Synthesis of novel poly(amidoxime) grafted multiwall carbon nanotube gel and uranium adsorption.

This is the first report on the synthesis of a new functional nanocomposite gel containing amidoxime functionalized multiwall carbon nanotube (AO-MWNT-FNC GEL). The surface morphology of AO-MWNT-FNC GEL was investigated by field emission scanning electron microscopy. The modification of gel with amidoxine groups was confirmed by Fourier transform infra red spectroscopy. The AO-MWNT-FNC GEL provides effective binding with uranium ions as was ascertained by X-ray photoelectron spectroscopy. The AO-MWNT-FNC GEL was utilized as the new adsorbent for the recovery of uranium ions from aqueous solution. UV-visible spectroscopy was used to monitor the adsorption capacity of the AO-MWNT-FNC GEL toward uranium ions. The influence of initial uranium ion concentration and solution pH on the adsorption capacity of the AO-MWNT-FNC GEL were studied in batch experiments. The new FNC-GEL designed in this study is distinguished by higher adsorption capacity for uranium ions due to the synergistic contributions from high surface area of MWNT and the functional AO groups in FNC-GEL and exhibits potential for efficient recovery of uranium ions.

Facile electrodeposition of flower like gold nanostructures on a conducting polymer support.

Herein, we report an attractive, simple and templateless synthetic method for the formation of anisotropic gold nanostructures. Gold 'mesoflowers' consisting of arrays of nanoplatelets were synthesized by electrochemical gold deposition on/off conditions onto a conducting poly(diphenylamine) (PDPA) support matrix. A possible forming mechanism of anisotropic gold nanostructures is presented. The electronic and electrochemical properties of gold nanoflower decorated PDPA in an assembled diode configuration were evaluated.

Differential localization of Rho GTPases in live cells: regulation by hypervariable regions and RhoGDI binding.

Determinants of membrane targeting of Rho proteins were investigated in live cells with green fluorescent fusion proteins expressed with or without Rho-guanine nucleotide dissociation inhibitor (GDI)alpha. The hypervariable region determined to which membrane compartment each protein was targeted. Targeting was regulated by binding to RhoGDI alpha in the case of RhoA, Rac1, Rac2, and Cdc42hs but not RhoB or TC10. Although RhoB localized to the plasma membrane (PM), Golgi, and motile peri-Golgi vesicles, TC10 localized to PMs and endosomes. Inhibition of palmitoylation mislocalized H-Ras, RhoB, and TC10 to the endoplasmic reticulum. Although overexpressed Cdc42hs and Rac2 were observed predominantly on endomembrane, Rac1 was predominantly at the PM. RhoA was cytosolic even when expressed at levels in vast excess of RhoGDI alpha. Oncogenic Dbl stimulated translocation of green fluorescent protein (GFP)-Rac1, GFP-Cdc42hs, and GFP-RhoA to lamellipodia. RhoGDI binding to GFP-Cdc42hs was not affected by substituting farnesylation for geranylgeranylation. A palmitoylation site inserted into RhoA blocked RhoGDI alpha binding. Mutations that render RhoA, Cdc42hs, or Rac1, either constitutively active or dominant negative abrogated binding to RhoGDI alpha and redirected expression to both PMs and internal membranes. Thus, despite the common essential feature of the CAAX (prenylation, AAX tripeptide proteolysis, and carboxyl methylation) motif, the subcellular localizations of Rho GTPases, like their functions, are diverse and dynamic.

Carboxyl methylation of Ras-related proteins during signal transduction in neutrophils.

In human neutrophils, as in other cell types, Ras-related guanosine triphosphate-binding proteins are directed toward their regulatory targets in membranes by a series of posttranslational modifications that include methyl esterification of a carboxyl-terminal prenylcysteine residue. In intact cells and in a reconstituted in vitro system, the amount of carboxyl methylation of Ras-related proteins increased in response to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (FMLP). Activation of Ras-related proteins by guanosine-5'-O-(3-thiotriphosphate) had a similar effect and induced translocation of p22rac2 from cytosol to plasma membrane. Inhibitors of prenylcysteine carboxyl methylation effectively blocked neutrophil responses to FMLP. These findings suggest a direct link between receptor-mediated signal transduction and the carboxyl methylation of Ras-related proteins.

Antigen-specific helper function of cell-free T cell products bearing TCR V beta 8 determinants.

