Deletions of portions of chromosomes 1p and 19q are closely associated with the oligodendroglioma histologic phenotype. In most cases, 1p and 19q are codeleted, yet the mechanism of dual loss is unexplained. We report 5 cases (World Health Organization grade III) in which metaphase cytogenetics identified a derivative chromosome consisting of what appears to be the whole arms of 1q and 19p forming a der(1;19)(q10;p10). Metaphase fluorescent in situ hybridization (FISH) confirmed the derivative chromosome was composed of 1q and 19p material in 3 cases; in 2 cases with few metaphases, FISH confirmed 19p material on the derivative chromosome. In all cases, interphase FISH showed net loss of 1p and 19q in 77% to 92% of cells, and microsatellite studies were consistent with 1p and 19q loss. We hypothesize the following: occurrence of a balanced whole-arm translocation between chromosomes 1 and 19 forming 2 derivative chromosomes, one composed of 1q and 19p, the other of 1p and 19q. Subsequent loss of the der(1;19)(p10;q10) then results in the simultaneous 1p and 19q loss observed in oligodendroglioma with retention of the der(1;19)(q10;p10) seen in these cases.

The myristoylated alanine-rich C kinase substrate (MARCKS) protein, a prominent cellular substrate for protein kinase C, is associated with membranes in various cell types. MARCKS is myristoylated at its amino terminus; this modification is thought to play the major role in anchoring MARCKS to cellular membranes. Recent studies have suggested that the protein's basic phosphorylation site/calmodulin binding domain may also be involved in the membrane association of MARCKS through electrostatic interactions. The present studies used mutations in the primary structure of the protein to investigate the nature of the association between MARCKS and cell membranes. In chick embryo fibroblasts, activation of protein kinase C led to a decrease in MARCKS membrane association as determined by cell fractionation techniques. Cell-free assays revealed that nonmyristoylated MARCKS exhibited almost no affinity for fibroblast membranes, despite readily demonstrable binding of the wild-type protein. Similar experiments in which the four serines in the phosphorylation site domain were mutated to aspartic acids, mimicking phosphorylation, decreased, but did not eliminate, membrane binding when compared to either the wild-type protein or a comparable tetra-asparagine mutant. Addition of calmodulin in the presence of Ca2+ also inhibited binding of the wild-type protein to membranes, presumably by neutralizing the phosphorylation site domain, or by physically interfering with its membrane association. Surprisingly, expression of a nonmyristoylatable mutant form of MARCKS in intact cells led to only a 46% decrease in its plasma membrane association, as determined by cell fractionation and immunoelectron microscopy. These results are consistent with a complex model of the interaction of MARCKS with cellular membranes, in which the myristoyl moiety, the positively charged phosphorylation site domain, and possibly other domains make independent contributions to membrane binding in intact cells.

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The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a widely expressed, prominent substrate for protein kinase C. MARCKS is largely associated with membranes in cells, and hydrophobic interactions involving the amino-terminal myristoyl moiety are thought to play a role in anchoring MARCKS to cellular membranes. In addition, experiments in cell-free systems have suggested that electrostatic interactions between the positively charged phosphorylation site/calmodulin binding domain (PSD) of MARCKS and negatively charged membrane lipids are also involved in this association. Although it has been inferred from phosphorylation experiments, the electrostatic nature of the interaction between the PSD and membranes has not been demonstrated directly in intact cells. We expressed human MARCKS mutated in the myristoylation site and the PSD in REF52 cells; the cells were then fractionated by ultracentrifugation. Both nonmyristoylatable MARCKS and MARCKS in which the four serines in the PSD were mutated to aspartic acids, mimicking phosphorylation, exhibited decreased membrane affinity when compared to the fully myristoylated, wild-type, tetra-Ser protein or a myristoylated, tetra-Asn mutant. A double mutant, nonmyristoylatable protein in which the four serines in the PSD were mutated to aspartic acids exhibited negligible membrane association. Similar results were obtained in 293 cells that stably expressed chicken MARCKS mutated in the same domains. The double mutant, nonmyristoylatable tetra-Asp chicken protein exhibited little membrane association as determined by both subcellular fractionation and immunoelectron microscopy. These results indicate that myristoylation and electrostatic interactions involving the PSD exert independent, essentially additive effects on the membrane association of MARCKS in intact cells.

