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Biomedical subjects

D E Macfarlane

Publications and source records attributed to D E Macfarlane.

At least 19 recordsLinked to original sources
PubMed1990

Identification of phosphoprotein NP33 as a nucleus-associated ribosomal S6 protein and its phosphorylation in hematopoietic cells.

Exposure of HL-60 promyelocytes to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate increased incorporation of 32P into a Mr approximately 33,000 protein (NP33) found in the nuclear matrices prepared by treating cells with Triton X-100, nucleases, and 2 M NaCl (D. E. Macfarlane, J. Biol. Chem., 261: 6947-6953, 1986). We now report that 12-O-tetradecanoylphorbol-13-acetate causes phosphorylation of NP33 in U937, K562, HEL, Molt-3, and Raji cell lines, all of which are rapidly proliferating cells of hematopoietic origin. 12-O-Tetradecanoylphorbol-13-acetate caused a lesser degree of NP33 phosphorylation in peripheral blood lymphocytes and monocytes and none in granulocytes or platelets. The incorporation of 32P into NP33 was complete in about 10 min, and it was prevented or reversed by staurosporin, indicating that NP33 is continuously phosphorylated and dephosphorylated. NP33 was purified to homogeneity from Triton X-100-washed nuclei or whole cells by extraction with H2SO4, acetone precipitation, and preparative two-dimensional gel electrophoresis. The amino-terminal amino acid sequence of NP33 appears to be the same as that of ribosomal S6 protein. NP33 appears to be S6 protein copurifying with the nuclear matrix.

Alkaloids↗
PubMed1990

Xanthoma disseminatum and Waldenström's macroglobulinemia.

A 71-year-old woman had a 7-month history of multiple asymptomatic papules on the upper part of the body. The clinical picture and histopathologic findings confirmed the diagnosis of xanthoma disseminatum. Further studies revealed Waldenström's macroglobulinemia. Treatment with chlorambucil and prednisone led to a dramatic reduction in her paraprotein level. In addition, cutaneous lesions improved and new lesions stopped appearing. Treatment of selected papules by cryotherapy or intralesional corticosteroid injection provided further improvement. This case is notable because (1) it is the first report of xanthoma disseminatum in a patient with Waldenström's macroglobulinemia and (2) there was an unusually good response to therapy.

Aged↗
PubMed1990

P47 phosphoprotein of blood platelets (pleckstrin) is a major target for phorbol ester-induced protein phosphorylation in intact platelets, granulocytes, lymphocytes, monocytes and cultured leukaemic cells: absence of P47 in non-haematopoietic cells.

Aggregating agents including phorbol esters which activate protein kinase C induce the rapid phosphorylation of a Mr = 47,000 cytosolic protein in blood platelets (P47 or pleckstrin). This protein is well resolved by analytical 16-BAC----SDS two-dimensional PAGE and was purified from platelets by preparative 16-BAC----SDS PAGE. Polyclonal antibodies were raised to the protein in mice and rabbits. These antisera detected a single protein with the migration of P47 on Western blots of platelet extracts, and the rabbit antisera immunoprecipitated 32P-labelled P47 from platelet cytosol. The presence of P47 in other haematopoietic cells was determined by prelabelling them with 32P and observing increased 32P incorporation into the location of P47 on autoradiographs of 16-BAC----SDS analytical PAGE of cells exposed to phorbol ester. The identity of the phosphoprotein found in this location was further established by probing Western blots of SDS PAGE gels of cultured cell lines with the P47 antisera. P47 was detected in peripheral blood lymphocytes, monocytes and granulocytes (including the granulocytes of two unrelated patients with X-linked chronic granulomatous disease). P47 was also found in HL-60 promyelocytes (especially after differentiation with retinoic acid), U937 histiocytes, HEL leukaemia cells, and Raji 'B' lymphoblasts. It was not detected in normal erythrocytes, K562 leukaemic cells, MOLT-3 'T' lymphoblasts, or in wide range of non-haematopoietic cell lines. We conclude that P47 is a major target for the action of phorbol ester induced phosphorylation in platelets, normal leucocytes and some haematopoietic cell lines. These cells have as their common feature the ability when stimulated to develop adhesive functions on their plasma membranes.

