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Analysis of the human Rab6A gene structure reveals the presence of a duplicated exon, and incorporation of either of the two exons by alternative splicing is shown to generate two Rab6 isoforms named Rab6A and Rab6A', which differ in only three amino acid residues located in regions flanking the PM3 GTP-binding domain of the proteins. These isoforms are ubiquitously expressed at similar levels, exhibit the same GTP-binding properties, and are localized to the Golgi apparatus. Overexpression of the GTP-bound mutants of Rab6A (Rab6A Q72L) or Rab6A' (Rab6A' Q72L) inhibits secretion in HeLa cells, but overexpression of Rab6A' Q72L does not induce the redistribution of Golgi proteins into the endoplasmic reticulum. This suggests that Rab6A' is not able to stimulate Golgi-to-endoplasmic reticulum retrograde transport, as described previously for Rab6A. In addition, Rab6A' interacts with two Rab6A partners, GAPCenA and "clone 1," but not with the kinesin-like protein Rabkinesin-6, a Golgi-associated Rab6A effector. Interestingly, we found that the functional differences between Rab6A and Rab6A' are contingent on one amino acid (T or A at position 87). Therefore, limited amino acid substitutions within a Rab protein introduced by alternative splicing could represent a mechanism to generate functionally different isoforms that interact with distinct sets of effectors.

Molecular pathology is an integral part of daily diagnostic pathology and used for classification of tumors, for prediction of prognosis and response to therapy, and to support treatment decisions. For these reasons, analyses in molecular pathology must be highly reliable and hence external quality assessment (EQA) programs are called for. Several EQA programs exist to which laboratories can subscribe, but they vary in scope, number of subscribers, and execution. The guideline presented in this paper has been developed with the purpose to harmonize EQA in molecular pathology. It presents recommendations on how an EQA program should be organized, provides criteria for a reference laboratory, proposes requirements for EQA test samples, and defines the number of samples needed for an EQA program. Furthermore, a system for scoring of the results is proposed as well as measures to be taken for poorly performing laboratories. Proposals are made regarding the content requirements of an EQA report and how its results should be communicated. Finally, the need for an EQA database and a participant manual are elaborated. It is the intention of this guideline to improve EQA for molecular pathology in order to provide more reliable molecular analyses as well as optimal information regarding patient selection for treatment.

No abstract

In order to identify the mucins synthesized and secreted in the rat colon, we studied their biochemical characteristics and biosynthesis and evaluated their analogy to human colonic mucins. Purified mucin from both species appeared similar with respect to composition, buoyant density and mobility on SDS/PAGE. Isolated rat colonic mucin (RCM) was used to elicit a polyclonal antiserum, which was used in metabolic labelling studies to identify mucins and mucin precursors. RCM is synthesized as a 600 kDa precursor protein, which oligomerizes before O-glycosylation. The mature, high-molecular mass mucin is secreted and displays an anomalous molecular mass on SDS/PAGE of approximately 650 kDa. Polymorphism in precursor size was found among different rats, suggesting genetic heterogeneity. Molecular mass, biosynthesis and secretion of RCM appeared similar to human MUC2. Moreover, RCM precursor could be immunoprecipitated using specific anti-(human MUC2) antisera, indicating that the RCM can be designated rat MUC2. This study describes the biosynthesis of two homologous mucins in two different species. The high degree of similarity suggests functional analogy.