Serum osmolality measured in fingertip capillary blood is comparable to serum osmolality measured in venous blood.

Blood osmolality is considered the gold standard hydration assessment, but has limited application for technical and invasive reasons. Paired antecubital-venous blood and fingertip-capillary blood were collected pre- and 30 min post-drinking 600 mL water in 55 male/female participants. No bias (0.2 mOsmo/kg, limits of agreement = -2.5 to 2.8 mOsmo/kg) was found between sampling methods, with high linear correlation (Spearman's r = 0.95, P < 0.001). Capillary blood sampling offers an accurate less-invasive method for determining serum osmolality than venous blood sampling.

Post-exercise skimmed milk, but not a sucrose beverage decreases energy intake at the next meal compared to a placebo beverage in active males.

This study compared the appetite and energy intake effects of three post-exercise beverages at a subsequent post-exercise meal. On three occasions, ten active males: (mean ± sd) age 21.3 ± 1.2 y, V˙ O2peak 58 ± 5 mL/kg/min) performed 30-min cycling at ∼60% V˙ O2peak and five 4-min intervals at 85% V˙ O2peak. Post-exercise, placebo (PLA: 57 kJ), skimmed milk (MILK: 1002 kJ) or sucrose (CHO: 1000 kJ) beverages (615 mL) were consumed. Sixty min post-beverage, subjects consumed an ad-libitum pasta lunch in a 30 min eating period. Subjective appetite and plasma acylated ghrelin and plasma glucose were determined pre-exercise, post-exercise and pre-meal, with sensory characteristics of beverages rated. Ad-libitum energy intake in MILK (6746 ± 2035) kJ) was lower than CHO (7762 ± 1921) kJ) (P = 0.038; dz = 0.98; large effect) and tended to be lower than PLA (7672 (2005) kJ) (P = 0.078; dz = 0.76; medium effect). Including energy consumed in beverages, energy intake was greater in CHO than PLA (P = 0.010; dz = 1.24; large effect) or MILK (P = 0.026; dz = 0.98; large effect), with PLA and MILK not different (P = 0.960; dz = 0.02; trial effect). Plasma ghrelin, plasma glucose and appetite were not different between trials. MILK was perceived thicker than CHO (P = 0.020; dz = 1.11; large effect) and creamier than PLA (P = 0.026; dz = 1.06; large effect). These results suggest that when energy balance is important for an exerciser, post-exercise skimmed milk ingestion reduces energy intake compared to a sucrose beverage and might therefore help facilitate recovery/adaptation without affecting energy balance.

The N-terminus of GPR37L1 is proteolytically processed by matrix metalloproteases.

GPR37L1 is an orphan G protein-coupled receptor expressed exclusively in the brain and linked to seizures, neuroprotection and cardiovascular disease. Based upon the observation that fragments of the GPR37L1 N-terminus are found in human cerebrospinal fluid, we hypothesized that GPR37L1 was subject to post-translational modification. Heterologous expression of GPR37L1-eYFP in either HEK293 or U87 glioblastoma cells yielded two cell surface species of approximately equivalent abundance, the larger of which is N-glycosylated at Asn105. The smaller species is produced by matrix metalloprotease/ADAM-mediated proteolysis (shown by the use of pharmacological inhibitors) and has a molecular weight identical to that of a mutant lacking the entire N-terminus, Δ122 GPR37L1. Serial truncation of the N-terminus prevented GPR37L1 expression except when the entire N-terminus was removed, narrowing the predicted site of N-terminal proteolysis to residues 105-122. Using yeast expressing different G protein chimeras, we found that wild type GPR37L1, but not Δ122 GPR37L1, coupled constitutively to Gpa1/Gαs and Gpa1/Gα16 chimeras, in contrast to previous studies. We tested the peptides identified in cerebrospinal fluid as well as their putative newly-generated N-terminal 'tethered' counterparts in both wild type and Δ122 GPR37L1 Gpa1/Gαs strains but saw no effect, suggesting that GPR37L1 does not signal in a manner akin to the protease-activated receptor family. We also saw no evidence of receptor activation or regulation by the reported GPR37L1 ligand, prosaptide/TX14A. Finally, the proteolytically processed species predominated both in vivo and ex vivo in organotypic cerebellar slice preparations, suggesting that GPR37L1 is rapidly processed to a signaling-inactive form. Our data indicate that the function of GPR37L1 in vivo is tightly regulated by metalloprotease-dependent N-terminal cleavage.

Metalloprotease cleavage of the N terminus of the orphan G protein-coupled receptor GPR37L1 reduces its constitutive activity.

Little is known about the pharmacology or physiology of GPR37L1, a G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor that is abundant in the cerebellum. Mice deficient in this receptor exhibit precocious cerebellar development and hypertension. We showed that GPR37L1 coupled to the G protein Gα(s) when heterologously expressed in cultured cells in the absence of any added ligand, whereas a mutant receptor that lacked the amino terminus was inactive. Conversely, inhibition of ADAMs (a disintegrin and metalloproteases) enhanced receptor activity, indicating that the presence of the amino terminus is necessary for GPR37L1 signaling. Metalloprotease-dependent processing of GPR37L1 was evident in rodent cerebellum, where we detected predominantly the cleaved, inactive form. However, comparison of the accumulation of cAMP (adenosine 3',5'-monophosphate) in response to phosphodiesterase inhibition in cerebellar slice preparations from wild-type and GPR37L1-null mice showed that some constitutive signaling remained in the wild-type mice. In reporter assays of Gα(s) or Gα(i) signaling, the synthetic, prosaposin-derived peptide prosaptide (TX14A) did not increase GPR37L1 activity. Our data indicate that GPR37L1 may be a constitutively active receptor, or perhaps its ligand is present under the conditions that we used for analysis, and that the activity of this receptor is instead controlled by signals that regulate metalloprotease activity in the tissue.

