Identifying N fertilizer management strategies to reduce ammonia volatilization: Towards a site-specific approach.

Concerns about ammonia (NH3) losses from nitrogen (N) mineral fertilizers have forced policymakers to set emission reduction commitments across Europe. Although best available techniques (BATs) have been recommended, large uncertainties still exist due to poorly targeted site-specific approaches that might compromise their effectiveness. Here we proposed and tested a conceptual framework designed to identify most effective BATs that reduce NH3 at the site-specific level. The study was conducted in the Veneto region, northeast Italy. After the mapping of NH3 emission potential areas, BATs and business-as-usual N fertilization scenarios were assessed using a modified version of the DNDC agroecosystem model and compared with urea broadcast distribution under different pedo-climatic conditions. The most promising practices were further tested in a field experiment using a wind tunnel combined with a FTIR gas analyzer. Results showed that closed-slot injection reduced NH3 emissions with any type of mineral or organic fertilizers. Injected application, with ammonium nitrate or organic fertilizers, reduced NH3 loss in maize by 75% and 96%, respectively, and in winter wheat by 87% and 98%, compared to surface broadcast. Injection was the most promising technology to support, being already available to farmers. However, some increase in nitrate leaching was observed, mostly in case of winter wheat (+24% for AN injection; +89% for organic fertilizers). By contrast, urea incorporation with hoeing, the most common technique used by farmers in spring crops, did not show satisfactory results, because the partial burial of urea caused strong NH3 emissions that were even higher compared to surface broadcast. Recommended NH3 reduction techniques should be tailored to local pedo-climatic and management conditions, and evaluated, in a holistic approach, considering all N fluxes in the environment.

Molecular genetics of mosquito resistance to malaria parasites.

Malaria parasites are transmitted by the bite of an infected mosquito, but even efficient vector species possess multiple mechanisms that together destroy most of the parasites present in an infection. Variation between individual mosquitoes has allowed genetic analysis and mapping of loci controlling several resistance traits, and the underlying mechanisms of mosquito response to infection are being described using genomic tools such as transcriptional and proteomic analysis. Malaria infection imposes fitness costs on the vector, but various forms of resistance inflict their own costs, likely leading to an evolutionary tradeoff between infection and resistance. Plasmodium development can be successfully completed onlyin compatible mosquito-parasite species combinations, and resistance also appears to have parasite specificity. Studies of Drosophila, where genetic variation in immunocompetence is pervasive in wild populations, offer a comparative context for understanding coevolution of the mosquito-malaria relationship. More broadly, plants also possess systems of pathogen resistance with features that are structurally conserved in animal innate immunity, including insects, and genomic datasets now permit useful comparisons of resistance models even between such diverse organisms.

Evidence for recurrent paralogous gene conversion and exceptional allelic divergence in the Attacin genes of Drosophila melanogaster.

Insects produce a limited variety of antibacterial peptides to combat a wide diversity of pathogens. These peptides are often conserved across evolutionarily distant taxa, but little is known about the level and structure of polymorphism within species. We have surveyed naturally occurring genetic variation in the promoter and coding regions of three Attacin antibacterial peptide genes from 12 lines of Drosophila melanogaster. These genes exhibit high levels of silent nucleotide variations (1-3% per nucleotide heterozygosity), but are not excessively polymorphic at the amino acid level. There is extensive variation in the Attacin promoters, some of which may affect transcriptional efficiency, and one line carries a deletion in the Attacin A coding region that renders this gene nonfunctional. Two of the genes, Attacins A and B, are arranged in tandem and show evidence of repeated interlocus gene conversion. Attacin C, more divergent and located 1.3 Mbp upstream of Attacins A and B, does not appear to have been involved in such exchanges. All three genes are characterized by divergent haplotypes, and one Attacin AB allele appears to have recently increased rapidly in frequency in the population.

Linkage disequilibria and the site frequency spectra in the su(s) and su(w(a)) regions of the Drosophila melanogaster X chromosome.

