Hyperthermic treatment of chromomycosis with disposable chemical pocket warmers

A case of chromomycosis in which hyperthermia proved effective is reported. The patient was a 56-year-old male bean curd maker who, without any previous history of minor trauma, developed on the extensor side of the left upper arm an eczematous lesion that underwent gradual radial expansion. The lesion showed a well-defined, 7×10 cm infiltrated erythematous plaque with the central area healed and, at the upper and lower borders, adherent scales and crusts on the surface. Histological examination revealed granulomatous changes in the dermis, as well as sclerotic cells within giant cells and microabscesses. On culturing,Fonsecaea pedrosoi was isolated. The patient was treated with disposable chemical pocket warmers, which were secured over the lesion with a rather tight elastic bandage, so that they kept the affected area warm for 24 hours a day. After a month of such hyperthermic treatment, the erythema and infiltration had decreased considerably, and microscopic examination and culture of the crusts both yielded negative results. Examination of biopsy specimens of the lesion after the third month showed that it had cicatrized. The treatment was stopped after 4 months, and no relapse occurred. We also summarize the published results of local hyperthermic treatment of chromomycosis in Japan.     

Identification of HLA-B44 subtypes associated with extended MHC haplotypes

The HLA class I antigen B44 is found in each of two different extended major histocompatibility haplotypes (allele combinations of HLA-B, HLA-DR, and complement genes BF, C2, C4A, and C4B in linkage disequilibrium). Using isoelectric focusing, two variants of HLA-B44 were identified. The basic variant was found in all cell lines with the extended haplotype HLA-B44, DR7, FC31, and the acidic variant in all cell lines with the extended haplotype HLA-B44, DR4, SC30. The occurrence of each antigen variant with a unique extended haplotype explains previous observations concerning the nonrandom association of B44 variants with DR antigens.     

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to rescue a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.     

Preparation of megabase-sized tomato DNA and separation of large restriction fragments by field inversion gel electrophoresis (FIGE)

The Schwartz and Cantor technique for releasing and fractionating megabase-sized DNA from agarose-embedded cells is beginning to bridge the gap in resoluation between classical genetics and current molecular DNA techniques, particularly in mammalian systems. As yet no conditions have been described for preparing plant DNA that is of sufficient length to allow similar long-range restriction mapping experiments in plant systems. In this report, we describe the application of the Schwartz and Cantor technique for preparing high molecular weight DNA from embedded tomato leaf protoplasts, as well as conditions for generating and fractionating large restriction fragments, by field inversion gel electrophoresis (FIGE). The bulk of DNA released from lysed protoplasts was at least 2 Mb in size and amenable to restriction digestion as shown by hybridizing Southern blots with, among others, a probe for the Adh-2 gene of tomato. Restriction fragments as large as 700 kb were detected. Chloroplast DNA is isolated intact, amenable to restriction analysis and, in its native form, not mobile in FIGE.     

Pulmonary paracoccidioidomycosis in immunized mice

Paracoccidioidomycosis was induced in immunized (IM) and non-immunized (NI) mice. The histopathology, the number of fungi in the lungs, the cellular (footpad test — FPT and macrophage inhibition factor assay — MIF) and humoral (immunodiffusion test) immune response were investigated serially postinfection. In the IM mice, at days 1 and 3, there was intense and predominant macrophagic-lymphocytic alveolitis with loose granulomatous reaction; at day 30, inflammation was mild. In the NI group, up to day 3, the lesions were focal; later there was formation of extensive epithelioid granuloma. The number of fungi in IM mice were always smaller than those of NI group. Immunization alone induced positive FPT and MIF indices with low titer of antibody. After infection, there was a significant decrease of the FPT indices in the IM group, which we interpreted as desensitization due to trapping of sensitized lymphocytes in the lungs. In conclusion, (1) The lesional pattern of pulmonary paracoccidioidomycosis in IM mice was similar to that of a hypersensitivity pneumonitis. This reaction was probably effective in reducing the extension of the infection and decrease the number of fungi. (2) In this model, pulmonary resistance against P. brasiliensis seems to be related to local and systemic delayed-type hypersensitivity reaction.     

Comparative effects of a recombinant and a mutein type of granulocyte colony stimulating factor on the growth of Meth-A fibrosarcoma with 5-fluorouracil chemotherapy

The present study was designed to evaluate the effects of a recombinant human G-CSF (rhG-CSF) and a mutein G-CSF(KW-2228) on leucopenia and tumor growth in mice treated with 5-fluorouracil (5-FU). In normal mice, the number of leucocytes (white blood cell, WBC) reached the peak 12 hours after a single injection of either type of G-CSF and decreased to the normal level after 24 hours. Daily administration induced a continuous increase in the WBC count, however, administrations at intervals did not. Meth-A fibrosarcoma was subcutaneously inoculated into the backs of syngeneic BALB/c mice. The mice were treated with 5-FU alone or with G-CSFs. Chemotherapy with 5-FU alone resulted in leucopenia and an insignificant inhibition of tumor growth. The conjunctive administration of G-CSFs with 5-FU resulted in a significantly augmented inhibition of tumor growth, and leukopenia was not seen. This augmenting effect was more prominent with KW-2228.These results suggest that in 5-FU chemotherapy G-CSFs may be beneficial in restoring the number of leucocytes from leucopenic state and in augmenting the tumor inhibitory effect. Furthermore, KW-2228 may be more beneficial than the natural type rhG-CSF.     

Acid phosphatase-11, a tightly linked molecular marker for root-knot nematode resistance in tomato: from protein to gene,using PCR and degenerate primers containing deoxyinosine

With a view to cloning the root-knot nematode resistance gene Mi in tomato by chromosome walking, we have developed a molecular probe for the tightly linked acid phosphatase-1 (Aps-1) locus. The acid phosphatase-1 allozyme (APS-1¹), encoded by the Aps-1 ¹ allele originating from Lycopersicon peruvianum, was purified to apparent homogeneity from tomato roots and suspension cells. Microsequencing of CNBr and tryptic peptides generated from APS-1¹ provided a partial amino acid sequence, which accounted for approximately 23% of the protein and revealed two stretches of homology with soybean proteins KSH3 and VSP27, comprising 22 matches within 26 amino acid residues. The partial amino acid sequence information enabled us to isolate a 2.4 kb genomic Aps-1 ¹ sequence by means of the polymerase chain reaction (PCR), primed by degenerate pools of oligodeoxyribonucleotides, synthesized on the basis of the amino acid sequences. Synthesis of the 2.4 kb PCR product was specific for genomic templates carrying the L. peruvianum Aps-1 ¹ allele. Crucial to the priming specificity and the synthesis of the 2.4 kb genomic sequence was the use of degenerate primer pools in which the number of different primer species was limited by incorporating deoxyinosine phosphate residues at three and four base ambiguities. In using cDNA as a template, a 490 bp sequence was obtained, indicating a high proportion of intron sequences in the 2.4 kb genomic Aps-1 ¹ sequence. The Aps-1 ¹ origin of the PCR product was confirmed by RFLP (restriction fragment length polymorphism) analysis, using both a chromosome 6 substitution line and a pair of nearly isogenic lines, differing for a small chromosomal region around the Aps-1/Mi loci.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.