Specific clinical signs and symptoms are predictive of clinical course in sporadic Creutzfeldt-Jakob disease.

BACKGROUND AND PURPOSE: Akinetic mutism is thought to be an appropriate therapeutic end-point in patients with sporadic Creutzfeldt-Jakob disease (sCJD). However, prognostic factors for akinetic mutism are unclear and clinical signs or symptoms that precede this condition have not been defined. The goal of this study was to identify prognostic factors for akinetic mutism and to clarify the order of clinical sign and symptom development prior to its onset. METHODS: The cumulative incidence of akinetic mutism and other clinical signs and symptoms was estimated based on Japanese CJD surveillance data (455 cases) collected from 2003 to 2008. A proportional hazards model was used to identify prognostic factors for the time to onset of akinetic mutism and other clinical signs and symptoms. RESULTS: Periodic synchronous discharges on electroencephalography were present in the majority of cases (93.5%). The presence of psychiatric symptoms or cerebellar disturbance at sCJD diagnosis was associated with the development of akinetic mutism [hazard ratio (HR) 1.50, 95% confidence interval (CI) 1.14-1.99, and HR 2.15, 95% CI1.61-2.87, respectively]. The clinical course from cerebellar disturbance to myoclonus or akinetic mutism was classified into three types: (i) direct path, (ii) path via pyramidal or extrapyramidal dysfunction and (iii) path via psychiatric symptoms or visual disturbance. CONCLUSIONS: The presence of psychiatric symptoms or cerebellar disturbance increased the risk of akinetic mutism of sCJD cases with probable MM/MV subtypes. Also, there appear to be sequential associations in the development of certain clinical signs and symptoms of this disease.

Temporal and regional variations in sporadic Creutzfeldt-Jakob disease in Japan, 2001-2010.

The objective of this study was to examine temporal and regional variations of sporadic Creutzfeldt-Jakob disease (sCJD) in a retrospective study using Japanese national surveillance data from 2001 to 2010. We calculated the incidence of sCJD by age and sex, derived the standardized incidence in each of the 47 prefectures, and performed spatial disease clustering analysis. The average annual incidence of sCJD was 1.026 per million in men (637 patients) and 1.132 per million in women (733 patients), a significant sex difference after adjustment for age (P = 0.001). The ratios of familial CJD to sCJD apparently increased between 2001-2005 and 2006-2010, possibly as a result of the nationwide introduction of genetic testing after 2006. Based on the data of 2006-2010, certain geographical clusters of sCJD were identified. The incidence of sCJD was higher in several specific prefectures compared to the national average. Thus, sCJD appears to have regional variations, suggesting the existence of genetic or region-specific factors affecting the incidence of the disease.

Deletion of platelet-derived growth factor receptor-ß improves diabetic nephropathy in Ca²âº/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice.

AIMS/HYPOTHESIS: The activation of platelet-derived growth factor receptor-ß (PDGFR-ß) signalling is increased in the glomeruli and tubules of diabetic animals. In this study, we examined the role of PDGFR-ß signalling during the development of diabetic nephropathy. METHODS: We recently generated pancreatic beta cell-specific Ca(2+)/calmodulin-dependent protein kinase IIα (Thr286Asp) transgenic mice (CaMKIIα mice), which show very high plasma glucose levels up to 55.5 mmol/l and exhibit the features of diabetic nephropathy. These mice were crossed with conditional knockout mice in which Pdgfr-ß (also known as Pdgfrb) was deleted postnatally. The effect of the deletion of the Pdgfr-ß gene on diabetic nephropathy in CaMKIIα mice was evaluated at 10 and 16 weeks of age. RESULTS: The plasma glucose concentrations and HbA(1c) levels were elevated in the CaMKIIα mice from 4 weeks of age. Variables indicative of diabetic nephropathy, such as an increased urinary albumin/creatinine ratio, kidney weight/body weight ratio and mesangial area/glomerular area ratio, were observed at 16 weeks of age. The postnatal deletion of the Pdgfr-ß gene significantly decreased the urinary albumin/creatinine ratio and mesangial area/glomerular area ratio without affecting the plasma glucose concentration. Furthermore, the increased oxidative stress in the kidneys of the CaMKIIα mice as shown by the increased urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion and the increased expression of NAD(P)H oxidase 4 (NOX4), glutathione peroxidase 1 (GPX1) and manganese superoxide dismutase (MnSOD) was decreased by Pdgfr-ß gene deletion. CONCLUSIONS/INTERPRETATION: The activation of PDGFR-ß signalling contributes to the progress of diabetic nephropathy, with an increase in oxidative stress and mesangial expansion in CaMKIIα mice.