Over the last decade, surveys of DNA sequence variation in natural populations of several Drosophila species and other taxa have established that polymorphism is reduced in genomic regions characterized by low rates of crossing over per physical length. Parallel studies have also established that divergence between species is not reduced in these same genomic regions, thus eliminating explanations that rely on a correlation between the rates of mutation and crossing over. Several theoretical models (directional hitchhiking, background selection, and random environment) have been proposed as population genetic explanations. In this study samples from an African population (n = 50) and a European population (n = 51) were surveyed at the su(s) (1955 bp) and su(w(a)) (3213 bp) loci for DNA sequence polymorphism, utilizing a stratified SSCP/DNA sequencing protocol. These loci are located near the telomere of the X chromosome, in a region of reduced crossing over per physical length, and exhibit a significant reduction in DNA sequence polymorphism. Unlike most previously surveyed, these loci reveal substantial skews toward rare site frequencies, consistent with the predictions of directional hitchhiking and random environment models and inconsistent with the general predictions of the background selection model (or neutral theory). No evidence for excess geographic differentiation at these loci is observed. Although linkage disequilibrium is observed between closely linked sites within these loci, many recombination events in the genealogy of the sampled alleles can be inferred and the genomic scale of linkage disequilibrium, measured in base pairs between sites, is the same as that observed for loci in regions of normal crossing over. We conclude that gene conversion must be high in these regions of low crossing over.

Antigen localization in immunoperoxidase-stained plastic-embedded soft tissues.

Immunoperoxidase stains were performed on normal and neoplastic tissue from prostate, colon, thyroid, lung, nerve, uterus, and placenta embedded in both plastic (glycolmethacrylate [GMA]) and paraffin. Positive results in plastic section were obtained for carcinoembryonic antigen (CEA), keratin, epithelial membrane antigen (EMA), thyroglobulins, S-100, prostate-specific antigen, human chorionic gonadotrophin (HCG), and beta-HCG. More delicate staining with more precise localization of antigens is noted. Superior (paraformaldehyde) fixation and cold processing followed by GMA polymerization (4 degrees C) allow for optimum antigen survival. After fixation, tissue processing involves a series of 0.1 mol/L phosphate buffer rinses with sucrose and ammonium chloride in a conventional dip-and-dunk processor placed in a 4 degrees C cold room. Acetone dehydrations are used before GMA infiltration, cold polymerization, and sectioning. Before immunoperoxidase staining, the plastic section is digested in .25% bovine trypsin for ten minutes. The immunoperoxidase methods described can be useful when small biopsies are routinely embedded in plastic to obtain improved histologic (hematoxylin-eosin) sections. There may also be research applications in quantifying antigen expression in benign, dysplastic, and neoplastic tissues by examining the stains under high power.

Renal cell carcinoma vs. renal oncocytoma. Report of a case with overlap features and review of the literature.

Although the salient features of renal oncocytomas and renal cell carcinomas have been discussed in the recent literature, renal masses with features of both entities will present diagnostic difficulty, especially when the cells are diffusely eosinophilic on microscopic examination. A case of a firm, tan, rounded mass replacing the lower pole of the kidney is discussed. The final diagnosis of renal cell carcinoma, granular cell type, was made after multiple sections of the tumor were examined, and after electron microscopy was performed. A thorough search by light microscopy should be made for clear cell foci, necrosis, mitotic activity, and vascular or capsular invasion, features generally accepted as pathognomonic for renal cell carcinoma. Cellular and especially nuclear pleomorphism is typically focal or mild in renal oncocytomas. True oncocytic tumors will be packed with mitochondria on electron microscopy; however, granular renal cell carcinomas will contain mitochondria as well as other cellular organelles, lipid, and glycogen. Electron microscopy should be performed on tumors suspected of being oncocytomas because eosinophilia on hematoxylin and eosin stain, as demonstrated by this case, is not a predictable measure of mitochondria content. Immunoperoxidase staining for vimentin in oncocytomas has recently been shown to be negative, and may offer a method of ruling out oncocytoma in vimentin-positive tumors, pending further studies.

Antigenic characterization of medullary carcinoma of the breast: HLA-DR expression in lymph node positive cases.

