CHIPS for genetic testing to improve a regional clinical genetic service.

In current practice of clinical genetics, molecular diagnosis has become more widely used than ever before. DNA diagnosis is important for appropriate medical care of the patient, and proper genetic counseling to the family. However, genetic testing of orphan disease cannot always be performed easily. In multiple congenital anomalies (MCA) syndromes by monogenic cause, the broad mutational spectrum and large size of responsible genes often make molecular diagnosis expensive and cumbersome. We solve this problem with on-demand genetic testing by CHIPS (CEL nuclease mediated heteroduplex incision with polyacrylamide gel electrophoresis and silver staining) technology, which is the ultimately conventional and economical mutation screening system. In this article, we show eight patients with MCA syndromes who were recently treated at our hospital, and demonstrate that CHIPS successfully offers efficient and inexpensive genetic testing and facilitates clinical genetic service in our local region.

Ischemic preconditioning upregulates vascular endothelial growth factor mRNA expression and neovascularization via nuclear translocation of protein kinase C epsilon in the rat ischemic myocardium.

Ischemic preconditioning (IP) exerts cardioprotection through protein kinase C (PKC) activation, whereas myocardial ischemia enhances vascular endothelial growth factor (VEGF) mRNA expression. However, the IP effect or the involvement of PKC on the VEGF expression is unknown in myocardial infarction. We investigated whether IP enhances VEGF gene expression and angiogenesis through PKC activation in the in vivo myocardial infarction model. Sprague-Dawley rats were assigned into the following 3 groups: the sham group; the IP group, which underwent 3 cycles of 3 minutes of ischemia and 5 minutes of reperfusion (IP procedure); and the non-IP group. The latter 2 groups were subsequently subjected to left anterior descending coronary artery occlusion. To examine the involvement of PKC, the PKC inhibitor chelerythrine (5 mg/kg) or bisindolylmaleimide (1 mg/kg) was injected intravenously before the IP procedures. PKCepsilon was translocated to the nucleus after 10 minutes of ischemia after the IP procedure but was not translocated in the non-IP and the sham groups. VEGF mRNA expression 3 hours after infarction was significantly higher in the IP group than in the non-IP and the sham groups. Capillary density in the infarction was significantly higher, whereas the infarct size was smaller in the IP group than in the non-IP group at 3 days of infarction. Chelerythrine but not bisindolylmaleimide blocked all of the IP effects on the nuclear translocation of PKCepsilon, enhancement of VEGF mRNA expression and angiogenesis, and infarct size limitation. These results show that IP may enhance VEGF gene expression and angiogenesis through nuclear translocation of PKCepsilon in the infarcted myocardium.

Sex differences in the play fighting activity of golden hamster infants.

Play fighting of homosexual and heterosexual dyads in golden hamsters was studied. The behaviors of male-male (MM), female-female (FF), and female-male (FM) dyads were recorded from the 25th to the 31st day of age. Play fighting activity of heterosexual dyads was depressed since MF and FM dyads exhibited a significantly high physical contact time and engaged in less play fighting, with a significantly lower time spent on such activity. Alternation ratio in the pinning posture was very high and subjects did not differ in the mean number of active (top) and passive pinning (under) in each dyad, showing an inexistence of sex dominance. In most cases, differences in the performances of MM and FF dyads were not significant. We have concluded that play fighting in golden hamsters may be predominantly modulated by environmental stimuli (sex of the partner) rather than by endogenous stimuli (androgen level).

Effects of blindness on play fighting in golden hamster infants.

The effects of bilateral optic enucleation on play fighting in golden hamster infants were studied. The behaviors of blind-blind (BB), blind-intact (BI) and intact-intact (II) dyads were recorded from the 30th to the 36th day of age. It has been noted that the differences in physical contact, play fighting and grooming time, as well as in the percentage of time spent in play fighting, the amount of locomotion and other activities of BB, BI and II were small and statistically insignificant. The mean number of active and passive pinning in all groups was equivalent and the alternation of active and passive pinning, or vice versa, was around 50%, showing that posture during play fighting is not an indicator of dominance. It has been concluded that vision is not important for either the initiation or the modulation of play fighting in golden hamsters.

Serum levels of VEGF and basic FGF in the subacute phase of myocardial infarction.

