Functional analysis of human MLH1 and MSH2 missense variants and hybrid human-yeast MLH1 proteins in Saccharomyces cerevisiae

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant inherited disease caused by defects in the process of DNA mismatch repair (MMR), and mutations in the hMLH1 or hMSH2 genes are responsible for the majority of HNPCC. In addition to clear loss-of-function mutations conferred by nonsense or frameshift alterations in the coding sequence or by splice variants, genetic screening has revealed a large number of missense codons with less obvious functional consequences. The ability to discriminate between a loss-of-function mutation and a silent polymorphism is important for genetic testing for inherited diseases like HNPCC where the opportunity exists for early diagnosis and preventive intervention. In this study, quantitative in vivo DNA MMR assays in the yeast Saccharomyces cerevisiae were performed to determine the functional significance of amino acid replacements observed in the human population. Missense codons previously observed in human genes were introduced at the homologous residue in the yeast MLH1 or MSH2 genes. This study also demonstrated feasibility of constructing genes that encode functional hybrid human-yeast MLH1 proteins. Three classes of missense codons were found: (i) complete loss of function, i.e. mutations; (ii) variants indistinguishable from wild-type protein, i.e. silent polymorphisms; and (iii) functional variants which support MMR at reduced efficiency, i.e. efficiency polymorphisms. There was a good correlation between the functional results in yeast and available human clinical data regarding penetrance of the missense codon. The results reported here raise the intriguing possibility that differences in the efficiency of DNA MMR exist between individuals in the human population due to common polymorphisms.     

Release of gastrin on stimulation of the sciatic and brachial nerves of the cat

The vasomotor response of dopamine and dopaminergic agonists was studied in vitro on middle cerebral arteries from cat and pial arteries from humans. The action of various inhibitors was tested in order to define the receptors involved. A contractile response could be obtained by epinine, apomorphine and dopamine in the mentioned order of potency. The effect was blocked by alpha-receptor as well as serotonin receptor antagonists. The mode of inhibition suggested that serotonin receptors rather than alpha-adrenoceptors mediated the dopamine-induced contraction. A dose-dependent dilatation could be evoked by the dopaminergic agonists on actively contracted pial arteries. The relative potency was epinine > dopamine > apomorphine. The order of potency for the agonists, together with blocking experiments (including a parallel shift in the log dose-response curve induced by bulbocapnine), indicated that the vasodilatation is mediated by specific dopamine receptors.     

Similarities and differences of human and experimental mouse lymphangiomas.

Lymphangioma is a disfiguring malformation of early childhood. A mouse lymphangioma model has been established by injecting Freund's incomplete adjuvant (FIA) intraperitoneally, but has not been compared with the human disease. We show that, in accordance with studies from the 1960s, the mouse model represents an oil-granuloma, made up of CD45-positive leukocytes and invaded by blood and lymph vessels. Several markers of lymphatic endothelial cells are expressed in both mouse and human, like CD31, Prox1, podoplanin, and Lyve-1. However, the human disease affects all parts of the lymphovascular tree. We observed convolutes of lymphatic capillaries, irregularly formed collectors with signs of disintegration, and large lymph cysts. We observed VEGFR-2 and -3 expression in both blood vessels and lymphatics of the patients, whereas in mouse VEGFR-2 was confined to activated blood vessels. The experimental mouse FIA model represents a vascularized oil-granuloma rather than a lymphangioma and reflects the complexity of human lymphangioma only partially.     

