Regulation of phosphorylation of the c-erbB-2/HER2 gene product by a monoclonal antibody and serum growth factor(s) in human mammary carcinoma cells.

Monoclonal antibody (MAb) 4D5 was used to analyze the phosphorylation of p185HER2, the gene product of c-erbB-2/HER2, in SK-BR-3 cells. Culture in the continuous presence of 4D5 reduced the in vivo steady-state levels of p185HER2 phosphorylation by 80% in a dose-dependent manner, suggesting that MAb 4D5 may have interfered with the activation of phosphorylation of p185HER2. The observed MAb-mediated reduction of p185HER2 phosphorylation could not be completely accounted for by down-regulation. When cultures were grown under serum-free conditions, the steady-state levels of p185HER2 phosphorylation were reduced by 56%, and addition of 4D5 further inhibited phosphorylation to 20% of steady-state levels. With continuous exposure to increasing concentrations of newborn calf serum in these cultures, there was a linear increase in tyrosine-specific phosphorylation of p185HER2, reaching a 5.4-fold increase with 10% newborn calf serum. Phosphorylation of p185HER2 in the presence of newborn calf serum was not attributable to stimulation of the epidermal growth factor receptor by epidermal growth factor or by transforming growth factor-alpha. Extension of these observations to two other mammary carcinoma cell lines. MDA-MB-453 and BT-474, also demonstrated a significant capacity of serum to induce p185HER2 phosphorylation. The demonstration of antibody-mediated partial inhibition of phosphorylation under serum-free conditions suggests that mammary carcinoma cells may also produce and secrete a factor or factors which may activate p185HER2. Our observation that growth-inhibitory MAb 4D5 is able to reduce the phosphorylation of p185HER2 by newborn calf serum and by a cellular-derived factor(s) suggests the existence of a growth factor(s) which uses phosphorylation of p185HER2 as a signal transduction pathway to regulate cell proliferation.     

Role of Ca2+ ion on Leishmania-macrophage attachment

Summary Leishmania donovani, the etiological agent for the disease visceral leishmaniasis, attach themselves to the macrophages for initiation of the disease. The attachment process has been found to be regulated by Ca²⁺ ions. Verapamil, a Ca²⁺-channel blocker inhibits Leishmania-macrophage attachment. The inhibitory effect is increased with time. Nifedipine, another Ca²⁺-channel blocker exhibits the same effect. The attachment process is stimulated by Ca²⁺-ionophore alone. The inhibitory effects of the calcium channel blockers are reversed by the ionophore.     

Luminescence of ruthenium(II) polypyridyls: evidence for intercalative binding to Z-DNA.

Photophysical studies have been undertaken to characterize the binding interactions of enantiomers of Ru(phen)3(2+), Ru(DIP)3(2+), and racemic Ru(bpy)2dppz2+ (where phen = 1,10-phenanthroline, DIP = 4,7-diphenylphenanthroline, and dppz = dipyridophenazine) with Z-form poly d(GC). Parallel enhancements in steady state luminescent intensity and a lengthening of luminescent lifetimes are seen for ruthenium enantiomers with Z-DNA as for B-DNA but with enantioselectivities reversed. Greater enhancements are seen for delta-isomers with the right-handed helix but for lambda-isomers with the left-handed helix. Ru(bpy)2dppz2+, an avid intercalator in B-DNA, displays no luminescence free in aqueous solution, but luminesces brightly bound to either B- or Z-poly d(GC). Stern-Volmer quenching studies also support the enantioselective preference in binding to B-DNA by delta-isomers and a reversal with binding to Z-DNA preferentially by the lambda-isomers. Steady state polarization studies indicate a rigid association of the complexes with both B- and Z-DNA on the time-scale of their emission and again with symmetrical enantioselectivities for the left and right-handed helices. Given the well characterized intercalative association of the complexes with B-DNA, the parallel results seen here with Z-DNA point strongly to a comparable intercalative association with the Z-form helix. That molecules may interact with Z-DNA through intercalation has not been demonstrated previously and now requires consideration in describing the range of interactions of small molecules and proteins with Z-DNA.     

Adenine photodimerization in deoxyadenylate sequences: elucidation of the mechanism through structural studies of a major d(ApA) photoproduct.

The mechanism of the photodimerization of adjacent adenine bases on the same strand of DNA has been elucidated by determining the structure of one of the two major photoproducts that are formed by UV irradiation of the deoxydinucleoside monophosphate d(ApA). The photoproduct, denoted d(ApA)*, corresponds to a species of adenine photodimer first described by P?rschke (P?rschke, D. (1973) J.Am.Chem.Soc. 95, 8440-8446). From a detailed examination of its chemical and spectroscopic properties, including comparisons with the model compound N-cyano-N1-(1-methylimidazol-5-yl)formamidine, it is deduced that d(ApA)* contains a deoxyadenosine unit covalently linked through its C(8) position to C(4) of an imidazole N(1) deoxyribonucleoside moiety bearing an N-cyanoformamidino substituent at C(5). On treatment with acid, d(ApA)* is degraded with high specificity to 8-(5-amino-imidazol-4-yl)adenine whose identity has been confirmed by independent chemical synthesis. It is concluded that the primary event in adenine photodimerization entails photoaddition of the N(7)-C(8) double bond of the 5'-adenine across the C(6) and C(5) positions of the 3'-adenine. The azetidine species thus generated acts as a common precursor to both types of d(ApA) photoproduct which are formed from it by competing modes of azetidine ring fission.     

