Binding of N-dansylgalactosamine to winged-bean tuber lectin: studies by fluorescence quenching titrations

The winged-bean tuber lectin binds to N-dansyl(5-dimethylaminonaphthalene-1-sulphonic acid)galactosamine, leading to a 12.5-fold increase in dansyl fluorescence with a concomitant 25 nm blue-shift in the emission maximum. The enhancement of fluorescence intensity was completely reversed by the addition of methyl α-galactopyranoside. The lectin has two binding sites per molecule for this fluorescent sugar and an association constant of 2.59 · 105 M−1 at 25° C. The binding of N-dansylgalactosamine to the lectin shows that it can accommodate a large hydrophobic substituent on the C-2 carbon of d-galactose. Studies with other sugars indicate that a hydrophobic substituent with α-conformation at the anomeric position increases the affinity of binding. The C-4 and C-6 hydroxyl groups are also critical for sugar binding to this lectin.

Serum testosterone levels during the menstrual-cycle and early-pregnancy in the bonnet monkey (macaca-radiata)

Abstract is not available.

Exergy analysis of a Gifford-McMahon cycle cryorefrigerator

Exergy analysis of a Gifford-McMahon cycle refrigerator is presented. Exergy losses occurring in various components are considered and the exergy balance is shown in tabular form. It is observed that the major losses occur in the compressor and at the cold end.

The Neurofibromatosis 2 tumor suppressor merlin in cytoskeleton organization and cell cycle regulation

Neurofibromatosis 2 (NF2) is a dominantly inherited disorder, which predisposes to multiple tumours of the nervous system, typically schwannomas and meningiomas. Biallelic inactivation of the NF2 gene occurs both in sporadic and NF2-related schwannomas and in most meningiomas. The NF2 gene product merlin (or schwannomin) is structurally related to the ERM proteins, ezrin, radixin and moesin, which act as molecular linkers between the actin cytoskeleton and the plasma membrane. Merlin is a tumor suppressor that participates in cell cycle regulation. Merlin s phosphorylation status appears to be associated with its tumour suppressor activity, i.e. non-phosphorylated merlin functions as a tumour suppressor, whereas protein phosphorylation results in loss of functional activity. This thesis study was initiated to investigate merlin s role as a tumor suppressor and growth inhibitor. These studies show, that like many other tumor suppressors, also merlin is targeted to the nucleus at some stages of the cell cycle. Merlin s nuclear localization is regulated by cell cycle phase, contact inhibition and adhesion. In addition, a potential nuclear binding partner for merlin was identified, Human Enhancer of Invasion 10 (HEI10), a cyclin B interacting protein. Many tumor suppressors interact with microtubules and this thesis work shows that also merlin colocalizes with microtubules in mitotic structures. Merlin binds microtubules directly, and increases their polymerization in vitro and in vivo. In addition, primary mouse Schwann cells lacking merlin displays disturbed microtubule cytoskeleton. Fourth part of this thesis work began from the notion that PKA phosphorylates an unidentified site from the merlin N-terminus. Our studies show that serine 10 is a target for PKA and modulation of this residue regulates cytoskeletal organization, lamellipodia formation and cell migration. In summary, this thesis work shows that merlin s role is much more versatile than previously thought. It has a yet unidentified role in the nucleus and it participates in the regulation of both microtubules and the actin cytoskeleton. These studies have led to a better understanding of this enigmatic tumor suppressor, which eventually will aid in the design of specific drugs for the NF2 disease.

Gifford-McMahon cycle- a theoretical analysis

A theoretical analysis of the Gifford-McMahon cycle is presented. Expressions for the ideal refrigeration produced and the figure of merit are developed. Various losses occurring in a real machine are considered and equations to account for these losses are derived. Results are presented in graphical form.

Heat balance analysis of single stage Gifford-McMahon cycle cryorefrigerator

A heat balance analysis of single stage Gifford-McMahon cycle cryorefrigerator is presented. Ideal refrigeration, actual refrigeration, net refrigeration and the various losses are tabulated. It is observed that pressure-volume losses account for a major fraction of the total losses.

Defects in tricarboxylic acid cycle enzymes Fumarate hydratase and Succinate dehydrogenase in cancer

