In Vitro Propagation of Gladiolus: Optimisation of Conditions for Shoot Multiplication

Effect of some physical and chemical factors on in vitro shoot multiplication in Gladioius was investigated. A modified MS medium with NH₄NO₃ reduced to half strength, KH₂PO₄ replaced with 300 mg/l of NaH₂PO₄ 2 H₂O and Kl omitted completely, was found to be better than the original MS. BAP was better than either kinetin or 2iP. Of the various physical factors studied, the propagule size, light and container volume influenced the rate of shoot multiplication. On the modified medium containing 0.5 mg/l BAP in dark, the propagules bearing 20 buds showed 8-fold multiplication.     

Formation of acetic acid from cellulosic substrates byFusarium oxysporum

Summary Four strains of Fusarium oxysporum and a strain of Monilia brunnae were screened for their ability to convert cellulosic substrates into ethanol/acetic acid. These strains were found to utilize cellulose and produce extracellular cellulases. However, only F. oxysporum 841 was found to convert glucose, xylose, and cellulose into ethanol and acetic acid as major end-products under microaerobic conditions. Acetic acid at a level of 4.7 g/l resulted in a single-step process on potato pulp medium, indicating the potential of the strain for converting cellulosic substrates into acetic acid. Offprint requests to: K. Schügerl     

Molecular cloning,characterization and expression of a nitrofuran reductase gene ofescherichia coli

Mini-mu derivatives carrying plasmid replicons can be used to clone genesin vivo. This method was adopted to generate phasmid clones which were later screened for their ability of restore nitrofurantoin sensitivity of a nitrofuran-resistant host by eliciting nitroreductase activity. One phasmid-derived clone (pAJ101) resulted in considerable increase in nitroreductase activity when introduced into a nitrofurantoin-resistant mutant ofEscherichia coli with reduced nitroreductase activity. Subsequently, a 1.8 kb fragment obtained from pAJ101 by partial digestion with 5au3A, was subcloned into pUC18 to yield pAJ102. The nitroreductase activity attributable to pAJ102 was capable of reducing both nitrofurantoin and nitrofurazone. The polypeptides encoded by pAJ102 were identified by the minicell method. A large, well-defined band corresponding to 37 kDa and a smaller, less-defined band corresponding to 35 kDa were detected. Tnl000 mutagenesis was used to delineate the coding segment of the 1.8 kb insert of pAJ102. A 0.8 kb stretch of DNA was shown to be part of the nitroreductase gene. The gene was mapped at 19 min on theEscherichia coli linkage map.     

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.     

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.     

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.