Although the T cell receptor (TCR) alpha beta heterodimer and its encoding genes have been characterized, a cell-free form of this receptor, which is needed for the study of functional or ligand-binding properties of the receptor, has not previously been isolated. When the cell-free supernatant products of activated cloned T helper (TH) cells were found to mediate helper activity with antigen specificity identical to that of intact T cells, experiments were carried out to determine whether this functional activity was mediated by a cell-free form of TCR-related material. A disulfide-linked dimer indistinguishable from the T cell surface alpha beta heterodimer was precipitated from cell-free supernatants of cloned TH cells with F23.1, a monoclonal antibody specific for a TCR V beta 8 determinant. Moreover, when cell-free TH products were bound to and eluted from immobilized F23.1, these affinity-purified materials had antigen-specific and major histocompatibility complex-restricted helper activity that synergized with recombinant lymphokines in the generation of B cell antibody responses. These findings suggest that the factor isolated from T cell supernatants is a cell-free form of the TCR alpha beta dimer.

Reduced meiotic recombination on the XY bivalent is correlated with an increased incidence of sex chromosome aneuploidy in men with non-obstructive azoospermia.

Both aberrant meiotic recombination and an increased frequency of sperm aneuploidy have been observed in infertile men. However, this association has not been demonstrated within individual men. The purpose of this study was to determine the association between the frequency of recombination observed in pachytene spermatocytes and the frequency of aneuploidy in sperm from the same infertile men. Testicular tissue from seven men with non-obstructive azoospermia (NOA) and six men undergoing vasectomy reversal (controls) underwent meiotic analysis. Recombination sites were recorded for individual chromosomes. Testicular and ejaculated sperm from NOA patients and controls, respectively, were tested for aneuploidy frequencies for chromosomes 9, 21, X and Y. There was a significant increase in the frequency of pachytene cells with at least one achiasmate bivalent in infertile men (12.4%) compared with controls (4.2%, P = 0.02). Infertile men also had a significantly higher frequency of sperm disomy than controls for chromosomes 21 (1.0% versus 0.24%, P = 0.001), XX (0.16% versus 0.03%, P = 0.004) and YY (0.12% versus 0.03%, P = 0.04). There was a significant correlation between meiotic cells with zero MLH1 foci in the sex body and total sex chromosome disomy (XX + YY + XY) in sperm from men with NOA (r = 0.79, P = 0.036).

The relationship between meiotic recombination in human spermatocytes and aneuploidy in sperm.

BACKGROUND: We have previously demonstrated that a decreased recombination frequency between human X and Y chromosomes is associated with the production of aneuploid 24,XY sperm. This study's aim was to determine the relationship between recombination frequency in human pachytene spermatocytes and aneuploidy frequencies in individual chromosomes in sperm from the same men. METHODS: Six previously fertile vasectomy reversal patients donated testicular tissue for meiotic analysis of pachytene spermatocytes using immunocytogenetic techniques for visualization of the synaptonemal complex and recombination sites (MLH1). Individual meiotic chromosomes were identified with centromere-specific multicolor fluorescence in situ hybridization (FISH), and the number of MLH1 signals was recorded for individual chromosomes. An ejaculated sperm sample was obtained from each patient 2-26 months post-reversal for FISH analysis of sperm aneuploidy frequencies of chromosomes 1, 9, 13, 21, X and Y. RESULTS: There was no significant correlation between meiotic recombination frequency and sperm aneuploidy for any individual chromosome. Similarly, there was no correlation between aneuploid sperm and bivalents with no recombination. CONCLUSIONS: The study provides unique data on intra-individual human recombination and aneuploidy events. It also demonstrated for the first time that men do not have an increased frequency of sperm aneuploidy 5-9 years post-vasectomy.

Out-of-pocket costs of AIDS care in China: are free antiretroviral drugs enough?

Financial access to HIV care and treatment can be difficult for many people in China, where the government provides free antiretroviral drugs but does not cover the cost of other medically necessary components, such as lab tests and drugs for opportunistic infections. This article estimates out-of-pocket costs for treatment and care that a person living with HIV/AIDS in China might face over the course of one year. Data comes from two treatment projects run by Médecins Sans Frontières in Nanning, Guangxi Province and Xiangfan, Hubei Province. Based on the national treatment guidelines, we estimated costs for seven different patient profiles ranging from WHO Clinical Stages I through IV. We found that patients face significant financial barriers to even qualify for the free ARV program. For those who do, HIV care and treatment can be a catastrophic health expenditure, with cumulative patient contributions ranging from approximately US$200-3939/year in Nanning and US$13-1179/year in Xiangfan, depending on the patient's clinical stage of HIV infection. In Nanning, these expenses translate as up to 340% of an urban resident's annual income or 1200% for rural residents; in Xiangfan, expenses rise to 116% of annual income for city dwellers and 295% in rural areas. While providing ARV drugs free of charge is an important step, the costs of other components of care constitute important financial barriers that may exclude patients from accessing appropriate care. Such barriers can also lead to undesirable outcomes in the future, such as impoverishment of AIDS-affected households, higher ARV drug-resistance rates and greater need for complex, expensive second-line antiretroviral drugs.

