Positional cloning without a genome map: using 'Targeted RFLP Subtraction' to isolate dense markers tightly linked to the regA locus of Volvox carteri.

The ability to isolate genes defined by mutant phenotypes has fueled the rapid progress in understanding basic biological mechanisms and the causes of inherited diseases. Positional cloning, a commonly used method for isolating genes corresponding to mutations, is most efficiently applied to the small number of model organisms for which high resolution genetic maps exist. We demonstrate a new and generally applicable positional cloning method that obviates the need for a genetic map. The technique is based on Restriction Fragment Length Polymorphism (RFLP) Subtraction, a method that isolates RFLP markers spanning an entire genome. The new method, Targeted RFLP Subtraction (TRS), isolates markers from a specific region by combining RFLP Subtraction with a phenotypic pooling strategy. We used TRS to directly isolate dense markers tightly linked to the regA gene of the eukaryotic green alga Volvox. As a generally applicable method for saturating a small targeted region with DNA markers, TRS should facilitate gene isolation from diverse organisms and accelerate the process of physically mapping specific regions in preparation for sequence analysis.     

Ultrastructural visualization of exocytosis in the pig pineal gland

Summary The pinealocytes of the pig contain conspicuous dense bodies, the nature and role of which are not yet fully elucidated. The aim of this study was to demonstrate whether or not these structures are involved in the secretion process. The tannic acid-Ringer incubation (TARI)-method, which allows a clear-cut ultrastructural study of secretory discharge by exocytosis, has been used. The results indicate that pig pinealocytes release the content of the dense bodies with an amorphous inner structure into the extracellular space via exocytosis and that this secretion is quantitatively important. The secreted material is proteinaceous in nature; this indicates that polypeptides are released by the pineal.     

Transient diabetes mellitus with ketoacidosis in a child during the treatment of acute lymphoblastic leukemia with L-asparaginase]

A case of a 11-year girl with the acute lymphoblastic leukemia is presented. Patient was treated with L-asparaginase and developed a transient but lasting several weeks diabetes mellitus with ketoacidosis as a sequelae of this therapy.     

Electrophoretic seed albumin patterns inVicia species of sectt.Hypechusa andPeregrinae (Fabaceae)

This work is a continuation of electrophoretic investigations aimed at revealing a wild relative ofVicia faba. Electrophoretic analysis (PAGE) of seed albumins covered 52 accessions representing eightVicia species of sect.Hypechusa and two species of sect.Peregrinae. Most of the examined species showed an intraspecific variation due to differences between accessions and/or individual variation within accessions. In spite of the intraspecific variation, marked interspecific differences were recorded. However, none of the investigated species displayed electrophoretic seed albumin patterns similar to those reported earlier forV. faba. Contribution of the obtained results to characterization of the examined taxa is discussed.     

Morphology of the pig's pineal gland from 8th to 16th week of foetal life

Pineal glands of the pig from the 8th to the 16th week of foetal life were examined by means of both light and electron microscopy, and changes in the pineal structure were observed. The pinealocyte structure manifests the secretory activity of the pineal gland at the time of examination.     

Avian thrombocyte thrombospondin

A high molecular weight glycoprotein (450,000) was obtained from thrombin-treated duck thrombocytes by barium citrate adsorption technique followed by heparin-agarose affinity chromatography. Amino acid composition (high number of acidic amino acids and cystine) as well as carbohydrate contents (1.3 per cent hexosamine, 0.9 per cent sialic acid and 1.5 per cent hexose) showed similarity to mammalian platelet thrombospondin.     

Crystal structure to 2.45 A resolution of a monoclonal Fab specific for the Brucella A cell wall polysaccharide antigen.

The atomic structure of an antibody antigen-binding fragment (Fab) at 2.45 A resolution shows that polysaccharide antigen conformation and Fab structure dictated by combinatorial diversity and domain association are responsible for the fine specificity of the Brucella-specific antibody, YsT9.1. It discriminates the Brucella abortus A antigen from the nearly identical Brucella melitensis M antigen by forming a groove-type binding site, lined with tyrosine residues, that accommodates the rodlike A antigen but excludes the kinked structure of the M antigen, as envisioned by a model of the antigen built into the combining site. The variable-heavy (VH) and variable-light (VL) domains are derived from genes closely related to two used in previously solved structures, M603 and R19.9, respectively. These genes combine in YsT9.1 to form an antibody of totally different specificity. Comparison of this X-ray structure with a previously built model of the YsT9.1 combining site based on these homologies highlights the importance of VL:VH association as a determinant of specificity and suggests that small changes at the VL:VH interface, unanticipated in modeling, may cause significant modulation of binding-site properties.     

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.