Developmental changes in the activity and cellular localization of hepatic casein kinase II in the rat

Abstract The activity and cellular localization of hepatic casein kinase II(CKII) was examined during late fetal development in the rat. Cultured fetal hepatocytes displayed constitutive CKII activity which was not further activated by growth factor exposure. Similarly, fetal liver CKII showed approximately fivefold greater activity than adult liver. The fetal hepatic activity was, to a large degree, localized to a nuclear fraction. Postnuclear cytosol preparations from fetal and adult liver showed similar CKII activity. In all cases, FPLC ion exchange chromatography followed by Western immunoblotting showed that immunoreactive CKII coincided with kinase activity. However, Parallel determinations of CKII activity and immunoreactive CKII levels showed a higher(five-to sixfold) CKII specific activity in nuclear extracts compared to cytosol. In summary, fetal hepatic CKII demonstrates coincident nuclear localization and activation. We hypothesize that the regulation of hepatic CKII is relevant to the mitogen-independent proliferation displayed by fetal rat hepatocytes. © Wiley-Liss, Inc.     

Isolation of a cDNA encoding a growth-arrest associated gene and characterization of its regulation

We are interested in understanding the molecular events associated with the growth-arrest of vascular SMCs. We constructed a subtracted cDNA library enriched in nucleotide sequences associated with quiescent SMCs. This library was screened with similarly subtracted ³²P-labeled cDNAs to identify growth-arrest associated cDNA clones. Characterization of 19 of these cDNA clones revealed that 9 hybridized to mRNAs that exhibited a 2–3 fold increase in growth-arrested SMCs. In addition, two other cDNAs hybridized to a 5 Kb mRNA that was elevated approximately 10-fold in high density growth-arrested SMCs. Genomic Southern blot hybridization and DNA sequencing analysis indicated that these cDNAs encoded the same gene (LG7) and that this gene may be a member of a multigene family or that it may contain a sequence shared by other unrelated genes. Augmented expression of LG7 was associated with both high cell density and serum deprivation induced growth-arrest. LG7 mRNA expression was down-regulated when SMCs were incubated with FBS or with reagents that arrest cells in early S-phase. Additional analysis with cell cycle specific inhibitors indicated that LG7 mRNA levels were also low when cells were blocked at the G₂ phase of the cell cycle but blockage at mitosis resulted in an elevated level of LG7 mRNA. We further demonstrated that the expression of LG7 was dependent on the presence of a relatively labile protein since protein synthesis inhibitors specifically blocked the expression of this mRNA but not the mRNA expression of α₁(III) collagen or ferritin H-chain. Finally, we demonstrated that Bt₂cAMP was able to induce mRNA expression of LG7 within 2 h, suggesting that this gene may be directly regulated via the cyclic-AMP-dependent protein kinase pathway.     

Establishment and characterization of a new,spontaneously immortalized,pancreatic ductal cell line from the Syrian golden hamster

Spontaneously immortal pancreatic cell lines are not available. By use of a defined culture medium, such a line (TAKA-1) was established from the Syrian golden hamster. Cytological, cytogenetic, molecular biological, enzymatic and receptor patterns as well as antigenicity were studied and were compared with those of the normal hamster pancreatic ductal cells in vivo. TAKA-1 cells grew exponentially in a monolayer on collagen gel in a defined medium but did not proliferate in soft agar. Ultrastructurally, the cells closely resembled the normal hamster pancreatic ductal cells. Similarities and dissimilarities were found between the normal ductal cells and TAKA-1 cells. Similarities included the presence of cytokeratin, carbonic anhydrase and some tumor-associated antigens. However, unlike the normal ductal cells, TAKA-1 cells expressed blood group A angigen and anti-vimentin, showed affinity to selected lectins, and an abnormality of chromosome 3, which is suggested to be associated with immortality. Moreover, unlike the hamster pancreatic ductal cancer cells but like the normal hamster pancreatic ductal cells, TAKA-1 cells did not have a c-Ki-ras mutation. EGF, TGF- and secretin, but not CCK or GRP, bound to the TAKA-1 cells. TAKA-1 cells produced TGF-, and their growth was stimulated by exogenous EGF in serum-free medium. This cell line presents a suitable model for biologic and pathologic study of the hamster pancreatic ductal cells in vitro.     

