EFhd2/Swiprosin-1 is a common genetic determinator for sensation-seeking/low anxiety and alcohol addiction.

In many societies, the majority of adults regularly consume alcohol. However, only a small proportion develops alcohol addiction. Individuals at risk often show a high sensation-seeking/low-anxiety behavioural phenotype. Here we asked which role EF hand domain containing 2 (EFhd2; Swiprosin-1) plays in the control of alcohol addiction-associated behaviours. EFhd2 knockout (KO) mice drink more alcohol than controls and spontaneously escalate their consumption. This coincided with a sensation-seeking and low-anxiety phenotype. A reversal of the behavioural phenotype with ß-carboline, an anxiogenic inverse benzodiazepine receptor agonist, normalized alcohol preference in EFhd2 KO mice, demonstrating an EFhd2-driven relationship between personality traits and alcohol preference. These findings were confirmed in a human sample where we observed a positive association of the EFhd2 single-nucleotide polymorphism rs112146896 with lifetime drinking and a negative association with anxiety in healthy adolescents. The lack of EFhd2 reduced extracellular dopamine levels in the brain, but enhanced responses to alcohol. In confirmation, gene expression analysis revealed reduced tyrosine hydroxylase expression and the regulation of genes involved in cortex development, Eomes and Pax6, in EFhd2 KO cortices. These findings were corroborated in Xenopus tadpoles by EFhd2 knockdown. Magnetic resonance imaging (MRI) in mice showed that a lack of EFhd2 reduces cortical volume in adults. Moreover, human MRI confirmed the negative association between lifetime alcohol drinking and superior frontal gyrus volume. We propose that EFhd2 is a conserved resilience factor against alcohol consumption and its escalation, working through Pax6/Eomes. Reduced EFhd2 function induces high-risk personality traits of sensation-seeking/low anxiety associated with enhanced alcohol consumption, which may be related to cortex function.

Equine CRISP-3: primary structure and expression in the male genital tract.

Although originally described in the male rodent genital tract, cysteine-rich secretory proteins (CRISPs) are expressed in a variety of mammalian tissue and cell types. The proteins of the male genital tract have been observed associated to spermatozoa and are believed to play a role in mammalian fertilization. Here we describe the identification and primary structure of the first equine member of the CRISP family. Equine CRISP-3 is transcribed and expressed in the stallion salivary gland, in the ampulla and the seminal vesicle. It displays all 16 conserved cysteine residues and shows 82% homology to human and 78% to guinea pig CRISP-2 (AA1, TPX 1) and 77% to human CRISP-3. In contrast to other mammalia, in the horse CRISP-3 is synthesized in great amounts in the accessory sexual glands, ampulla and seminal vesicle, thus allowing the isolation of equine CRISP-3 in amounts suitable for biochemical, physiological and structural studies from stallion seminal plasma.

Biochemical and conformational characterisation of HSP-3, a stallion seminal plasma protein of the cysteine-rich secretory protein (CRISP) family.

HSP-3 is a member of the cysteine-rich secretory protein (CRISP) family from stallion seminal plasma. We report a large-scale purification protocol for native HSP-3. This protein is a non-glycosylated polypeptide chain with a pI of 8-9 and an isotope-averaged molecular mass of 24987 +/- 3 Da. The molecular mass of HSP-3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP-3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9-9 million HSP-3 molecules/cell coat the postacrosomal and mid-piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP-3 was assessed by combination of circular dichroism and Fourier-transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP-3 may belong to the alpha+beta class of proteins. Thermal denaturation of HSP-3 is irreversible and follows a non-two state transition characterised by a Tm of 64 degrees C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP-3 molecules and suggests the monomer as the cooperative unfolding unit.

Preferential expression of cystein-rich secretory protein-3 (CRISP-3) in chronic pancreatitis.

BACKGROUND: Chronic pancreatitis (CP) is a progressive inflammatory process resulting in exocrine and endocrine pancreatic insufficiency in advanced stages. Cysteine-rich secretory protein (CRISP-3) has been identified as a defense-associated molecule with predominant expression in the salivary gland, pancreas and prostate. AIMS: In this study, we investigated CRISP-3 expression in normal pancreatic tissues, chronic pancreatitis tissues, pancreatic cancer tissues and pancreatic cancer cell lines, as well as in other gastrointestinal organs. MATERIALS AND METHODS: 15 normal pancreatic tissues, 14 chronic pancreatitis tissues and 14 pancreatic cancer tissues as well as three pancreatic cancer cell lines were analyzed. Moreover, hepatocellular carcinoma and esophageal, stomach and colon cancers were also analyzed together with the corresponding normal controls. RESULTS: CRISP-3 was expressed at moderate to high levels in chronic pancreatitis tissues and at moderate levels in pancreatic cancer tissues but at low levels in normal pancreatic tissues, and was absent in three pancreatic cancer cell lines. CRISP-3 expression was below the level of detection in all cancerous gastrointestinal tissues and in all normal tissues except 2 of 16 colon tissue samples. CRISP-3 mRNA signals and immunoreactivity were strongly present in the cytoplasm of degenerating acinar cells and in small proliferating ductal cells in CP tissues and CP-like lesions in pancreatic cancer tissues. In contrast, CRISP-3 expression was weak to absent in the cytoplasm of cancer cells as well as in acinar cells and ductal cells in pancreatic cancer tissues and normal pancreatic tissues. CONCLUSION: These results reveal that the distribution of CRISP-3 in gastrointestinal tissues is predominantly in the pancreas. High levels of CRISP-3 in acinar cells dedifferentiating into small proliferating ductal cells in CP and CP-like lesions in pancreatic cancer suggests a role of this molecule in the pathophysiology of CP.

Expression of CRISP proteins in the male equine genital tract.

Cysteine rich secretory proteins (CRISPs) have been detected immunochemically in the equine male genital tract. CRISPs are secretory products of the epididymis, the ampulla and the seminal vesicle. A particular feature of the horse is the abundance of CRISPs in seminal plasma. CRISPs can also be detected in extracts of testicular, epididymal and ejaculated spermatozoa in increasing amounts. Unlike other seminal plasma proteins, they cannot be removed completely from spermatozoa by high salt treatment. The remaining CRISP antigens are localized on the midpiece, and the postacrosomal and equatorial region of the sperm head. Tissue distribution and localization of CRISPs on equine spermatozoa point to a role of these proteins in epididymal sperm maturation and equine reproduction.