Major fragment of soluble peptidoglycan released from growing Bordetella pertussis is tracheal cytotoxin.

Bordetella pertussis is known to release a factor which promotes the loss of ciliated respiratory epithelium and copurifies with a soluble peptidoglycan (PG) fragment termed tracheal cytotoxin (TCT). The objective of this study was to determine whether pertussis organisms turn over and release PG derivatives in addition to TCT. B. pertussis Tohama (phase III) was grown in liquid Stainer-Scholte medium containing [3H]diaminopimelic acid (DAP) to label PG specifically, washed to remove free label, and suspended in fresh medium without [3H]DAP. Molecular sieve chromatography of supernatants obtained from such cultures revealed a single included peak of 3H, the elution volume of which corresponded roughly to a disaccharide peptide monomer standard (ca. 10(3) daltons). This material (i) contained [3H]DAP in acid-hydrolyzable linkage, (ii) comigrated with 1,6-anhydro-N-acetylmuramic acid-containing disaccharide peptides on paper chromatography, (iii) was resistant to degradation by mild alkali, and (iv) was indistinguishable from authentic TCT by high-voltage paper electrophoresis and two reversed-phase high-performance liquid chromatography systems. Together, the data suggest that B. pertussis releases a markedly homogeneous set of PG fragments, consisting principally of TCT, and that TCT is possibly a nonreducing, anhydromuramic acid-containing fragment or a cyclic PG derivative.     

Regional difference in the appearance of apoptotic cell death in the ligamentum flavum of the human cervical spine

Ossification or calcification of the ligamentum flavum (LF) is relatively common in the middle and lower cervical, thoracic, and lumbar spine but extremely rare in the upper cervical region. This clinical fact suggests that there exist local factors promoting or preventing ossification or calcification of LF. However, little is known about the differences in the ultrastructure and cellular alterations of the LF between the different spinal levels, even in the cervical spine. With electron microscopy, we examined samples of LF collected surgically from the upper and lower cervical spine regions; we then studied the apoptotic appearance of ligament cells using a preferential labeling method. We found direct evidence of apoptosis of ligament cells in the LF. Apoptosis was more apparent in the upper region samples than in the lower region samples. The spaces around the normal fibroblasts were filled with thick collagen fibrils, but the collagen fibrils disappeared around the apoptotic bodies and thin fibrils were formed. The difference of the level of apoptosis may correlate to the ultrastructual difference of LF, and our data will benefit further investigations seeking to clarify the mechanism of various pathological conditions in the human LF.     

Pathogenic role of polyclonal and polymeric IgA in a murine model of mesangial proliferative glomerulonephritis with IgA deposition.

Molecular size and charge distribution of IgA of sera and glomerular eluates were investigated in ddY mice, spontaneously developing mesangial proliferative glomerulonephritis (GN) with IgA deposition after 40 weeks of age. Serum IgA levels were increased in aged ddY mice more than 40 weeks old with a significant increase (P less than 0.01) at the age of 60 weeks, comparing with those of BALB/c mice. The isoelectric focusing (IEF) spectrotype of pooled serum IgA in 60-week-old mice ranged from 4.2 to 5.5, being similar to those in younger ddY (16 weeks old) and control BALB/c mice (12 weeks old) without enhanced expression of specific IgA peaks. However, IgA in the glomerular eluate from the 60-week-old mice showed limited anionic spectrotypes from pH 4.2 to 4.8. HPLC of IgA in pooled sera and glomerular eluates of 16-, 40- and 60-week-old ddY mice, revealed markedly increased ratios of the dimeric IgA (dIgA) and polymeric IgA (pIgA) in the total IgA with age. In the contrast to serum profiles, monomeric IgA (mIgA) was always detected as the smallest peak of the IgA fractions in glomerular eluates. Furthermore, aged mice with severe GN showed a higher percentage of dIgA and pIgA in total IgA (80%) in the sera than that of the mice with mild GN (64%). HPLC analysis under acid condition of glomerular IgA from 40-week-old ddY mice showed a similar pattern of dIgA and pIgA peaks in neutral buffer without the appearance of mIgA. These findings suggest that there is a selective mechanism for glomerular accumulation of more acid IgA among the polyclonally expanded IgA in old ddY mice, and that the polymeric form of IgA plays a pathogenic role in the development of mesangial proliferative GN in these mice.     