Blood Platelets↗
PubMed1989

Absence of phorbol ester-induced down-regulation of myc protein in the phorbol ester-tolerant mutant of HL-60 promyelocytes.

The human promyelocytic leukemia cell line HL-60 has an amplified number of copies of the protooncogene c-myc. It is induced to differentiate by exposure to the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA). We have developed a mutant phorbol ester-tolerant (PET) line of HL-60 which undergoes a transient growth arrest but does not differentiate when exposed to TPA (Macfarlane et al., Br. J. Haematol., 68: 291-302, 1988). The defect is not due to a general failure of TPA-induced phosphorylation. In this paper, we show that exposing phorbol ester-sensitive (S) HL-60 cells to TPA caused the disappearance of the c-myc protein antigen (detected on Western blots) in 4 h, whereas TPA had no effect on the c-myc protein content of PET cells. Dimethyl sulfoxide caused the rapid disappearance of the myc antigen in both cells. PET cells had slightly more copies of the c-myc gene detected on Southern blots than S cells. c-myc mRNA was equally unstable in both cells, as determined by Northern blots following actinomycin D. TPA induced the down-regulation of c-myc mRNA in S cells to a greater extent than in PET cells. Dimethyl sulfoxide caused a rapid down-regulation of c-myc mRNA in both cell lines. This shows that PET cells have a defect in the mechanism by which protein kinase C regulates c-myc transcription. Our results provide further evidence that reduction in c-myc expression is necessary for differentiation to occur in HL-60 cells.

Blotting, Southern↗
PubMed1989

Two dimensional benzyldimethyl-n-hexadecylammonium chloride----sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution technique for the purification of proteins from complex mixtures.

A two dimensional preparative polyacrylamide gel electrophoresis system is described which has both high resolving power and high loading capacity (approximately 100 mg protein). The first dimension is electrophoresis toward the cathode at acid pH in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC), and the second dimension is electrophoresis toward the anode in the presence of sodium dodecyl sulfate. Proteins purified in this way have been sequenced and used successfully as antigens.

Amino Acid Sequence↗
PubMed1988

A phorbol ester tolerant (PET) variant of HL-60 promyelocytes.

The promyelocytic leukaemia cell line HL-60 differentiates to a macrophage-like cell when exposed to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) and other agents which activate protein kinase C. To investigate this phenomenon we developed an HL-60 variant which does not differentiate when exposed to TPA. HL-60 cells were exposed to the mutagen ethyl methanesulphonate and were cloned in soft agar in the presence of a normally lethal concentration of TPA. One colony of cells that proliferated in TPA was obtained. The cells of this phorbol ester tolerant (PET) line have retained their resistance to TPA for several years without selective pressure. They are somewhat larger than their phorbol ester sensitive (S) parent, but they are otherwise morphologically similar. When PET-cells are exposed to TPA their growth is arrested for approximately 48 h. Thereafter, they resume their original rate of replication at all concentrations of TPA tested. S-cells undergo changes typical of HL-60 when exposed to TPA; they aggregate, stop growing, adhere to the flask and die. The PET-cells appeared to be as sensitive as S-cells to other agents which differentiate HL-60 such as retinoic acid, dimethysulphoxide, and 1,25-dihydroxyvitamin D3, as determined by rate of proliferation in culture, Wright's stain, nitroblue tetrazolium reduction, and induction of the ectoenzyme NAD-glycohydrolase. TPA-induced protein phosphorylation was studied using one- and two-dimensional polyacrylamide gel electrophoresis. Several proteins increased their incorporation of 32P when S- and PET-cells were exposed to TPA, the most prominent of which were the two previously described nuclear matrix proteins of 80 kd and 33 kd. There was no difference in the protein phosphorylation pattern in S- and PET-cells, nor in how this pattern changed on TPA exposure. Fluorescent activated cell sorting and karyotypic analysis revealed PET-cells to be a hypotetraploid variant of S-cells, with approximately 80 chromosomes, including a marker chromosome iso(1p) not found in the S-cells. Identification of the biochemical lesion responsible for this TPA resistance in PET cells will provide clues concerning the mechanism of this important pathway for the induction of cell differentiation.

Cell Differentiation↗
PubMed1987

Depletion of arachidonic acid from GH3 cells. Effects on inositol phospholipid turnover and cellular activation.

