Identification of a novel MHC class I gene, Mamu-AG, expressed in the placenta of a primate with an inactivated G locus

Maternal tolerance of the fetal allograft remains poorly understood. In humans, expression of the highly polymorphic classical HLA-A and HLA-B loci is suppressed, while expression of the nonclassical HLA-G locus is up-regulated at the maternal-fetal interface. Like other nonclassical MHC class I molecules, HLA-G exhibits limited diversity, but certain characteristics of HLA-G distinguish it from other nonclassical MHC class I molecules: it has a truncated cytoplasmic domain, it is the product of alternatively spliced mRNAs, and it is expressed primarily in the placenta. We have examined MHC class I expression in the placenta of the rhesus monkey to determine whether this animal is a suitable model in which to study the function of HLA-G. Although the rhesus monkey possesses orthologs of many MHC class I and II loci found in humans, the HLA-G ortholog is a pseudogene in this nonhuman primate species. In this study, we report the identification of a novel nonclassical MHC class I locus expressed in the placenta of the rhesus monkey, Mamu-AG (Macaca mulatta-AG). Although unrelated to HLA-G, Mamu-AG encodes glycoproteins with all of the characteristics of HLA-G. These Mamu-AG glycoproteins are limited in their diversity, possess truncated cytoplasmic domains, are the products of alternatively spliced mRNAs, and their expression is restricted to the placenta. Taken together, these data suggest that convergent evolution may have resulted in the expression of a unique nonclassical MHC class I molecule in the rhesus monkey placenta, and that the common structural features of Mamu-AG and HLA-G may be functionally significant.     

Establishment of an optimal hypo-osmotic swelling test by examining single spermatozoa in four different hypo-osmotic solutions

In order to find an optimal hypo-osmotic swelling test (HOST) and to identify viable sperm cells from patients with asthenozoospermia for intracytoplasmic sperm injection (ICSI), we tested single motile and non-motile spermatozoa in four hypo-osmotic solutions by micromanipulation. The four solutions were: A, H2O; B, 50 mOsm NaCl; C, 150 mOsm NaCl and D, 150 mOsm sodium citrate and fructose. Eosin Y staining was then carried out for evaluation of viability of the spermatozoa after HOST. Using motile spermatozoa, no statistical difference was found in HOST-positive spermatozoa between these four solutions. There were more viable sperm cells after HOST in solutions C and D, as noted by Eosin Y staining. After non-motile spermatozoa were incubated for 1 min in the four solutions, HOST with solution C gave the best results for identification of viable sperm cells compared to the other three solutions. When motile spermatozoa were incubated in solution C or solution D for 30 min, the result of HOST with solution C (10.8% dead spermatozoa) was superior to that of solution D (49.1% dead spermatozoa). In conclusion, the HOST protocol using 150 mOsm NaCl (solution C) for 1 min yielded the best results for selection of viable spermatozoa. This procedure should be used for selection of viable spermatozoa for ICSI in patients with 100% non-motile spermatozoa.     

Proton NMR assignments and magnetic axes orientations for wild-type yeast iso-1-ferricytochrome c free in solution and bound to cytochrome c peroxidase

Extensive proton hyperfine-shifted resonance assignments have been made for wild-type yeast iso-1-ferricytochrome c when it is free in solution and when it is noncovalently complexed to resting state cytochrome c peroxidase. Complete heme proton resonance assignments were made for free iso-1-ferricytochrome c, while for CcP-complexed iso-1-ferricytochrome c, 70% of heme proton assignments were made. Additional proton resonance assignments were made for hyperfine-shifted protons of amino acids near the heme. These assignments allowed identification of the most extensive set of complex-induced proton shifts yet reported for CcP/cytochrome c complexes. Several purely dipolar-shifted resonances from heme vicinity amino acid protons were also assigned in both free and complexed iso-1-ferricyt c. Both sets of resonance assignments allowed assessment of the origin of proton complex-induced shifts. Using the assigned dipolar-shifted proton resonances as a basis, the orientations of the principal axis systems of the paramagnetic susceptibility tensors for free and cytochrome c peroxidase-bound iso-1-ferricytochrome c were elucidated. The results indicated that the iso-1-ferricytochrome c magnetic axis system orientation shifts significantly upon complex formation. The direction of the complex-induced shifts for heme proton resonances is largely accounted for by the magnetic anisotropy changes. However, analysis of heme complex-induced shifts also reveals local changes in magnetic environment for two heme substituents, presumably through a specific structure change.     

Phase II study of mitoguazone, cyclophosphamide, doxorubicin, vincristine and prednisone for patients with diffuse histologic subtypes of non-Hodgkin's lymphoma: an Eastern Cooperative Oncology Group Study (PE481)

Mitoguazone, an investigational agent with significant activity in advanced lymphoma, was added to a modified CHOP regimen (COPA) in an effort to improve the activity of standard therapy in 66 previously untreated patients with stages II-IV lymphoma and diffuse histology of intermediate or high grade other than lymphoblastic in this phase II pilot study. The regimen was well tolerated and the complete response rate in diffuse large cell lymphoma was 55%. Sixty-five percent of all complete responders were in complete response for at least one year. Despite these excellent results. it is unlikely that the addition of mitoguazone improved results compared with those obtained with standard therapy alone, since similar results have been frequently reported with the latter.     