Structural organization and epithelial cell types of the intestinal diverticula (protopancreas) of ammocoetes of southern hemisphere lampreys: functional and phylogenetic implications

Larvae of the two southern hemisphere lamprey genera, Mordacia and Geotria, possess one and two intestinal diverticula, respectively, each originating at the oesophageal-intestinal junction. These diverticula comprise an inner layer of simple columnar epithelium composed solely of zymogen and mucous cells, a middle layer consisting mainly of a blood sinus, and an outer serosa layer covered by a simple squamous epithelium (mesothelium). The inner surface is highly folded only in Mordacia. The secretion of mucus probably protects the epithelium from the effects of digestive enzymes secreted by the zymogen cells and/or bile, which enters the diverticulum at its tip. Unlike the situation in southern hemisphere lampreys, the zymogen cells of the larvae of holarctic lampreys are located in the anterior intestine, a condition considered to be primitive. It is thus proposed that intestinal diverticula were developed during the evolution of southern hemisphere lampreys. The relocation of zymogen cells in the diverticula increases the area for these cells, and thus the capacity for the synthesis and secretion of digestive enzymes, particularly in Mordacia where the inner surface is folded.     

Detection of oligoclonal T lymphocytes in lymph nodes draining from advanced non-small-cell lung cancer

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-smallcell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. in an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.     

Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer

Data from an ongoing clinical radioimmunoscintigraphy trial indicate that^99mTc-labeled monoclonal antibody (mAb) E48 is highly capable of selectively targeting squamous cell carcinoma of the head and neck (HNSCC). The percentage of the injected dose per gram of tumor tissue was found to be high, rendering mAbE48 a promising candidate mAb for therapeutic purposes. We now describe the construction of a chimeric (moouse/human) mAb E48 by recombinant DNA technology. The genes encoding the variable domains of the heavy and light chain were cloned and ligated into experession vectors containing the human 1 heavy-chain gene and the human k lightchain gene respectively. Biological properties of the resulting chimeric mAb E48 were compared to the murine form in vitro and in vivo. The reactivities of chimeric (c)mAb and murine (m)mAb E48 with HNSCC, as assessed by immunohistochemical staining as well as immuno-blotting were shown to be similar. The affinity constant appeared to be 0.9×10¹⁰ M^–1 and 1.6×10¹⁰ M^–1 for the mmAb and cmAb respectively. The biodistribution of both antibodies was tested by simultaneous injection into nude mice bearing human HNSCC xenografts. cmAb E48 was found to be cleared more rapidly from the blood than mmAb E48, resulting in a 30% lower tumor uptake but similar tumor to non-tumor ratios, 3 days after injection. Moreover, it was shown that cmAb E48 is highly capable of lysing HNSCC targets in ADCC assays in vitro, whereas the mmAb appeared to be almost incative. These data indicate that cmAb E48 has potential as a targeting agent for the eradication of HNSCC in man.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Mauthner cell-initiated abrupt increase of the electric organ discharge in the weakly electric fishGymnotus carapo

Stimulation of the spinal cord of the electric fish Gymnotus carapo, evoked an abrupt increase in the discharge rate of the electric organ. At the maximum of this response, the rate increased an average of 26 ± 11.8%. The duration of the response was 4.9 ± 2.12 s; its latency was 10.4 ± 1.1 ms. Activation of the Mauthner axon played a decisive role in this phenomenon as indicated by the following: (1) recordings from the axon cap of the Mauthner cell demonstrated that the response was evoked if the Mauthner axon was antidromically activated and (2) a response that was similar to that produced by spinal cord stimulation, was elicited by intracellular stimulation of either Mauthner cell. Stimulation of the eighth nerve could also increase the discharge rate of the electric organ. The effect was greater if a Mauthner cell action potential was elicited. The findings described in the present report, indicate the existence of a functional connection between the Mauthner cell and the electromotor system in Gymnotus carapo. This connection may function to enhance the electrolocative sampling of the environment during Mauthner-cell mediated behaviors. This is a novel function for the Mauthner cell.Abbreviations EHP extrinsic hyperpolarizing potential - EOD electric organ discharge - M-AIR Mauthner initiated abrupt increase in rate - M-cell Mauthner cell - M-axon Mauthner axon - PM pacemaker nucleus - PM-cell pacemaker cell - PPn prepacemaker nucleus - SPPn sublemniscal prepacemaker nucleus