The myristoylated alanine-rich C kinase substrate, or MARCKS protein, is a widely expressed, prominent substrate for protein kinase C. Although the exact function of MARCKS has not been elucidated, targeted disruption of the MARCKS gene (Macs) in mice has shown that MARCKS plays a crucial role in the development of the central nervous system. Mice deficient in MARCKS exhibited universal perinatal death with defects in neurulation, fusion of the cerebral hemispheres, formation of the great forebrain commissures, and retinal and cortical lamination (Stumpo et al., Proc. Natl. Acad. Sci. USA 92, 944-948, 1995). In the present studies, a transgene consisting of approximately 3.4 kb of promoter from the human MARCKS gene (MACS), with an epitope tag sequence inserted at the carboxyl terminus of the MARCKS coding region, was able to complement completely MARCKS deficiency in mice. Thus, the human transgene contained all of the elements necessary for normal developmental expression of MARCKS. To test the importance of MARCKS myristoylation to its developmental role, an otherwise identical transgene was constructed in which the glycine at the amino terminus of MARCKS was mutated to an alanine. This mutation, which resulted in the expression of nonmyristoylated MARCKS, was successful in partially rescuing the Macs null phenotype. Specifically, about 25% of these mice survived the perinatal period; these survivors appeared to develop normally except for slightly decreased body size. In both the survivors and the nonsurvivors, all of the known anatomical defects associated with MARCKS deficiency were corrected by expression of the nonmyristoylated human protein. These results indicate that myristoylation of MARCKS is not required for the protein to correct many of the developmental abnormalities characteristic of its deficiency.

Screening patients at high risk of glucose-6-phosphate dehydrogenase (G6PD) deficiency (individuals of African and Mediterranean descent) is recommended prior to initiation of rasburicase. However, a high index of suspicion of G6PD deficiency is also crucial in a patient who develops methaemoglobinaemia or haemolytic anaemia following rasburicase therapy, irrespective of ethnic origin. We report a 56-year-old Hispanic male with mantle cell lymphoma with bulky lymphadenopathy who presented to the emergency department with dyspnoea and chest discomfort. Forty-eight hours prior to presentation, he had received chemotherapy with R-CHOP (rituximab, cyclophosphamide, doxorubicin, vincristine, prednisone) with pegfilgrastim support and two 6 mg doses of intravenous rasburicase for the prevention of tumour lysis syndrome. Physical examination was unremarkable. Oxygen saturation (SpO 2 ) was 85% on 100% oxygen via a non-rebreather mask at 10 l/min. Arterial blood gases showed a partial oxygen pressure (PaO 2 ) of 216 mmHg. A full blood count showed haemoglobin concentration (Hb) 93 g/l and white blood cell count 77Á9 9 10 9 /l (0Á8% lymphocytes). Serum creatinine was 73Á37 lmol/l, uric acid 35Á69 lmol/l and methaemoglobin 8Á4%. Because of the presence of methaemoglobinaemia, intravenous methylene blue was administered. Eight hours later, the patient complained of diaphoresis and lightheadedness. SpO 2 had dropped to 80% and Hb to 66 g/l. Total and direct bilirubin were 41Á05 and 6Á84 lmol/l respectively, haptoglobin was less than 10 mg/l and lactate dehydrogenase was elevated at 864 u/l (normal range 94-202), consistent with acute haemolytic anaemia. A peripheral blood film showed irregularly contracted cells and retraction of haemoglobin from the red cell membrane (left). Heinz bodies were demonstrated on supravital staining with new methylene blue (right). The patient was managed with supportive treatment and four units of packed red blood cells were transfused. By 24 h, methaemoglobin levels had dropped to zero. The suspicion of G6PD deficiency was confirmed by enzyme assay with an enzyme level of 1Á5 u/g Hb (8Á8-13Á4). G6PD deficiency is associated with NADPH deficiency, and methylene blue is dependent on NADPH to reduce methaemoglobin to haemoglobin. This renders methylene blue ineffective in G6PD-deficient patients, and it may aggravate methaemoglobinaemia at high doses by oxidizing haemoglobin. Its use should be avoided.

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Context:Pathologists grade follicular lymphoma (FL) cases by selecting 10, random high power fields (HPFs), counting the number of centroblasts (CBs) in these HPFs under the microscope and then calculating the average CB count for the whole slide. Previous studies have demonstrated that there is high inter-reader variability among pathologists using this methodology in grading.Aims:The objective of this study was to explore if newly available digital reading technologies can reduce inter-reader variability.Settings and Design:In this study, we considered three different reading conditions (RCs) in grading FL: (1) Conventional (glass-slide based) to establish the baseline, (2) digital whole slide viewing, (3) digital whole slide viewing with selected HPFs. Six board-certified pathologists from five different institutions read 17 FL slides in these three different RCs.Results:Although there was relative poor consensus in conventional reading, with lack of consensus in 41.2% of cases, which was similar to previously reported studies; we found that digital reading with pre-selected fields improved the inter-reader agreement, with only 5.9% lacking consensus among pathologists.Conclusions:Digital whole slide RC resulted in the worst concordance among pathologists while digital whole slide reading selected HPFs improved the concordance. Further studies are underway to determine if this performance can be sustained with a larger dataset and our automated HPF and CB detection algorithms can be employed to further improve the concordance.