In vitro evidence that carbohydrate moieties derived from uromodulin, an 85,000 dalton immunosuppressive glycoprotein isolated from human pregnancy urine, are immunosuppressive in the absence of intact protein

Our laboratory recently reported the purification of a unique immunosuppressive glycoprotein isolated from human pregnancy urine (7). This glycoprotein, which we term uromodulin, has a m.w. of 85,000 as assessed on SDS-PAGE and is 30% carbohydrate. Uromodulin blocks in vitro antigen-specific T cell proliferation to recall antigens such as tetanus toxoid at concentrations as low as 100 pM. This glycoprotein also blocks the in vitro generation of spontaneous monocyte-mediated cytotoxicity (7, 36). Recent evidence strongly suggests that the primary action of uromodulin is to act as a specific ligand and modulator of IL 1 (10, 33). We now report additional biochemical characterization of uromodulin, and based on three independent lines of evidence, find that its immunologic activity appears to result from its glycosylation. First, measures to alter the tertiary folding of the protein backbone of uromodulin, including succinylation or reduction and carboxymethylation, fail to significantly affect its in vitro bioactivity. Second, after extensive digestion of intact uromodulin with pronase, the majority of the in vitro bioactivity can be recovered in a single carbohydrate-rich fraction. Finally, digestion with N-glycanase (N-glycosidase F-, an enzyme specific for N-asparagine-linked oligosaccharides) and subsequent purification on thin layer chromatography yields a single complex oligosaccharide that appears to be responsible for the majority of the in vitro immunosuppression mediated by uromodulin. These data suggest that uromodulin displays N-linked carbohydrate sequences capable of down-regulating antigen-specific T cell responses in vitro. It has been suggested that endogenous lectins may play an important role as recognition molecules in mammalian, as well as more primitive immune systems (23, 24). Our in vitro biologic data strongly suggest that the carbohydrate portion of uromodulin is an excellent candidate to function as a potential lectin receptor.     

Lysophosphatidylcholine disrupts the acrosome of tammar wallaby (macropus eugenii) spermatozoa

The acrosomal status of wallaby spermatozoa was evaluated by light and electron microscopy after incubation in 1–100 μM lysophosphatidylcholine (LPC) for up to 120 min. Treatment with 1 and 10 μM LPC for 120 min did not lead to acrosomal loss, or detectable alteration to the acrosome, as detected by Bryan's staining and light microscopy. Incubation with 25 μM LPC had little effect on acrosomal loss, however statistically significant changes (P < 0.05) in the acrosomal matrix (altered) were detected after 10-min incubation by light microscopy. Around 50% of acrosomes were altered after 20-min incubation in 50 μM LPC (P < 0.001), and 40% of spermatozoa had lost their acrosome after 60-min incubation (P < 0.001). Treatment with 75 and 100 μM LPC led to rapid acrosomal loss from around 50% of spermatozoa within 10 min (P < 0.001), and by 60 min acrosomal loss was 70–80%. LPC, like the diacylglycerol DiC₈ (1,2-di-octanoyl-sn-glycerol), is thus an effective agent to induce loss of the relatively stable wallaby sperm acrosome, and it also induces changes within the acrosomal matrix. Ultrastructure of the LPC-treated spermatozoa revealed that the plasma membrane and the acrosomal membranes were disrupted in a manner similar to that seen after detergent treatment (Triton X-100). There was no evidence of point fusion between the plasma membrane overlying the acrosome and the outer acrosomal membrane. The plasma membrane was the first structure to disappear from the spermatozoa. The acrosomal membranes and matrix showed increasing disruption with time and LPC concentration. Wallaby spermatozoa incubated with LPC at concentrations that induced significant acrosomal loss also underwent a rapid decline in motility that suggested that acrosomal loss may be due to cell damage, rather than a physiological AR. This study concluded that LPC-induced acrosomal loss from tammar wallaby spermatozoa is due to its action as a natural detergent and not as a phosphoinositide pathway intermediate. The study further demonstrates the unusual stability of the marsupial acrosomal membranes. © 1993 Wiley-Liss, Inc.     