Experimental evaluation of photocrosslinkable chitosan hydrogel as injection solution for endoscopic resection.

BACKGROUND AND STUDY AIMS: Saline as an injection solution for endoscopic resection techniques has several disadvantages such as a short-lasting effect leading to a potentially higher risk of bleeding and perforation. The new substance of photocrosslinkable chitosan hydrogel in a DMEM/F12 medium (PCH) can be converted into an insoluble hydrogel by ultraviolet irradiation for 30 s, and was evaluated in two sets of animal experiments. METHODS: 18 pigs were used in the two parts of the study. First, mucosal resections were done with either PCH or hypertonic saline; the effects of both agents on wound healing were examined endoscopically and histologically. Second, in vivo degradation of PCH was examined using six pig stomachs. RESULT: PCH injection led to a longer-lasting elevation with clearer margins, compared with hypertonic saline, thus enabling precise endoscopic submucosal dissection (ESD) along the margins of the elevated mucosa. The endoscopic appearance after ESD was similar in both groups. PCH biodegradation was completed within 8 weeks according to endoscopic and histologic analyses. CONCLUSION: PCH is a promising agent for submucosal injection prior to various techniques of endoresection. It should be evaluated in clinical trials after biocompatibility testing for PCH is completed.

Osteopontin expression in normal and hypobaric hypoxia-exposed rats.

AIM: Experimental pulmonary hypertension induced in a hypobaric hypoxic environment (HHE) is characterized by structural remodelling of the heart and pulmonary arteries. Osteopontin (OPN) has emerged as a key factor in cardiovascular remodelling in response to pressure or volume overload. We studied the possible effects of HHE on the OPN synthesis system. METHODS: One hundred and forty-eight male Wistar rats were housed in a chamber with conditions equivalent of an altitude of 5500 m for up to 21 days. RESULTS: Plasma OPN protein level was found to be significantly decreased on day 0.5 of exposure to HHE, as was the level in the adrenal gland (which secreted highest levels of OPN protein). In the right ventricle of the heart (mRNA) and the lung (protein), OPN expression was found to be significantly increased only on day 1 and day 5, respectively, of exposure to HHE. By immunohistochemistry, the distribution and intensity of OPN protein in several organs were found to alter during exposure to HHE. However, these changes in OPN synthesis did not coincide with the moderate increase in pulmonary arterial pressure (PAP) (maximal mean PAP, 24.5 mmHg) during HHE. CONCLUSION: Pulmonary hypertension in HHE with conditions equivalent of an altitude of 5500 m may induce little or no OPN in heart and lung. Sustained induction may require a more severe PAP overload.

Long-term interleukin-1alpha treatment inhibits insulin signaling via IL-6 production and SOCS3 expression in 3T3-L1 adipocytes.

Proinflammatory cytokines are well-known to inhibit insulin signaling to result in insulin resistance. IL-1alpha is also one of the proinflammatory cytokines, but the mechanism of how IL-1alpha induces insulin resistance remains unclear. We have now examined the effects of IL-1alpha on insulin signaling in 3T3-L1 adipocytes. Prolonged IL-1alpha treatment for 12 to 24 hours partially decreased the protein levels as well as the insulin-stimulated tyrosine phosphorylation of IRS-1 and Akt phosphorylation. mRNA for SOCS3, an endogenous inhibitor of insulin signaling, was dramatically augmented 4 hours after IL-1alpha treatment. Concomitantly, the level of IL-6 in the medium and STAT3 phosphorylation were increased by the prolonged IL-1alpha treatment. Addition of anti-IL-6 neutralizing antibody to the medium or overexpression of dominant-negative STAT3 decreased the IL-1alpha-stimulated STAT3 activation and SOCS3 induction, and ameliorated insulin signaling. These results suggest that the IL-1alpha-mediated deterioration of insulin signaling is largely due to the IL-6 production and SOCS3 induction in 3T3-L1 adipocytes.

Submucosal injection, for endoscopic mucosal resection, of photocrosslinkable chitosan hydrogel in DMEM/F12 medium.

