Identification of genes expressed preferentially in the honeybee mushroom bodies by combination of differential display and cDNA microarray

To clarify the molecular basis underlying the neural function of the honeybee mushroom bodies (MBs), we identified three genes preferentially expressed in MB using cDNA microarrays containing 480 differential display-positive candidate cDNAs expressed locally or differentially, dependent on caste/aggressive behavior in the honeybee brain. One of the cDNAs encodes a putative type I inositol 1,4,5-trisphosphate (IP(3)) 5-phosphatase and was expressed preferentially in one of two types of intrinsic MB neurons, the large-type Kenyon cells, suggesting that IP(3)-mediated Ca(2+) signaling is enhanced in these neurons.     

Effect of carnosine and related compounds on the inactivation of human Cu,Zn-superoxide dismutase by modification of fructose and glycolaldehyde

Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.     

CHO1, a mammalian kinesin-like protein, interacts with F-actin and is involved in the terminal phase of cytokinesis

CHO1 is a kinesin-like protein of the mitotic kinesin-like protein (MKLP)1 subfamily present in central spindles and midbodies in mammalian cells. It is different from other subfamily members in that it contains an extra approximately 300 bp in the COOH-terminal tail. Analysis of the chicken genomic sequence showed that heterogeneity is derived from alternative splicing, and exon 18 is expressed in only the CHO1 isoform. CHO1 and its truncated isoform MKLP1 are coexpressed in a single cell. Surprisingly, the sequence encoded by exon 18 possesses a capability to interact with F-actin, suggesting that CHO1 can associate with both microtubule and actin cytoskeletons. Microinjection of exon 18-specific antibodies did not result in any inhibitory effects on karyokinesis and early stages of cytokinesis. However, almost completely separated daughter cells became reunited to form a binulceate cell, suggesting that the exon 18 protein may not have a role in the formation and ingression of the contractile ring in the cortex. Rather, it might be involved directly or indirectly in the membrane events necessary for completion of the terminal phase of cytokinesis.     

Age-related changes of element contents in human tendon of the iliopsoas muscle and the relationships among elements

To elucidate compositional changes of the tendons with aging, the authors investigated age-related changes of element contents in the tendon of the iliopsoas muscle by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 14 men and 14 women, ranging in age from 4 to 102 yr. The insertion tendons of the iliopsoas were removed from the cadavers after ordinary dissection and also surgically from patients with congenital dislocation of the hip. It was found that both Mg and P in the tendon of the iliopsoas muscle increased significantly with aging, but the other elements, such as S, Ca, Na, Zn, Fe, and Si, hardly changed with aging. With regard to the relationship among element contents, extremely significant correlations were found between P and S contents, between Mg and Na contents, and between S and Zn contents. In addition, very significant correlations were found between Si and either S or Zn contents. However, there was no significant correlation between Ca and P contents. These results revealed that with regard to age-related changes of element contents and the relationships among element contents, the tendon of the iliopsoas muscle was different from the Achilles tendon, in which both Mg and P decreased significantly with aging and there were significant correlations each other among the contents of S, Mg, and P.     

Electron microscopic examination of the orexin immunoreactivity in the dorsal raphe nucleus

The ultrastructure and the synaptic relationships of the orexin-A-like immunoreactive fibers in the dorsal raphe nucleus were examined with an immunoelectron microscopic method. At the electron microscopic level, most of the immunoreactive fibers, a varicosity appearance at the light microscopic level, were found as axon terminals. The large dense-cored vesicles contained in the immunoreactive axon terminals were the most intensely immunostained organellae. These axon terminals were often found to make synapses. While the axo-dendritic synapses were usually asymmetric in appearance, the axo-somatic synapses were symmetric. Orexin-A-like immunoreactive processes with no synaptic vesicles were also found. These processes often received asymmetric synapses. With less frequency, the synapses were found between the orexin-like immunoreactive processes. The results suggest that the orexin peptides are stored in the large dense-cored vesicles; the orexin-containing fibers may have influences on the physiological activities of the dorsal raphe nucleus through direct synaptic relationships.     

