Lymphatic architecture of the human gingival interdental papilla.

Many studies have investigated the lymphatic architecture of head and neck using experimental animals, confirming the existence of lymphatic networks beneath the epithelium in gingival tissue. In this study, we investigated the use of these lymphatics as a drug delivery route by studying the architecture of lymphatic vessels in human interdental papilla. Serial cryosections were cut using the film-transfer method. To identify lymphatics, the sections were stained using enzyme histochemical and immunohistochemical techniques and three-dimensional images of lymphatics were reconstructed using 3D visualization software. Capillary lymphatic networks were observed in the lamina propria beneath the epithelium in human interdental papilla, and they joined with lymphatic networks beneath the epithelium in free gingiva. The networks consisted of a single layer of large irregular, hexagonal meshes and precollecting lymphatic vessels heading toward collecting lymphatic vessels that exited on the periosteum of the alveolar crest. These findings suggest that lymphatic flow from the interdental papilla drains into collecting lymphatic vessels running buccolingually on the alveolar crest of the interdental papilla. This may be an important anatomical feature during inflammation throughout the oral cavity in that the drainage function is maintained by part of lymphatic flow that is not impaired during the healing process.

The role of sexual steroid hormones in the direct stimulation by Kisspeptin-10 of the secretion of luteinizing hormone, follicle-stimulating hormone and prolactin from bovine anterior pituitary cells.

The aims of the present study were to clarify the effect of Kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and prolactin (PRL) from bovine anterior pituitary (AP) cells and evaluate the ability of sex steroids to enhance the sensitivity of gonadotropic and lactotropic cells to Kp10. AP cells prepared from 7-week-old male calves were incubated for 12h with estradiol (E(2); 10(-8)M), progesterone (P(4); 10(-8)M), testosterone (T; 10(-8)M), or vehicle only (control), and then for 2h with Kp10 (10(-6)M). The amounts of LH, FSH and PRL released into the culture medium after the 2-h incubation period were examined. Kp10 significantly stimulated the secretion of LH from the AP cells treated with E(2) and T (P<0.05), but not from the P(4)-treated cells. In contrast, Kp10 had no effect on the secretion of FSH regardless of the steroid treatment. Kp10 significantly stimulated the secretion of PRL (P<0.05), the sexual steroid hormones having no effect. The LH- or FSH-releasing response to gonadotropin-releasing hormone (GnRH; 10(-8)M) and PRL-releasing response to thyrotropin-releasing hormone (TRH; 10(-8)M) were significantly greater than those to Kp10 (P<0.05). The present results suggest that E(2) and T, but not P(4), enhance the sensitivity of gonadotropic cells to the secretion of LH in response to Kp10. However, Kp10 had no stimulatory effect on the secretion of FSH regardless of the effect of sex steroids. Kp10 directly stimulates the secretion of PRL from the pituitary cells, and sex steroids do not enhance the sensitivity of lactotropic cells to Kp10. Furthermore, the LH- and FSH-releasing effect and the PRL-releasing effect of Kp10 are less potent than that of GnRH and TRH, respectively.

Characteristics of prolactin-releasing response to salsolinol in vivo in cattle.

The aims of the present study were to clarify the effect of salsolinol (SAL), a dopamine (DA)-derived endogenous compound, on the secretion of prolactin (PRL) in cattle. The experiments were performed from April to June using calves and cows. A single intravenous (i.v.) injection of SAL (5mg/kg body weight [BW]) or sulpiride (a DA receptor antagonist, 0.1mg/kg BW) significantly stimulated the release of PRL in male and female calves (P<0.05), though the response to SAL was smaller than that to sulpiride. The secretory pattern of PRL in response to SAL or sulpiride in female calves resembled that in male calves. A single i.v. injection of SAL or sulpiride significantly stimulated the release of PRL in cows (P<0.05). There was no significant difference in the PRL-releasing response between the SAL- and sulpiride-injected groups in cows. A single intracerebroventricular injection of SAL (10mg/head) also significantly stimulated the release of PRL in castrated calves (P<0.05). These results show that SAL is involved in the regulatory process for the secretion of PRL, not only in male and female calves, but also in cows. The results also suggest that the potency of the PRL-releasing response to SAL differs with the physiological status of cattle.

Characteristics of stimulation of gonadotropin secretion by kisspeptin-10 in female goats.

