Heterozygosity with respect to Zfp148 causes complete loss of fetal germ cells during mouse embryogenesis.

Zfp148 belongs to a large family of C2H2-type zinc-finger transcription factors. Zfp148 is expressed in fetal germ cells in 13.5-d-old (E13.5) mouse embryos. Germ-line transmission of mutations were not observed in chimeric Zfp148(+/-) mice, and some of these mice completely lacked spermatogonia. The number of primordial germ cells in Zfp148(+/-) tetraploid embryos was normal until E11.5, but declined from E11.5 to E13.5 and continued to decline until few germ cells were present at E18.5. This phenotype was not rescued by wild-type Sertoli or stromal cells, and is therefore a cell-autonomous phenotype. These results indicate that two functional alleles of Zfp148 are required for the normal development of fetal germ cells. Recent studies have shown that Zfp148 activates p53, which has an important role in cell-cycle regulation. Primordial germ cells stop proliferating at approximately E13.5, which correlates with induction of phosphorylation of p53 and its translocation to the nucleus. Phosphorylation of p53 is impaired in Zfp148(+/-) embryonic stem cells and in fetal germ cells from chimeric Zfp148(+/-) embryos. Thus, Zfp148 may be required for regulating p53 in the development of germ cells.

Characterization of a motif for specific binding to hyaluronan in chicken SPACR.

The chicken sialoprotein associated with cones and rods (SPACR) binds to hyaluronan (HA) in the interphotoreceptor matrix space, but the motif for HA binding is still unknown. This study was conducted to determine the critical site required for specific binding to HA. Western blotting study showed that SPACR binds biotinylated HA, and this interaction was specifically inhibited by unlabeled HA. A series of GST fusion proteins covering whole SPACR was prepared, and reactivity with HA was individually screened to narrow down the region for the binding. Further, putative HA-binding motif found near the carboxyl-terminus of SPACR was mutated by site-directed mutagenesis to identify the critical binding site. Finally, we showed that native SPACR derived from retina similarly binds to HA-affinity column under both reducing and non-reducing conditions. These results revealed that the specific putative HA-binding motif is located near the carboxyl-terminus of chicken SPACR, and suggested that a structural integrity such as folded structure is not largely involved in the HA binding.

Medial horn supporting ligament in Asian upper eyelids.

The purpose of this study was to describe the medial horn supporting ligament (MHSL) attached to the medial horn of the levator aponeurosis. The authors examined 3 different groups of specimens: gross cadaveric samples, microscopic cadaveric samples and intraoperative samples from levator resections. In all eyelids in the gross cadaveric samples, the MHSL was attached from the superior to the medial surfaces of the medial horn, and ran deeper in the direction of the trochlea in the superior region. Furthermore, the MHSL continued to the Whitnall ligament and was partially attached to the trochlea. In the microscopic samples, the MHSL was located adjacent to the levator aponeurosis in all specimens. The MHSL was constituted by comparatively thick fibers with few smooth muscle fibers. Under the MHSL, another fibrous structure existed with minute fibers including much smooth muscle. In the intraoperative samples from levator resections, the MHSL was also observed in all specimens. The MHSL was released from the medial horn in the cases of lateral tarsal shifts, but not in cases without. The MHSL, an indicator of the medial margin of the levator aponeurosis, may provide support, tension and suspension to the medial margin of the medial horn, strengthening its fragility.

Occlusal tooth wear in female F344/N rats with aging.

OBJECTIVES: This study was conducted to ascertain whether laboratory rats are an adequate animal model for aging oral cavity research, especially on occlusal tooth wear (OTW), which progresses with aging and causes abnormal occlusions. Mastication has been reported to relate to cognition in the elderly. Thus, it is important to care for the oral cavity, especially in the frail elderly, for the maintenance of all-round quality of life. Adequate and appropriate animal models are essential for basic and clinical research on the oral cavity. METHODS: Dried maxilla and mandible specimens from 98 young, aging or aged female F344/N rats were used. RESULTS: The levels of OTW of all molars were monitored with aging. The molar tooth began to wear at 1-month old (M) and progressed rapidly till 12M. Subsequently, OTW progressed slowly till 30M, and then rapidly again after 35M. CONCLUSIONS: This study showed that progress of OTW is well correlated with the entire life span of the rat, and suggested that the rat aged over 12M would be an adequate animal model for research on OTW in middle-aged and elderly people.

