BSTRACTMeiosis I (MI), the division that generates haploids, is prone to errors that lead to aneuploidy in females. Haspin is a kinase that phosphorylates histone H3 on threonine 3, thereby recruiting Aurora kinase B (AURKB) and the chromosomal passenger complex (CPC) to kinetochores to regulate mitosis. Haspin and AURKC, an AURKB homolog, are enriched in germ cells, yet their significance in regulating MI is not fully understood. Using inhibitors and overexpression approaches, we show a role for haspin during MI in mouse oocytes. Haspin-perturbed oocytes display abnormalities in chromosome morphology and alignment, improper kinetochoremicrotubule attachments at metaphase I and aneuploidy at metaphase II. Unlike in mitosis, kinetochore localization remained intact, whereas the distribution of the CPC along chromosomes was absent. The meiotic defects following haspin inhibition were similar to those observed in oocytes where AURKC was inhibited, suggesting that the correction of microtubule attachments during MI requires AURKC along chromosome arms rather than at kinetochores. Our data implicate haspin as a regulator of the CPC and chromosome segregation during MI, while highlighting important differences in how chromosome segregation is regulated between MI and mitosis.

Numerous proteins are modified by sumoylation in human sperm; excessive sumoylation is a marker of defective spermatozoa.

Small ubiquitin-like modifiers (SUMO) proteins have been implicated in cellular stress response in different tissues, but whether sumoylation has a similar role during spermatogenesis is currently unknown. In this study, changes in the levels of both free SUMO isoforms and high-molecular weight (HMW) SUMO conjugates were monitored before and after the induction of different types of cellular stresses. Using cell lines and primary cells freshly isolated from mouse testes, significant changes were detected in the levels of SUMO1 and SUMO2/3 conjugates following short exposure of the cells to heat stress and oxidative stress. While high concentrations of H 2 O 2 caused an increase in protein sumoylation, low concentrations of H 2 O 2 mostly caused protein desumoylation. Immunofluorescence studies localized SUMO to the sites of DNA double-strand breaks in stressed germ cells and during meiotic recombination. To study the effect of oxidative stress in vivo, animals exposed to tobacco smoke for 12 weeks were used. Changes in sumoylation of HMW proteins were consistent with their oxidative damage in the tobacco-exposed mice. Our results are consistent with the important roles of different SUMO isoforms in stress responses in germ cells. Furthermore, this study identified topoisomerase 2 a as one of the targets of sumoylation during normal spermatogenesis and under stress.

A new radiopharmaceutical, 99m Tc-Tetraethylenepentamine(TEPA)-Folate has been synthesized introducing TEPA to the γ-carboxyl group of folic acid. This binds 99m Tc with high efficiency at ambient temperature. The resulting 99m Tc-N 5 -Folate is stable under physiological conditions at least for 24 h after radiocomplexation. TEPA is a known open chain pentamine (N 5 ) chelator, its four-nitrogen act as the binding site for 99m Tc. The folate membrane receptor binding of the 99m Tc-TEPA-Folate by established human tumor cell lines (KB, U-87MG and MDA-MB-468) showed K d in µM range in normal DMEM (10% serum, 10 µM folic acid). The blood kinetic studies showed more than 70% clearance within five minutes from the circulation. The KB cell line tumors in mice were readily identifiable in the γ images and revealed major accumulation of radiotracer in liver, kidneys and intestines. High tumor uptake was shown in the tumor bearing nude mice; tumorto-blood ratios reached 2.68 ± 0.52 and 5.5 ± 1.47 at 1 and 4 h after post injection respectively. Surviving fractions as obtained in clonogenic assay were 1.02 ± 0.07 and 1.03 ± 0.05 in U-87MG and MDA-MB-468 cell lines respectively. The 99m Tc-N 5 -Folate conjugate have promising utility as a receptor specific radiopharmaceutical for imaging neoplastic tissues known to over express folate-binding protein. INTRODUCTIONFolate receptor system is an interesting molecular target for selective targeted delivery of drugs. 1-5 Overexpression of folate receptor in a wide variety of tumors, including breast, cervical, colorectal, renal, nasopharyngeal and ovarian cancers, 6-8 has prompted search for promising folate-conjugated radiometal chelates as potential imaging and therapeutic agents. Conjugates of folic acid are preferred targeting ligands to radiolabel monoclonal antibody against folate receptors mainly due to their small size, lack of immunogenicity, rapid clearance from the blood stream and well-defined simple conjugation chemistry. Folic acid conjugates have been shown to bind the folate receptors with high affinity and are taken up by tumor cells via folate receptor mediated endocytosis. 9-13 Folate-conjugation, thus presents a useful method for receptor-mediated drug delivery into receptor-positive tumor cells. Radiolabeled folate and their conjugates are progressing through clinical trials and very soon expected to become the part of diagnostic and therapeutic armamentarium of oncology.Several folate receptor systems have been targeted with low molecular weight folatechelate conjugates. [14][15][16][17][18][19] However, only 111 In-DTPA-Folate derivatives are exploited for clinical application. 19,20 The field of radiodiagnostic imaging has been transitioning towards using 99m Tc-based probes as it decays more rapidly and decreases the patient's exposure time to the radioactive agent. Moreover, 99m Tc-based radiodiagnostic agents are less expensive, safer and produce high quality images. Consequently, several 99m Tc-based folate conjugates have been developed such a...

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