Composition and molecular organization of lipids and proteins in the envelope of mycoplasmavirus MVL2.

MVL2 virus was purified from culture supernatants of infected Acholeplasma laidlawii cells by differential centrifugation, followed by velocity centrifugation in sucrose gradients. The purified virus contained 0.08 to 0.1 mumol of lipid phosphorous per ml of viral protein. Thin-layer chromatography of viral lipids revealed the presence of phospho-, glyco-, and phosphoglycolipids identical with those found in the host cell membrane, but the relative amount of phosphatidylglycerol was much lower than that in the virus. The fatty acid composition of lipids incorporated into the virus included lipids synthesized before and after infection. The freedom of motion of spin-labeled fatty acids in MVL2 depended markedly on temperature and on the position of the nitroxide group on the hydrocarbon chain of the probe, suggesting that the local environment of the probe has the properties of a lipid bilayer. Nevertheless, the lipid hydrocarbon chains in MVL2 appear to be less mobile than those in membranes of the host cells. Polyacrylamide gel electrophoresis of purified MVL2 revealed four major and about five minor polypeptide bands. None of the polypeptide bands gave a positive periodic acid-Schiff reaction. Lactoperoxidase-mediated iodination, followed by proteolytic digestion of intact MVL2 particles, revealed that at least two major polypeptides are localized on the external surface of the viral envelope.     

Cell size in aging monolayer cultures

Summary Changes in the size of the area covered by individual cultured WI-38 cells as the cultures age have been studied by using a new microphotographic paper cutout technique. This method is nondestructive and nonintrusive and avoids a number of artifacts which can occur in the measurement of suspended cells. The measurements reveal that the decreased cell yield of late passage cultures-reflects not only the appearance of a subpopulation of larger cells but also the failure of the cells to utilize all the growth surface available to them. This work was supported in part by USPHS research grant AG-00378 and by a fellowship, AG-05019, from the National Institute on Aging.     

Early induction of ribonucleotide reductase gene expression by transforming growth factor beta 1 in malignant H-ras transformed cell lines.

Previous investigations have indicated that the suppression of proliferation by transforming growth factor (TGF) beta 1 is often lost upon cellular transformation, and that proliferation of some tumors is stimulated by TGF-beta. The present study provides the first observation of a link between TGF-beta 1 regulation of this process and alterations in the expression of ribonucleotide reductase, a highly controlled rate-limiting step in DNA synthesis. A series of radiation and T24-H-ras-transformed mouse 10T1/2 cell lines exhibiting increasing malignant potential was evaluated for TGF-beta 1 induced alterations in ribonucleotide reductase M1 and M2 gene expression. Early increases in M1 and/or M2 message and protein levels were observed only in malignant cell lines. The TGF-beta 1 induced changes in M1 and/or M2 gene expression occurred prior to any detectable changes in the rates of DNA synthesis, supporting the novel concept that ribonucleotide reductase gene expression can be elevated by TGF-beta 1 without altering the proportion of cells in S phase. T24-H-ras-transformed 10T1/2 cells were transfected with a plasmid containing the coding region of TGF-beta 1 under the control of a zinc-sensitive metallothionein promoter. When these cells were cultured in the presence of zinc, a large induction of TGF-beta 1 message was observed within 1 h. Both M1 and M2 genes were also induced, with increased mRNA levels appearing 2 h after zinc treatment, or 1 h after TGF-beta 1 message levels were clearly elevated. In total, the data suggests a mechanism of autocrine stimulation of malignant cells by TGF-beta 1, in which early alterations in the regulation of ribonucleotide reductase may play an important role.     

CD4+ T cell clones specific for the human p97 melanoma-associated antigen can eradicate pulmonary metastases from a murine tumor expressing the p97 antigen.

p97 is a human tumor-associated Ag present on most melanoma cells that represents a possible target for immunologic attack. To evaluate the capacity of T cells reactive with this protein to promote elimination of melanoma cells expressing p97, a murine model was developed by transfecting a C3H/HeN melanoma with the p97 cDNA, generating p97-specific CD4+ T cells by in vivo immunization of C3H/HeN mice with a vaccinia/p97 recombinant virus followed by in vitro cloning with soluble p97 protein, and determining whether these CD4+ T cells could mediate rejection of pulmonary metastases. Characterization of the T cell clones demonstrated the presence of both I-Ak and I-Ek-restricted clones, although the majority of clones recognized p97 in the context of I-Ek. Analysis of clonal specificity using truncated p97 proteins revealed that at least three epitopes were immunogenic, and further studies with overlapping 15-amino acid peptides from a region of the p97 molecule defined by these truncated proteins identified an immunodominant epitope responsible for the majority of the I-Ek response. The T cell clones were not capable of directly recognizing the p97-expressing melanoma cells but responded to the tumor if syngeneic APC were present to process the tumor-derived p97 Ag. The therapeutic efficacy of these CD4+ T cell clones was evaluated in an adoptive therapy model in which mice bearing metastatic pulmonary lesions were treated by i.v. administration of the p97-specific cells. Despite the inability of the CD4+ clones to directly respond to or lyse the tumor cells, the clones were effective in promoting tumor eradication. In vitro studies demonstrated that this may have reflected secretion of lymphokines that activated macrophages to lyse the tumor. The results suggest that noncytolytic p97-specific CD4+ T cell clones can be effective in therapy of pulmonary melanoma metastases. Moreover, if human T cells reactive with the p97 protein could be generated, the expression of this tumor-associated Ag in melanoma cells might be adequate for such T cells to mediate a therapeutic antitumor response.     