Age-dependent decreases of phosphorus and magnesium in human achilles’ tendons

To elucidate changes of human tendons with aging, the authors studied age-related changes of elements in human Achilles’ tendons by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of seven men and seven women, ranging in age from 61 to 97 yr. It was found that the content of calcium increased progressively with aging in the Achilles’ tendons, whereas the contents of phosphorus and magnesium decreased gradually with aging. The previous investigations demonstrated that the content of calcium and phosphorus increased progressively with aging in most, but not all, human tissues, except for the bones. In ligaments, such as the anterior cruciate ligament and the ligament of the head of the femur, which are histologically similar to the Achilles’ tendon, it was previously found that both the contents of calcium and phosphorus increased with aging in the ligaments. It should be noted that the content of phosphorus in the Achilles’ tendons decreased during the aging process. In addition, it was found that there was a very high direct correlation between phosphorus and magnesium contents in the tendons, but not between calcium and phosphorus contents.     

Correlations of calcium accumulations in arteries, veins, cartilages, ligaments, and bones in single humans

To show the relationships of calcium accumulation in the thoracic aorta to the other tissues, calcium contents were determined with a microwave-induced plasma-atomic emission spectrometer on arteries, veins, cartilages, ligaments, and bones. These tissues were resected from 18 individuals, consisting of 11 men and 7 women who died in the age range 59–91 yr. As thoracic and abdominal aortas are routinely used for radiographic examination of arterial calcification, they appear to be standard tissues of the calcium accumulation. The calcium accumulations were determined in the femoral artery, the superior and inferior venae cavae, the internal jugular vein, cartilages of the articular disk of the temporomandibular joint and the intervertebral disk, both the ligaments of the anterior cruciate ligament and the ligamentum capitis femoris, and the calcaneus, in contrast with the thoracic aorta. As calcium increased in the thoracic aorta, it increased in the femoral artery, the articular disk of the temporomandibular joint, the intervertebral disk, both ligaments of the anterior cruciate ligament, and the ligamentum capitis femoris, but it did not increase in veins, such as the superior and inferior venae cavae and the internal jugular vein. In contrast, it decreased in the calcaneus.     

Age-related changes of element contents in the human meniscus

The relative contents (RCs) of elements in the human menisci from 23 subjects in the age range between 65 and 93 yr were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of sulfur, calcium, and phosphorus in menisci increased progressively until the 80s, being the highest in the 80s, and thereafter decreased. The RCs of magnesium in menisci increased progressively until the 90s. Regarding the medial and lateral menisci, higher RCs of magnesium and iron, and a lower RC of phosphorus were found in lateral menisci in comparison with those in medial menisci. There were sexual differences in the RCs of calcium and phosphorus of medial and lateral menisci. The RCs of calcium and phosphorus were about 50% higher in women’s menisci than in men’s. Histological examinations showed that structureless mucoid masses were observed in the menisci, with very high RCs of calcium and phosphorus being detected.     

Melting Curve Analysis after T Allele Enrichment (MelcaTle) as a Highly Sensitive and Reliable Method for Detecting the JAK2V617F Mutation

Detection of the JAK2V617F mutation is essential for diagnosing patients with classical myeloproliferative neoplasms (MPNs). However, detection of the low-frequency JAK2V617F mutation is a challenging task due to the necessity of discriminating between true-positive and false-positive results. Here, we have developed a highly sensitive and accurate assay for the detection of JAK2V617F and named it melting curve analysis after T allele enrichment (MelcaTle). MelcaTle comprises three steps: 1) two cycles of JAK2V617F allele enrichment by PCR amplification followed by BsaXI digestion, 2) selective amplification of the JAK2V617F allele in the presence of a bridged nucleic acid (BNA) probe, and 3) a melting curve assay using a BODIPY-FL-labeled oligonucleotide. Using this assay, we successfully detected nearly a single copy of the JAK2V617F allele, without false-positive signals, using 10 ng of genomic DNA standard. Furthermore, MelcaTle showed no positive signals in 90 assays screening healthy individuals for JAK2V617F. When applying MelcaTle to 27 patients who were initially classified as JAK2V617F-positive on the basis of allele-specific PCR analysis and were thus suspected as having MPNs, we found that two of the patients were actually JAK2V617F-negative. A more careful clinical data analysis revealed that these two patients had developed transient erythrocytosis of unknown etiology but not polycythemia vera, a subtype of MPNs. These findings indicate that the newly developed MelcaTle assay should markedly improve the diagnosis of JAK2V617F-positive MPNs.