severe dry eye. We hypothesize that androgen deficiency, which reportedly occurs in primary and secondary Sjögren's syndrome (e.g., systemic lupus erythematosus, rheumatoid arthritis), is a critical etiologic factor in the pathogenesis of dry eye syndromes. We further hypothesize that androgen treatment to the ocular surface will promote both lacrimal and meibomian gland function and alleviate both "aqueous-deficient" and "evaporative" dry eye. Our results demonstrate that androgens regulate both lacrimal and meibomian gland function, and suggest that topical androgen administration may serve as a safe and effective therapy for the treatment of dry eye in Sjögren's syndrome.

Identification of androgen receptor protein and 5alpha-reductase mRNA in human ocular tissues.

BACKGROUND/AIMS: Androgens have been reported to influence the structural organisation, functional activity, and/or pathological features of many ocular tissues. In addition, these hormones have been proposed as a topical therapy for such conditions as dry eye syndromes, corneal wound healing, and high intraocular pressure. To advance our understanding of androgen action in the eye, the purpose of the present study was twofold: firstly, to determine whether tissues of the anterior and posterior segments contain androgen receptor protein, which might make them susceptible to hormone effects following topical application; and, secondly, to examine whether these tissues contain the mRNA for types 1 and/or 2 5alpha-reductase, an enzyme that converts testosterone to the very potent metabolite, dihydrotestosterone. METHODS: Human ocular tissues and cells were obtained and processed for histochemical and molecular biological procedures. Androgen receptor protein was identified by utilising specific immunoperoxidase techniques. The analysis of type 1 and type 2 5alpha-reductase mRNAs was performed by the use of RT-PCR, agarose gel electrophoresis, and DNA sequence analysis. All immunohistochemical evaluations and PCR amplifications included positive and negative controls. RESULTS: These findings show that androgen receptor protein exists in the human lacrimal gland, meibomian gland, cornea, bulbar and forniceal conjunctivae, lens epithelial cells, and retinal pigment epithelial cells. In addition, our results demonstrate that the mRNAs for types 1 and 2 5alpha-reductase occur in the human lacrimal gland, meibomian gland, bulbar conjunctiva, cornea, and RPE cells. CONCLUSION: These combined results indicate that multiple ocular tissues may be target sites for androgen action.

Latent structure of the symptomatology of hospitalized patients with bipolar mania.

Several studies have attempted to understand the dimensions of psychiatric symptoms in manic episodes, but only a few have been able to model the latent structure of mania in bipolar disorder patients using confirmatory factor analysis. The objective of the present study was to search for the best model of the symptomatology of hospitalized manic patients. To achieve this goal, 117 manic inpatients during a manic crisis participated in this research. Exploratory factor analysis was conducted followed by confirmatory factor analysis using an exploratory factor analysis solution and three other theory-based models. The exploratory factor analysis results revealed a six-factor structure: depression, suicide, insomnia, mania, psychosis, and anxiety. This solution also presented the best fit to the data when tested with confirmatory factor analysis. A five-factor solution, without suicide as a separate dimension, appeared to be more theoretically suitable. Another important finding was that anxiety was an independent dimension in mania. Some hypotheses are discussed in light of contemporary theories, and future studies should investigate this aspect further.

Activation of Dictyostelium myosin light chain kinase A by phosphorylation of Thr166.

Phosphorylation of the regulatory light chain is an important mechanism for the activation of myosin in non-muscle cells. Unlike most myosin light chain kinases (MLCKs), MLCK-A from Dictyostelium is not activated by Ca2+/calmodulin. Autophosphorylation increases activity, but only to a low level, suggesting that there is an additional activation mechanism. Here, we show that MLCK-A is autophosphorylated on Thr289, which is C-terminal to the catalytic domain. Phosphorylation of MLCK-A increases in response to concanavalin A (conA) treatment of cells, which was previously shown to activate MLCK-A. However, a mutant kinase with an alanine at position 289 (T289A) is also phosphorylated in vivo, indicating that there is an additional phosphorylated residue. Based on comparisons with other protein kinases, we tested whether phosphorylation of Thr166 drives activation of MLCK-A. Our data indicate that phosphorylation of Thr289 occurs in vivo, but is not associated with conA-induced activation, whereas phosphorylation of Thr166 by some as yet unidentified kinase is associated with activation. Replacement of Thrl66 with glutamate results in a 12-fold increase in activity as compared with the wild-type enzyme, supporting the idea that phosphorylation of Thr166 increases MLCK-A activity.

