Delivery of RANKL-Binding Peptide OP3-4 Promotes BMP-2-Induced Maxillary Bone Regeneration.

Although bone morphogenetic protein 2 (BMP-2) is known to stimulate osteogenesis, there is evidence that high doses of BMP-2 can lead to side effects, including inflammation and carcinogenesis. The supplementation of other bone-augmenting agents is considered helpful in preventing such side effects by reducing the amount of BMP-2 required to obtain a sufficient amount of bone. We recently showed that a receptor activator of nuclear factor κB ligand (RANKL)-binding peptide promotes osteoblast differentiation. In the present study, we aimed to investigate whether OP3-4, a RANKL-binding peptide, promotes BMP-2-induced bone formation in the murine maxilla using an injectable gelatin hydrogel (GH) carrier. A GH carrier containing OP3-4 with BMP-2 was subperiosteally injected into the murine maxillary right diastema between the incisor and the first molar. The mice were sacrificed 28 d after the injections. The local bone formation in the OP3-4-BMP-2-injected group was analyzed in comparison to the carrier-injected, BMP-2-injected, and control-peptide-BMP-2-injected groups. The GH carrier containing OP3-4 with BMP-2 enlarged the radio-opaque area and increased the bone mineral content and density in the radiological analyses in comparison to the other experimental groups. Interestingly, fluorescence-based histological analyses revealed that the mineralization had started from the outside, then proceeded inward, suggesting that the size of the newly formed bone had already been set before calcification started and that the effects of OP3-4 might be involved in accelerating the early steps of osteogenesis. Actually, OP3-4 enhanced the BMP-2-induced 5-bromo-2'-deoxyuridine (BrdU)-positive cell numbers at the injected site on day 7 and the expression of Runx2 and Col1a1, which are early osteogenic cell markers, on day 10 after the subperiosteal injections. In summary, we demonstrated, for the first time, that the application of OP3-4 by subperiosteal injection promoted BMP-2-induced bone formation, which could lead to the development of an easy and noninvasive means of promoting alveolar ridge formation.

A novel single-nucleotide polymorphism in the 3'-untranslated region of the human dihydrofolate reductase gene with enhanced expression.

A novel single-nucleotide polymorphism (SNP), 829C-->T in the 3'-untranslated region of the human dihydrofolate reductase (DHFR) gene transcript, was identified in the study population of 37 patients with childhood leukemias/lymphomas and 83 healthy Japanese children. Frequencies of the DHFR 829C/C, 829C/T, and 829T/T genotypes were 83.8, 10.8, and 5.4%, respectively, in the cases and 74.7, 19.3, and 6.0% in the controls, showing no significant difference in genotype frequencies between the cases and controls. When determined by real-time quantitative reverse transcription-PCR analysis, the highest expression of the DHFR transcript was demonstrated in the samples with a DHFR 829T/T polymorphism (P < 0.001). Direct association of the presence of the SNP with methotrexate-related adverse events in each patient was not demonstrated in this limited analysis. These data suggest that the novel DHFR 829 polymorphism is associated with a positive role in gene expression and provide evidence of a functional SNP in the 3' regulatory region of the gene.

(99m)Tc-HYNIC-derivatized ternary ligand complexes for (99m)Tc-labeled polypeptides with low in vivo protein binding.

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ((99m)Tc)-labeled polypeptides with tricine as a coligand. However, (99m)Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [(99m)Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare (99m)Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [(99m)Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [(99m)Tc](HYNIC-Fab)(tricine)(2.) When injected into mice, [(99m)Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [(99m)Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [(99m)Tc](HYNIC-NGA)(tricine)(2.) In addition, [(99m)Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [(99m)Tc](HYNIC-NGA)(tricine)(2.) Similar results were observed with (99m)Tc-labeled Fab fragments where [(99m)Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [(99m)Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [(99m)Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [(99m)Tc](HYNIC)(tricine)(BP) complex attractive as a radiolabeling reagent for targeted imaging.

Changes in expressions of proinflammatory cytokines IL-1beta, TNF-alpha and IL-6 in the brain of senescence accelerated mouse (SAM) P8.

The senescence-accelerated mouse (SAM) is known to be a murine model for accelerated aging. The SAMP8 strain shows age-related deterioration of learning and memory at an earlier age than control mice (SAMR1). In the present study, we investigated the changes in expressions of interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in the brain of SAMP8. In the hippocampus of 10 months old SAMP8, the expression of IL-1 mRNA was significantly elevated in comparison with that of SAMR1. In both strains of SAMs, increases in IL-1beta protein in the brain were observed at 10 months of age compared with 2 and 5 months. The only differences found between the strain in protein levels were at 10 months and were elevations in IL-1beta in the hippocampus and hypothalamus, and in TNF-alpha and IL-6 in the cerebral cortex and the hippocampus in SAMP8 as compared with SAMR1. However, lipopolysaccharide-induced increases in the expression of these cytokines in brain did not differ between SAMP8 and SAMR1. Increases in expression of proinflammatory cytokines in the brain may be involved in the age-related neural dysfunction and/or learning deficiency in SAMP8.

Intra-individual differences between technetium-99m-HMPAO and technetium-99m-ECD in the normal medial temporal lobe.

UNLABELLED: Regional distributions of 99mTc-hexamethyl propyleneamine oxime (99mTc-HMPAO) and 99mTc-ethyl cysteinate dimer (99mTc-ECD) were compared in the normal brain. METHODS: Six paid, healthy volunteers (mean age 26 yr) had high-resolution neuroperfusion SPECT using both 99mTc-HMPAO and 99mTc-ECD on separate days. RESULTS: Regional distribution of the two tracers differed. Technetium-99m-HMPAO accumulated more in the thalamus, frontal lobe, temporal lobe and cerebellum than 99mTc-ECD, which accumulated more in the occipital and parietal lobes. There was a considerable difference in the accumulation of the two tracers in the medial temporal lobe. The percent accumulations of 99mTc-HMPAO and 99mTc-ECD in the medial temporal lobe compared with the mean global cerebral cortical accumulation were 93.9% +/- 2.4% and 83.1% +/- 4.1% (mean +/- s.d.), respectively. CONCLUSION: The results suggest that 99mTc-HMPAO and 99mTc-ECD require specific and separate criteria for diagnosing temporal lobe pathologies, such as dementia and temporal lobe epilepsy.

[A case of rectal carcinoma that disappeared following preoperative chemotherapy with an FT-207 suppository--confirmed by a thorough histologic examination of the resected specimen].

Presented is the case of a patient, a 56-year-old female, who had complained of bloody stool and constipation. A barium enema and endoscopic examination revealed a tumor (Type 2) with a crater surrounded by a thick embankment, extending from the anterior wall to the left wall of the lower rectum. Biopsy specimens of the tumor disclosed a well-differentiated adenocarcinoma. A FT-207 suppository (1500 mg/day) was administered preoperatively for 50 days (total dose 75 g). On February 16, 1987, the patient underwent an abdominoperineal excision. The resected specimen took on the appearance of a chronic ulcer with an irregular depression, measuring 2.0 x 3.0 cm in size, in the lower rectum. The histology of the lesion also indicated a chronic ulcer, the base of which consisted of fibrosis covered with regenerative mucosa. No cancer cells or nests were demonstrated even serial tissue sections. As far as the rectal carcinoma is concerned, there has been no reported case of its disappearance by preoperative chemotherapy. The above results suggest that preoperative adjuvant chemotherapy can be quite effective against an advanced rectal carcinoma.