Up-regulation of the iC3b receptor (CR3) is neither necessary nor sufficient to promote neutrophil aggregation.

The iC3b receptor (CR3) is required for neutrophil adhesive functions, including homotypic aggregation. Because stimuli that enhance neutrophil adhesion also induce up-regulation of surface CR3, it is widely held that these two responses are causally related. We have dissociated CR3 display (immunofluorescence) from CR3 function (aggregation). Neutrophils isolated at 4 degrees C and rewarmed to 37 degrees C up-regulated surface CR3 twofold, but did not aggregate. The kinetics of FMLP-induced CR3 up-regulation were discordant with those of aggregation. In the absence of extracellular divalent cations, CR3 expression increased twofold after exposure to FMLP, but neutrophils did not aggregate. FMLP elicited 3.5-fold more aggregation than the ionophore A23187, yet less than one-half as much CR3 up-regulation. 3 mM sodium salicylate inhibited aggregation 55 +/- 4%, but had no effect on CR3 up-regulation. Conversely, 1 mM tetracaine completely inhibited CR3 up-regulation, while significantly enhancing aggregation. Neutroplasts expressed CR3, but did not up-regulate the receptor; in contrast, FMLP induced CR3-dependent aggregation of neutroplasts. We conclude that, although constitutive surface CR3 is required for neutrophil aggregation, the up-regulation of CR3 is neither necessary nor sufficient to promote cell-cell adhesion.

Integrin-dependent homotypic adhesion of neutrophils. Arachidonic acid activates Raf-1/Mek/Erk via a 5-lipoxygenase- dependent pathway.

AA stimulates integrin-dependent neutrophil adhesion, a critical early step in acute inflammation. However, neither the signaling pathway(s) of AA-stimulated adhesion, nor whether AA acts directly or through the generation of active metabolites, has been elucidated. Previously, we have observed a tight association between neutrophil Erk activation and homotypic adhesion in response to chemoattractants acting through G protein-linked receptors. We now report a similar association between homotypic adhesion and Erk activation in response to AA. Erk activation was cyclooxygenase independent and required AA metabolism to 5(S)- hydroperoxyeicosatetraenoic acid (5-HpETE) via 5-lipoxygenase, but not the further lipoxygenase-dependent metabolism of 5-HpETE to leukotrienes. AA stimulation of Erk was accompanied by Raf-1 activation and was sensitive to inhibitors of Raf-1 and Mek. Whereas activation of Erk by AA was pertussis toxin sensitive, [3H]-AA binding to neutrophils was not saturable, suggesting that an AA metabolite activates a G protein. Consistent with this hypothesis, Erk activation by 5(S)-hydroxyeicosatetraenoic acid (5-HETE; lipoxygenase-independent metabolite of 5-HpETE) was also pertussis toxin sensitive. These data suggest that a 5-lipoxygenase metabolite of AA, e.g., 5-HETE, is released from AA-treated cells to engage a plasma membrane-associated, pertussis toxin-sensitive, G protein-linked receptor, leading to activation of Erk and adhesion via the Raf-1/Mek signal transduction pathway.

Outside-in signaling in the chondrocyte. Nitric oxide disrupts fibronectin-induced assembly of a subplasmalemmal actin/rho A/focal adhesion kinase signaling complex.