Natural dental caries in molars of osteogenic disorder Shionogi rats.

Osteogenic disorder Shionogi (ODS) rats are genetically defective in ascorbic acid biosynthesis. They exhibit a gait abnormality due to dysfunctional bone formation and display various dental abnormalities. Conditions of the oral cavity and tooth quality both influence the development of dental caries. This study was designed to determine the characteristics of dental caries in ODS/ ShiJclod/od rats. Caries were scored and compared among ODS/ShiJclod/od, ODS/ShiJcl+/+, and Jcl:Wistar retired breeders. Among male rats, the caries scores of the ODS/ShiJclod/od and ODS/ShiJcl+/+ groups were similar to each other but greater than those in Jcl:Wistar rats, whereas among female rats, caries scores in ODS/ShiJclod/od animals were equivalent to or somewhat greater than those in ODS/ShiJcl+/+ rats, whose scores were markedly greater than those of Jcl:Wistar rats. The results suggest that ODS/ShiJcl rats were more susceptible to dental caries than were Jcl:Wistar rats. Under the conditions of the study, caries scores between ODS/ ShiJclod/od and ODS/ShiJcl+/+ rats differed only among parous females.

The lower eyelid retractor consists of definite double layers.

PURPOSE: To examine whether the lower eyelid retractor consists of a single layer or multiple layers. DESIGN: Retrospective clinical case series and dissectional study. PARTICIPANTS: Fifty-one lower eyelid retractors (31 right, 20 left) of 44 patients (ages, 63-95 years) during entropion surgeries and 10 lower eyelids (5 right, 5 left) of 5 Oriental cadavers (73-91 years old at death) were observed macroscopically. Specimens from 20 postmortem lower eyelids of 12 Orientals (11 right, 9 left; 66-96 years old at death) were used for microscopic observations. METHODS: Macroscopically, we bluntly or, in parts, sharply dissected lower eyelid retractors into 2 layers during entropion surgeries. Cadaveric lower eyelids also were used to investigate relationships between the lower eyelid retractor and the Lockwood ligament. Cadaveric lower eyelids with sagittal full-thickness sections of the central part were examined microscopically using Masson trichrome staining. MAIN OUTCOME MEASURES: Anatomical findings in the lower eyelid retractor. RESULTS: Lower eyelid retractors easily were detached into 2 layers. Macroscopically, the anterior layer was defined as a coarse tissue continuing from the Lockwood ligament, which joined with the lower orbital septum, and constituted the lower conjoined fascia. The posterior layer appeared to be a dense fibrous sheet with a lustrous surface. Microscopically, the lower eyelid retractor consisted of a definite double layer. The anterior layer, a coarse fibrous tissue, consisted of the suborbicularis fascial layer, orbital septum, and superficial part of the capsulopalpebral fascia, which continued to the anterior lamellae of the lower eyelids. The posterior layer consisted of dense fibers of the capsulopalpebral fascia with smooth muscle continuing to the tarsus. CONCLUSIONS: The lower eyelid retractor consists of a definite double layer. The posterior dense layer containing smooth muscle is the main tractional component of the lower eyelid retractor.

An anomalous muscle linking superior and inferior rectus muscles in the orbit.

Dissections of the bilateral orbits in a 45-year-old female cadaver, who had no ocular movement disorders in her lifetime, revealed anomalous muscles linking the superior and inferior rectus muscles. The muscles, situated between the optic nerve and the lateral rectus muscle, originated from the annulus of Zinn and branched off two heads; one inserted into the medial inferior side of the superior rectus muscle and the other inserted into the central superior side of the inferior rectus muscle. Each insertion was located on a distal site of the myoneural junction of each rectus muscle. Histological investigations showed that the muscles had a striated muscle structure. No definite nerve insertion was observed in the muscles. Although this type of anomalous muscle has been reported in a few Caucasian cases, the present study is the first report in an Asian person. Anomalous orbital structures, which are a rare cause of strabismus, are important in the differential diagnosis of intra-orbital space-occupying lesions, rather than the differential diagnosis of strabismus.