BACKGROUND AND STUDY AIMS: We studied the ability of a photocrosslinkable chitosan in DMEM/F12 medium to maintain submucosal thickness and to reduce bleeding after mucosal resection. We also investigated the behavior of chitosan hydrogels with regard to wound healing. METHODS: The gastric submucosal layer of heparinized rats was injected with the photocrosslinkable chitosan in medium (which was then irradiated with ultraviolet light to form a hydrogel), or with sodium hyaluronate, or hypertonic saline, and three investigations were done, using three different sets of rats. The first and second were measurement of the thickness of the layer, and of the amount of bleeding induced by mucosal resection, respectively. Thirdly, the effects of the chitosan hydrogel on wound healing were examined histologically. RESULTS: Gastric submucosal layers of chitosan hydrogel-treated animals remained significantly thicker than those of other groups for at least 6 h after injection. The total amount of bleeding 20 min after mechanical mucosal resection was 170.0 +/- 20.0 mg, 678.3 +/- 226.3 mg, and 1020.0 +/- 104.1 mg in the chitosan hydrogel, sodium hyaluronate, and hypertonic saline groups, respectively. Histological study revealed that the focus of bleeding was surrounded by chitosan hydrogel and that almost all the hydrogel was biodegraded within 4 weeks. Furthermore, a discernible, but not statistically significant effect of the chitosan hydrogel on wound healing was observed. CONCLUSIONS: The chitosan hydrogel produced mucosal elevation after submucosal injection with ultraviolet irradiation, and it significantly reduced bleeding after mucosal resection. Our newly developed chitosan hydrogel in medium might be a promising submucosal agent for endoscopic mucosal resection.

Role of CD14 expression in the differentiation-apoptosis switch in human monocytic leukemia cells treated with 1alpha,25-dihydroxyvitamin D3 or dexamethasone in the presence of transforming growth factor beta1.

Transforming growth factor beta (TGF-beta) enhanced the growth-inhibitory activities of dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (VD3) on human monocytoid leukemia U937 cells. TGF-beta and VD3 synergistically increased the expression of differentiation-associated markers such as the CD11b and CD14 antigens, whereas TGF-beta and Dex did not. On the other hand, TGF-beta and Dex synergistically increased the number of Apo2.7-positive cells, which represents the early stage of apoptosis, whereas TGF-beta and VD3 did not, suggesting that TGF-beta enhanced apoptosis with Dex and enhanced monocytic differentiation with VD3. In the presence of TGF-beta, the retinoblastoma susceptibility gene product, pRb, was synergistically dephosphorylated by Dex as well as VD3. TGF similarly enhanced the expression of the p21Waf1 gene in U937 cells treated with Dex and VD3. TGF-beta dose-dependently increased the expression of Bcl-2 and Bad and decreased the expression of Bcl-X(L) in U937 cells. Dex enhanced the down-regulation of Bcl-X(L) expression in TGF-beta-treated cells, whereas VD3 blocked this down-regulation of Bcl-X(L). However, the down-regulation of Bcl-X(L) by treatment with the antisense oligomer did not affect the apoptosis or differentiation of U937 cells. The apoptosis of CD14-positive cells was suppressed in the VD3 plus TGF-beta-treated cultures. These results suggest that the expression of CD14 is involved in the survival of differentiated cells.

Vesnarinone and glucocorticoids cooperatively induce G1 arrest and have an anti-tumour effect on human non-small cell lung carcinoma cells grown in nude mice.

Vesnarinone, an oral cardiotonic, inhibited the growth of several human non-small cell lung carcinoma cell lines, and its anti-proliferative effects in vitro and in vivo were greatly enhanced by combination with glucocorticoids, but not other steroids. Simultaneous treatment with vesnarinone and dexamethasone is the most effective to evoke the synergistic effect in the growth inhibition of lung carcinoma EBC-1 cells. Dexamethasone and other glucocorticoids induced morphological changes in EBC-1 cells and these agents together with vesnarinone induced alkaline phosphatase activity, which is a typical marker of type II pneumocyte maturation. This treatment arrested the growth of the cells at the G1 phase, indicating that this treatment is cytostatic rather than cytotoxic. These results suggest that vesnarinone plus glucocorticoid might be useful in lung cancer therapy.

Elevated expression of differentiation inhibitory factor nm23 mRNA in monoblastic crisis of a patient with chronic myelogenous leukemia.