The aims of the present study were to clarify the effect of kisspeptin-10 (Kp10) on the secretion of luteinizing hormone (LH), follicle stimulating hormone (FSH), growth hormone (GH) and prolactin (PRL) in goats, and compare the characteristics of any response with those of the response to gonadotropin-releasing hormone (GnRH). The experiments were performed using four female goats (4-5 years old) in the luteal phase of estrous cycle. A single intravenous (i.v.) injection of 1, 5 and 10 microg/kg b.w. (0.77, 3.85 and 7.69 nmol/kg b.w.) of Kp10 stimulated the release of LH. Maximum values were observed 20-30 min after the injection. On the other hand, Kp10 did not alter plasma GH and PRL concentrations significantly. Three consecutive i.v. injections of Kp10 (5 microg/kg b.w.) or GnRH (5 microg/kg b.w.: 4.23 nmol/kg b.w.) at 2-h intervals increased both plasma LH and FSH levels after each injection (P<0.05); however, the responses to Kp10 were different from a similar level of GnRH. The rate of decrease in LH and FSH levels following the peak was attenuated in Kp10-treated compared to GnRH-treated animals. These results show that Kp10 can stimulate the release of LH and FSH but not GH and PRL in female goats and suggest that the LH- and FSH-releasing effect of the i.v. injection of Kp10 is less potent than that of GnRH.

Expression pattern of adhesion molecules in junctional epithelium differs from that in other gingival epithelia.

BACKGROUND AND OBJECTIVE: The gingival epithelium is the physiologically important interface between the bacterially colonized gingival sulcus and periodontal soft and mineralized connective tissues, requiring protection from exposure to bacteria and their products. However, of the three epithelia comprising the gingival epithelium, the junctional epithelium has much wider intercellular spaces than the sulcular epithelium and oral gingival epithelium. Hence, the aim of the present study was to characterize the cell adhesion structure in the junctional epithelium compared with the other two epithelia. MATERIAL AND METHODS: Gingival epithelia excised at therapeutic flap surgery from patients with periodontitis were examined for expression of adhesion molecules by immunofluorescence. RESULTS: In the oral gingival epithelium and sulcular epithelium, but not in the junctional epithelium, desmoglein 1 and 2 in cell-cell contact sites were more abundant in the upper than the suprabasal layers. E-cadherin, the main transmembranous molecule of adherens junctions, was present in spinous layers of the oral gingival epithelium and sulcular epithelium, but was scarce in the junctional epithelium. In contrast, desmoglein 3 and P-cadherin were present in all layers of the junctional epithelium as well as the oral gingival epithelium and sulcular epithelium. Connexin 43 was clearly localized to spinous layers of the oral gingival epithelium, sulcular epithelium and parts of the junctional epithelium. Claudin-1 and occludin were expressed in the cell membranes of a few superficial layers of the oral gingival epithelium. CONCLUSION: These findings indicated that the junctional epithelium contains only a few desmosomes, composed of only desmoglein 3; adherens junctions are probably absent because of defective E-cadherin. Thus, the anchoring junctions connecting junctional epithelium cells are lax, causing widened intercellular spaces. In contrast, the oral gingival epithelium, which has a few tight junctions, functions as a barrier.

Partial purification and characterization of an esterase acting on the anticancer pro-drugs, 7-ethylcamptothecin derivatives.

A hydrolytic enzyme which catalyzes hydrolysis of the ester-linkage of a series of 17-O-acyl derivatives of 7-ethylcamptothecin-21-(2-dimethylamino)ethylamide [acyl derivatives of 22E] was purified from rat liver and its properties were characterized. It hydrolyzed the ester-linkage of all 22E derivatives tested as well as p-nitrophenyl acetate at pH 8-9 but had no effect on 7-ethyl-10-[4-(piperidino)-1-piperidino] carbonyloxycamptothecin (CPT-11: irinotecan), unlike CPT-11 converting carboxylesterase, which was previously purified from rat serum [Tsuji T. et al., J. Pharmacobio-Dyn., 14, 341 (1991)]. The enzyme had no effect on either acetyl choline or butyrylcholine. It was inhibited by several organophosphorous compounds such as diisopropyl fluorophosphate (DFP), bis-(p-nitrophenyl)phosphate and paraoxon, but was insensitive to inhibitors specific for choline esterases. These results indicate that this liver esterase is clearly distinct from choline esterase and serum CPT-11 converting enzyme and is able to convert pro-drugs, O-acyl derivatives of 22E, to an antitumor agent.

Synthesis and antitumor activity of 20(S)-camptothecin derivatives. A-ring-substituted 7-ethylcamptothecins and their E-ring-modified water-soluble derivatives.