Lateral canthal support system in Japanese.

The present study aimed to elucidate microscopically the precise structure of the generally termed 'lateral canthal tendon' (LCT). Specimens from 9 post-mortem lower eyelids of 6 Japanese aged from 72 to 91 years old at death were fixed in 10% buffered formalin, and microscopically examined. Specimens were excised as exenterated samples including an area 5 mm wider than the orbital aperture. The removed contents were further incised longitudinally on the central eyelid and also incised parallel to the upper eyelid margin on the site 3 mm from its margin. After the preparation of microscopical examination, sections of all 9 eyelids were stained with Hematoxylin and Eosin. We found that the structure generally termed LCT consisted of two definitive different layers microscopically. The superficial layer was only an orbital septum (septal band). It was mainly constituted of thick fibers between adipose-rich tissues. The deep layer continued from the tarsus and projected posteriorly; which was a ligament (tarsoligamentous band). This tissue was constituted by thin, minute fibers with little adipose tissues. The structure generally termed LCT is not a tendon but a complex constitution of an orbital septum and a ligament; which we named, in a mass, 'lateral canthal bands', cooperatively supporting the lateral canthus.

The lacrimal canaliculus and sac bordered by the Horner's muscle form the functional lacrimal drainage system.

PURPOSE: To clarify the relationship between the orbicularis oculi muscle, lacrimal canaliculus, and lacrimal sac and to propose a new theory of the lacrimal drainage system. DESIGN: Dissection study. PARTICIPANTS: Sixteen Asian cadavers (29 upper eyelids). METHODS: Gross dissections of the orbicularis oculi muscle in the medial canthus of 10 Asian cadavers were performed, and the relationship between the orbicularis oculi muscle, lacrimal canaliculus, and lacrimal sac was examined. The upper eyelids of the remaining 6 cadavers were dissected grossly, and sections of the lacrimal sac, common lacrimal canaliculus, and upper canaliculus were examined microscopically. The relationship between the lacrimal passage and its surrounding tissues also was examined. RESULTS: The lacrimal canaliculus and sac both were divided into 2 distinct compartments in relation to the Horner's muscle. The upper half of the lacrimal sac was related to the Horner's muscle through the posterior branch of the medial canthal tendon and connective tissue, and the lower half was related to the capsulopalpebral fascia and orbital fat. The lateral part of the lacrimal canaliculus was covered with the Horner's muscle. The medial part of the lacrimal canaliculus, namely the common lacrimal canaliculus, was not covered by the Horner's muscle, but was covered by the preseptal and orbital parts of the orbicularis oculi muscle on the anterior surface. CONCLUSIONS: The lacrimal canaliculus and sac were divided into 2 distinct compartments by the Horner's muscle, and it is possible that both contribute to the lacrimal drainage system.

Significant growth inhibition of human lung cancer cells both in vitro and in vivo by the combined use of a selective cyclooxygenase 2 inhibitor, JTE-522, and conventional anticancer agents.

This study reports that a selective COX-2 inhibitor JTE-522inhibits both in vitro and in vivo growth of human lung cancer cells as a single agent. Furthermore, the adjunct use of JTE-522 is shown to significantly enhance treatment efficacy of conventional anticancer drugs not only in vitro but also in vivo without causing any noticeable side effects. Indeed, IC(50)s of various anticancer agents in vitro were reduced by up to 70%, whereas the combination therapy of JTE-522 with docetaxel and vinorelbine inhibited tumor growth in vivo by 65 and 55%, respectively. Taken together, these findings suggest that the use of a selective COX-2 inhibitor in the treatment of lung cancer may be promising, especially because of its enhancement of the treatment efficacy of conventional anticancer agents without compromising quality of life.

Aging changes in the periodontal bone of F344/N rat.