Differentiation inhibitory factor nm23 gene has been found to be expressed in high quantities in acute myelogenous leukemia (AML), especially in acute monocytic leukemia (AML-M5) and is suggested as a new prognostic factor in AML-M5. We report an example of elevated expression of nm23 mRNA in a patient with chronic myelogenous leukemia (CML) who developed monoblastic crisis. Relative levels of nm23-H1 and -H2 mRNA extracted from the patient's peripheral blood mononuclear cells and bone marrow mononuclear cells were measured by quantitative reverse transcriptase polymerase chain reaction. The level of nm23-H1 mRNA in CML cells at the chronic phase was as high as that in bone marrow cells from healthy volunteers. The mRNA level of nm23-H2 was slightly below the normal level. At blastic crisis, however, expression of both nm23-H1 and -H2 mRNA was elevated to about three to nine times of that at the chronic phase. Proliferated blastic cells were positive for non-specific esterase, and the serum lysozyme level was elevated and diagnosed as monoblastic crisis. The patient received combined chemotherapy but response was partial. These findings are compatible with our previous report that nm23 gene is overexpressed in monocytic leukemia.

A novel uracil analog, 6-chloro-5-(2-propenyl)uracil, preferentially enhances growth inhibition and differentiation of myeloid leukemia cells induced by 1alpha,25-dihydroxyvitamin D3.

The novel uracil analog, 6-chloro-5-(2-propenyl)uracil (TI90), inhibited the growth of myeloid leukemia cells and induced morphologic and functional differentiation of the cells. Although TI90 was a weak inducer of differentiation, it greatly enhanced the growth inhibition and differentiation of the leukemia cells previously induced by 1alpha,25-dihydroxyvitamin D3 (VD3) or all-trans retinoic acid (ATRA). TI90 cooperated with VD3 much more effectively than with ATRA in inhibiting cell growth and inducing differentiation. It also decreased the effective concentration of VD3 to the 10(-10) M level. On the other hand, there was no significant synergy between VD3 and the other uracil analogs. TI90 did not affect VD3 metabolism or the number and affinity of VD3 receptors (VDR) in HL-60 cells. Signals from VD3 are predominantly mediated by VDR and the ligand-activated binding of VDR to vitamin D-responsive element (VDRE) as a heterodimer with the retinoid X receptor (RXR). According to the results of a gel shift assay, TI90 enhanced the intensity of the retarded band with synthetic VDRE oligomer in the presence of VD3, suggesting that TI90 increases the number of phosphorylated receptors by inhibiting phosphatase activity, and also stimulates the formation of a functional complex of VDR with RXR.

Differanisole A, a novel antitumor antibiotic, enhances growth inhibition and differentiation of human myeloid leukemia cells induced by 9-cis retinoic acid.

Differanisole A, 3,5-dichloro-2-hydroxy-4-methoxy-6-n-propylbenzoic acid, inhibited growth of human myeloid leukemia cells. The compound induced G1 arrest and granulocytic differentiation of HL-60 cells, although the differentiation-inducing effect was modest. Differanisole A and 9-cis retinoic acid (9cisRA) synergistically inhibited the growth and induced functional and morphologic differentiation of HL-60 and NB4 cells, whereas the combined treatment with differanisole A and all-trans retinoic acid or 1alpha,25-dihydroxyvitamin D3 was less effective. Similar results were obtained in primary culture of leukemia cells from a patient with acute promyelocytic leukemia. The synergistic effect on growth inhibition and induction of differentiation required simultaneous treatment with differanisole A and 9cisRA. Differanisole A and an RXR-specific ligand (Ro47-5944) cooperatively inhibited the cell growth, while the combined effect of differanisole A and an RAR-specific ligand Am80 was just additive. Differanisole A in combination with 9cisRA may have implications for therapy of acute promyelocytic leukemia patients.

Neplanocin A, a potent inhibitor of S-adenosylhomocysteine hydrolase, potentiates granulocytic differentiation of acute promyelocytic leukemia cells induced by all-trans retinoic acid.