Myosin light chain kinase (MLCK) gene disruption in Dictyostelium: a role for MLCK-A in cytokinesis and evidence for multiple MLCKs.

We have created a strain of Dictyostelium that is deficient for the Ca2+/calmodulin-independent MLCK-A. This strain undergoes cytokinesis less efficiently than wild type, which results in an increased frequency of multinucleate cells when grown in suspension. The MLCK-A-cells are able, however, to undergo development and to cap crosslinked surface receptors, processes that require myosin heavy chain. Phosphorylated regulatory light chain (RLC) is still present in MLCK-A-cells, indicating that Dictyostelium has one or more additional protein kinases capable of phosphorylating RLC. Concanavalin A treatment was found to induce phosphorylation of essentially all of the RLC in wild-type cells, but RLC phosphorylation levels in MLCK-A-cells are unaffected by concanavalin A. Thus MLCK-A is regulated separately from the other MLCK(s) in the cell.

MLCK-A, an unconventional myosin light chain kinase from Dictyostelium, is activated by a cGMP-dependent pathway.

Dictyostelium myosin II is activated by phosphorylation of its regulatory light chain by myosin light chain kinase A (MLCK-A), an unconventional MLCK that is not regulated by Ca2+/calmodulin. MLCK-A is activated by autophosphorylation of threonine-289 outside of the catalytic domain and by phosphorylation of threonine-166 in the activation loop by an unidentified kinase, but the signals controlling these phosphorylations are unknown. Treatment of cells with Con A results in quantitative phosphorylation of the regulatory light chain by MLCK-A, providing an opportunity to study MLCK-A's activation mechanism. MLCK-A does not alter its cellular location upon treatment of cells with Con A, nor does it localize to the myosin-rich caps that form after treatment. However, MLCK-A activity rapidly increases 2- to 13-fold when Dictyostelium cells are exposed to Con A. This activation can occur in the absence of MLCK-A autophosphorylation. cGMP is a promising candidate for an intracellular messenger mediating Con A-triggered MLCK-A activation, as addition of cGMP to fresh Dictyostelium lysates increases MLCK-A activity 3- to 12-fold. The specific activity of MLCK-A in cGMP-treated lysates is 210-fold higher than that of recombinant MLCK-A, which is fully autophosphorylated, but lacks threonine-166 phosphorylation. Purified MLCK-A is not directly activated by cGMP, indicating that additional cellular factors, perhaps a kinase that phosphorylates threonine-166, are involved.

Temporal and spatial distribution of the variants of merozoite surface protein-1 (MSP-1) in Plasmodium falciparum populations in Brazil.

The polymorphic, merozoite surface protein-1 (MSP-1) of Plasmodium falciparum, an antigen of the parasite's asexual blood-stages, is a major malaria-vaccine candidate. Nucleotide sequences of each variable domain or block of this antigen may be grouped into one of three possible allelic types (K1, MAD20 and RO33), and 24 major types of the msp-1 gene may be defined, as unique combinations of allelic types in these variable blocks. Isolates collected from the Brazilian Amazon, over a period of 14 years, have now been investigated, by PCR-based typing of the msp-1 gene. Thirteen of the 24 possible gene-types were identified, and 336 P. falciparum clones were fully typed among 239 isolates. Most parasites (87%) belonged to one of the seven most frequent gene-types. Marked temporal variation in the distribution of msp-1 variants was found when comparing parasites sampled in the same sites at intervals of at least 5 years. Spatial variations were also found when comparing parasites from both neighbouring and distant sites within the Amazon Basin. The between-population variance in the frequencies of msp-1 allelic types found in Brazil, as estimated by Wright's FST statistic, is of similar magnitude to that found in previous world-wide comparisons. The potential implications of these findings for the development of an MSP-1-based, multivalent malaria vaccine are discussed.

Impact of gender on exocrine gland inflammation in mouse models of Sjögren's syndrome.