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a recently characterized cancer syndrome which predisposes to cutaneous and uterine leiomyomas as well as renal cell carcinoma (RCC). Uterine leiomyosarcoma (ULMS) has also been observed in certain Finnish HLRCC families. The predisposing gene for this syndrome, fumarate hydratase (FH), was identified in 2002. The well-known function of FH is in the tricarboxylic acid cycle (TCAC) in the energy metabolism of cells. As FH is a novel cancer gene, the role of FH mutations in tumours is in general unknown. Similarly, the mechanisms through which defective FH is associated with tumourigenesis are unclear. The loss of a wild type allele has been observed in virtually all HLRCC patients tumours and the FH enzyme activities are either totally lost or remarkably reduced in the tissues of mutation carrier patients. Therefore, FH is assumed to function as a tumour suppressor. Mutations in genes encoding subunits of other TCAC enzyme SDH have also been reported recently in tumours: mutations in SDHB, SDHC, and SDHD genes predispose to paraganglioma and pheochromocytoma. In the present study, mutations in the SDHB gene were observed to predispose to RCC. This was the first time that mutations in SDHB have been detected in extra-paraganglial tumours. Two different SDHB mutations were observed in two unrelated families. In the first family, the index patient was diagnosed with RCC at the age of 24 years. Additionally, his mother with a paraganglioma (PGL) of the heart and his maternal uncle with lung cancer were both carriers of the mutation. The RCC of the index patient and the PGL of his mother showed LOH. In the other family, an SDHB mutation was detected in two siblings who were both diagnosed with RCC at the ages of 24 and 26 years. Both of the siblings also suffered PGL. All these tumours showed LOH. Therefore, we concluded that mutations in SDHB predispose also for RCC in certain families. Several tumour types were analysed for FH mutations to define the role of FH mutations in these tumour types. In addition, patients with a putative cancer phenotype were analysed to identify new HLRCC families. Three FH variants were detected, of which two were novel. One of the variants was observed in a patient diagnosed with ULMS at the age of 41 years. However, LOH was not detected in the tumour tissue. The FH enzyme activity of the mutated protein was clearly reduced, being 43% of the activity of the normal protein. Together with the results from an earlier study we calculated that the prevalence of FH mutations in Finnish non-syndromic ULMS is around 2.4%. Therefore, FH mutations seem to have a minor role in the pathogenesis on non-syndromic ULMS. Two other germline variants were detected in a novel tumour type, ovarian mucinous cystadenoma. However, tumour tissues of the patients were not available for LOH studies and therefore LOH status remained unclear. Therefore, it is possible that FH mutations predispose also for ovarian tumours but further studies are needed to verify this result. A novel variant form of the FH gene (FHv) was identified and characterized in more detail. FHv contains an alternative first exon (1b), which appeared to function as 5 UTR sequence. The translation of FHv is initiated in vitro from exons two and three. The localization of FHv is both cytosolic and nuclear, in contrast to the localization of FH in mitochondria. FHv is expressed at low levels in all human tissues. Interestingly, the expression was induced after heat shock treatment and in chronic hypoxia. Therefore, FHv might have a role e.g. in the adaptation to unfavourable growth conditions. However, this remains to be elucidated.

Thermodynamic and kinetic studies on saccharide binding to soya-bean agglutinin.

The fluorescence of N-dansylgalactosamine [N-(5-dimethylaminonaphthalene-1-sulphonyl)galactosamine] was enhanced 11-fold with a 25 nm blue-shift in the emission maximum upon binding to soya-bean agglutinin (SBA). This change was used to determine the association constants and thermodynamic parameters for this interaction. The association constant of 1.51 X 10(6) M-1 at 20 degrees C indicated a very strong binding, which is mainly due to a relatively small entropy value, as revealed by the thermodynamic parameters: delta G = -34.7 kJ X mol-1, delta H = -37.9 kJ X mol-1 and delta S = -10.9 J X mol-1 X K-1. The specific binding of this sugar to SBA shows that the lectin can accommodate a large hydrophobic substituent on the C-2 of galactose. Binding of non-fluorescent ligands, studied by monitoring the fluorescence changes when they are added to a mixture of SBA and N-dansylgalactosamine, indicates that a hydrophobic substituent at the anomeric position increases the affinity of the interaction. The C-6 hydroxy group also stabilizes the binding considerably. Kinetics of binding of N-dansylgalactosamine to SBA studied by stopped-flow spectrofluorimetry are consistent with a single-step mechanism and yielded k+1 = 2.4 X 10(5) M-1 X s-1 and k-1 = 0.2 s-1 at 20 degrees C. The activation parameters indicate an enthalpicly controlled association process.

Chemical Modification Studies On Ricinus-Communis (Castor Bean) Agglutinin

Ricinus communis agglutinin was subjected to various chemical treatments and the effect on its hemagglutinating and saccharide-binding properties was studied. Acetylation, succinylation and citraconylation led to a complete loss in the activity of the agglutinin, whereas reductive methylation had no effect on the activity, showing that charged amino groups were involved in the hemagglutinating and saccharide-binding activity of Ricinus agglutinin. Modification of tryptophyl, arginyl and carboxyl-group-containing residues did not lead to any loss in the activity of the agglutinin. Acetylation of tyrosyl groups with N-acetylimidazole strongly reduced the hemagglutinating and saccharide-binding property of Ricinus agglutinin. The loss in activity was restored on deacetylation of the tyrosyl groups. Modification of tyrosyl residues also led to a change in the immunological properties of the agglutinin. The initial rate of modification of tyrosyl and amino groups and the concomitant loss of activity was reduced in the presence of lactose.