Twenty-six novel A-ring-modified 7-ethylcamptothecins (6) were synthesized by Friedländer's condensation of the chiral tricyclic ketone (5) with aminopropiophenones (4). The compounds substituted with fluorine at the 11 position showed strong cytotoxicity to KB and L1210 cells. The 11-fluoro derivatives also exhibited strong inhibitory activity on DNA topoisomerase I. Nine compounds 6 with four to ten times stronger cytotoxicity than that of camptothecin were selected and converted into water-soluble 17-O-acyl amide derivatives (8). Compounds 8e (10-Me, O-COCH2CH2SCH3) and 8f (11-F, O-COC2H5) showed activity towards Meth A in mice that was comparable to that of CPT-11, at lower doses than CPT-11.

Chemical modification of antitumor alkaloids, 20(S)-camptothecin and 7-ethylcamptothecin: reaction of the E-lactone ring portion with hydrazine hydrate.

The structure of the N-amino pyridone (4a) obtained by the reaction of camptothecin (1a) with hydrazine was determined by X-ray crystallography. A mixture of 7-ethylcamptothecin (1b) and hydrazine hydrate was stirred at room temperature, and the hydrazide (2b) was isolated as its diacetate 2c. Treatment of the 17-O-acetyl amide (5a) with hydrazine gave 1b (74% yield) and the N-amino lactam 6 (11% yield). Compounds with bulky acyl groups, 5c--e, gave 6 in modest yields. The N-amino lactam 6 was smoothly dehydrated into the pyridone 4d by refluxing in hydrazine hydrate.

Chemical modification of an antitumor alkaloid, 20(S)-camptothecin: E-lactone ring-modified water-soluble derivatives of 7-ethylcamptothecin.

7-Ethylcamptothecin (1d), a model which does not have any site on the A-ring for further modification was converted into water-soluble derivatives by opening the E-ring lactone. 1d was heated in N,N-dimethylenediamine to yield amide 2a, and this was then acylated to furnish 3a-q, which were soluble in water as their HCl salts. The propionyl (3b), butyryl (3c) and methylthiopropionyl (3h) derivatives showed higher activity than the sodium salt of 1d. The acyl group makes the derivatives more lipophilic, and ease of hydrolysis of amide 2a to 1d is thought to be necessary for significant activity.

Chemical modification of an antitumor alkaloid, 20(S)-camptothecin: glycosides, phosphates and sulfates of 7-ethyl-10-hydroxycamptothecin.

Water-soluble derivatives having the lactone ring intact were synthesized starting from 7-ethyl-10-hydroxycamptothecin (1). Glycosides (2) of the phenolic hydroxyl group of 1 were obtained by reaction with acetylated alpha-bromosugars in acetone or aqueous acetone in the presence of potassium carbonate, followed by deprotection. Phosphates (3) were prepared by reaction of 1 with phosphoryl chloride in pyridine or with dibenzylchlorophosphoridate. Sulfates (4) were obtained by reaction of 1 with sulfur trioxide-pyridine complex in the presence of a tertiary amine. The organic ammonium salts of monophosphate (3p) and sulfates (4a and 4b) showed significant activity against L1210 in vivo.

[Effect of hydroxylapatite particles during healing of experimental furcation involvement in beagle dogs].

The present study was performed to evaluate the effects of hydroxylapatite (HAP) on tissue regeneration in various types of furcation involvement. Upper premolar tooth sites of 9 beagle dogs (3-6 years old) were used. HAP particles were implanted into three types of furcation defects: Class III lesion (by Glickman) in 2nd premolar sites, artificially caused by intrafurcal suturing or spontaneously developed about 3 months after extraction of the 1st premolar; Class IV lesion in 4th premolar sites artificially produced 2 months before implantation; and through-and-through furcal bony defects in 3rd premolar sites at the time of HAP implantation. Macroscopic, radiographic and microscopical investigations of the postoperative status were carried out at intervals of 1, 2, 3 and 6 months. The appearance of the gingiva in bony defect sites was almost the same as that in the presurgical status 2 weeks after implantation, although gingival inflammation was persistent in Class III and IV lesions. Most particles in Class III and IV lesions exfoliated until 2 weeks after implantation, and the junctional space between recipient bone and particles could not be distinguished, probably as a result of incorporation of HAP and osseous tissue. Histological observation revealed that HAP particles were surrounded by new bone located at the top of the alveolar crest in all defects. However, there were no obvious signs of coronal bone formation or connective tissue attachment in Class III and IV lesions up to 3 months after the operation. At 6 months there was evidence of new bone formation over the presurgical crests in Class III and IV lesions. On the other hand, there were obvious signs of connective tissue attachment and bone formation in bony defect sites, probably induced by the tissue of the periodontal ligament remaining after the surgical procedure, and nonphysiological ankylosis between root and bone was frequently observed. There were notable findings, such as newly formed osteoid tissue intervening with the adhesive particles, calcified tissue intervening between HAP particles and root surface showing ankylosis, and peripheral osteoid formation in the HAP particles. It is postulated that HAP has no apparent role in induction of bone formation, although there is chemical affinity to calcified tissue, and it is effective in yielding a volume of bone-like tissue where osseous repair could be performed. However, HAP did not enhance regeneration of lost periodontal structures including connective tissue attachment.