The distribution of coastal fish eDNA sequences in the Anthropocene

Aim

Coastal fishes have a fundamental role in marine ecosystem functioning and contributions to people, but face increasing threats due to climate change, habitat degradation and overexploitation. The extent to which human pressures are impacting coastal fish biodiversity in comparison with geographic and environmental factors at large spatial scale is still under scrutiny. Here, we took advantage of environmental DNA (eDNA) metabarcoding to investigate the relationship between fish biodiversity, including taxonomic and genetic components, and environmental but also socio-economic factors.

Location

Tropical, temperate and polar coastal areas.

Time period

Present day.

Major taxa studied

Marine fishes.

Methods

We analysed fish eDNA in 263 stations (samples) in 68 sites distributed across polar, temperate and tropical regions. We modelled the effect of environmental, geographic and socio-economic factors on α- and β-diversity. We then computed the partial effect of each factor on several fish biodiversity components using taxonomic molecular units (MOTU) and genetic sequences. We also investigated the relationship between fish genetic α- and β-diversity measured from our barcodes, and phylogenetic but also functional diversity.

Results

We show that fish eDNA MOTU and sequence α- and β-diversity have the strongest correlation with environmental factors on coastal ecosystems worldwide. However, our models also reveal a negative correlation between biodiversity and human dependence on marine ecosystems. In areas with high dependence, diversity of all fish, cryptobenthic fish and large fish MOTUs declined steeply. Finally, we show that a sequence diversity index, accounting for genetic distance between pairs of MOTUs, within and between communities, is a reliable proxy of phylogenetic and functional diversity.

Main conclusions

Together, our results demonstrate that short eDNA sequences can be used to assess climate and direct human impacts on marine biodiversity at large scale in the Anthropocene and can further be extended to investigate biodiversity in its phylogenetic and functional dimensions.     

Thyroglobulin interactions with thyroid membranes. Relationship between receptor recognition of N-acetylglucosamine residues and the iodine content of thyroglobulin preparations

Bovine thyroglobulin has been subjected to sequential glycohydrolase treatment in order to define further the components of the carbohydrate chain which are important in binding of the glycoprotein to bovine thyroid membranes. Preparations of asialoagalactothyroglobulin exhibit the best binding, suggesting that exposed N-acetylglucosamine residues on the B carbohydrate chain of thyroglobulin play an important role in the interaction of thyroglobulin with the thyroid membranes. Enhanced binding of asialoagalactothyroglobulin to microsomal, lysosomal, and Golgi membranes, as well as to thyroid cells in culture, was also observed. Isopycnic rubidium chloride gradient centrifugation, a procedure used in the isolation of thyroglobulin molecules with a low iodine content, also isolates thyroglobulin molecules with a low sialic acid content and with an increased ability to interact with wheat germ agglutinin, a lectin which recognizes exposed N-acetylglucosamine residues. The studies further indicate that there is a correlation between iodine content, exposed N-acetylglucosamine residues, and the binding of thyroglobulin to thyroid membranes.     