Medullary carcinoma of the breast has attracted attention because of its relatively good prognosis, in spite of its high cytologic grade. It has, by definition, a consistent, florid tumor infiltrating lymphocyte (TIL) population, probably the result of cytotoxic T-lymphocytes recognizing tumor cells in an HLA-DR-restricted manner. HLA-DR tends to be more highly expressed on primary medullary carcinoma cells than on ductal carcinoma cells; however, the MHC-class II antigenicity of the tumor cells themselves has not been analyzed extensively, and as yet there has been no comparative study of HLA-DR expression in medullary and ductal carcinomas metastatic to lymph nodes. Eleven cases of medullary carcinoma and 15 cases of ductal carcinoma, primaries, and respective lymph node metastases were analyzed by immunoperoxidase staining for HLA-DR and lymphocytes antigens. Polymerase chain reaction (PCR) analysis to identify HLA-DR subtypes from the paraffin blocks was performed on selected cases of primaries and nodal metastases of both tumor types. Immunoperoxidase staining for HLA-DR antigen revealed a marked difference in antigen expression between medullary and ductal carcinomas. In the medullary carcinomas, the mean percentage of cells staining for HLA-DR was 74.5% in the primary tumors and 67.3% in the nodal metastases. For the ductal carcinomas, the mean percentage of cells staining was 17.7% in the primaries and 7% in the metastases. There was a tendency for the level of HLA-DR expression to remain high in medullary carcinoma metastatic to nodes, whereas whatever HLA-DR was present within ductal primaries tended to diminish when cells metastasized to regional nodes. PCR analysis of the HLA-DR within the two tumor types revealed no emerging subtype or variant that could be associated with either the medullary or the ductal carcinomas. Medullary carcinoma cells express much greater quantities of HLA-DR, on the whole, than ductal carcinomas. Expression of HLA-DR is retained on medullary carcinoma cells that have spread to lymph nodes, whereas the smaller quantities of HLA-DR present within ductal primaries tend to diminish even further when the tumor cells are found in lymph nodes. No discernible HLA-DR mutations or predominant subtypes emerged on PCR analysis, and the authors therefore conclude that it is the quantity and not the quality of HLA-DR expression in medullary carcinoma that maintains the characteristic TIL infiltrate, not seen in ductal carcinomas.

Visualization of the ribosome-lamella complex in plastic-embedded biopsy specimens as an aid to diagnosis of hairy-cell leukemia.

Hairy-cell leukemia is a lymphoid leukemia of B-cell lineage, the cells of which are characteristically tartrate resistant acid phosphatase positive on blood and bone marrow smears. However, because hairy-cell leukemia is frequently associated with abundant marrow stroma, dry marrow taps, and peripheral pancytopenia, the diagnosis may rest on the appearance of the bone marrow biopsy specimen alone. The ribosome-lamella complex has been associated with hairy-cell leukemia, and can be visualized by light microscopy using l-micron sections of plastic-embedded bone marrow specimens stained with toluidine blue. We describe the findings in a case in which bone marrow and liver biopsy specimens were positive for hairy cells containing ribosome-lamella complex, which were visualized with both electron microscopy and light microscopy. Reliable light microscopic identification of ribosome-lamella complex may provide an easy and inexpensive method of aiding in the diagnosis of hairy-cell leukemia when aspirate material is not available for tartrate-resistant acid phosphatase stain.

A modified Brown and Hopps stain for identification of gram-positive and gram-negative microorganisms in glycol methacrylate-embedded tissues.

Plastic embedding is a recently developed technique that has been shown to be superior to conventional paraffin embedding in the histopathologic identification of microorganisms in tissue specimens. This report describes a modification of the Brown and Hopps stain for the differentiation of gram-positive and gram-negative microorganisms in 3-micron-thick plastic sections. This technique is easily performed and has a rapid turnaround time. This staining method permits a more precise histopathologic diagnosis of infectious agents than is possible in paraffin-embedded tissues.

P53 and Ki-67 antigen expression in small oral biopsy specimens of salivary gland tumors.

OBJECTIVE: To investigate the possibility that various salivary gland tumors that look histologically similar could express p53 oncoprotein and Ki-67 proliferation antigen differentially and possibly aid in distinguishing benign from malignant lesions. STUDY DESIGN: Intraoral paraffin-embedded biopsy specimens of salivary gland tumors were used. Thirty-eight pleomorphic adenomas, 17 adenoid cystic carcinomas, 23 monomorphic adenomas, and 17 polymorphous low-grade adenocarcinomas were stained with p53 and Ki-67 antibodies by using an immunoperoxidase detection system. Each case was evaluated in terms of staining intensity and percentage of cells staining. RESULTS: Ki-67 and p53 antigens are expressed in generally low levels in the histologically well-differentiated salivary tumors that were studied here, both benign and malignant. Only 1 solid-type adenoid cystic carcinoma showed a high percentage of cells expressing p53. CONCLUSIONS: The histologically well-differentiated salivary tumors studied do not show differential expression of p53 and Ki-67, in spite of their differing courses of biologic behavior. These antibodies should not be relied on to distinguish benign from malignant lesions of the salivary glands; however, they might be markers for those lesions that are dedifferentiating histologically and, therefore, might be displaying more aggressive behavior.