Several neplanocin A analogs were synthesized and their growth-inhibiting and differentiation-inducing activities on myelogenous leukemia cells were examined. An adenosine kinase-ineffective analog of neplanocin A was effective in inducing differentiation, suggesting that phosphorylation of the nucleoside is not essential for inducing the differentiation of leukemia cells. Neplanocin A induced functional and morphological differentiation of HL-60 cells, but did not effectively induce differentiation of NB4, a cell line derived from a leukemia patient with t(15;17). However, these cells have been known to undergo granulocytic differentiation upon treatment with all-trans retinoic acid (ATRA), and are used as a model for differentiation therapy in acute promyelocytic leukemia. Preexposure of NB4 cells to low concentrations of neplanocin A greatly enhanced the ATRA-induced differentiation of the cells, whereas representative antileukemic drugs such as cytosine arabinoside and daunomycin did not enhance this differentiation. A clinical strategy that combines intermittent treatment with neplanocin A analogs and a low dose of ATRA may increase the clinical response and decrease the adverse effects of ATRA.

An aggressive nasal lymphoma accompanied by high levels of soluble Fas ligand.

Fas ligand (FasL), either in the membrane bound form or soluble form, has cytotoxic activity against Fas-expressing cells. We report a case of nasal lymphoma accompanied by liver damage and pancytopenia. The serum level of soluble FasL (sFasL) was very high on admission, but rapidly decreased to normal levels after chemotherapy for lymphoma. Liver damage and pancytopenia also improved with the decrease in serum sFasL. Since Fas is expressed on both hepatocytes and haemopoietic cells, these facts suggest that FasL was expressed on lymphoma cells and directly associated with pathogenesis of liver damage and pancytopenia through its cytotoxic activity.

Reversible differentiation of human monoblastic leukemia U937 cells by ML-9, an inhibitor of myosin light chain kinase.

Human monoblastic leukemia U937 cells are induced to differentiate into monocytes and macrophages by various agents. We have shown that 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-9), an inhibitor of myosin light chain kinase, induces differentiation of monocytoid leukemia cell lines U937 and THP-1 but not of myeloblastic leukemic ML-1 cell or erythroleukemia K562 cells. In the present study, we further analyzed the effect of ML-9 in comparison with that of 1 alpha, 25-dihydroxyvitamin D3 (VD3) a typical inducer of monocytic differentiation. ML-9 induced nitroblue tetrazolium (NBT)-reducing activity of U937 cell more rapidly than VD3: This differentiation marker was induced significantly after incubation with ML-9 and VD3 for 4 hours and 1 day, respectively. ML-9 also induced alpha-naphthyl acetate esterase (ANAE) activity, another monocytic differentiation marker, more rapidly than VD3. The maximum levels of these markers induced by ML-9 were comparable to those induced by VD3, but after removal of ML-9 from the medium by washing the cells, the expressions of theses markers decreased within 4 hours and reached basal levels in 1 day, indicating that ML-9's induction of expression of differentiation-associated phenotypes was reversible. The growth inhibition of U937 cells by ML-9 was also reversible. Similar effects were observed in another line of human monoblastic cells, THP-1. ML-9 had little or no effect on the morphology of U937 cells but increased the expression of monocyte-macrophage lineage-associated surface antigen, CD14, to some extent. Irreversible terminal differentiation induced by VD3 is associated with down regulation of the expression of c-myc and upregulation of the expression of c-fos and c-jun, but ML-9 did not affect the expression of these oncogenes appreciably. ML-9-induced differentiation was also reversible when the cells were cultured with cultured with ML-9 plus an anti-cancer drug such as 1-beta-D-arabino-furanosylcytosine or daunomycin. it became irreversible, however, upon simultaneous treatment with dexamethasone and transforming growth factor-beta 1 (TGF-beta 1), which did not induce differentiation of U937 cells but caused growth arrest of the cells in the G0/G1 phase of the cell cycle. These results suggest that ML-9 should be useful for studying the mechanisms of monocytic differentiation.

Enhancement of activity of 1alpha, 25-dihydroxyvitamin D3 for growth inhibition and differentiation induction of human myelomonocytic leukemia cells by tretinoin tocoferil, an alpha-tocopherol ester of all-trans retinoic acid.

