Recent trends in research on differentiation of hematopoietic cells and lymphokines (hemopoietins).

A recent trend in hematological research fields has been to isolate and characterize hematopoietic stem cells/progenitors and their growth factors (hemopoietins) to gain a much better understanding of the nature of the stem cell and the mechanisms regulating its development. It is generally accepted that all the various types of blood cells develop from a single progenitor called a hematopoietic stem cell. Quantitative studies of the function of hemopoietic stem cells began two decades ago with the development of a spleen colony assay, and then, clonal cell culture techniques for committed progenitors were developed with several models for hematopoietic differentiation being proposed. Within the last few years, some hormones have been discovered that are known as hematopoietic growth hormones or hemopoietins, each of which is of protein nature and causes specific classes of blood cells to be made and primed. These hormones also enhance the function of the mature cells, the genes of which have recently been cloned. On the other hand, long-term bone marrow culture has recently permitted detailed investigations of the relationship between hematopoietic cells and the microenvironment in which they are found, e.g. stromal cells, in vitro, relating to the regulation of cell proliferation and differentiation. Further, in hematological fields, other bioactive factors including differentiation-inducing compounds, e. g. bioactive glycosphingolipids, and leukocyte-endothelial cell recognition molecules (adhesion receptors) have been discovered, the molecular mechanism(s) of which have yet to be elucidated. This communication focuses on recent advances in research on soluble hemopoietins and other bioactive factors relating to differentiation-induction and to cell-to-cell recognition.     

Sequence analysis of replication origin of plasmid pLS11 of Bacillus subtilis IFO 3022.

The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4%) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.     

Acute pulmonary alveolar hypoxia increases lung and plasma endothelin-1 levels in conscious rats.

To investigate the effect of pulmonary alveolar hypoxia on the synthesis and release of endothelin (ET)-1, ET-1-like immunoreactivity (-LI) levels of the lung and plasma were measured in conscious unrestrained rats under hypoxic conditions. Sixty-min exposure to alveolar hypoxia (10% O2 or 5% O2) increased the ET-1-LI level in the lung. The plasma ET-1-LI level in hypoxic rats also increased significantly. The increase of plasma and lung ET-1-LI levels were parallel to the severity of hypoxia. These results demonstrates that acute pulmonary alveolar hypoxia increases lung and plasma ET-1-LI levels in conscious unrestrained rats, suggesting a possible physiological or pathophysiological significance of ET in alveolar hypoxia.     

Purification and characterization of dipeptidyl peptidase IV from Streptococcus salivarius HHT.

Dipeptidyl peptidase IV (DAP IV) was purified from Streptococcus salivarius HHT by anion-exchange chromatography, gel filtration and affinity chromatography after lysis of cell walls with N-acetylmuramidase. DAP IV was purified 114-fold with a yield of 16.6% from total activity of the crude extract. The purified enzyme was shown to be homogeneous by disc gel electrophoresis. The molecular weight of the enzyme was estimated to be about 109,000 by gel filtration and 47,000 by sodium dodecylsulphate SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a dimeric form. The optimum pH for the reaction was 8.7 in Gly-NaOH buffer, and the isoelectric point of the enzyme was pH 4.2. The enzyme hydrolysed specifically N-terminal X-Pro from X-Pro-p-nitroanilides. The enzyme activity was hardly affected by various cations, sulphydryl-blocking reagents and metal chelators. The enzyme activity was markedly inhibited by 1 mM diisopropylfluoride, and the desialysed enzyme was attacked by proteinases.     

Microsurgical toenail transfer to the hand

Free nonvascularized toenail grafts have been used to reconstruct congenital or traumatic nail defects of the thumb or finger. Unfortunately, these transfers often result in deformity or atrophy. To avoid these undesirable results, microsurgical free vascularized toenail transfer was performed in 10 patients, 3 for congenital nail absence and 7 for traumatic nail defects. Patient age averaged 17 years (range 2 to 32 years). In contrast with previous reports, the whole big or second toenail complex without pulp was used in reconstruction. All 10 nails were successfully transferred with complete survival. No digits required reexploration. There were no donor- or recipient-site problems. Follow-up averaged 3 years, with a range of 14 months to 5 years and 4 months. Appropriate nail growth occurred in the congenital patients. No atrophy of the nail complex was found as long as sufficient bony support was present (9 of 10 cases). Whole free vascularized toenail transfers for reconstruction of congenital and traumatic nailbed defects achieve excellent aesthetic results while maintaining normal hand function.     

Silence of streptomycin 6-phosphotransferase gene derived by incubation at a high temperature inStreptomyces griseus

Summary The bald mutants from streptomycin (SM)-producingStreptomyces griseus 2247 obtained by incubation at high temperature (36° C), designated as HT strains, lost resistance to their own antibiotic and scarcely produced the antibiotic. Although SM susceptibility in the mutant was due to loss of SM 6-phosphotransferase activity produced in the cell, the gene coding for the enzyme cloned from an HT strain was surely expressed inS. lividans 1326 as a host. Northern blot analysis showed that the corresponding RNA is not detected in the mutant, indicating that though the gene encoding SM 6-phosphotransferase, at least, the structural gene is not deleted in the cell, the expression is silent.     

An establishment of ELISA for murine IL-6

Summary Murine interleukin-6 (mIL-6) was expressed inEscherichia coli as human growth hormone (hGH) fusion protein. The products were cleaved by thrombin to liberate mIL-6. Monoclonal and polyclonal antibodies specific to mIL-6 were prepared by immunizing rats with mIL-6 thus obtained. ELISA for the quantitation of mIL-6 was also established, which could detect mIL-6 in a quantity as low as 2 ng/ml.     

Structural and functional analysis of a polyoma-related mammalian plasmid (L factor): the enhancer activity and plasmid establishment.

L factor is a unique plasmid DNA which was originally discovered in a subclone (B822) of mouse L cells at a high copy number (more than 5,000 copies/cell). The presence of L factor caused no detectable abnormalities to the plasmid-bearing cells. We determined the total DNA sequence of the L factor I (and a part of L factor II) and compared it with that of polyoma DNA. Both DNA are common to the general construction of DNA frames such as early, late and noncoding regions, suggesting the two to be closely related. On the other hand, the L factor DNA sequences differ substantially from that of polyoma in the DNA sequences corresponding to the polyoma large T antigen, capsid proteins and a portion of the enhancer region. In order to investigate the mechanism of plasmid establishment of L factor, we compared the enhancer activity, capacity of DNA replication and efficiency of plasmid establishment of L factor with those of polyoma. The results indicate that L factor enhancer activity and DNA replication capacity were considerably lower than those of polyoma, suggesting that these altered (lowered) activities associated with L factor contribute to the plasmidal establishment and stable maintenance of L factor.