Molecular biology techniques as clinical laboratory tests

The quality of results obtained with molecular biology techniques depends on the control of preanalytical and analytical error associated with such techniques. Preanalytical error can be introduced during the isolation of DNA and RNA. The type of detergent used in cell lysis can affect the amplification of DNA by techniques such as the polymerase chain reaction(PCR). Ribonuclease(RNase) contamination is a serious problem in the isolation of undegraded RNA, and, thus, this enzyme should be inhibited. Anticoagulants used for blood collection can affect the quality of results with molecular biology techniques. The control of contamination from the working environment is essential to the minimization of preanalytical, analytical and postanalytical error. Molecular biology techniques for a wide range of clinical laboratory tests have been established in hospitals such as clinical laboratory tests for infections, molecular diagnoses of leukemia and aberrant genes in metabolism.     

Morphological observations of the replication of herpesvirus tamarinus in RL-33 cells

Summary The replication in RL-33 cells (rabbit lung cell line) of herpesvirus tamarinus isolated from cotton-topped marmosets(Saguinus oedipus) was investigated by electron microscopy. In the early stages of infection, ring-shaped and granular structures, and fibrillar materials were recognized in the nucleus. Immature particles were often found in such nuclei. The envelope of the virus was formed by budding through intracytoplasmic membranes, the inner nuclear membrane or the membrane of intracytoplasmic vacuoles. Virus particles which appeared to be budding through the plasma membrane were also observed. Aberrant viral forms were produced by independent budding of both the inner and outer nuclear membranes. The mature particles once enveloped acquired a second envelope by budding through intracytoplasmic double membranes or the outer nuclear membrane. Unusual virus-associated structures were observed in the cytoplasm and nucleus. Virus particles appeared to be released by the process of reverse phagocytosis.With 19 Figures     

Reaction mechanisms of beta1H globulin.

The reaction mechanisms of beta1H were studied. The generation of alternative pathway C3 and C5 convertases on the cell surface as well as in the fluid phase was inhibited by beta1H globulin. The cell preparation bearing the C3b site could bind beta1H with little effect on the C3b hemolytic activity. Bound beta1H was dissociated by the action of C3bINA and C3bINA-treated C3b bearing cell did not bind beta1H anymore. Cell-bound beta1H was also dissociated by the action of B (or Bb). From these and other results, the following conclusions were obtained. The C3b site-bearing cell could bind beta1H on the C3c region of C3b molecules facilitating the C3bINA action on C3b, and beta1H shared the same binding site with B (or Bb) inhibiting the generation of the alternative pathway convertases competitively.     

Claudin‐7 Expressed on Lateral Membrane of Rat Epididymal Epithelium does not Form Aberrant Tight Junction Strands

Claudins are integral membrane proteins at tight junctions (TJs) and form TJ strands. In the present study, we found that claudin‐7 was localized along the entire lateral membranes of epididymal epithelium, including the apical junctional region throughout the epididymis, but claudin‐8 was restricted to the apical junctional region. This finding raises the possibility that aberrant TJ strands may be formed on lateral membranes. Thus, we focused on examining whether TJ strands exist on lateral membranes of epididymal epithelium. Freeze‐fracture electron microscopy showed that aberrant TJ strands were observed in only a few principal cells in all segments of the epididymis except for the initial segment, indicating that the occurrence of aberrant strands is very rare. Aberrant TJ strands were smooth and not subdivided into individual particles in the protoplasmic face, and complementary grooves in the extracellular face were almost free of particles. Aberrant TJ strands in the distal caput and corpus epididymis were accompanied by many vesicle‐like structures but those in the proximal caput and cauda epididymis were not. These results suggest that most of claudin‐7 in lateral membranes may exist in a nonpolymerized form and may play some different roles other than the formation of TJ strands, for example, in the formation of a pool of claudin proteins or in the reinforcement of cell adhesion. Anat Rec, 1431‐1438, 2007. © 2007 Wiley‐Liss, Inc.     