Tretinoin tocoferil is an alpha-tocopherol ester of all-trans retinoic acid (RA) and safely used in the treatment of skin ulcer. Tretinoin tocoferil inhibited proliferation of human promyelocytic leukemia HL-60 cells and induced granulocytic differentiation of the cells, but less than RA. alpha-Tocopherol did not affect differentiation of HL-60 cells, but at high concentrations enhanced its nitroblue tetrazolium (NBT)-reducing activity and expression of surface antigen CD11b, which are markers of myelomonocytic differentiation induced by RA. Tretinoin tocoferil increased NBT reduction in HL-60 cells treated with RA. It also enhanced the differentiation of HL-60 cells induced by dimethyl sulfoxide, phorbol-12-myristate 13-acetate or 1alpha,25-dihydroxyvitamin D3 (VD3). In combination with a low concentration of VD3, it induced the NBT-reducing activity of human monoblastic U937 cells very effectively. Moreover, it enhanced the differentiation of human myelomonocytic ML-1, THP-1, P39/TSU, and P31/FUJ cells induced by VD3. In combination with VD3, it synergistically inhibited the proliferation of HL-60, U937, ML-1, THP-1, P39/TSU, and P31/FUJ cells and decreased the effective concentration of VD3 to a 10(-10) mol/L level. Because tretinoin tocoferil was reported to induce neither retinoid-related toxicity nor teratogenicity, the therapeutic advantage of the use of it in treatment of myelomonocytic leukemia is suggested.

Transforming growth factor beta and dexamethasone cooperatively enhance c-jun gene expression and inhibit the growth of human monocytoid leukemia cells.

Glucocorticoids inhibit the proliferation of lymphoid leukemia cells, whereas most myeloid leukemia cells are resistant to glucocorticoids. However, this study showed that glucocorticoids significantly and preferentially inhibited growth of monocytoid leukemia cells in combination with a low concentration of transforming growth factor beta (TGF beta). Combined 1 alpha,25-dihydroxyvitamin D3 and TGF beta markedly induced monocytic differentiation of U937 cells, whereas dexamethasone (Dex) and TGF beta essentially did not, although both combinations similarly inhibited the growth of U937 cells. The growth inhibition was accompanied by a block in the cell cycle progression from G1 to S phase (G1 arrest). Expression of glucocorticoid receptors was not affected by TGF beta, although they are induced during the monocytic differentiation of myelogenous leukemia cells and have increased sensitivity to glucocorticoids. The expression of TGF beta receptors also was not enhanced by Dex. TGF beta significantly stimulated glucocorticoid responsive element-mediated transcription activity. Combined Dex and TGF beta stimulated the expression of c-jun and c-fos early responsive genes in U937 cells, although Dex or TGF beta alone did not. The combination synergistically induced expression of c-jun gene, reaching a maximum level at 24 h. On the other hand, expression of c-fos gene was induced by TGF beta alone and increased additively in combination with Dex. Treatment with antisense oligonucleotide complementary to the first exon of c-jun mRNA reduced the growth-inhibitory effect of Dex and TGF beta in a dose-dependent manner. However, exposure of U937 cells to the sense oligomer of c-jun mRNA or an antisense oligomer of c-fos mRNA did not affect the growth inhibition. These results suggested that the preferential expression of c-jun and stimulation of glucocorticoid responsive element-mediated transactivation are closely associated with the growth arrest of U937 cells incubated with Dex and TGF beta.

Structural analysis of proteoglycan macrophage colony-stimulating factor.

Proteoglycan macrophage colony-stimulating factor (PG-M-CSF) was recently reported as a high molecular type of macrophage colony-stimulating factor (M-CSF). We analyzed its structure by determining the expression of mutant M-CSF cDNA in Chinese hamster ovary cells. PG-M-CSF contained two types of molecules, a homodimeric 150-200-kDa subunit and a heterodimeric form of a 43-kDa subunit and the 150-200-kDa subunit. The 150-200-kDa subunit carries a chondroitin sulfate chain, and its amino-terminal amino acid sequence was identical to that of the 43-kDa subunit, which is known to form the conventional M-CSF molecule (85-kDa M-CSF). The results obtained with the carboxyl-terminal deleted mutants showed that the PG-M-CSF-specific 150-200-kDa subunit had a large part of the precursor sequence at its carboxyl terminus removed in the 43-kDa subunit by proteolytic processing. The expression of mutagenized cDNA, in which Arg220 was replaced by an alanine residue, resulted in the disappearance of the 43-kDa subunit but not that of the 150-200-kDa subunit, indicating that Arg220-Pro-Pro-Arg is essential to process PG-M-CSF to 85-kDa M-CSF. Truncated mutation analysis showed that the carboxyl terminus of the 150-200-kDa subunit lay downstream of Arg412. We also showed that the chondroitin sulfate binding site in the 150-200-kDa subunit was Ser277, since conversion of Ser277 to the alanine residue resulted in complete loss of the chondroitin sulfate substitution.