Scleroblast cultures from the gorgonianLeptogorgia virgulata (Lamarck) (Coelenterata: Gorgonacea)

Summary Scleroblasts were separated from fragmented tissue of growing tips ofLeptogorgia virgulata and cultured using a modification of the technique of Rannou. Replacement of fetal bovine serum with horse serum seemed to increase scleroblast viability. Cell adhesion occurred from 14 to 43 d. Cultured scleroblasts demonstrated cell aggregation, spicule formation, and extrusion of spicules into the external medium. Cells showing spicules in the process of being extruded appeared on the average after 24 d of culture. Variability among cultures was marked with respect to both division and spicule formation. Healthy cultures were maintained for more than 4 mo. This work was supported by National Science Foundation grants PCM8201389 and DCB8502698. This is contribution No. 674 of Belle W. Baruch Institute for Marine Biology and Coastal Research, University of South Carolina.     

An attempt to isolate genes responsible for spontaneous and experimental metastasis in the mouse model

Cancer develops and progresses as genetic alterations occur subsequently. Onset process of cancer has become well understood in some types of cancer, such as colorectal cancers. In this process, responsible alterations were identified in numbers of oncogenes such as k-ras, and tumor suppressor genes such as p53, as Vogelstein proposed earlier in the multistage carcinogenesis theory. In contrast, our understanding remains short to draw such an adequate diagram for the process during which cancer becomes more malignant, i.e., metastatic. To examine the molecular basis for this progression step, mouse metastasis models have been established where tumor cell lines are inoculated into mice and metastasize to specific organs. The model using B16 melanoma cells is one of the most developed. BL6 subline, one of the most metastatic, was obtained from F10 subline simply through six rounds of in vitro selection. Nonetheless, BL6 cells metastasize lungs much more heavily than F10 cells when injected subcutaneously. The difference in gene expression between the two sublines is considered rather small but relevant for spontaneous metastasis. We began our research by elaborating a method for the construction of subtracted cDNA libraries, and made it applicable to BL6 and F10 cells. As a result, we were able to isolate a couple of genes that were expressed differently between the two sublines. As might be expected, each of the genes appeared to play a role more or less in distinct aspects of spontaneous metastasis of B16 melanoma cells. Moreover, similar roles were expected for the genes in the process by which human melanoma cells metastasize.     

A novel small protein of Bacillus subtilis involved in spore germination and spore coat assembly

Two small genes named sscA (previously yhzE) and orf-62, located in the prsA-yhaK intergenic region of the Bacillus subtilis genome, were transcribed by SigK and GerE in the mother cells during the later stages of sporulation. The SscA-FLAG fusion protein was produced from T(5) of sporulation and incorporated into mature spores. sscA mutant spores exhibited poor germination, and Tricine-SDS-PAGE analysis showed that the coat protein profile of the mutant differed from that of the wild type. Bands corresponding to proteins at 59, 36, 5, and 3 kDa were reduced in the sscA null mutant. Western blot analysis of anti-CotB and anti-CotG antibodies showed reductions of the proteins at 59 kDa and 36 kDa in the sscA mutant spores. These proteins correspond to CotB and CotG. By immunoblot analysis of an anti-CotH antibody, we also observed that CotH was markedly reduced in the sscA mutant spores. It appears that SscA is a novel spore protein involved in the assembly of several components of the spore coat, including CotB, CotG, and CotH, and is associated with spore germination.     

Variations in concentrations of bacterial metabolites, enzyme activities, moisture, pH and bacterial composition between and within individuals in faeces of seven healthy adults

Variations between and within individuals, and correlations between concentrations of bacterial metabolites, including putrefactive products, ammonia and short chain fatty acids (SCFAs), enzyme activities, moisture and pH, as well as bacterial composition, were studied in faecal samples from seven healthy adults over a period of 7 months. Large variations, both between and within individuals, were observed in concentrations of putrefactive products. Although values for ammonia, SCFAs, enzyme activities, moisture and pH were generally variable, significant person-to-person differences were observed.
While ranges of log viable counts of the predominant bacteria such as eubacteria, bifidobacteria and bacteroides in each subject remained between 0·2 and 1·3, those of enterobacteria, streptococci (including enterococci) and lecithinase-negative clostridia varied between 0·4 and 3·0. Levels of bifidobacteria, enterobacteria, streptococci and total aerobic bacteria showed inter-individual variations. Correlations were found among certain of the parameters: moisture correlated negatively with p -cresol ( r = -0·707), pH ( r = -0–671) and β-glucosidase activity (GS) ( r = -0·608), and positively with acetic acid ( r = 0·621), while negative correlations were observed in pH with acetic and butyric acids ( r = -0·690 and -0·623, respectively).
No significant correlations were found between bacterial compositions, and other faecal factors such as pH, moisture, metabolic enzyme activities and concentrations of putrefactive products.     

X-ray Crystallographic Study of an HIV-1 Fusion Inhibitor with the gp41 S138A Substitution

The S138A substitution of fusion inhibitory peptides derived from the C-terminal heptad repeat (C-HR) of the human immunodeficiency virus type 1 (HIV-1) gp41 leads to enhanced binding affinity to the N-terminal heptad repeat (N-HR). As such, these peptides exhibit highly potent anti-HIV-1 activity. X-ray crystallographic analysis was performed to understand the effect of the substitution on binding affinity. The comparison of the native and S138A crystal structures indicated that the increase in the hydrophobicity of the S138A substitution may aid the stabilization of the N-HR/C-HR complex through additional hydrophobic contacts. Free-energy calculations suggest that the difference between the desolvation free energies of the C-HR-derived peptides with and without the S138A mutation dominates the observed difference in anti-HIV-1 activity.     

Preparation of antibodies against a novel immunosuppressant, FTY720, and development of an enzyme immunoassay for FTY720

FTY720 (1) is a novel immunosuppressant (immunomodulator), derived from ISP-I (2: myriocin and thermozymocidin). To clarify the pharmacokinetic properties of 1, antibodies against 1 were prepared and a competitive enzyme immunoassay (EIA) was developed. Two kinds of haptens, 3 and 4, for 1 were synthesized and coupled to ovalbumin (OVA). Rabbits were immunized with 3-OVA or 4-OVA, and corresponding antibodies were obtained. Both antibodies recognized the 2-amino-2-(2-phenylethyl)propane-1,3-diol moiety in 1. Using the anti-3-OVA antibody, a competitive EIA for 1 was developed and evaluated. The range of quantification by the EIA was 0.06-10 ng/mL. The application of the EIA has enabled us to measure the FTY720 concentration in serum after oral administration of 1 (1mg/kg) to rats.     

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal‐regulated kinase 1/2

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an ‘acute’ phase (0–15 min) and ‘tonic’ phase (16–120 min), which is accompanied by significant phosphorylation of extracellular signal‐regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post‐formalin injection. To uncover a possible relationship between the slow‐onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 μg/mL in microdialysis fiber, 1 μL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of ‘tonic’ phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre‐treatment with NK1 receptor antagonist CP96345 (10 μM) blocked the effect of SP infusion. However, formalin‐induced ‘tonic’ phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the ‘tonic’ phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP‐NK1 receptor–ERK1/2 system.