Elevated levels of fibronectin (Fn) in articular cartilage have been linked to the progression of both rheumatoid and osteoarthritis. In this study, we examined intracellular events which follow ligation of Fn to its receptor, the integrin alpha5beta1. In addition, we examined the regulatory influence of nitric oxide on these events, since this free radical has been implicated in cartilage degradation. Exposure of chondrocytes to Fn-coated beads resulted in the circumferential clustering of the alpha5beta1 integrin receptor, which was accompanied by the subplasmalemmal assembly of a focal activation complex comprised of F-actin, the tyrosine kinase, focal adhesion kinase (FAK), the ras related G protein rho A, as well as tyrosine-phosphorylated proteins. Treatment with exogenous nitric oxide (NO) or catabolic cytokines which induce nitric oxide synthase blocked the assembly of F-actin, FAK, rho A and tyrosine-phosphorylated proteins while not affecting the total number of beads bound per cell nor the clustering of alpha5beta1 integrin. Use of a cGMP antagonist (Rp-8-Br cGMPS) or cGMP agonist (Sp-cGMPS) either abolished or mimicked the NO effect, respectively. Adherence of chondrocytes to fibronectin enhanced proteoglycan synthesis by twofold (vs. albumin). In addition, basic fibroblast growth factor (FGF) and insulin growth factor (IGF-1) induced proteoglycan synthesis in chondrocytes adherent to Fn but not albumin suggesting a costimulatory signal transduced by alpha5betal and the FGF receptor. Both constitutive and FGF stimulated proteoglycan synthesis were completely inhibited by nitric oxide. These data indicate that the ligation of alpha5beta1 in the chondrocyte induced the intracellular assembly of an activation complex comprised of the cytoplasmic tail of alpha5beta1 integrin, actin, and the signaling molecules rho A and FAK. We show that NO inhibits the assembly of the intracellular activation complex and the synthesis of proteoglycans, but has no effect on the extracellular aggregation of alpha5beta1 integrin. These observations provide a basis by which nitric oxide can interfere with chondrocyte functions by affecting chondrocyte-matrix interactions.

ERK1/2-induced phosphorylation of R-Ras GTPases stimulates their oncogenic potential.

The Ras-related (R-Ras) isoforms TC21, R-Ras and M-Ras are members of the Ras superfamily of small GTPases. R-Ras family proteins are frequently overexpressed in human cancers, and expression of activated mutants of these GTPases is sufficient to induce cell transformation. Unlike Ras, few activating mutations of R-Ras proteins have been reported in human cancer, and very little is known about the regulation of their activity. In this study, we report that TC21 and R-Ras are phosphorylated on a conserved serine, Ser186 and Ser201, respectively, in intact cells. This residue is located in the C-terminal hypervariable region of the proteins and is not conserved in M-Ras. We show that the MAP kinases ERK1/2 phosphorylate TC21 and R-Ras on this C-terminal serine residue both in vitro and in vivo. Phosphorylation of R-Ras proteins does not affect their subcellular localization or stability but rather stimulates their activation. Phosphorylation-defective mutants of R-Ras and TC21 are compromised in their ability to promote cancer cell adhesion and migration/invasion, respectively. Importantly, we show that phosphorylation of TC21 and R-Ras potentiates their tumorigenic activity in immunodeficient mice. Our results identify a novel regulatory mechanism of the small GTPases TC21 and R-Ras that controls their oncogenic potential.

Isolation of tissue-type plasminogen activator-inhibitor complexes from human plasma. Evidence for a rapid plasminogen activator inhibitor.

Plasminogen activator-inhibitor complexes were analyzed by SDS-polyacrylamide gel electrophoresis and enzymography. The complexes appeared as fibrinolytically active bands in the fibrin-indicator gel. A high-molecular-weight t-PA form comigrating with a t-PA-inhibitor complex (Mr 95 000-135 000) from cultured human endothelial cells was purified from plasma by immunoadsorption on anti-t-PA-Sepharose followed by gel filtration on Sephadex G-150. The high-molecular-weight t-PA form was fibrinolytically inactive when assayed by the fibrin-plate method. It was converted to a form with the same electrophoretic mobility as t-PA (Mr 72 000) when treated with 1.5 M NH4OH/39 mM SDS. These observations suggested that the plasma high-molecular-weight t-PA form was an enzyme-inhibitor complex. The complex did not show immunological cross-reactivity with a number of known plasma serine proteinase inhibitors. Both t-PA and u-PA rapidly formed complexes with an inhibitor which was present in plasma in pmolar concentrations. p-Aminobenzamidine blocked the reaction, indicating that the active center of the activator was indeed implicated in complex formation. The complex between the plasma inhibitor and t-PA and the high-molecular-weight t-PA had the same electrophoretic mobilities. The rapid plasminogen activator inhibitor in plasma showed remarkable similarity to a plasminogen activator inhibitor from cultured human endothelial cells. In addition to the high-molecular-weight t-PA form described above, three other t-PA forms were isolated from plasma. Our results indicated that they represented free t-PA and t-PA in complex with respectively C1-esterase inhibitor and alpha 2-antiplasmin.

Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex.

Serum-free conditioned media and cell extracts from cultured human umbilical vein endothelial cells were analyzed for plasminogen activator by SDS-polyacrylamide gel electrophoresis and enzymography on fibrin-indicator gels. Active bands of free and complexed tissue-type plasminogen activator (t-PA) or urokinase-type plasminogen activator (u-PA) were identified by the incorporation of specific antibodies against, respectively, t-PA or u-PA in the indicator gel. The endothelial cells predominantly released a high-molecular-weight t-PA (95 000-135 000). This t-PA form was converted to Mr-72 000 t-PA by 1.5 M NH4OH/39 mM SDS. A component with high affinity for both t-PA and u-PA could be demonstrated in serum-free conditioned medium and endothelial cell extract. The complex between this component and Mr-72 000 t-PA comigrated with high-molecular-weight t-PA. From the increase in Mr of t-PA or u-PA upon complex formation, the Mr of the endothelial cell component was estimated to be 50 000-70 000. The reaction between t-PA or u-PA and the plasminogen activator-binding component was blocked by 5 mM p-aminobenzamidine, while the complexes, once formed, could be cleaved by 1.5 M NH4OH/39 mM SDS. These observations indicated that the active center of plasminogen activator was involved in the complex formation. It was further noted that serum-free conditioned medium or endothelial cell extract inhibited plasminogen activator activity when assayed by the fibrin-plate method. Evidence is provided that the plasminogen activator-binding component was different from a number of the known plasma serine proteinase inhibitors, the placenta inhibitor and the fibroblast surface protein, proteinase-nexin. We conclude that cultured endothelial cells produce a rapid inhibitor of u-PA and t-PA as well as a t-PA-inhibitor complex.

An essential arginyl residue in yeast hexokinase.

The inactivation of yeast hexokinase A (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) by phenylglyoxal obeys pseudo first-order kinetics. Formation of a reversible enzyme-reagent complex prior to modification is suggested by the observed saturation kinetics. Loss of activity correlates with the incorporation of 1 mol of [14C]phenylglyoxal per mol 50 000 dalton subunit. No significant conformational change occurs concomitantly. Inactivation is attributable to modification of an arginyl residue. The pattern of protection by substrates and analogs favors an interaction of this essential residue with the terminal phosphoryl group of ATP or glucose 6-phosphate.

Yeast 3-phosphoglycerate kinase. Essential arginyl residues at the 3-phosphoglycerate binding site.

Yeast 3-phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phospho-transferase, EC 2.7.2.3) is inactivated by phenylglyoxal. Loss of activity correlates with the modification of two arginyl residues, both of which are protected by all of the substrates. The modification is not accompanied by any significant conformational change as determined by optical rotatory dispersion. Ultraviolet difference spectrophotometry indicates that the inactivated enzyme retains its capacity for binding the nucleotide substrates whereas the spectral perturbation characteristic of 3-phosphoglycerate binding is abolished in the modified enzyme. The data suggest that at least one of the two essential arginyl residues is located at or near the 3-phosphoglycerate binding site. A likely role of this residue could be its interaction with the negatively charged phosphate or carboxylate groups of 3-phosphoglycerate.

Differential phosphorylation of the beta2 integrin CD11b/CD18 in the plasma and specific granule membranes of neutrophils.

Neutrophil aggregation is mediated by the beta2 integrin CD11b/CD18, which has limited expression on the surface membrane of resting cells but is recruited from intracellular organelles after cell activation. We have previously found that CD11b/CD18 newly translocated to the plasma membrane does not contribute to adhesion but must be modified to be functional. Because neutrophil aggregation induced by phorbol myristate acetate (PMA) is accompanied by de novo phosphorylation of the CD18 cytoplasmic tail, we sought to determine whether CD11b/CD18 phosphorylation is separately regulated in the different cellular compartments. Accordingly, [32P]-labeled CD11b/CD18 was immunoprecipitated from purified neutrophil-specific granule or plasma membrane lysates. In plasma membrane fractions, as in whole cell lysates, CD18 became phosphorylated in cells exposed to PMA but not in untreated cells or cells treated with N-formyl-methionyl-leucyl-phenylalanine (fMLP). The alpha chain, CD11b, was phosphorylated under all conditions. In contrast, only marginal phosphorylation of specific granule-associated CD18 or CD11b was observed. Calyculin A, an inhibitor of serine/threonine phosphatases (pp1 > pp2a), induced strong phosphorylation of CD18 in the plasma membrane but not in the specific granules. Addition of intact specific granule membranes to the plasma membranes from PMA-treated neutrophils markedly decreased phosphorylation in both CD11b and CD18 subunits. These data suggest that the phosphorylation of CD11b/CD18, which accompanies neutrophil activation, is limited to plasma membrane-associated molecules. Phosphorylation, either constitutive or induced, is absent in the specific granule membranes. The difference may be due to a specific granule-associated phosphatase, probably distinct from ppl. Therefore adhesion-competent plasma membrane CD11b/CD18 and adhesion-incompetent specific granule CD11b/CD18 differ in their state of phosphorylation.

