The distinction between Burkitt lymphoma and diffuse large B-Cell lymphoma with c-myc rearrangement.

To compare immunophenotypic and molecular features between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) with c-myc rearrangements (c-mycR DLBCL), we analyzed 18 cases of B-cell non-Hodgkin's lymphoma with c-mycR that were confirmed by chromosomal and/or Southern blotting analyses. The cases were histologically classified into 10 BLs and five DLBCLs. The remaining three cases could not be classified because of suboptimal quality of the surgical materials. BLs were from five adults and five children, whereas all DLBCLs were from adults. BLs were positive for CD20 (10/10 cases examined), CD10 (9/10), Bcl-2 (1/9), and Bcl-6 (10/10), whereas they were negative for CD3 (0/10) and EBV (0/8), by Epstein-Barr virus (EBV) EBER-1 RNA in situ hybridization. c-MycR DLBCLs were positive for CD20 (5/5), CD10 (2/5), Bcl-2 (3/4), and Bcl-6 (4/4), whereas none of them were positive for CD3 and EBV. A mean of MIB-1 index (MIB-1+ cells/neoplastic cells, %) of BLs (98.1%) was higher than that of c-mycR DLBCLs (66.3%; P <.0001). Somatic mutation of immunoglobulin heavy-chain gene variable region (VH gene) in BLs (four cases) ranged from 0.7 to 4.9% with an average value of 2.3%, whereas those in DLBCLs (three cases) from 8.2 to 32.0% with an average value of 17.0%. It is, therefore, concluded that a growth fraction of nearly 100%, as well as a monotonous proliferation of medium-sized cells and c-myc(R), should be of value in the diagnosis of BL, which is probably different from c-myc(R) DLBCL. In addition, CD10+, Bcl-2-, and low frequency of mutation of the VH gene could be helpful for the histologic distinction of BL from (c-mycR) DLBCL.     

Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase; however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.     

Saliva-binding region of Streptococcus mutans surface protein antigen.

A 190-kDa surface protein antigen (PAc) of Streptococcus mutans binds to human salivary components. For detection of specific binding of the PAc protein to human salivary components, a simple sandwich assay was used. Microtiter plates precoated with recombinant PAc (rPAc), PAc fragments, or S. mutans whole cells were allowed to react with human whole saliva and then were incubated with biotinylated rPAc. The biotinylated rPAc bound to salivary components was detected by use of alkaline phosphatase-conjugated streptavidin and p-nitrophenylphosphate. In this assay, the binding of whole cells of S. mutans and purified rPAc to salivary components was confirmed. For determination of a saliva-binding region of the PAc molecule, 14 truncated PAc fragments were constructed by use of the polymerase chain reaction and an expression vector, pAX4a+. The binding of these truncated PAc fragments to human salivary components was determined by the sandwich assay. Among the truncated PAc fragments, fragments corresponding to residues 39 to 864 and residues 39 to 1000 of PAc showed a high ability to bind to salivary components. Shorter recombinant fragments corresponding to residues 39 to 217, residues 200 to 481, residues 470 to 749, and residues 688 to 864 did not exhibit any binding ability. The fragment that corresponds to a proline-rich repeating region (residues 828 to 1000) bound directly to the PAc protein. These results suggest that residues 39 864 of the PAc molecule are important in the binding of the surface protein to human salivary components, and the proline-rich repeating region of the PAc protein may contribute to spontaneous self-aggregation of the PAc protein.     

Alpha-fetoprotein-producing immature mediastinal teratoma showing rapid and massive recurrent growth in an adult.

A case of immature mediastinal teratoma in a 43-year-old Japanese man is reported. The tumor, which was multicystic with solid zones and measured 12 x 6 x 8 cm, arose in the anterior mediastinum. The serum alpha-fetoprotein (AFP) level was elevated to 5,114 ng/ml before surgery. Histologically, the solid zones showed an admixture of irregular glands lined by columnar or cuboidal epithelium set in a spindle cell stroma, some foci of primitive neural tissue, and scattered small nests of hepatoid cells. Immunohistochemically, the hepatoid cells and epithelia lining some of the cysts showed a strongly positive reaction for AFP. Eight months after surgery, the patient died of respiratory failure caused by a rapidly growing massive recurrent tumor, which measured 40 x 24 x 13 cm, in the left thoracic cavity. However, the elevated serum AFP level had been decreasing during the course of the recurrence in response to chemotherapy. The recurrent tumor showed remarkable proliferation of loose mesenchymal tissue without primitive neural tissue. These findings suggest that immature mediastinal teratoma in adults is highly malignant, and that non-AFP-producing mesenchymal tissue played a critical role in forming the rapidly growing massive recurrent tumor in the present case.     

The ultrasound examination of the deep vein thrombosis

Ultrasonography is very useful for detection of deep vein thrombosis. The purpose of this paper is to show a method for detecting them efficiently by high resolution transducer and color Doppler system. We examined patients in the supine and prone positions. To detect the venous flow easily and differentiate thrombi from simple venous dilatation, some maneuvers are useful; one is pushing the vein area using the transducer on examination, the second is breathing overload, and the last is so-called milking. We can find throombi in the external iliac or femoral veins of patients who have symptoms of lower leg swelling, however, we need to better detect venous thrombi in the lower leg in patients with a history of pulmonary embolism. Because deep venous thrombi are increasing, the role of ultrasound will expand in the future.     