Different corticostriatal projections from two parts of the cortical masticatory area in the rabbit

The cortical masticatory area (CMA) elicits rhythmic jaw movements in response to repetitive stimulation and is involved in the control of mastication. Based on jaw movement patterns, the CMA is divided into two parts. One is the part of the CMA in which a T-pattern similar to jaw movements during food transport in natural mastication is evoked by electrical stimulation. The other is more dorsomedially located, and during chewing a C-pattern similar to jaw movements can be induced. However, it is still not known which region of the putamen receives projections from the CMA and whether projections originate from both parts of the CMA. In this study, electrophysiological and histological experiments were undertaken in rabbits to investigate projections from the CMA to the putamen. Both experiments showed that the ventral region of the putamen received projections from the CMA. The density of the projections from the CMA area inducing the T-pattern seemed to be higher than that from the area inducing the C-pattern. Furthermore, the peak latency of the evoked potentials from stimulation of the CMA area inducing the T-pattern was shorter than that from stimulation of the area inducing the C-pattern. The data obtained from the present study indicate the functional role of the ventral region of the putamen in the regulation of mastication, and further suggest that the corticostriatal pathway is involved in the transition between behavioral jaw movement patterns.     

Crucial role of donor-derived stromal cells in successful treatment for intractable autoimmune diseases in mrl/lpr mice by bmt via portal vein

We have recently established a new bone marrow transplantation (BMT) method for the treatment of intractable autoimmune diseases in MRL/lpr mice; the method consists of fractionated irradiation (5.5 Gy x 2), followed by BMT of whole bone marrow cells (BMCs) from allogeneic C57BL/6 mice via the portal vein (abbreviated as 5.5 Gy x 2 + PV). In the present study, we investigate the mechanisms underlying the early engraftment of donor-derived cells in MRL/lpr mice by this method. In the mice treated with this method, the number of donor-derived cells possessing the mature lineage (Lin) markers rapidly increased in the BM, spleen, and liver; almost 100% were donor-derived cells by 14 days after the treatment. The number of donor-derived hemopoietic progenitor cells (defined as c-kit(+)/Lin(-) cells) increased in the BMCs, hepatic mononuclear cells, and especially spleen cells by 14 days after the treatment. Simultaneously, hemopoietic foci adjoining donor-derived stromal cells were observed in the liver when injected via the PV, but not via the peripheral vein (i.v.). When adherent cell-depleted BMCs were injected via the PV, recipients showed a marked reduction in the survival rate. However, when mice were transplanted with adherent cell-depleted BMCs with cultured stromal cells, all the recipients survived. These findings suggest that not only donor hematopoietic stem cells (HSCs) but also donor stromal cells administered via the PV were trapped in the liver, resulting in the early engraftment of donor HSCs in cooperation with donor-derived stromal cells. This new strategy to facilitate the early recovery of hemopoiesis would therefore be of great advantage in human application.     

Gastric juvenile polyposis associated with germline SMAD4 mutation

We treated a 39-year-old woman with hypoproteinemia and anemia who had profuse gastric polyposis. Radiographic and endoscopic examination showed numerous polyps restricted to the stomach. The patient had pulmonary arteriovenous malformations in the left lung. Histological examination of the resected stomach revealed the gastric polyposis to be composed of cystic dilatation of the glands with small areas of adenocarcinoma. These findings were compatible with gastric juvenile polyposis (GJP) accompanied by gastric cancer. Analysis of genomic DNA revealed that the patient had truncating mutation of SMAD4, a responsible gene for juvenile polyposis (JP). Our case suggests that SMAD4 is possibly a responsible gene for GJP.     

A possible pathogenic role of CD8+ T cells and their derived cytokine, IL-16, in SLE

Current investigations into the role of CD8+ T cells and their derived cytokine, interleukin (IL)-16, in the induction of CD4+ T cell abnormalities in systemic lupus erythematosus (SLE) were reviewed and discussed on the basis of results mainly obtained in our laboratory.