IgG-KAPPA-PRODUCING PRIMARY PLASMACYTOMA OF THE THYROID GLAND WITH PREOPERATIVE SERUM M PROTEIN

A case of primary plasmacytoma of the thyroid gland which occurred in a 63-year-old woman is reported. Histologic and ultramicroscopic examination revealed that the excised thyroid tumor was plasmacytoma superimposed on lymphocytic thyroiditis. Immunohistological study showed that the tumor cells produced intracytoplasmic immunoglobulin (IgG-kappa). Electropho-retic and immunoelectrophoretic studies disclosed the presence of monoclonal immunoglobulin (IgG-kappa) in samples of the patient's serum which had been obtained preoperatively. After completion of irradiation therapy to the neck following tumor removal, the serum monoclonal immunoglobulin disappeared. The patient is currently alive and well without any evidence of the tumor three years after surgery.     

Changes in the nuclear uptake and retention of 3H-estrogen in gonadotrophs and lactotrophs as a function of age

A quantitative autoradiographic immunocytochemical study was performed in which the nuclear uptake and retention of 3H-estradiol (3H-E2) by luteinizing hormone (LH) and prolactin (PRL) cells was examined in 19-21-year-old baboons. 3H-E2 concentrating cells were found in all of the three lobes of the pituitary in varying percentages (38.7%, pars distalis; 17.1%, pars intermedia; 6.3%, pars nervosa). Approximately 80% of PRL cells and nearly 100% of LH cells were labeled. A count of the number of silver grains over nuclei revealed a marked variation of the accumulation of 3H-E2 by LH cells and to a lesser extent in PRL cells. These results suggest functional heterogeneity among LH and PRL cells. The present results are discussed in relation to the physiological state of old animals.     

The determination of serum and urine myoglobin

We have reviewed the current methods of determining serum and urine myoglobin. Qualitative determination of urine myoglobin has been possible for over 20 years, but the quantitative determination is a recent development. Between 1963 (spectrophotometry) and 1978 (radioimmunoassay, RIA), efforts have been made to develop a more sensitive determination method. The RIA method thus developed has a sensitivity as high as 1 ng/ml. Such methods are valuable in the diagnosis of acute myocardial infarction. Recently, simple, rapid, and non-isotopic method such as enzyme immunoassay (EIA) and latex agglutination test have been developed and made rapid determination possible. We applied the "Mb-latex [Seiken]", one such latex agglutination method to the TBA-30 R single-multi type automated analyzer. We obtained good results in the fundamental study such as in precision and accuracy. The reference ranges obtained using serum and urine samples corresponded to the values reported by others with the exception of the lower limit of the ranges. Proficiency testing data using the same reagents and same samples at five laboratories revealed some systematic biases. The reasons for such interlaboratory differences is not clear. However, these systematic biases may be due to the type of equipment, the type of calibration procedure and others including reagents and standard solutions. We are continuing our effort to improve the reliability of interlaboratory data.     

VDE-initiated intein homing in Saccharomyces cerevisiae proceeds in a meiotic recombination-like manner

BACKGROUND: Inteins and group I introns found in prokaryotic and eukaryotic organisms occasionally behave as mobile genetic elements. During meiosis of the yeast Saccharomyces cerevisiae, the site-specific endonuclease encoded by VMA1 intein, VDE, triggers a single double-strand break (DSB) at an inteinless allele, leading to VMA1 intein homing. Besides the accumulating information on the in vitro activity of VDE, very little has been known about the molecular mechanism of intein homing in yeast nucleus. RESULTS: We developed an assay to detect the product of VMA1 intein homing in yeast genome. We analysed mutant phenotypes of RecA homologs, Rad51p and Dmc1p, and their interacting proteins, Rad54p and Tid1p, and found that they all play critical roles in intein inheritance. The absence of DSB end processing proteins, Sae2p and those in the Mre11-Rad50-Xrs2 complex, also causes partial reduction in homing efficiency. As with meiotic recombination, crossover events are frequently observed during intein homing. We also observed that the absence of premeiotic DNA replication caused by hydroxyurea (HU) or clb5delta clb6delta mutation reduces VDE-mediated DSBs. CONCLUSION: The repairing system working in intein homing shares molecular machinery with meiotic recombination induced by Spo11p. Moreover, like Spo11p-induced DNA cleavage, premeiotic DNA replication is a prerequisite for a VDE-induced DSB. VMA1 intein thus utilizes several host factors involved in meiotic and recombinational processes to spread its genetic information and guarantee its progeny through establishment of a parasitic relationship with the organism.