Method for In Situ Evaluation of Superoxide Production by Pulmonary Macrophages in the Rat

In order to investigate superoxide production by pulmonary macrophages in the rat, a route was created by ligating both the inferior and superior venae cavae and resecting the aorta after cannulation through the inferior vena cava into the right atrium of the heart. Lung perfusion was performed via this route with nitro blue tetrazolium. Although there was no formazan deposition throughout the lung, it became detectable in both alveolar and interstitial macrophages when phorbol myristate acetate was added to the perfusate. This deposition was markedly enhanced by previous injection of Corynebacterium parvum. The deposition disappeared after further addition of Cu(Lys)₂, a scavenger of superoxide anions. This procedure may be useful for estimating in situ the ability of pulmonary macrophages to produce superoxide in the rat.     

Association of Actinobacillus actinomycetemcomitans leukotoxin with nucleic acids on the bacterial cell surface.

Actinobacillus actinomycetemcomitans, a periodontopathic gram-negative bacterium, produces a leukotoxin that is a member of the RTX cytotoxin family. Although genes may function in toxin secretion, the leukotoxin is not secreted extracellularly but remains associated with the bacterial cell surface. We report here that this toxin-cell surface association is mediated by nucleic acids and directly demonstrate that the extracellular secretion of toxin occurs in growing cultures with increased ionic strength of medium. All examinations were performed with freshly harvested A. actinomycetemcomitans 301-b from anaerobic fructose-limited chemostat cultures. The occurrence of cell surface-localized DNA was shown by directly digesting whole cells with the restriction endonuclease EcoRI or HindIII, which yielded many DNA fragments. The cell surface DNA constituted about 20% of the total cellular DNA. The leukotoxin was released from the whole cells by digestion with DNase I as well as restriction endonucleases. Because the leukotoxin binds ionically to DNA, it is dependent on the ionic strength of buffers or media. Accordingly, the toxin was released from cells suspended in saline at pH 7.5 in the presence of increasing amounts of MgCl2 (0 to 10 mM) or NaCl (0 to 50 mM). Moreover, a considerable quantity of leukotoxin was detected in the culture supernatant of fructose-limited chemostat cultures when sodium succinate solution was pumped into the steady state as an additional salt (30 and then 50 mM). This toxin-DNA association was also found in well-characterized strains including not only the leukotoxin-producing ATCC 29522 but also the toxin production-variable ATCC 29523 and the non-leukotoxin-producing ATCC 33384 when these strains were grown in the chemostat culture.     

Activation dependence of isotonic transient in response to step tension reduction in cardiac muscle segment during barium contracture

Summary To clarify the activation-dependence of dynamic mechanical characteristics of contracting cardiac muscle, we analysed the healthy central segment length (SL) response to step decrease in tension at two different levels of barium contracture (0.2 mM and 0.5 mM Ba²⁺) in rat papillary muscles with a fixed initial SL. The time course of this response is thought to reflect the kinetics of actin-myosin interaction. The muscle was released stepwise from the steady contracture tension (T_c) to new steady tension levels (T_r) of varying magnitudes at 22° C. The SL responses consisted of four phases at T_r/T_c > 0.3. The amplitude of shortening in the second phase, after the initial rapid and minute shortening in the first phase, increased with an increase in amplitude of step tension reduction, and was greater at the higher activation level when compared at an identical amount of T_r/T_c. The fourth phase, after the remarkable lengthening in the third phase, was an extremely slow and minute shortening toward a new steady SL under the new tension. The duration of the second and third phase was quite insensitive to activation level at T_r/T_c > 0.85, but became longer at the higher activation level with larger amounts of tension reduction. The velocity measured from the initial quasi-steady SL shortening in the second phase increased significantly with the increase in activation level. These results are discussed in terms of cross-bridge kinetics underlying the isotonic SL transients at two different activation levels.     

Functional expression of the ascofuranone-sensitive Trypanosoma brucei brucei alternative oxidase in the cytoplasmic membrane of Escherichia coli

Trypanosome alternative oxidase (TAO) is the terminal oxidase of the respiratory chain of long slender bloodstream forms (LS forms) of African trypanosoma, which causes sleeping sickness in human and nagana in cattle. TAO is a cytochrome-independent, cyanide-insensitive quinol oxidase and these properties are quite different from those of the bacterial quinol oxidase which belongs to the heme-copper terminal oxidase superfamily. Only little information concerning the molecular structure and enzymatic features of TAO have been available, whereas the bacterial enzyme has been well characterized. In this study, a cDNA encoding TAO from Trypanosoma brucei brucei was cloned into the expression vector pET15b (pTAO) and recombinant TAO was expressed in Escherichia coli. The growth of the transformant carrying pTAO was cyanide-resistant. A peptide with a molecular mass of 37 kDa was found in the cytoplasmic membrane of E. coli, and was recognized by antibodies against plant-type alternative oxidases from Sauromatum guttatum and Hansenula anomala. Both the ubiquinol oxidase and succinate oxidase activities found in the membrane of the transformant were insensitive to cyanide, while those of the control strain, which contained vector alone, were inhibited. This cyanide-insensitive growth of the E. coli carrying pTAO was inhibited by the addition of ascofuranone, a potent and specific inhibitor of TAO ubiquinol oxidase. The ubiquinol oxidase activity of the membrane from the transformant was sensitive to ascofuranone. These results clearly show the functional expression of TAO in E. coli and indicate that ubiquinol-8 in the E. coli membrane is able to serve as an electron donor to the recombinant enzyme and confer cyanide-resistant and ascofuranone-sensitive growth to E. coli. This system will facilitate the biochemical characterization of the novel terminal oxidase, TAO, and the understanding on the mechanism of the trypanocidal effect of ascofuranone.