Image segmentation using a neural network

An object extraction problem based on the Gibbs Random Field model is discussed. The Maximum a'posteriori probability (MAP) estimate of a scene based on a noise-corrupted realization is found to be computationally exponential in nature. A neural network, which is a modified version of that of Hopfield, is suggested for solving the problem. A single neuron is assigned to every pixel. Each neuron is supposed to be connected only to all of its nearest neighbours. The energy function of the network is designed in such a way that its minimum value corresponds to the MAP estimate of the scene. The dynamics of the network are described. A possible hardware realization of a neuron is also suggested. The technique is implemented on a set of noisy images and found to be highly robust and immune to noise.     

Cytopathology of peritoneal endometriosis caused by ruptured ovarian cysts

The cytopathologic features of two cases of peritoneal endometriosis (secondary to ruptured ovarian endometrial cysts) are described. Both patients presented with abdominal distension and tenderness and were clinically thought to have an abdominal tumor. Preoperative cytologic examination of peritoneal fluids gave a diagnosis of endometriosis in both cases. The endometrial tissue was present in the smears as honeycombs, syncytia and tight clusters of both epithelial and stromal cells. Subsequent surgery confirmed the cytodiagnosis in both cases. These cases emphasize the need to include endometriosis in the differential diagnosis of peritoneal effusions, especially in women.     

Three-dimensional structure of proteinase K at 0.15-nm resolution

The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39--His69--Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34--Cys123 and Cys178--Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel beta-sheet decorated by six alpha-helices, three short antiparallel beta-sheets, 18 beta-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132--Gly136), probably associated with a cis----trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132--Leu133--Gly134 on one side and Gly100--Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39--His69--Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent.     

Dynamic state of site-specific DNA methylation concurrent to altered prolactin and growth hormone gene expression in the pituitary gland of pregnant and lactating rats

The regulatory mechanism for the hormonal control of prolactin (PRL) and growth hormone (GH) gene expression in rat pituitary gland during gestation and lactation is verified in this study. The level of PRL-specific mRNA (mRNAPRL) sequences in the pituitary gland is elevated in the later part of gestation and more prominently so in lactation. In contrast the expression of GH gene is inhibited in the same tissue during gestation and lactation resulting in the dramatic decrease in the level of GH-specific mRNA (mRNAGH) sequences. We now demonstrate the influence of a tissue-specific altered DNA methylation pattern on the temporal modulation of expression of PRL and GH genes in the pituitary gland during alternate physiological states. An altered methylation pattern of specific "-C-" residues only in the coding region of PRL and GH gene can be detected concurrently with the altered level of expression of these genes in the pituitary gland during gestation and lactation. These results also demonstrate the dynamic state of methylation of specific -C- residues during the transition of these two genes from one state of expression to another in the same tissue. A correlation between site-specific DNA methylation and tissue-specific expression of PRL and GH gene in pituitary gland is reported. Thus a role of DNA methylation in the hormonal control of PRL and GH gene expression in physiological states such as pregnancy and lactation is proposed.     

Insulin effect on [14C]-valine incorporation and its relation to hexokinase activity in developing brain

Using minced brain cortex from fetal and postnatal rats, we studied the incorporation of [14C]-valine into protein in the presence of insulin. We also assayed the "particle bound" and soluble hexokinase in these tissues. Insulin significantly stimulated the incorporation of [14C]-valine into brain proteins from fetal stage upto 2 days of life. After this period the insulin effect was minimal, with no effect by day 5. The "particle bound" (40,000g pellet) brain hexokinase, on the other hand, remained low till about 2 days of life and then increased to almost adult level by 5 days. Our results show that there is an inverse relation between this anabolic effect of insulin and the "particle bound" hexokinase activity in the cortex of developing rat brain.     

Reconstituted U1 small nuclear ribonucleoprotein complex restores 5' splice site cleavage activity

Functional reconstitution of U1 small nuclear ribonucleoprotein particle (U1 snRNP) was performed using in vitro transcribed U1 snRNA. Hela cell nuclear extract was depleted of its constituent snRNPs by centrifugation at 100,000 X g. The supernatant was devoid of snRNAs and lacked cleavage activity in splicing reactions using in vitro transcribed beta-globin pre-mRNA as substrate. The resulting pellet which contained the snRNAs, retained 5' splice site cleavage activity in a similar splicing reaction. Supplementation of the inactive supernatant fraction with in vitro transcribed U1 snRNA, partially restored 5' splice site cleavage activity thereby demonstrating the specific requirement of U1 snRNP in the initial stage of pre-mRNA splicing.