Phil Marsh

Glycogen synthase kinase-3beta phosphorylation of MAP1B at Ser1260 and Thr1265 is spatially restricted to growing axons.

Recent experiments show that the microtubule-associated protein (MAP) 1B is a major phosphorylation substrate for the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) in differentiating neurons. GSK-3β phosphorylation of MAP1B appears to act as a molecular switch regulating the control that MAP1B exerts on microtubule dynamics in growing axons and growth cones. Maintaining a population of dynamically unstable microtubules in growth cones is important for axon growth and growth cone pathfinding. We have mapped two GSK-3β phosphorylation sites on mouse MAP1B to Ser1260 and Thr1265 using site-directed point mutagenesis of recombinant MAP1B proteins, in vitro kinase assays and phospho-specific antibodies. We raised phospho-specific polyclonal antibodies to these two sites and used them to show that MAP1B is phosphorylated by GSK-3β at Ser1260 and Thr1265 in vivo. We also showed that in the developing nervous system of rat embryos, the expression of GSK-3β phosphorylated MAP1B is spatially restricted to growing axons, in a gradient that is highest distally, despite the expression of MAP1B and GSK-3β throughout the entire neuron. This suggests that there is a mechanism that spatially regulates the GSK-3β phosphorylation of MAP1B in differentiating neurons. Heterologous cell transfection experiments with full-length MAP1B, in which either phosphorylation site was separately mutated to a valine or, in a double mutant, in which both sites were mutated, showed that these GSK-3β phosphorylation sites contribute to the regulation of microtubule dynamics by MAP1B.

Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis

The contribution of cis-regulatory mutations to human disease remains poorly understood. Whole-genome sequencing can identify all noncoding variants, yet the discrimination of causal regulatory mutations represents a formidable challenge. We used epigenomic annotation in human embryonic stem cell (hESC)-derived pancreatic progenitor cells to guide the interpretation of whole-genome sequences from individuals with isolated pancreatic agenesis. This analysis uncovered six different recessive mutations in a previously uncharacterized ∼400-bp sequence located 25 kb downstream of PTF1A (encoding pancreas-specific transcription factor 1a) in ten families with pancreatic agenesis. We show that this region acts as a developmental enhancer of PTF1A and that the mutations abolish enhancer activity. These mutations are the most common cause of isolated pancreatic agenesis. Integrating genome sequencing and epigenomic annotation in a disease-relevant cell type can thus uncover new noncoding elements underlying human development and disease.

E-cadherin Interactions Regulate β-Cell Proliferation in Islet-like Structures

Islet function is dependent on cells within the islet interacting with each other. E-cadherin (ECAD) mediates Ca2+-dependent homophilic cell adhesion between b-cells within islets and has been identified as a tumour suppressor. We generated clones of the MIN6 β-cell line that stably over- (S) and under-express (αS) ECAD. Modified expression of ECAD was confirmed by quantitative RT-PCR, immunoblotting and immunocytochemistry. Preproinsulin mRNA, insulin content and basal rates of insulin secretion were higher in S cells compared to aS and control (V) cells. However, stimulated insulin secretory responses were unaffected by ECAD expression levels. ECAD expression did affect proliferation, with enhanced ECAD expression being associated with reduced proliferation and vice versa. Formation of islet-like structures was associated with a significant reduction in proliferation of V and S cells but not αS cells. These data suggest that ECAD expression levels do not modulate insulin secretory function but are consistent with a role for ECAD in the regulation of β-cell proliferation.

Neonatal and Late-Onset Diabetes Mellitus Caused by Failure of Pancreatic Development: Report of 4 More Cases and a Review of the Literature

OBJECTIVE. Permanent neonatal diabetes mellitus caused by developmental failure of the pancreas is rare. Thus far, only a few genetic causes have been reported. We now report the clinical and genetic aspects of 4 more cases of permanent neonatal diabetes mellitus caused by pancreatic agenesis or hypoplasia. PATIENTS AND METHODS. All 4 of the patients were from consanguineous kinships, and all presented with diabetes mellitus and pancreatic exocrine insufficiency. Three patients had pancreatic agenesis, and 1 had pancreatic hypoplasia on computed tomography scan. DNA was extracted from blood samples of patients and unaffected family members. Specific genes were amplified by polymerase chain reaction and characterized by DNA sequencing. RESULTS. Several genes that encode transcription factors that have known roles in pancreas development were characterized in the affected children and unaffected family members. These genes include Pdx1, the master regulator of pancreas development and β-cell differentiation, and other transcription factors that are expressed early in pancreas development, namely, Ptf1a, Sox9, Sox17, Hnf6, and HlxB9. Several novel polymorphisms were found in our patients. However, these were also present in unaffected individuals. No disease-causing mutations were found in any of these genes. CONCLUSIONS. These findings add to the 4 cases already in the literature in which the Pdx1 structural gene has been found to be normal in patients with pancreatic agenesis or hypoplasia. The analysis here has been extended to include the screening of 4 other candidate genes in addition to promoter elements upstream of the Pdx1. Two of the cases occurred in a sibling pair, and 2 were isolated, so there may be more than 1 etiology in the cases reported here.