Niclosamide blocks glucagon phosphorylation of Ser552 on ß-catenin in primary rat hepatocytes via PKA signalling.

Recently, it has been found that glucagon is able to activate the ß-catenin signalling pathway leading to increased cyclin D1 and c-Myc expression in liver. Therefore the main aim of the present study is to determine whether the effect of glucagon activating ß-catenin signalling leading to increased target gene expression is mediated through cAMP activation of PKA (protein kinase A). Primary rat hepatocytes were incubated with insulin, glucagon or adrenaline (epinephrine) and a range of inhibitors of PI3K (phosphoinositide 3-kinase), Wnt, mitochondrial uncoupler (niclosamide) or PKA inhibitors to dissect out the pathway leading to increased Ser(552) phosphorylation on ß-catenin following glucagon exposure. In primary rat hepatocytes, we found that short exposure to glucagon or adrenaline caused a rapid increase in Ser(552) phosphorylation on ß-catenin that leads to increased cyclin D1 and c-Myc expression. A range of PI3K and Wnt inhibitors were unable to block the effect of glucagon phosphorylating ß-catenin. Interestingly, both niclosamide and the PKA inhibitor H89 blocked the glucagon effect on ß-catenin signalling, leading to a reduction in target gene expression. Likewise, niclosamide inhibited cAMP levels and the direct addition of db-cAMP (dibutyryl-cAMP sodium salt) also resulted in Ser(552) phosphorylation of ß-catenin. We have identified a new pathway via glucagon signalling that leads to increased ß-catenin activity that can be reversed with the antihelminthic drug niclosamide, which has recently shown promise as a potential treatment of T2D (Type 2 diabetes). This novel finding could be useful in liver cancer treatment, particularly in the context of T2D with increased ß-catenin activity.

Vaginal immunization of monkeys against urinary tract infection with a multi-strain vaccine.

Cynomolgus monkeys were treated with a vaccine containing 10 heat-killed uropathogenic bacteria including 6 Escherichia coli strains. The multi-strain vaccine was administered either as a vaginal surface immunogen or intramuscularly. Following an induced E. coli cystitis, bladder infections were significantly reduced compared with controls at 1 and 2 weeks (intramuscular route) or 1 week (vaginal route) after UTI. This vaccine has been shown to be efficacious against cystitis in humans when given parenterally and has now proved efficacious in nonhuman primates by the vaginal mucosal route.

Congenital immunodeficiencies in mice increase susceptibility to urinary tract infection.

Severe combined immunodeficient (SCID), T cell deficient, and immunocompetent mice were challenged intravesically with viable uropathogenic Escherichia coli. In comparison to immunocompetent controls, SCID mice had significantly greater numbers of viable E. coli in their bladders and kidneys 7 days after inoculation. Splenic anti-E. coli antibody-forming cells (AFC) were virtually absent in SCID mice at 7.0 days after infection. Adoptive transfer of spleen cells from E. coli-immunized immunocompetent mice to SCID mice enhanced their resistance to urinary tract infection (UTI), as evidenced by lower bacterial counts in bladder and kidneys following an induced infection. Congenitally T cell deficient nude mice and immunocompetent heterozygous controls had equivalent bladder and kidney infection levels at 2 and 7 days after UTI. Immunocompetency thus appears to play a significant role in resistance to E. coli UTI in this animal model. Since mice deficient only in T cells did not show increased UTI susceptibility, T cell-independent antibody responses may be an important immunologic defense mechanism.

Immunization against urinary tract infection with a multi-valent vaginal vaccine.

Systemic and local immune responses are thought to play an important role in susceptibility to urinary tract infection. In an attempt to boost local immunity, a vaccine was administered parenterally or vaginally to two mouse strains. Both routes of immunization increased the number of splenic antibody-forming cells against the bacterial strains in the vaccine. Following vaginal or parenteral immunization and subsequent induction of cystitis with live E. coli, immunized animals had fewer viable bladder E. coli than non-immunized animals.

Selective killing of transformed baby hamster kidney (BHK) cells.

We report here that certain drugs can protect Syrian baby hamster kidney cells (BHK) in culture against the lethal agents cytosine arabinonucleoside, hydroxyurea, and colcemid. Polyoma virus-transformed BHK cells (PyBHK) are killed under the same conditions. The protective drugs include caffeine and streptovitacin A. Kinetic studies show that these drugs act specifically in G1, and that they shift BHK cells from G1 into the G0 state at the restriction point, similar to the effects of high cell density or serum deprivation. These drugs do not block the growth of PyBHK cells nearly as effectively, consistent with a reduced effectiveness of restriction point control in virus-transformed cells. Consequently, the transformed cells around their cycle and are killed by the cell cycle phase-specific toxic agents, in contrast to the arrested BHK cells. These findings provide a model for studies on differential killing of tumor versus normal cells in vivo.