Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.     

Site of reovirus replication in liver is determined by the type of hepatocellular insult.

Reovirus type 1 strain Lang is restricted from replicating in adult murine livers. In noninjured livers, approximately 1% of hepatocytes express reovirus antigen during infection. However, hepatocytes can be induced to express reovirus antigen if challenged with either toxins or trauma. We used selective hepatotoxins or surgical trauma to demonstrate that reovirus antigen localization in liver is determined by the site of hepatocellular insult and the timing of the virus inoculum.     

Assembly, intracellular processing, and expression at the cell surface of the human alpha beta T cell receptor/CD3 complex. Function of the CD3-zeta chain

The TCR/CD3 complex is a multimeric protein complex composed of a minimum of seven transmembrane chains (TCR alpha beta-CD3 gamma delta epsilon zeta 2). Whereas earlier studies have demonstrated that both the TCR-alpha and -beta chains are required for the cell surface expression of the TCR/CD3 complex, the role of the CD3 chains for the TCR/CD3 expression have not been experimentally addressed in human T cells. In this study the function of the CD3-zeta chain for the assembly, intracellular processing, and expression of the TCR/CD3 complex in the human leukemic T cell line Jurkat was investigated. The results indicate that: 1) CD3-zeta is required for the cell surface expression of the TCR/CD3 complex; 2) the pentameric form (TCR alpha beta-CD3 gamma delta epsilon) of the TCR/CD3 complex and single TCR chains associated with CD3 (TCR alpha-CD3 gamma delta epsilon and TCR beta-CD3 gamma delta epsilon) are produced in the endoplasmic reticulum in the absence of CD3-zeta; 3) the CD3-zeta does not associate with TCR alpha-CD3 gamma delta epsilon or TCR beta-CD3 gamma delta epsilon complexes; 4) CD3-zeta associate with the pentameric form of the TCR/CD3 complex in the endoplasmic reticulum to form the heptameric complex (TCR alpha beta-CD3 gamma delta epsilon----TCR alpha beta-CD3 gamma delta epsilon 2); and 5) CD3-zeta is required for the export of the TCR/CD3 complex from the endoplasmic reticulum to the Golgi apparatus for subsequent processing.     

Short chain alcohols activate guanine nucleotide-dependent phosphoinositidase C in turkey erythrocyte membranes

The ability of alcohols to regulate inositol lipid-specific phospholipase C (phosphoinositidase C) was examined in turkey erythrocyte ghosts prepared by cell lysis of erythrocytes which were prelabeled with [3H] inositol. Guanosine 5'-[gamma-thiotriphosphate] GTP[S] stimulated the production of both [3H]inositol bisphosphate (18-fold) and [3H]inositol trisphosphate (6-fold) in this system. The accumulation of [3H]inositol bisphosphate and [3H]inositol trisphosphate was linear up to 8 min following an initial lag period of 1-2 min. Ethanol (300 mM) reduced the lag period for [3H]inositol phosphate accumulation at submaximal GTP[S] concentrations and caused a shift to the left (3-fold) in the dose-response curve. Other short chain alcohols, methanol (300 mM), 1-propanol (200 mM), and 1-butanol (50 mM) also enhanced the accumulation of [3H] inositol phosphates in the presence of submaximal GTP[S] concentrations. Receptor activation by the purinergic agonist adenosine 5'-[beta-thio]disphosphate (ADP[S]) (10 microM) also reduced the lag period for [3H] inositol phosphate formation and shifted the GTP[S] dose response to the left (10-fold). In addition, ADP[S] increased the response to maximal GTP[S] concentrations. The formation of [3H]inositol phosphates induced by GTP[S] was associated with a concomitant decrease in labeling of both [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate, but no decrease in [3H]phosphatidylinositol was observed. All of the alcohols tested enhanced the breakdown of [3H]polyphosphoinositides in the presence of GTP[S]. The dose response to guanosine 5'-[beta gamma-imino]triphosphate for [3H]inositol phosphate formation was displaced to the left by ethanol (300 mM) and ADP[S] (10 microM) (2- and 7-fold), respectively. ADP[S] also enhanced the maximal response to guanosine 5'-[beta gamma-imino]triphosphate. The [3H]inositol phosphate formation produced in response to NaF was unaffected by either ethanol or receptor activation. These results indicate that alcohols initiate an activation of phosphoinositidase C, mediated at the level of the regulatory guanine nucleotide-binding protein.     

Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters

We have shown previously that chick muscle cells transformed with Rous sarcoma virus are unable to form clusters of acetylcholine receptors (AChRs) (Anthony, D. T., S. M. Schuetze, and L. L. Rubin. 1984. Proc. Natl. Acad. Sci. USA. 81:2265-2269) and are missing a 37-KD tropomyosin-like protein (TM-2) (Anthony, D. T., R. J. Jacobs-Cohen, G. Marazzi, and L. L. Rubin. 1988. J. Cell Biol. 106:1713-1721). In an attempt to clarify the role of TM-2 in the formation and/or maintenance of AChR clusters, we have microinjected a monoclonal antibody specific for TM-2 (D3-16) into normal chick muscle cells in culture. D3-16 injection blocks the formation of new clusters but does not affect the preexisting ones. In addition, TM-2 is concentrated at rat neuromuscular junctions. These data suggest that TM-2 may play an important role in promoting the formation of AChR clusters.     

Maternal diabetes and cardiovascular malformations: predominance of double outlet right ventricle and truncus arteriosus

Most studies on the relationship of maternal diabetes to cardiovascular malformations (CVM) have been prospective investigations of pregnancy outcome and therefore could not identify associations with rare cardiac lesions. The results of a retrospective study shed new light on the risks of specific cardiac defects in diabetic pregnancies. The Baltimore-Washington Infant Study, a population-based case-control investigation of CVM, provides information on maternal diabetes reported in personal interviews. Among 2259 mothers of cases, 35 (1.5%) reported diabetes present before pregnancy (called "overt") and 95 (4.2%) reported diabetes only during pregnancy (called "gestational"). Among 2,801 mothers of controls, 14 (0.5%) had overt diabetes and 83 (3.0%) had gestational diabetes. Malformation-specific risks were expressed as odds ratios (OR) with 99.5% confidence intervals (CI). The strongest associations with overt maternal diabetes were found with double outlet right ventricle (OR 21.33; 99.5% CI 3.34, 136.26), and truncus arteriosus (OR 12.81; 99.5% CI 1.43, 114.64). No significant diagnosis-specific associations were found with gestational diabetes. Non-cardiac malformations were present in 23% of infants with CVM whose mothers had overt diabetes and in 26% of infants with CVM whose mother had gestational diabetes, in 32% of infants with CVM whose mothers did not have diabetes, and in 4% of controls. Double outlet right ventricle and truncus arteriosus are malformations dependent upon neural-crest-cell-derived ectomesenchymal tissues; these are precisely the conotruncal abnormalities that result from experimental ablation of the neural crest in chick embryos. The association with diabetes suggests a further etiologic link between these two lesions.     

Ethanol inhibits thrombin-induced secretion by human platelets at a site distinct from phospholipase C or protein kinase C.

Ethanol is known to inhibit the activation of platelets in response to several physiological agonists, but the mechanism of this action is unclear. The addition of physiologically relevant concentrations of ethanol (25-150 mM) to suspensions of washed human platelets resulted in the inhibition of thrombin-induced secretion of 5-hydroxy[14C]tryptamine. Indomethacin was included in the incubation buffer to prevent feedback amplification by arachidonic acid metabolites. Ethanol had no effect on the activation of phospholipase C by thrombin, as determined by the formation of inositol phosphates and the mobilization of intracellular Ca2+. Moreover, ethanol did not interfere with the thrombin-induced formation of diacylglycerol or phosphatidic acid. Stimulation of platelets with phorbol ester (5-50 nM) resulted in 5-hydroxy[14C]tryptamine release comparable with those with threshold doses of thrombin. However, ethanol did not inhibit phorbol-ester-induced secretion. Ethanol also did not interfere with thrombin- or phorbol-ester-induced phosphorylation of myosin light chain (20 kDa) or a 47 kDa protein, a known substrate for protein kinase C. By electron microscopy, ethanol had no effect on thrombin-induced shape change and pseudopod formation, but prevented granule centralization and fusion. The results indicate that ethanol does not inhibit platelet secretion by interfering with the activation of phosphoinositide-specific phospholipase C or protein kinase C by thrombin. Rather, the data demonstrate an inhibition of a Ca2(+)-mediated event such as granule centralization.