Screening of a HUVEC cDNA library with transplant‐associated coronary artery disease sera identifies RPL7 as a candidate autoantigen associated with this disease

A HUVEC cDNA library was screened with sera from two patients who had developed transplant‐associated coronary artery disease (TxCAD) following cardiac transplantation. A total of six positive clones were isolated from a primary screen of 40 000 genes. Subsequent DNA sequence analysis identified these to be lysyl tRNA synthetase, ribosomal protein L7, ribosomal protein L9, β transducin and TANK. Another gene whose product could not be identified showed homology to a human cDNA clone (DKFZp566M063) derived from fetal kidney. Full‐length constructs of selected genes were expressed as his‐tag recombinant fusion proteins and used to screen a wider patient base by ELISA to determine prevalence and association with TxCAD. Of these ribosomal protein L7 showed the highest prevalence (55·6%) with TxCAD sera compared to 10% non‐CAD.

Eukaryotic integral membrane protein expression utilizing the Escherichia coli glycerol-conducting channel protein (GlpF).

A fusion protein expression system is described that allows for production of eukaryotic integral membrane proteins in Escherichia coli (E. coli). The eukaryotic membrane protein targets are fused to the C terminus of the highly expressed E. coli inner membrane protein, GlpF (the glycerol-conducting channel protein). The generic utility of this system for heterologous membrane-protein expression is demonstrated by the expression and insertion into the E. coli cell membrane of the human membrane proteins: occludin, claudin 4, duodenal ferric reductase and a J-type inwardly rectifying potassium channel. The proteins are produced with C-terminal hexahistidine tags (to permit purification of the expressed fusion proteins using immobilized metal affinity chromatography) and a peptidase cleavage site (to allow recovery of the unfused eukaryotic protein).

Involvement of Tis11b, an AU-rich binding protein, in induction of apoptosis by rituximab in B cell chronic lymphocytic leukemia cells

B-cell chronic lymphocytic leukemia is the commonest form of leukemia in the Western world, characterized by an accumulation of monoclonal CD5+ B cells in the peripheral blood and lymphoid organs. It is clinically a heterogenous disease with overall variable response to many drugs used either alone or in combination, and is currently untreatable.

Post-Transcriptional Regulation of BCL2 mRNA by the RNA-Binding Protein ZFP36L1 in Malignant B Cells

The human ZFP36 zinc finger protein family consists of ZFP36, ZFP36L1, and ZFP36L2. These proteins regulate various cellular processes, including cell apoptosis, by binding to adenine uridine rich elements in the 3′ untranslated regions of sets of target mRNAs to promote their degradation. The pro-apoptotic and other functions of ZFP36 family members have been implicated in the pathogenesis of lymphoid malignancies. To identify candidate mRNAs that are targeted in the pro-apoptotic response by ZFP36L1, we reverse-engineered a gene regulatory network for all three ZFP36 family members using the ‘maximum information coefficient’ (MIC) for target gene inference on a large microarray gene expression dataset representing cells of diverse histological origin. Of the three inferred ZFP36L1 mRNA targets that were identified, we focussed on experimental validation of mRNA for the pro-survival protein, BCL2, as a target for ZFP36L1. RNA electrophoretic mobility shift assay experiments revealed that ZFP36L1 interacted with the BCL2 adenine uridine rich element. In murine BCL1 leukemia cells stably transduced with a ZFP36L1 ShRNA lentiviral construct, BCL2 mRNA degradation was significantly delayed compared to control lentiviral expressing cells and ZFP36L1 knockdown in different cell types (BCL1, ACHN, Ramos), resulted in increased levels of BCL2 mRNA levels compared to control cells. 3′ untranslated region luciferase reporter assays in HEK293T cells showed that wild type but not zinc finger mutant ZFP36L1 protein was able to downregulate a BCL2 construct containing the BCL2 adenine uridine rich element and removal of the adenine uridine rich core from the BCL2 3′ untranslated region in the reporter construct significantly reduced the ability of ZFP36L1 to mediate this effect. Taken together, our data are consistent with ZFP36L1 interacting with and mediating degradation of BCL2 mRNA as an important target through which ZFP36L1 mediates its pro-apoptotic effects in malignant B-cells.

MECP2 mutations in Serbian Rett syndrome patients.

Background –  Rett syndrome is a severe neurodevelopmental X‐linked dominant disorder affecting 1/15,000 girls worldwide. Eight years ago, the MECP2 gene was associated with the devastating clinical features observed in Rett syndrome patients.