The aim of this research was to determine whether a rat was an adequate laboratory animal model for periodontal research on elderly humans. Thirty-two F344/NSlc female rats ranged between 30 and 1000 days of age were used. The alveolar bone loss around the molars was assessed by a morphometric method. A significant correlation was found between age and the amount of alveolar bone loss. For further analysis, the rats were grouped into four by age; 30-60 days, 220-430 days, 640-850 days, and more than 850 days. The means of alveolar bone loss were compared between age groups. It was found that the resorption of the alveolar bone around the molars of the rats continued until they were 1000-days-old, and this trend was stronger in the mandible than the maxilla. It was suggested that rats could be used as adequate laboratory animals for periodontal research.

Posterior aspect of the orbital septum is reinforced by ligaments.

PURPOSE: To investigate the ligaments reinforcing the posterior aspect of the orbital septum. METHODS: Sixteen upper eyelids of eight cadavers of Asians were dissected. Ten were subjected to gross dissections to investigate the ligaments attached to the posterior aspect of the orbital septum and to investigate the relationships with the associated ligamentous structures, and six were used for histological sections to elucidate the ligament anchoring sites in the septum. RESULTS: The ligaments were attached to the posterior aspect of the orbital septum in both upper and lower eyelids in all cases. Some septa in the upper eyelids were supported by the lower-positioned transverse ligament in the lateral area. In all cases, part of the Lockwood ligament was attached to the posterior aspect of the orbital septum in the lower eyelids. Histologically, the ligaments were anchored to the posterior aspect of the orbital septum. CONCLUSIONS: The ligaments were attached to the posterior aspect of the orbital septum. These ligaments, in cooperation with the associated ligaments, are thought to complement the fragility of the orbital septum.

Identification of genes preferentially expressed in articular cartilage by suppression subtractive hybridization.

Suppression subtractive hybridization is very effective to enrich differentially expressed genes in two different tissues or cells. We therefore used the technique to identify characteristic genes expressed in rat knee joint articular cartilage as compared to rat costal cartilage. In this study, we revealed that several genes were enriched in a subtracted articular cartilage cDNA library. The most enriched gene is lubricin that is a putative key molecule for joint lubrication. The second gene is milk fat globule epidermal growth factor (EGF) factor 8, MFG-E8 whose expression has never been observed in cartilage. Other enriched genes are known to be expressed in cartilage, however their differential expressions in cartilages have not been necessarily common. The preferential expression of characteristic genes in articular cartilage would provide unique properties to the tissue. Our findings will provide a new view of articular cartilage.

Fibrous connective tissue between Müller's muscle and the palpebral conjunctiva as a reinforcement structure and a natural barrier for the upper eyelid.

This study was performed to illustrate and discuss the significance of fibrous connective tissue between the Müller's muscle and the palpebral conjunctiva. Nine upper eyelids of 6 Oriental cadavers were microscopically examined; ages at death ranged from 72 to 91 years. Tissue of the posterior lamella of the upper eyelid was removed without the orbital septum and orbital fat. Removed eyelids were incised perpendicularly at the center of the eyelid. After pretreatment, sliced sections were stained with Hematoxylin and Eosin and examined microscopically. A thick fibrous connective tissue was found to exist between the Müller's muscle and the palpebral conjunctiva. The connective tissue continued proximally to the intermuscular transverse ligament and was distally attached to the posterior site of the upper aspect of the tarsus. All cases showed infiltration of lymphocytes from the conjunctiva; however, these were completely blocked by the fibrous connective tissue and never reached Müller's muscle. This connective tissue supports eyelid traction and is a natural barrier for the Muller's muscle against conjunctivitis.

Survival curve modified through dietary restriction (DR) in male Donryu rats.

It is well known that dietary restriction (DR) can prolong the life of certain inbred strains of laboratory rodents. This prolongation can be usually shown in a survival curve in parallel to that of non-DR groups. On an outbred strain, Donryu, however, DR resulted in a unique pattern of survival curve. In DR group, the first three quarters of male rats survived longer in a form parallel to non-DR, while the last quarter of them survived far longer in a quite different shape from controlled group. DR may select animals that are capable of longer survival within Donryu. The selection of a quarter animal indicates the parameter for selection may be regulated by a single recessive locus.