Involvement of Toll-like receptor 4 in the embryogenesis of the rodent filaria <Emphasis Type="Italic">Litomosoides sigmodontis</Emphasis>

To examine the role that lipopolysaccharide (LPS)-like molecules from the filarial intracellular endobacteria Wolbachia might play in the development of filarial infections, a natural infection in the LPS-nonresponsive C3H/HeJ mouse strain was compared to that of the LPS-responsive C3H/HeN mouse strain. C3H/HeN mice have been shown to be susceptible to the rodent filarial nematode Litomosoides sigmodontis, with the development of adult worms including females containing mature microfilariae (first stage larvae) in the uterine tubes. However, free microfilariae are not detected. In this study the worm burden and worm length were not significantly different between the C3H/HeN and C3H/HeJ mice. However, the fertility of worms from CeH/HeJ mice was found to be higher than those from C3H/HeN mice. Significantly, mature microfilariae were found at the site of infection only in C3H/HeJ mice. These results indicate a role for TLR4 signaling in the immune response that inhibits worm embryogenesis and prevents the release of microfilariae or directly kills released microfilariae.     

Biochemical effects and drug levels in rats after long-term treatment with the specific 5-HT-uptake inhibitor,citalopram

The effects in rats of long-term administration of the potent, specific 5-HT uptake inhibitor citalopram have been investigated. Citalopram hydrobromide (MW=405) was given in the diet, 99 or 25 mol/kg daily, for 13 days or orally, 49 mol/kg twice a day, for 14 days. High plasma and brain levels of citalopram were found during the treatment period, whereas negligible amounts were found 24 h after withdrawal. The 5-HT uptake mechanism in blood platelets was completely blocked, since levels of whole blood 5-HT during and shortly (2 days) after treatment were decreased by 75–90%. The drug load after the two highest doses in terms of plasma drug levels was the same as in depressed patients treated with citalopram. Receptor binding technique ex vivo was applied to different brain parts to measure receptor parameters for several neurotransmitters. All data were evaluated by Eadie-Hoffstee analysis. No changes were seen in B _max and K _d for -receptors (³H-dihydroalprenolol) in frontal cortex, occipital+temporal cortex, whole cortex and limbic structures, 5-HT₂ receptors (³H-spiroperidol) in frontal and whole cortex, ₁-receptors (³H-prazosin) in rest of brain and DA D-2 receptors (³H-spiroperidol) in corpus striatum and limbic structures. The uptake mechanism for 5-HT as well as the inhibitory effect of citalopram on this uptake remained unaffected in brain synaptosomes derived from control and from citalopram (99 mol/kg)-treated rats. Thus long-term treatment with citalopram does not induce changes in neurotransmitter receptors as seen with most tricyclic as well as newer atypical antidepressants. Most striking is the lack of - and 5-HT₂ receptor down-regulation. Since citalopram clinically shows clear antidepressant activity, this down-regulation does not seem to be a prerequisite of antidepressant activity.     

Changes in rat dopamine- and serotonin function in vivo after prolonged administration of the specific 5-HT uptake inhibitor,citalopram

The effect of prolonged administration of the potent and specific 5-HT uptake inhibitor citalopram on behavioural measures of dopaminergic and serotonergic activity has been studied in rats. Administration of citalopram in the diet at a daily dose of 99 mol/kg led to supersensitivity to d-amphetamine-induced hypermotility and stereotypy and to subsensitivity to apomorphine-induced hypomotility 2 h after withdrawal. Forepaw clonus induced by 5-methoxy-N,N-dimethyltryptamine was decreased 2 h and 24 h after withdrawal and the number of head shakes induced by 1-5-HTP and citalopram were decreased 24 h after withdrawal. The d-amphetamine potentiation was still seen after 24 h, whereas the response had returned to normal 3 and 7 days after withdrawal. The content of amphetamine in three different brain regions was about 50% higher compared with controls 24 h after withdrawal of prolonged citalopram administration. At this time citalopram had been eliminated, and citalopram itself could not affect amphetamine metabolism. Other experiments indicated a linear relation between d-amphetamine brain concentration and motility level. Thus, a 50% increase in citalopram-treated rats cannot alone account for 3-fold increase in d-amphetamine-induced motility. Potentiation of d-amphetamine-induced hypermotility was also found after citalopram in a daily dietary dose of 25 mol/kg for 13 days and after oral bolus injection (49 mol/kg twice daily for 14 days). Acute citalopram injection had no effect in any of these models. The results suggest increased responsiveness of dopaminergic mechanisms mediating hypermotility, and decreased sensitivity of dopamine receptors mediating sedation (proposed autoreceptors). Sensitivity of 5-HT receptors was also decreased. The mechanisms by which citalopram induces d-amphetamine supersensitivity as well as subsensitivity to apomorphine and 5-HT agonists are presently unknown, since no changes in dopaminergic and serotonergic receptor binding have been found after an identical dose regimen.     