We have adapted rat pituitary GH3 cells to grow in delipidated culture medium. In response, esterfied linoleic acid and arachidonic acid become essentially undetectable, whereas eicosa-5,8,11-trienoic acid accumulates and oleic acid increases markedly. These changes occur in all phospholipid classes, but are particularly pronounced in inositol phospholipids, where the usual stearate/arachidonate profile is replaced with oleate/eicosatrienoate (n - 9) and stearate/eicosatrienoate (n - 9). Incubation of arachidonate-depleted cells with 10 microM-arachidonic acid for only 24 h results in extensive remodelling of phospholipid fatty acids, such that close-to-normal compositions and arachidonic acid content are achieved for the inositol phospholipids. In comparison studies with arachidonic acid-depleted or -repleted cells, it was found that the arachidonate content does not affect thyrotropin-releasing-hormone (TRH)-stimulated responses measured at long time points, including [32P]Pi labelling of phosphatidylinositol and phosphatidic acid, stimulation of protein phosphorylation, and basal or TRH-stimulated prolactin release. However, transient events such as stimulated breakdown of inositol phospholipids and an initial rise in diacylglycerol are enhanced by the presence of arachidonate. These results show that arachidonic acid itself is not required for operation of the phosphatidylinositol cycle and is not an obligatory intermediate in TRH-mediated GH3 cell activation. It is possible that any structural or functional role of arachidonic acid in these processes is largely met by replacement with eicosatrienoate (n - 9). However, since arachidonate in inositol phospholipids facilitates their hydrolysis upon stimulation by TRH, arachidonic acid apparently may have a specific role in the recognition of these lipids by phospholipase C.

Animals↗
PubMed1987

Neonatal group B streptococcal septicaemia in a developing country.

During a 3-year study at the neonatal intensive care unit, University Hospital of the West Indies, the incidence of group B streptococcal (GBS) septicaemia was 13.6/1,000 admissions and 1.4/1,000 live births. GBS accounted for 35% of blood culture isolates and was the most frequent cause of septicaemia. Underlying disease or other possible predisposing factors were identified in 16 of 25 neonates with GBS septicaemia. Comparison of early onset and late onset disease indicated an increased incidence of prematurity, prolonged rupture of membranes, and respiratory distress in the former and a predominance of meningitis associated cases in the latter. Mortality was 50% for early onset cases, 29% for late onset cases and 36% overall. Antimicrobial therapy and preventive measures which may be appropriate for a developing country are discussed.

Developing Countries↗
PubMed1987

Cryptosporidiosis in well-nourished and malnourished children.

During a 5-month period, 513 stool samples submitted to the enteric laboratory at the University Hospital of the West Indies were examined for Cryptosporidium. Oocysts were detected in 4.9% of all stools, 7.3% of diarrhoeal stools, 19.5% of stools from malnourished children and 23.7% of stools from malnourished children with diarrhoea. Cryptosporidium was the sole pathogen detected in all 25 positive stools, and was the second most frequent enteric isolate. All cases of cryptosporidiosis occurred in children less than 2.5 years of age. All 15 malnourished children were admitted to hospital where they presented with dehydration (87%), vomiting (93%), fever (100%) and diarrhoea which lasted an average of 15.3 days. Two of these children died. In contrast, dehydration (20%), vomiting (40%) and fever (50%) were less common and diarrhoea less protracted in well-nourished children, four of whom were admitted to hospital. This preliminary report suggests that cryptosporidial gastroenteritis presents with increased frequency and severity in malnourished compared with well-nourished Jamaican children.

Child, Preschool↗
PubMed1986

Phorbol diester-induced phosphorylation of nuclear matrix proteins in HL60 promyelocytes. Possible role in differentiation studied by cationic detergent gel electrophoresis.