Difference in average survival between F344/Du and F344/N rats is not due to genetic contamination.

The average survival was found to be different between substrains of rats, F344/Du and F344/N. One of the causes of this difference could have been genetic contamination by crossbreeding with other strain(s) after the divergence of substrains. These two strains were examined by PCR-SSLP (simple sequence length polymorphism) procedure using microsatellite (Mit) markers to identify the contamination. All 288 Mit markers distributed throughout the genome exhibited no size polymorphisms between these two strains. This indicated that there was no genetic contamination between these two strains. The phenotypic difference in average survival, thus, is not due to genetic contamination.

The medial canthal tendon is composed of anterior and posterior lobes in Japanese eyes and fixes the eyelid complementarily with Horner's muscle.

PURPOSE: To report that the medial canthal tendon (MCT) is not simply the aggregate of the orbicularis oculi muscle (OOM) and its tendon. METHODS: Twenty eyelids of 10 cadavers were used. The cadavers, seven male and three female, were all Japanese, with an average age of death of 76.2 years. The relationship between the MCT and the OOM, and between the tarsus and Horner's muscle were investigated. Histological findings were obtained with hematoxylin and eosin staining. RESULTS: The MCT was structured with an anterior lobe, the tendon from the tarsal area of the OOM, and a posterior lobe, the muscle-tendon transition area in the orbital area of the OOM. The nasal aspect of the tarsus was fixed by Horner's muscle. CONCLUSIONS: The MCT and Horner's muscle are located in an important area of the eyelid; therefore, it is essential to understand their precise anatomy.

No raphe identified in the orbicularis oculi muscle.

This study was performed to elucidate whether the raphe of the orbicularis oculi muscle (raphe) exists or not. Nine upper eyelids of 6 Oriental cadavers with ages at death ranging from 72 to 91 years were dissected; 6 for gross dissections and 3 for histological slice sections. After removing the lateral half of the eyelid skin, the lateral part of the orbicularis oculi muscle and its subjacent tissue were observed macroscopically. The full layered tissue of the 8 mm lateral part from the orbital rim was incised perpendicularly and sections sliced, which were then observed microscopically after staining with the hematoxylin and eosin. The raphe was not identified macroscopically or microscopically. The lateral part of the orbicularis oculi muscle was continuous without the tendinous intercalation; under it, fibrous connective tissue corresponding to the lateral orbital thickening was observed, and in which the band configuration, microscopically the tendinous structure, was formed. The raphe was not identified. The physiological role of the lateral part of the orbicularis oculi muscle is maintained by a less tight attachment of the lateral orbital thickening, but not by the raphe.

Hyaluronan dynamics during retinal development.

We have already shown the regulated expressions of hyaluronan binding molecules including versican, SPACR, and SPACRCAN, during retinal development. Here we analyzed the profiles of hyaluronan during these periods. Hyaluronan and hyaluronan synthase (HAS) expressions in chicken retinas at different ages were assessed by slot blot and real-time PCR, respectively. The molecular size of hyaluronan was determined using a Superose-6 column, and histochemistry was used to localize hyaluronan expression. Hyaluronan degradation activity was measured after adding hyaluronan to retinal extracts. In the embryonic retina, hyaluronan was mainly present in the inner plexiform and photoreceptor layers. Expression in the inner plexiform layer decreased with development and aging while expression in the photoreceptor layer remained constant. The molecular size of hyaluronan decreased from embryonic day 12 (E12) to postnatal day 1 (P1) with increasing retinal hyaluronan degradation activity. In adult retinas, hyaluronan was only present in the photoreceptor layer, and its molecular size was the highest. HAS-2 and HAS-3 expressions increased from E15 to P1, but both were reduced in adulthood. Thus hyaluronan was specifically detected where versican, SPACR, and SPACRCAN were previously demonstrated, and the synthesis and degradation of hyaluronan are regulated. Hyaluronan together with versican, SPACR, and SPACRCAN are closely related during retinal development, and may be important for retinal physiology.