Deposition of Poly(diphenylamine-co-3-aminobenzonitrile)/Palladium Nanocomposite Film and Evaluation of Electrocatalytic Activity Toward Borohydride Oxidation.

New nanocomposites, poly(diphenylamine-co-3-aminobenzonitrile)/palladium (P(DPA-co-3ABN)/Pd) and poly(diphenylamine)/palladium (PDPA/Pd), have been prepared by pulse potentiostatic method and used as electrocatalysts for borohydride oxidation. Linear sweep voltammogram of P(DPA-co-3ABN)/Pd-ME exhibited the oxidation wave between -0.8 V and 0.4 V that corresponds to the direct, potentially four-electron, oxidation of borohydride ions. The peak current for borohydride oxidation is much higher at P(DPA-co-3ABN)/Pd-ME electrode as compared to PDPA/Pd-ME. The incorporation of 3ABN units augments electrocatalytic behavior and thermal stability for the P(DPA-co-3ABN)/Pd catalyst.

Prenylcysteine analogs to study function of carboxylmethylation in signal transduction.

Carboxylmethylation of ras-related proteins is stimulated immediately on exposure of myeloid cells to inflammatory agonists. When the methylation reaction is inhibited with prenylcysteine analogs, G-protein-mediated signal transduction responses are disrupted, but responses to phorbol ester, calcium ionophore, and phospholipase C (PLC) remain intact. Furthermore, prenylcysteine analogs block GTP gamma S-induced aggregation of permeabilized platelets. Together, these results suggest that protein prenylcysteine methylation can play a role in signal transduction. A number of studies with AdoMet antagonists have suggested a role for methylation in cell-cycle regulation and stimulus-response coupling. Because the compounds generally inhibit all cellular methylation events, however, their effects have been difficult to interpret. On the other hand, prenylcysteine analogs have proved to be specific inhibitors of protein prenylcysteine methylation, as opposed to other types of methylation reactions. This enables the segregation of the role of methylation at C-terminal prenylcysteine residues from methylation at other sites, such as the carboxyl terminus of the catalytic subunit of PP2A. It should be emphasized, however, that prenylcysteine tails of proteins may interact with other target sites in addition to the methyltransferase enzyme(s), and prenylcysteine analogs may compete for these sites as well. One cannot assume that the inhibition of a response by the drugs necessarily implicates the involvement of a prenylcysteine methylation reaction. Studies with the analogs must be interpreted in conjunction with other results to ascertain the locus of their effects.

The effect of cold storage on recombination frequencies in human male testicular cells.

Meiotic recombination is essential for the segregation of homologous chromosomes and formation of normal haploid gametes. Decreased recombination is associated with the production of aneuploid sperm in humans. MLH1, a DNA mismatch repair protein, was recently found to mark the sites of recombination in humans. Newly developed immunofluorescence techniques to identify MLH1 foci on synaptonemal complexes (SCs) in pachytene cells from testicular tissue have opened up a new avenue of research on meiotic recombination. Future studies on normal and abnormal recombination in early meiosis will further research in human reproduction and genetics. However, the availability of testicular material will always be a major limiting factor in this kind of study. In order to obtain an adequate number of samples and samples of particular research interest, it is often of benefit to obtain samples from distant regions. Therefore, it is necessary to determine whether the quality of samples and accuracy of MLH1 frequencies change after transporting testicular samples from a distance. In the present study, we examined the recombination frequencies (numbers of MLH1 foci using immunofluorescence techniques) in 6 normal testicular samples. Each sample was split and analyzed in the fresh state and after storage on ice for two days, mimicking overnight courier air transport. The results showed no significant difference in the quality of the SC preparations or in the number of MLH1 foci between these two groups. These results demonstrate that testicular specimens may be shipped on ice without compromising data on chromosome pairing and recombination in early meiosis.