Immortal HL60 promyelocytes are induced to differentiate to mortal adherent cells by a variety of agents which activate protein kinase C, including 12-O-tetradecanoylphorbol 13-acetate (TPA). In order to investigate the mechanism of this effect, we incubated HL60 cells with [32P]orthophosphate with or without TPA and extracted their proteins with the cationic detergent benzyldimethyl-n-hexadecylammonium chloride prior to electrophoresis in a discontinuous polyacrylamide gel system in the first dimension. In this system, proteins migrate toward the cathode as a function of their molecular weight, and they are separated from other radioactive components which can obscure the pattern of protein phosphorylation on sodium dodecyl sulfate (SDS) gels. SDS gel electrophoresis was used in the second dimension, resulting in the clear resolution of a large number of proteins. TPA caused many changes in the pattern of protein phosphorylation in intact cells. Two proteins which prominently increased their incorporation of 32P were investigated in particular, and they were both found to be retained in the nuclear matrix following successive extraction of cells with Triton, digestion with DNase and RNase, and extraction with 2 M NaCl. These proteins migrated with apparent molecular weights of 80,000 and 33,000 on SDS gels, and are designated NP80 and NP33, respectively. NP80 was half-maximally phosphorylated after 7 min exposure to TPA, and half-maximally phosphorylated by 10 nM TPA. NP80 co-migrated with a faint Coomassie Blue-stained protein, and NP33 co-migrated with a more prominent protein. Several proteins incorporated less 32P when the cells were exposed to TPA, including one which was extracted from nuclei with the core histones and which co-migrated with histone H2A. Further study will be needed to determine whether the differentiation of HL60 induced by TPA is mediated via phosphorylation of these nuclear matrix proteins.

Antigens, Nuclear↗
PubMed1986

Multiresistant Salmonella ohio infections at the University Hospital of the West Indies.

During 1982-83 there was a substantial increase in the number of S. ohio infections at the University Hospital of the West Indies, which coincided with the appearance of strains resistant to chloramphenicol, cotrimoxazole, ampicillin, neomycin and carbenicillin. Multiresistant strains of S. ohio accounted for 19.3% of all salmonella isolates during this period and all of 40 strains tested were able to transfer resistance determinants to E. coli K12 J 53-2. S. ohio was cultured from stool (60), blood (5), wounds and abscesses (4) and postmortem material (2). Eighty-six per cent of S. ohio infections occurred in children of 3 years old or less. There was a high incidence of gastroenteritis in malnourished children, a 14% incidence of localizing infections and a 7% incidence of septicaemia. Two infants with severe gastroenteritis and bronchopneumonia died. There were a number of unusual infections including two cases of septicaemia in children receiving chloramphenicol for Haemophilus influenzae meningitis, a scrotal abscess secondary to extravasation of urine and infected scabies in a child with marasmic kwashiorkor.

Adolescent↗
PubMed1985

Bacteraemia at the University Hospital of the West Indies--a report of 222 cases.

A total of 222 cases of septicaemia was recorded at the University Hospital of the West Indies between June 1982 and June 1983. This gave an overall incidence of 16.1 per 1000 admissions. The 233 bacterial strains isolated comprised 100 Gram-positive and 133 Gram-negative organisms with Klebsiella pneumoniae, Streptococcus pneumoniae and Staphylococcus aureus being the most frequent. Highest rates of septicaemia were recorded in patients less than 1 year and over 50 years of age. Septicaemia caused by Gram-positive organisms was predominantly a disease of children whereas that caused by Gram-negative organisms arose more often in neonates and in patients over 50 years of age. A predisposing factor was noted in 104 patients of whom 42 had neoplastic disease. The most frequently identified initial sites of infection were the respiratory tract, the gastro-intestinal tract and the meninges. Most blood stream infections were community-acquired, three quarters of all septicaemic patients being admitted to the departments of medicine or paediatrics. There were 11 cases of polymicrobial septicaemia caused predominantly by Gram-negative organisms in patients with underlying disease. Appropriate antimicrobial drugs were administered to 57% of septicaemic patients whereas 17% received superfluous antimicrobial therapy. In those patients who received inappropriate antimicrobial therapy there was a marked increase in mortality. Forty of 61 deaths were attributed to septicaemia. Mortality from septicaemia caused by Gram-negative organisms was 21% compared with 13% for that caused by Gram-positive organisms. The organisms associated with the highest case fatality rates were Escherichia coli, 53%; Enterobacter sp., 27%; and beta-haemolytic streptococci 24%. There were no deaths from septicaemia caused by Haemophilus influenzae, Salmonella sp. or Serratia sp. The highest mortality rates were associated with neoplastic disease, diabetes, polymicrobial septicaemia, urinary tract infections and old age.

Adolescent↗