Granulocyte colony-stimulating factor (G-CSF) is a member of the CSF family of hormone-like glycoproteins that regulate haematopoietic cell proliferation and differentiation, and G-CSF almost exclusively stimulates the colony formation of granulocytes from committed precursor cells in semi-solid agar culture. Recently, Nomura et al. have established a human squamous carcinoma cell line (designated CHU-2) from a human oral cavity tumour which produces large quantities of CSF constitutively, and the CSF produced by CHU-2 cells has been purified to homogeneity from the conditioned medium. We have now determined the partial amino-acid sequence of the purified G-CSF protein, and by using oligonucleotides as probes, have isolated several clones containing G-CSF complementary DNA from the cDNA library prepared with messenger RNA from CHU-2 cells. The complete nucleotide sequences of two of these cDNAs were determined and the expression of the cDNA in monkey COS cells gave rise to a protein showing authentic G-CSF activity. Furthermore, Southern hybridization analysis of DNA from normal leukocytes and CHU-2 cells suggests that the human genome contains only one gene for G-CSF and that some rearrangement has occurred within one of the alleles of the G-CSF gene in CHU-2 cells.

The application of an orthostatic stress such as lower body negative pressure (LBNP) has been proposed to minimize the effects of weightlessness on the cardiovascular system and subsequently to reduce the cardiovascular deconditioning. The KAATSU training is a novel method to induce muscle strength and hypertrophy with blood pooling in capacitance vessels by restricting venous return. Here, we studied the hemodynamic, autonomic nervous and hormonal responses to the restriction of femoral blood flow by KAATSU in healthy male subjects, using the ultrasonography and impedance cardiography. The pressurization on both thighs induced pooling of blood into the legs with pressure-dependent reduction of femoral arterial blood flow. The application of 200 mmHg KAATSU significantly decreased left ventricular diastolic dimension (LVDd), cardiac output (CO) and diameter of inferior vena cava (IVC). Similarly, 200 mmHg KAATSU also decreased stroke volume (SV), which was almost equal to the value in standing. Heart rate (HR) and total peripheral resistance (TPR) increased in a similar manner to standing with slight change of mean blood pressure (mBP). High-frequency power (HF(RR)) decreased during both 200 mmHg KAATSU and standing, while low-frequency/high-frequency power (LF(RR)/HF(RR)) increased significantly. During KAATSU and standing, the concentration of noradrenaline (NA) and vasopressin (ADH) and plasma renin activity (PRA) increased. These results indicate that KAATSU in supine subjects reproduces the effects of standing on HR, SV, TPR, etc., thus stimulating an orthostatic stimulus. And, KAATSU training appears to be a useful method for potential countermeasure like LBNP against orthostatic intolerance after spaceflight.

A colony‐stimulating factor (CSF) has been purified to homogeneity from the serum‐free medium conditioned by one of the human CSF‐producing tumor cell lines, CHU‐2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O‐linked glycosides. Amino acid sequence determination of the molecule gave a single NH2‐terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte‐lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non‐adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G‐CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.

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Two different cDNAs for human granulocyte colony‐stimulating factor (G‐CSF) were isolated from a cDNA library constructed with mRNA prepared from human squamous carcinoma cells, which produce G‐CSF constitutively. The nucleotide sequence analysis of both cDNAs indicated that two polypeptides coded by these cDNAs are different at one position where three amino acids are deleted/inserted. When the two cDNAs were introduced into monkey COS cells under the SV40 early promoter, both of them produced proteins having authentic G‐CSF activity and some difference in the specific activity was suggested. A human gene library was then screened with the G‐CSF cDNA and the DNA fragment containing the G‐CSF chromosomal gene was characterized by the nucleotide sequence analysis. The human G‐CSF gene is interrupted by four introns and a comparison of the structures of the two G‐CSF cDNAs with that of the chromosomal gene indicated that the two mRNAs are generated by alternative use of two 5′ splice donor sequences in the second intron of the G‐CSF gene. When the G‐CSF chromosomal gene was expressed in monkey COS cells by using the SV40 enhancer two mRNAs were detected by S1 mapping analysis.