High-Protein,Low-Glycaemic Meal Replacement Decreases Fasting Insulin and Inflammation Markers—A 12-Month Subanalysis of the ACOORH Trial

Lifestyle interventions, including meal replacement, are effective in the prevention and treatment of type-2-diabetes and obesity. Since insulin is the key weight regulator, we hypothesised that the addition of meal replacement to a lifestyle intervention reduces insulin levels more effectively than lifestyle intervention alone. In the international multicentre randomised controlled ACOORH (Almased Concept against Overweight and Obesity and Related Health Risk) trial, overweight or obese persons who meet the criteria for metabolic syndrome (n = 463) were randomised into two groups. Both groups received nutritional advice focusing on carbohydrate restriction and the use of telemonitoring devices. The intervention group substituted all three main meals per day in week 1, two meals per day in weeks 2–4, and one meal per day in weeks 5–26 with a protein-rich, low-glycaemic meal replacement. Data were collected at baseline and after 1, 3, 6 and 12 months. All datasets providing insulin data (n = 446) were included in this predefined subanalysis. Significantly higher reductions in insulin (−3.3 ± 8.7 µU/mL vs. −1.6 ± 9.8 µU/mL), weight (−6.1 ± 5.2 kg vs. −3.2 ± 4.6 kg), and inflammation markers were observed in the intervention group. Insulin reduction correlated with weight reduction and the highest amount of weight loss (−7.6 ± 4.9 kg) was observed in those participants with an insulin decrease > 2 µU/mL. These results underline the potential for meal replacement-based lifestyle interventions in diabetes prevention, and measurement of insulin levels may serve as an indicator for adherence to carbohydrate restriction.     

Inflammation and the Association of Vitamin D and Depressive Symptomatology

Depression and vitamin D deficiency are major public health problems. The existing literature indicates the complex relationship between depression and vitamin D. The purpose of this study was to examine whether this relationship is moderated or mediated by inflammation. A community sample (n = 7162) from the LIFE-Adult-Study was investigated, for whom depressive symptoms were assessed via the German version of CES-D scale and serum 25-hydroxyvitamin D (25(OH)D) levels and inflammatory markers (IL-6 and CRP levels, WBC count) were quantified. Mediation analyses were performed using Hayes’ PROCESS macro and regression analyses were conducted to test moderation effects. There was a significant negative correlation between CES-D and 25(OH)D, and positive associations between inflammatory markers and CES-D scores. Only WBC partially mediated the association between 25(OH)D levels and depressive symptoms both in a simple mediation model (ab: −0.0042) and a model including covariates (ab: −0.0011). None of the inflammatory markers showed a moderation effect on the association between 25(OH)D levels and depressive symptoms. This present work highlighted the complex relationship between vitamin D, depressive symptoms and inflammation. Future studies are needed to examine the effect of vitamin D supplementation on inflammation and depressive symptomatology for causality assessment.     

Experimental SARS-CoV-2 Infection of Bank Voles

After experimental inoculation, severe acute respiratory syndrome coronavirus 2 infection was confirmed in bank voles by seroconversion within 8 days and detection of viral RNA in nasal tissue for up to 21 days. However, transmission to contact animals was not detected. Thus, bank voles are unlikely to establish effective transmission cycles in nature.