PHYTOPLANKTON LIPIDS: INTERSPECIFIC DIFFERENCES AND EFFECTS OF NITRATE,SILICATE AND LIGHT-DARK CYCLES1

The lipid content of various phytoplankton species was measured in response to nitrogen and silicon limitation and over the cell cycle in synchronized cultures. In a survey of 30 species it was found that during log-phase growth, green algae contained an average of 17.1% total lipids (% of total dry weight), whereas diatoms contained an average of 24.5%. Nitrogen deprivation for 4 to 9 days resulted in 2- to 3-fold increases in the lipid content of green algae, whereas both increases and decreases were noted in diatoms, depending on the species. The greatest lipid content measured in the study was 72% in Monallantus salina (strain GSB Sticho) which had been deprived of nitrogen for 9 days. Nitrate replenishment in a nitrogen starved culture of Oocystis polymorpha Groover & Bold showed that the excess cellular lipids do not rapidly disappear during recovery, until cell division occurs. A silicate deprivation experiment with Cyclotella cryptica Reimann, Lewin & Guillard (strain 7c) showed an increase in the total cellular lipid fraction from. 30 to 42% of dry weight within 6 h of the onset of silicon limitation, while the mass of lipid material per cell doubled within 12 h. The total lipid fraction in O. polymorpha was found to remain constant over the cell cycle in synchronized cultures regardless of the light regime. The data presented provided the first internally consistent study of phytoplankton lipids for a wide range of species and several growth conditions.     

Regulation of Muscarinic Acetylcholine Receptor Concentration in Cloned Neuroblastoma Cells

Abstract: The concentration of muscarinic acetylcholine receptors in the neuroblastoma cell line NIE-115 is regulated by receptor activation. Muscarinic agonists cause a time and dose-dependent loss of [³H]quinuclidinyl benzilate binding sites from cultured cells. Muscarinic antagonists have no effect on receptor concentration and block agonist-induced regulation. The maximum decrease in steady state receptor levels is 80% and occurs within 9 h. The altered steady state concentration persists as long as agonist remains present. Upon withdrawal of agonist, the concentration of receptors returns to control levels. This increase requires protein synthesis. Kinetically, the increase in receptors following withdrawal of agonist is slower than the decrease caused by addition of agonist, suggesting that bursts of receptor activation could lower receptor levels. In harmony with this prediction, cycles in which receptors are active for 15 min and then inactive for 15 min cause a 50% decrease in receptor concentration in a 6-h period.     

Binding of thyroglobulin to bovine thyroid membranes. Role of specific amino acids in receptor recognition

Bovine thyroglobulin was treated with increasing ratios of succinic anhydride, trinitrobenzene sulfonic acid, tetranitromethane, and N-acetylimidazole in an attempt to assess the role of lysine or tyrosine residues in binding to thyroid membrane receptors. Extensive succinylation results in dissociation to 12 S thyroglobulin with retention of a considerable portion of the three-dimensional structure. Only 25% of the lysine residues can be modified by trinitrophenylation without affecting inter-subunit interactions. Succinylation as well as trinitrophenylation increases the affinity of thyroglobulin for the membrane receptor by a factor of 2. The binding of thyroglobulin to the membrane was reduced after nitration of 30% of the tyrosyl residues with tetranitromethane. O-Acetylation of 40-70% of the tyrosyl residues by N-acetylimidazole nearly abolished the ability of thyroglobulin to bind to the membrane. Removal of the O-acetyl group with hydroxylamine restored the binding properties. The results indicate that tyrosyl residues play an important role in thyroglobulin interactions with thyroid membranes.     

5-alkylvinyl-1,2,4-triazole nucleosides: Synthesis and biological evaluation

Abstract

Some 5-substituted ribavirin analogues have a high antiviral and anticancer activity, but their mechanisms of action are obviously not the same as their parent compound. The SAR studies performed on 3 (5)-substituted 1,2,4-triazole nucleosides have shown a high dependency between the structure of the 3 (5)-substituent and the level of antiviral/anticancer activity. The most active substances of the row contain coplanar with the 1,2,4-triazole ring aromatic substituent which is connected by a rigid ethynyl bond. However, the compounds with the trans-vinyl linker also had antiviral activity. We decided to study the antitumor activity of ribavirin analogues with alkyl/aryl vinyl substituents in the 5th position of the 1,2,4-triazole ring. Protected nucleoside analogues with various 5-alkylvinyl substituents were obtained by Horner-Wadsworth-Emmons reaction from the common precursor and converted to the nucleosides. Arylvinyl nucleosides were synthesised according the reported procedures. All compounds did not show significant antiproliferative activity on several tumour cell lines. Coplanar aromatic motif in the 5-substituent for the anticancer activity manifestation was confirmed.