Analogs of thyrotropin-releasing hormone using an aminoglycine-based template

Novel thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2) analogs, made by solid phase, were derived from the general scaffold pGlu-(D/L)Agl(X)-Pro-NH2 where Agl = aminoglycine. Analogs ranged from X being a proton to an acylating agent derived from substituted (aromatic heterocyclic rings) formic or acetic acids or an aminotriazolyl moiety (3'-amino-1H-1',2',4'-triazolyl) built on N(alpha) of aminoglycine or Nbeta of alpha,beta-diaminoproprionic acid (Dpr). X was expected to mimic the electronic and structural characteristics of the imidazole ring of histidine. Analogs were purified by HPLC, characterized by mass spectrometry and isolated as either diastereoisomeric mixtures or pure isomers. Analogs, tested for their binding affinity to mouse pituitary TRH receptors, have apparent equilibrium inhibitory constants > 1 microM.     

Diagnostic features of clinical neurologic feline infectious peritonitis

Feline infectious peritonitis (FIP) is a fatal Arthus-type immune response of cats to infection with FIP virus, a mutant of the ubiquitous feline enteric coronavirus (FECV). The disease may occur systemically or in any single organ system, and primary neurologic disease is a common subset of such manifestations. We examined 16 domestic cats with clinical neurologic FIP and 8 control cats with nonneurologic FIP, with the intention of identifying the ante- and postmortem diagnostic tests that most contribute to accurate diagnosis. Of the 16 cats with neurologic FIP, 15 were less than 2 years of age and all 16 originated from large multiple-cat households. The most useful antemortem indicators of disease were positive anti-coronavirus IgG titer in cerebrospinal fluid, high serum total protein concentration, and findings on magnetic resonance imaging suggesting periventricular contrast enhancement, ventricular dilatation, and hydrocephalus. Postmortem diagnosis was facilitated by FIP monoclonal antibody staining of affected tissue and coronavirus-specific polymerase chain reaction. Most cats with neurologic and ocular forms of FIP had patchy, focal lesions, suggesting that recently developed technologies described in this report may be useful for evaluation of cats with suspected FIP.     

Extended visual fixation in young infants: look distributions, heart rate changes, and attention

Feeding a high-carbohydrate (CHO) diet and administration of alkalinizing agents have both been shown to improve performance in high-intensity exercise. The effect of these treatments in combination was investigated in the present study. Six healthy male subjects exercised to exhaustion on an electrically braked cycle ergometer at a power output equivalent to 100% of their maximum oxygen uptake (VO2,max) on four separate occasions. Each subject consumed either a diet with the same composition as his normal diet (termed the experimental normal (N) diet; 54 +/- 7% CHO, 13 +/- 2% protein, 33 +/- 7% fat) or a high-CHO diet (81 +/- 2% CHO, 13 +/- 2% protein, 6 +/- 1% fat) that had the same energy and protein content for the 3 days prior to the exercise tests. Subjects then ingested either a placebo (CaCO3) or trisodium citrate (0.3 g (kg body mass)-1) 3 h before exercise. Time to fatigue was not different between experimental conditions. Consumption of the high-CHO diet had no effect on blood acid-base status, but the ingestion of sodium citrate induced a mild metabolic alkalosis after both the N diet and the high-CHO diet. This alkalinizing effect was also evident after exercise, since blood pH, plasma bicarbonate and blood base excess were higher (P < 0.05) after the ingestion of sodium citrate than under the placebo conditions. The changes in blood lactate, pyruvate and glucose and plasma glycerol after exercise were similar for all experimental conditions. Blood lactate, glucose and pyruvate and plasma glycerol concentrations increased from resting values (P < 0.01) following exercise but this increase was similar under all experimental conditions. These data demonstrate that when the energy and protein content of the diets is the same, exercise capacity and the metabolic response to intense exercise are similar following consumption either of a high-CHO diet or a more normal diet. Acute ingestion of sodium citrate prior to exercise resulted in a reduction in post-exercise acidosis despite a blood lactate concentration that was similar to that observed after the ingestion of a placebo, but did not affect exercise performance under the conditions of this study.     

Immunocytochemical characterisation of rabbit and mouse embryonic fibroblasts

By using indirect immunofluorescence we demonstrated the localisation of extracellular matrix (ECM) proteins (laminin--LAM, collagen IV--COL IV, fibronectin--FN) and the basic fibroblast growth factor (bFGF) in rabbit and mouse primary embryonic fibroblasts (PEF). Proliferating mitotically arrested PEF (by mitomycin C) were compared in both species. The stability of protein expression was ascertained during the first five successive passages. In addition, STO cells (i.e. permanent line of irradiated mouse fibroblasts) were similarly analysed. Rabbit PEF showed very high extracellular staining for FN and a negligible cytoplasmic positivity for LAM and COL IV. A totally reversed staining pattern for ECM proteins was found in mouse PEF. A dense cytoplasmic granulation (concentrated around the nucleus) was revealed for LAM and COL IV and almost no reaction for FN. The staining patterns were very stable at the culture conditions we applied. They were maintained during the first five successive passages in proliferating as well as non-proliferating mouse and rabbit PEF and were independent of cell concentration (individually dispersed cells versus cells in a confluent layer). STO cells showed the same staining for ECM proteins as the mouse PEF, thus confirming their origin from the same animal species. Light granular staining for bFGF was found in the cytoplasm of proliferating and mitotically arrested rabbit and mouse PEF and STO cells. The differences in expression of ECM proteins between the rabbit and mouse PEF, as well as the synthesis of bFGF, should be taken into consideration when these cells are used in vitro as a feeder layer for various cells (e.g. embryonic stem cells).     

Design and construction of novel thrombin inhibitors featuring P3-P4 quaternary lactam dipeptide surrogates

Potent serine protease inhibitor 1a featuring a hybrid P3-P4 quaternary lactam dipeptide surrogate was prepared based upon SAR and molecular modeling investigations and in order to further probe the S2/S3 thrombin and FXa subsites. An efficient and concise synthetic route to the key aminolactam intermediate 4 was developed. The design, synthesis, and biological activity of this target and its P3-P4 diastereomer 1b is presented.