A multi-faceted approach for quantifying the estuarine-nearshore transition in the life cycle of the bull shark, Carcharhinus leucas.

Understanding the ontogenetic habitat linkages of sharks is important for conservation and managing human interactions. We used acoustic telemetry, catch data, elemental and stable isotope signatures and dietary analyses to investigate ontogenetic habitat use in south-east Queensland, Australia, by the bull shark Carcharhinus leucas, a IUCN 'near-threatened' species that is implicated in many shark attacks on humans in urban estuaries. Sequential analyses for delta(15)N and delta(13)C of vertebrae from five adult C. leucas and laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) for elemental composition from 23 C. leucas, including a pregnant female, were also used to trace ontogenetic habitat dependence. Acoustic telemetry indicated large juvenile and subadult C. leucas remained in estuarine habitats. delta(15)N values across shark vertebrae showed an ontogenetic shift in diet with total length (TL), confirmed by stomach contents. LA-ICPMS data reflected the ontogenetic movements of C. leucas from natal habitats. Differences among adults were gender related. Shifts in habitat use by subadults were correlated with a sigmoidal delta(13)C relationship with TL. C. leucas have a multipartite, stage-specific dependency in their transition between habitats along the freshwater-estuarine-marine continuum, making them particularly susceptible to the habitat alteration that is occurring globally.

Molecular and clinical characteristics of tricarboxylic acid cycle-associated tumors

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a rare, dominantly inherited tumor predisposition syndrome characterized by benign cutaneous and uterine (ULM) leiomyomas, and sometimes renal cell cancer (RCC). A few cases of uterine leiomyosarcoma (ULMS) have also been reported. Mutations in a nuclear gene encoding fumarate hydratase (FH), an enzyme of the mitochondrial tricarboxylic acid cycle (TCA cycle), underlie HLRCC. As a recessive condition, germline mutations in FH predispose to a neurological defect, FH deficiency (FHD). Hereditary paragangliomatosis (HPGL) is a dominant disorder associated with paragangliomas and pheochromocytomas. Inherited mutations in three genes encoding subunits of succinate dehydrogenase (SDH), also a TCA cycle enzyme, predispose to HPGL. Both FH and SDH seem to act as tumor suppressors. One of the consequences of the TCA cycle defect is abnormal activation of HIF1 pathway ( pseudohypoxia ) in the HLRCC and HPGL tumors. HIF1 drives transcription of genes encoding e.g. angiogenetic factors which can facilitate tumor growth. Recently hypoxia/HIF1 has been suggested to be one of the causes of genetic instability as well. One of the aims of this study was to broaden the clinical definers of HLRCC. To determine the cancer risk and to identify possible novel tumor types associated with FH mutations eight Finnish HLRCC/FHD families were extensively evaluated. The extension of the pedigrees and the Finnish Cancer Registry based tumor search yielded genealogical and cancer data of altogether 868 individuals. The standardized incidence ratio-based comparison of HLRCC/FHD family members with general Finnish population revealed 6.5-fold risk for RCC. Moreover, risk for ULMS was highly increased. However, according to the recent and more stringent diagnosis criteria of ULMS many of the HLRCC uterine tumors previously considered malignant are at present diagnosed as atypical or proliferative ULMs (with a low risk of recurrence). Thus, the formation of ULMS (as presently defined) in HLRCC appears to be uncommon. Though increased incidence was not observed, interestingly the genetic analyses suggested possible association of breast and bladder cancer with loss of FH. Moreover, cancer cases were exceptionally detected in an FHD family. Another clinical finding was the conventional (clear cell) type RCC of a young Spanish HLRCC patient. Conventional RCC is distinct from the types previously observed in this syndrome but according to these results, FH mutation may underlie some of young conventional cancer cases. Secondly, the molecular pathway from defective TCA cycle to tumor formation was intended to clarify. Since HLRCC and HPGL tumors display abnormally activated HIF1, the hypothesis on the link between HIF1/hypoxia and genetic instability was of interest to study in HLRCC and HPGL tumor material. HIF1α (a subunit of HIF1) stabilization was confirmed in the majority of the specimens. However, no repression of MSH2, a protein of DNA mismatch repair system, or microsatellite instability (MSI), an indicator of genetic instability, was observed. Accordingly, increased instability seems not to play a role in the tumorigenesis of pseudohypoxic TCA cycle-deficient tumors. Additionally, to study the putative alternative functions of FH, a recently identified alternative FH transcript (FHv) was characterized. FHv was found to contain instead of exon 1, an alternative exon 1b. Differential subcellular distribution, lack of FH enzyme activity, low mRNA expression compared to FH, and induction by cellular stress suggest FHv to have a role distinct from FH, for example in apoptosis or survival. However, the physiological significance of FHv requires further elucidation.