Tumor antigen expression in compound dysplastic nevi and superficial spreading melanoma defined by a panel of nevomelanoma monoclonal antibodies.

Studies of antigen expression in dysplastic nevi have been limited to some extent by difficulties in obtaining frozen nevi with which to react monoclonal antibodies. Accordingly, we obtained a panel of antibodies that binds to antigens preserved in paraffin-embedded tissue. This panel of monoclonal antibodies, raised against nevomelanoma antigens, was used on 26 dysplastic compound nevi and additionally on 14 invasive superficial spreading melanomas. Among the dysplastic nevi, two basic dermal staining patterns emerged. One pattern designated "Type 1" shows a histologically well-developed dermal component that tends to be antigenically well stratified, with the cells in the upper dermis expressing the most antigen, and gradual loss of antigenicity in the lower dermis as maturation of nevic cells occurs. A second ("Type 2") pattern was seen in which nevic cells tended to remain in the upper dermis, with less downgrowth, and to express antigen in a diffuse or patchy, but nonstratified distribution. Some differential distribution of the antigens was noted, with antibodies 404-101, HMB-45, and ME 109 binding to the activated junctional zone, but showing lower binding affinity within the dermis. ME 491, NKI-C3, and 506 bind to antigens abundant in the junctional zone as well as the dermis. The antibody ME 67-6 binds to both junction and dermis, but is more useful for delineation of antigenic stratification and presence of abnormal "clones."

[AIDS and the new therapies: course of research]. / L'AIDS e le nuove terapie: un percorso di ricerca.

The Authors notice the impact of AIDS on all fields of the social life. After outlining a short story of the disease and of the different therapies applied till now, they review the new ones and point out the necessity to provide nurses with specific and continuous education.

Frequencies of the pyrethroid resistance alleles of Vssc1 and CYP6D1 in house flies from the eastern United States.

House flies were collected from four dairies in Maine, New York, North Carolina, and Florida, where high levels of resistance to permethrin have been documented. Regions of two genes, CYP6D1 and Vssc1, having alleles that confer resistance to permethrin (and other pyrethroids) were analysed from individuals at each collection site. The combinations of resistance alleles for Vssc1 and CYP6D1 were highly variable between each state. The resistance allele CYP6D1v1 was found at a high frequency (0.63-0.91) at all sites. Individuals homozygous susceptible for CYP6D1 were very rare and detected only at the dairy in Maine. In addition to the typical Vssc1 mutation responsible for resistance, kdr (L1014F), we also identified individuals with a L1014H mutation. Although house flies homozygous for the L1014H mutation had a lower level of resistance to permethrin, compared to L1014F, the H1014 resistance allele was frequently detected. No individuals with the super-kdr allele (M918T + L1014F) were detected from the field collections. The intron 3 bp downstream of the kdr mutation was found to be extremely variable, providing an opportunity to reconstruct a phylogeny of Vssc1 alleles. Based on this analysis it appears the kdr-his mutation had multiple evolutionary origins, but that the kdr mutation may have had a single origin. The impacts of these findings on resistance management are discussed.

Chemotherapy in metastatic male breast cancer.

14 male patients with metastatic breast cancer received a total of 20 chemotherapeutic trials with 2 basic combination regimens, namely, variants of the Cooper's regimen CMFVP and adriamycin-containing combinations. Overall, a 35% response rate was observed. Our data suggest that cancer of the breast in the male is responsive to the same combinations used in the female, with regimens including adriamycin being probably superior to regimens without it.

Y chromosomal fertility factors kl-2 and kl-3 of Drosophila melanogaster encode dynein heavy chain polypeptides.