Sjögren's syndrome is a complex autoimmune disorder, that occurs almost exclusively in females, induces extensive lymphocyte accumulation in lacrimal and salivary glands, and represents one of the leading causes of dry eye and mouth in the world. The purpose of this study was to determine whether the profound, gender-related differences observed in the magnitude of exocrine gland inflammation in Sjögren's syndrome may also be found in tissues of mouse models of this disorder. Lacrimal and submandibular glands were obtained from adult MRL/lpr, MRL+/+ (MRL+), NZB/NZW F1 (F1), C3H/lpr, C3H/gld (gld), C57BL/6-lpr/lpr [B6/lpr; with (bcl-2(+)/lpr) or without (bcl-2(-)/lpr) bcl-2 transgene insertion] and nonobese diabetic (NOD) mice after the onset of autoimmune disease, and processed for microscopy and image analysis. Our results showed that: (1) the extent of inflammation was significantly greater in lacrimal glands of female MRL/lpr, MRL+, F1, C3H/lpr and gld mice, and salivary glands of female MRL+, F1 and gld mice, relative to those of males; (2) the severity of inflammation in NOD mice showed a tissue-specific pattern: inflammation was far worse in lacrimal glands of males, whereas immune pathology was far greater in salivary tissues in females; and (3) no gender-related variations were present in the degree of inflammation in lacrimal glands of bcl-2(+)/lpr and bcl-2(-)/lpr mice or in submandibular tissues of MRL/lpr, C3H/lpr, bcl-2(+)/lpr and bcl-2(-)/lpr mice. Our findings demonstrate that gender-, strain- and tissue-related differences exist in the extent of inflammation in several mouse models of Sjögren's syndrome.

An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp.

The ahp genes encoding the two proteins (F52a and C22) that make up an alkyl hydroperoxide reductase were mapped and cloned from Salmonella typhimurium and Escherichia coli. Two classes of oxidant-resistant ahp mutants which overexpress the two proteins were isolated. ahp-1 was isolated in a wild-type background and is dependent on oxyR, a positive regulator of defenses against oxidative stress. ahp-2 was isolated in an oxyR deletion background and is oxyR independent. Transposons linked to ahp-1 and ahp-2 or inserted in ahp mapped the genes to 13 min on the S. typhimurium chromosome, 59% linked to ent. Deletions of ahp obtained in both S. typhimurium and E. coli resulted in hypersensitivity to killing by cumene hydroperoxide (an alkyl hydroperoxide) and elimination of the proteins F52a and C22 from two-dimensional gels and immunoblots. ahp clones isolated from both S. typhimurium and E. coli complemented the cumene hydroperoxide sensitivity of the ahp deletion strains and restored expression of the F52a and C22 proteins. A cis-acting element required for oxyR-dependent, rpoH-independent heat shock induction of the F52a protein was present at the S. typhimurium but not the E. coli ahp locus.

Sequence diversity and linkage disequilibrium within the merozoite surface protein-1 (Msp-1) locus of Plasmodium falciparum: a longitudinal study in Brazil.

The merozoite surface protein-1 (MSP-1) is a major vaccine candidate for the asexual blood stage of malaria. We examined both the extent of sequence diversity in block 17, the 3' end of Msp-1 gene coding for a 19-kDa polypeptide (MSP-1(19)) putatively involved in red blood cell binding, and the patterns of linkage disequilibrium between polymorphic sites throughout the Msp-1 locus. The parasite population sample consisted of Plasmodium falciparum isolates collected between 1985 and 1998 in Rondjnia, an area of hypoendemic malaria transmission in the southwestern Brazilian Amazon. Results were summarized as follows. (1) Seven block-17 sequence variants or haplotypes were found among 130 isolates, including two new haplotypes (novel combinations of previously reported amino acid replacements), here named Brazil-1 (E-TSR-F) and Brazil-2 (Q-TSR-F). (2) As previously shown for other Msp-1 polymorphisms, frequencies of block-17 haplotypes displayed significant temporal variation. (3) Extensive linkage disequilibrium was demonstrated between neighboring dimorphic sites within block 17, as well as between polymorphisms at the 5' and 3' ends of Msp-1 (map distance range: 3.83-4.99 kb). (4) The overall patterns of linkage disequilibrium within Msp-1 remained stable over a period of nearly one decade, and examples of possible 'epidemic' expansion of parasites carrying particular Msp-1 alleles were found in the 1980s and 1990s. These results are discussed in relation to the population biology of P. falciparum and the development of malaria vaccines based on MSP-1.