We examined serial changes in serum levels of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) measured by ELISAs in 45 patients with acute myocardial infarction (AMI) who received heparin intravenously for 3 to 5 days after the onset and in 30 control subjects with an old myocardial infarction. To evaluate the effect of heparin on these serum levels, heparin was administered intravenously in 10 patients with AMI on day 21. Blood samples were obtained from all AMI patients on days 1, 2, 3, 7, 14, 21, and 28 and from 10 AMI patients before and 1 h after heparin administration. Serum VEGF level was significantly reduced after heparin administration (P<0.001). Serum samples from day 1 to 3 were therefore excluded from the subsequent analysis. Serum VEGF level in AMI patients was significantly higher on day 7 than in the control subjects (P<0.0001), and then decreased over time (P<0.0001). The serum VEGF level on day 7 was independently associated with the peak serum CK level (P<0.05). The serum bFGF level did not differ significantly between the AMI patients and the control subjects. In conclusion, the serum VEGF level may be selectively elevated during the healing process after AMI.

Vascular endothelial growth factor mRNA synthesis by peripheral blood mononuclear cells in patients with acute myocardial infarction.

We studied vascular endothelial growth factor (VEGF) mRNA synthesis by peripheral blood mononuclear cells (PBMCs) in 30 patients with acute myocardial infarction (AMI) and 20 healthy individuals. PBMCs were isolated from all patients on days 3 and 14 after the onset of aMI, and from all of control individuals. To prepare samples containing identical amounts of GAPDH cDNA, competitive PCR was performed by co-amplifying serial dilutions of GAPDH mutant templates. Next, to measure VEGF cDNA quantitatively in the samples containing identical amounts of GAPDH, we also used competitive PCR by co-amplifying mutant templates of VEGF. The serum VEGF concentrations on day 14 in patients with aMI were measured by an ELISA method. Higher levels of VEGF mRNA in PBMCs were present on day 14 than either on day 3 or in the control group. Serum VEGF concentrations correlated with the VEGF mRNA levels of PBMCs on day 14. Peak serum CK levels correlated well with VEGF mRNA levels of PBMCs on day 14. The present findings suggest that PBMCs may be one of the candidates responsible for elevated circulatory VEGF protein following aMI. In addition, VEGF mRNA may be overexpressed in PBMCs in response to cardiac muscle damage.

Play fighting of juvenile golden hamsters (Mesocricetus auratus): effects of two types of social deprivation and days of testing.

The effects of social deprivation schedules and days of testing on the play-fighting of juvenile golden hamsters were investigated. In Experiment 1, subjects totally (individually housed) or partially isolated (individually housed, but separated by a wire mesh 2 cm from another animal) for a period of 48 h spent significantly increased peer-peer bodily contact, play fighting, and play-fighting relative time (% of bodily contact time spent in play fighting), but exhibited decreased locomotor activity on the 1st day of testing. In Experiment 2, subjects reared since the 10th postnatal day with their mothers alone (chronically deprived of peer-peer social interactions) exhibited similar performances, except that locomotor activity was not affected. All dyads exhibited increased pinning turbulence (number of pinnings divided by time spent in play fighting) on the 1st day of testing, and the interactions between variables (deprivation schedules and days of testing) were statistically significant in most cases, suggesting that unfamiliarity between interactors also affect the performances of animals. We conclude that golden hamsters are sensitive to social deprivation, mothers do not substitute peer companions, animals reared in single-infant litters basically behave like socially-isolated animals, and the effects of induced social deprivation are reversible and disappear on days following testing.

Cross-fostering between two species of marmosets (Callithrix jacchus) and Callithrix penicillata).

Cross-fostering technique can be defined as adoption of infants by adults of other species. This phenomenon is poorly investigated because very young animals have few opportunities to interact peacefully with non-conspecific adults, either in captivity or in natural conditions. This study describes the induction of cross-fostering in captivity between white tufted-ear (Callithrix jacchus) and black tufted-ear marmosets (Callithrix penicillata). We conclude that this technique can be very useful for preserving the life of rejected by parents or orphan infants, mainly in the case of species with low reproduction rate in captivity or those threatened by extinction, as well for investigating the environmental effects on the typical behavior of species (courtship, food preference, vocalization patterns, e.g.).

Cross-fostering between two species of marmosets (Callithrix jacchus) and Callithrix penicillata)

Cross-fostering technique can be defined as adoption of infants by adults of other species. This phenomenon is poorly investigated because very young animals have few opportunities to interact peacefully with non-conspecific adults, either in captivity or in natural conditions. This study describes the induction of cross-fostering in captivity between white tufted-ear (Callithrix jacchus) and black tufted-ear marmosets (Callithrix penicillata). We conclude that this technique can be very useful for preserving the life of rejected by parents or orphan infants, mainly in the case of species with low reproduction rate in captivity or those threatened by extinction, as well for investigating the environmental effects on the typical behavior of species (courtship, food preference, vocalization patterns, e.g.).