Morphological Differences Between Radopholus citrophilus and R. similis.

SEM observations of the external morphology of populations of Radopholus citrophilus and R. similis revealed several diagnostic differences. The cloaco-spicular orifice on males of R. citrophilus had three to seven genital papillae (anterior hypoptygmata), whereas males of R. similis were either smooth or had one or two shorter genital papillae (anterior hypoptygmata). Females of R. citrophilus had four annules in the region of the vulval opening, but R. similis had five annules in the same region. The labial disc and lateral lips appeared to be of diagnostic significance, but these areas were more susceptible to artifacts due to fixation. An unknown population of Radopholus from Puerto Rico with a chromosome number of n = 4 was morphologically similar to R. similis. These morphological differences provide additional support that R. citrophilus and R. similis are distinct species.

Nucleotide sequence of the adenovirus type 40 inverted terminal repeat: close relation to that of adenovirus type 5.

Human adenovirus type 40 (Ad40) is a pathogen that causes acute infantile gastroenteritis. Ad40 has the distinct characteristic of being difficult to propagate in conventional cultured human cells. The nucleotide sequence of the inverted terminal repeat (ITR) of Ad40, which includes the origin of adenoviral DNA replication, was determined using recombinant plasmid DNA. By using our newly developed program to express the ITR homologies simply, we found that the ITR of Ad40, which is 163 nucleotides long, was related most closely to that of adenovirus type 5, which replicates efficiently.

Sequence analysis in the E1 region of adenovirus type 4 DNA.

Adenovirus type 4 (Ad4) is the sole member of adenovirus group E based on overall DNA sequence homology, restriction endonuclease cleavage patterns, and the size of capsid proteins. We cloned the BamHI-F fragment from the left end of Ad4 in pUC13-1 between the SalI and BamHI sites in order to carry out the structural analysis of the E1A region of Ad4. The complete sequence of the BamHI-F fragment (2042 bp) has been determined. From the DNA sequence, the splice sites for the putative 12 S and 13 S mRNAs, encoded by the E1A region of Ad4 were deduced. If protein synthesis initiates at the first available AUG triplet (position 575), these 12 S and 13 S mRNAs would code for polypeptides containing 226 and 257 amino acids, respectively. Comparison of Ad4- and Ad7-13 S mRNA-coded polypeptides indicates that there is 57% homology, whereas the homology is only 38% with Ad12 and 31% with Ad2-13 S mRNA-coded polypeptides. The structural analysis in the E1 region of Ad4 also includes the coding region for the E1B 19-kDa protein. Ad4 and Ad7 shows 65% homology in the coding regions for E1B 19-kDa protein. Comparison of the DNA sequence of Ad4 with those of Ad2, Ad7, and Ad12 by using a dot matrix computer program and by Southern hybridization revealed that Ad4 bears a stronger homology with Ad7 than with Ad2 and Ad12 in this region. Hydropathy plots and alignments of the putative polypeptides coded by this region in Ad4 with those from the corresponding regions of different serotypes to reveal the highly conserved domains also support the above conclusion.

Partial sequence analysis of cloned dengue virus type 2 genome.

Dengue virus (DEN) is a member of flaviviruses and contains a single, (+)-strand RNA of approx. 11 kb. Complementary DNA copy of the RNA was synthesized using reverse transcriptase and oligo(dT) as primer. The double-stranded DNA copy was cloned at the PstI site of pUC13'-1 vector and was used to transform Escherichia coli JM83. Eleven transfomants were found to contain DEN insert as screened by colony hybridization. Three clones were chosen for further characterization by nucleotide (nt), sequence analysis. Two of these clones overlapped by 470 bp. Sequences of these three clones totalling about 4.6 kb were obtained. Translation of this DNA in all possible reading frames revealed the presence of long open reading frames spanning the entire length of the cDNA clones. The putative polypeptides derived from the nt sequence are 885 and 643 amino acids in length and show homology to the region of polyprotein coded by the yellow fever virus genome corresponding to the non-structural proteins [Rice et al., Science 229 (1985) 726-733]. The significant homology between these two viruses in the regions coding for the non-structural proteins NS3 and NS5 suggests an important role for these two proteins in the life cycle of these viruses.