Suppression of splenic macrophage interleukin-1 secretion following intracerebroventricular injection of interleukin-1 beta: evidence for pituitary-adrenal and sympathetic control.

Intracerebroventricular (ICV) injections of interleukin-1 beta (IL-1 beta) produced a dose-dependent increase in plasma corticosterone and adrenocorticotropic hormone (ACTH) within 2 hr of injection and then declined over the next 24 hr. Using a potent steroidogenic dose of IL-1 beta (5 ng), ICV injection resulted in suppression of splenic macrophage IL-1 secretion following stimulation by LPS in vitro. Macrophage TGF-beta secretion was not affected, indicating a differential action of ICV IL-1 beta on macrophage cytokine production. Following adrenalectomy (ADX), the suppressive effect of ICV IL-1 beta was reversed and resulted in stimulation of macrophage IL-1 secretion, indicating that the suppression was mediated by adrenocorticol activation. However, surgical interruption of the splenic nerve to eliminate autonomic innervation of the spleen also prevented the macrophage suppressive signal in rats given ICV IL-1 beta. Furthermore, the combination of ADX and splenic nerve section resulted in a potent stimulatory effect of ICV IL-1 beta on splenic macrophage IL-1 secretion which was greater than either ADX or splenic nerve section alone. These results support the concept of a negative feedback on macrophage IL-1 secretion by the central action of IL-1 beta and indicate that both the hypothalamic-pituitary-adrenal axis and the sympathetic nervous system mediate this effect.     

High-level expression and secretion of a lysine-containing analog of Escherichia coli heat-stable enterotoxin'

The mechanism of action of the heat-stable enterotoxin STa secreted from enterotoxigenic forms of Escherichia coli has remained elusive, in part due to a tedious, low-yield purification procedure. We report here a method for obtaining large amounts of a biologically active lysine-containing analog of STa. Initial attempts to express the toxin using an expression vector that did not encode a signal sequence resulted in no biologically active material being recovered either from lysed cells or as a secretory product. However, use of the secretion vector pJAL36, which contains the STII enterotoxin signal sequence, allowed large amounts of an STa derivative containing the additional sequence Ser-Thr-Lys at the amino terminus of the mature enterotoxin to be readily purified from culture supernatants. This enterotoxin analog, known as KSTa-1, was equal in biological and receptor binding activity to the native toxin STa. The lysine residue present in KSTa-1 promises to be useful as a reactive amino acid that is readily derivatized to allow coupling of the enterotoxin to supports for affinity chromatography and antigenic conjugates. Additionally, the insertion of the lysine residue carboxy terminal to the Ser-Thr sequence adds a reversible “handle” to the toxin sequence in that the Ser-Thr-Lys segment can be removed by treatment with trypsin, releasing the native form of STa.     

Molecular analysis of the Smith-Magenis syndrome: a possible contiguous-gene syndrome associated with del(17)(p11.2).

We undertook clinical evaluation (32 cases) and molecular evaluation (31 cases) of unrelated patients affected with Smith-Magenis syndrome (SMS) associated with an interstitial deletion of band p11.2 of chromosome 17. Patients were evaluated both clinically and electrophysiologically for peripheral neuropathy, since markers showing close linkage to one form of Charcot-Marie-Tooth disease (CMT1A) map to this chromosomal region. The common clinical findings were broad flat midface with brachycephaly, broad nasal bridge, brachydactyly, speech delay, and hoarse, deep voice. Fifty-five percent of the patients showed clinical signs (e.g., decreased or absent deep tendon reflexes, pes planus or pes cavus, decreased sensitivity to pain, and decreased leg muscle mass) suggestive of peripheral neuropathy. However, unlike patients with CMT1A, these patients demonstrated normal nerve conduction velocities. Self-destructive behaviors, primarily onychotillomania and polyembolokoilamania, were observed in 67% of the patients, and significant symptoms of sleep disturbance were observed in 62%. The absence of REM sleep was demonstrated by polysomnography in two patients. Southern analysis indicated that most patients were deleted for five 17p11.2 markers--FG1 (D17S446), 1516 (D17S258), pYNM67-R5 (D17S29), pA10-41 (D17S71), and pS6.1-HB2 (D17S445)--thus defining a region which appears to be critical to SMS. The deletion was determined to be of paternal origin in nine patients and of maternal origin in six patients. The apparent random parental origin of deletion documented in 15 patients suggests that genomic imprinting does not play a role in the expression of the SMS clinical phenotype. Our findings suggest that SMS is likely a contiguous-gene deletion syndrome which comprises characteristic clinical features, developmental delay, clinical signs of peripheral neuropathy, abnormal sleep function, and specific behavioral anomalies.