The molecular identity and function of the Drosophila melanogaster Y-linked fertility factors have long eluded researchers. Although the D. melanogaster genome sequence was recently completed, the fertility factors still were not identified, in part because of low cloning efficiency of heterochromatic Y sequences. Here we report a method for iterative blast searching to assemble heterochromatic genes from shotgun assemblies, and we successfully identify kl-2 and kl-3 as 1beta- and gamma-dynein heavy chains, respectively. Our conclusions are supported by formal genetics with X-Y translocation lines. Reverse transcription-PCR was successful in linking together unmapped sequence fragments from the whole-genome shotgun assembly, although some sequences were missing altogether from the shotgun effort and had to be generated de novo. We also found a previously undescribed Y gene, polycystine-related (PRY). The closest paralogs of kl-2, kl-3, and PRY (and also of kl-5) are autosomal and not X-linked, suggesting that the evolution of the Drosophila Y chromosome has been driven by an accumulation of male-related genes arising de novo from the autosomes.

Hormonal treatment of disseminated male breast cancer.

14 males with disseminated cancer of breast received a total of 35 endocrine trials, mainly in the form of hormonal supplementation. Overall, a 43% response rate was observed. In particular, remissions occurred in 7 of 11 instances with cyproterone acetate, in 3 of 7 with tamoxifen, in 2 of 5 with estrogens, in 2 of 5 with aminoglutethimide, in none of 3 with high-dose medroxyprogesterone acetate, in none of 1 with androgens, and in 1 of 3 with castration. The response to additive hormonal therapy was 44%. Median overall response duration was 10 months, 11 months following additive hormonal therapy. Median survival from start of therapy was longer in responding than in nonresponding patients (23.5 vs. 11 months). A disease-free interval did not appear to influence hormonal response. Patients responding to one form of hormonal treatment had a greater likelihood of responding to subsequent hormonal manipulations. Additive hormonal therapy may provide effective palliation in males with advanced breast cancer, and should be considered as a valid alternative to orchiectomy.

Phase II trial with oral idarubicin in advanced breast cancer.

Idarubicin, a new analogue of daunorubicin, was administered to 27 patients with advanced breast cancer in a phase II trial. The drug was given orally at a dose of 30-35 mg/m2 every 3 weeks. Twenty-two patients were evaluable for response. All evaluable patients were previously treated with one or more chemotherapeutic regimens, including an anthracycline in more than 50% of the cases. Partial remissions were obtained in 5 patients, for a response rate of 23%. The median duration of response was 191 days. Mild nausea and vomiting were common. Diarrhea, which occurred in less than 50% of the patients, was usually short-lived. Alopecia was generally minimal. Myelosuppression was the dose-limiting toxic effect. Leukopenia was frequently seen, with full recovery by day 28 in 81% of the courses. Thrombocytopenia was less common than leukopenia. Four cases of grade 1 acute cardiac toxicity were recorded. This study suggests that idarubicin can induce regressions in advanced carcinoma of the breast, and justifies further studies in combination with other agents.

Contrasting patterns of nucleotide polymorphism at the alcohol dehydrogenase locus in the outcrossing Arabidopsis lyrata and the selfing Arabidopsis thaliana.

Nucleotide variation at the alcohol dehydrogenase locus (Adh) was studied in the outcrossing Arabidopsis lyrata, a close relative of the selfing Arabidopsis thaliana. Overall, estimated nucleotide diversity in the North American ssp. lyrata and two European ssp. petraea populations was 0.0038, lower than the corresponding specieswide estimate for A. thaliana at the same set of nucleotide sites. The distribution of segregating sites across the gene differed between the two species. Estimated sequence diversity within an A. lyrata population with a large sample size (0.0023) was much higher than has previously been observed for A. thaliana. This North American population has an excess of sites at intermediate frequencies compared with neutral expectation (Tajima's D = 2.3, P < 0.005), suggestive of linked balancing selection or a recent population bottleneck. In contrast, an excess of rare polymorphisms has been found in A. thaliana. Polymorphism within A. lyrata and divergence from A. thaliana appear to be correlated across the Adh gene sequence. The geographic distribution of polymorphism was quite different from that of A. thaliana, for which earlier studies of several genes found low within-population nucleotide site polymorphism and no overall continental differentiation of variation despite large differences in site frequencies between local populations. Differences between the outcrossing A. lyrata and the selfing A. thaliana reflect the impact of differences in mating system and the influence of bottlenecks in A. thaliana during rapid colonization on DNA sequence polymorphism. The influence of additional variability-reducing mechanisms, such as background selection or hitchhiking, may not be discernible.