Imaging out-of-plane polarized emission patterns on gap mode SERS substrates: from high molecular coverage to the single molecule regime.

Gap mode surface-enhanced Raman scattering (SERS) substrates are created when a single nanoparticle is deposited on a thin metal film, creating a region of significant electromagnetic field enhancement in the gap between the nanoparticle and the film due to excitation of a vertically-oriented, out-of-plane dipole plasmon mode, e.g. the gap plasmon. When molecules are located in the gap and couple to the gap plasmon mode, the resulting emission is polarized perpendicular to the thin film, generating SERS emission patterns that have a characteristic donut shape. We analyze these SERS emission patterns using a dipole emission model and extract out-of-plane and in-plane emission angles associated with the gap plasmon mode. Fluctuations in both of these angles reveal dynamic heterogeneity due to molecular motion within the hot spot that changes as a function of molecular coverage. We also reveal static heterogeneity associated with structural defects in the thin film component of the gap mode substrates, indicating that even nanometer-scale surface roughness can impact the quality of gap mode emission.

Studies on copper-yttria nanocomposites: high-energy ball milling versus chemical reduction method.

Oxide dispersion-strengthened copper-base composites are widely used for applications demanding high tensile strength, high hardness along with good electrical and thermal conductivity. Oxides of metals like aluminium, cerium, yttrium and zirconium are often used for this purpose as fine and uniformly distributed dispersoid particles in soft and ductile copper matrix. Such composites find applications as electrical contacts, resistance-welding tips, lead wires, continuous casting moulds, etc. In this investigation an attempt has been made to produce copper-yttria nanocomposites using two different morphologies of copper powder and two different processing routes namely, high-energy milling and in-situ chemical reduction. The synthesized powders were characterized by X-ray diffraction (XRD) and scanning electron microscopy (SEM) for their phase identification and morphological study. The nanocomposite powders in each case were subsequently processed to obtain bulk solids by classical powder metallurgy route of press-sinter-repress. The resultant bulk solid compacts were subjected to property evaluation. The study revealed that the properties of Cu-Y2O3 nanocomposites depend on the processing route used and in turn on the resultant powder morphology.

Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer.

Inefficient nuclear delivery of plasmid DNA is thought to be one of the daunting hurdles to gene transfer, utilizing a nonviral delivery system such as polycation-DNA complex. Following its internalization by endocytosis, plasmid DNA has to be released into the cytosol before its nuclear entry can occur. However, the stability of plasmid DNA in the cytoplasm, that may play a determinant role in the transfection efficiency, is not known. The turnover of plasmid DNA, delivered by microinjection into the cytosol, was determined by fluorescence in situ hybridization (FISH) and quantitative single-cell fluorescence video-image analysis. Both single- and double-stranded circular plasmid DNA disappeared with an apparent half-life of 50-90 min from the cytoplasm of HeLa and COS cells, while the amount of co-injected dextran (MW 70,000) remained unaltered. We propose that cytosolic nuclease(s) are responsible for the rapid-degradation of plasmid DNA, since (1) elimination of plasmid DNA cannot be attributed to cell division or to the activity of apoptotic and lysosomal nucleases; (2) disposal of microinjected plasmid DNA was inhibited in cytosol-depleted cells or following the encapsulation of DNA in phospholipid vesicles; (3) generation and subsequent elimination of free 3'-OH ends could be detected by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling assay (TUNEL), reflecting the fragmentation of the injected DNA; and finally (4) isolated cytosol, obtained by selective permeabilization of the plasma membrane, exhibits divalent cation-dependent, thermolabile nuclease activity, determined by Southern blotting and 32P-release from end-labeled DNA. Collectively, these findings suggest that the metabolic instability of plasmid DNA, caused by cytosolic nuclease, may constitute a previously unrecognized impediment for DNA translocation into the nucleus and a possible target to enhance the efficiency of gene delivery.

Cationic lipid-mediated transfection of cells in culture requires mitotic activity.

Cationic lipid-based delivery systems such as lipoplexes or stabilized plasmid-lipid particles (SPLP) represent a safer alternative to viral systems for gene therapy applications. We studied the impact of cell cycle status on the efficiency of transfection of human ovarian carcinoma tumor cells using two cationic-lipid based delivery systems. Cells arrested in the G1 phase of the cell cycle by treatment with aphidicolin were compared with an asynchronous dividing population of cells. Treatment of the cells with aphidicolin had no effect on the rate of internalization of the lipid formulated DNA or on the level of gene expression observable in stably transfected cells. However, cells treated with aphidicolin exhibited 20-fold lower reporter gene activity than asynchronous control cells upon incubation with lipoplexes. When cells arrested in the G1 phase were allowed to proceed through the cell cycle in the presence of the lipoplex or SPLP, transgene expression was found to coincide with the transition of cells from the G2/M phase into the G1 phase of the subsequent cell cycle. In addition, higher levels of reporter gene expression were observed when the cells were incubated with lipoplexes or SPLP during, or just before, mitosis. These results suggest that it may be possible to augment cationic lipid-mediated transfection by manipulating the cell cycle status of the target cells.

Differentially expressed Leishmania major gp63 genes encode cell surface leishmanolysin with distinct signals for glycosylphosphatidylinositol attachment.

The Leishmania cell surface metalloproteinase, leishmanolysin or GP63, is expressed in all stages of Leishmania major. Initial studies reported that in L. major the gp63 genes were arranged as five homologous, tandemly repeated genes (gp63 genes 1-5) and a sixth, less conserved gp63 gene located 8 kb downstream of gp63 gene 5. This study compared the sequences of L. major gp63 gene 1 and gp63 gene 6 and identified a seventh L. major gp63 gene located downstream from gp63 gene 6. The L. major gp63 genes exhibited stage-specific differences in their expression: gp63 genes 1-5 were expressed in promastigotes only, gp63 gene 6 was expressed in promastigotes and amastigotes, while gp63 gene 7 was expressed predominantly in stationary phase promastigotes and in amastigotes. Analysis of the predicted protein sequence of gp63 gene 6 (GP63-6) and gp63 gene 1 (GP63-1) showed that these two proteins were homologous in terms of overall predicted domain structure. L. major GP63-1 has been reported to contain a glycosylphosphatidylinositol (GPI) membrane anchor while sequence analysis predicted that GP63-6 contained a different hydrophobic C-terminus that may act as a transmembrane region. Transfection studies using L. major gp63 gene 1 and gp63 gene 6 expressed in L. donovani promastigotes showed that GP63-6 was expressed at the cell surface and that the distinct GP63-6 C-terminus was capable of mediating GPI anchor attachment.

Targeted gene deletion of Leishmania major genes encoding developmental stage-specific leishmanolysin (GP63).

The major surface glycoprotein of Leishmania major is a zinc metalloproteinase of 63 kDa referred to as leishmanolysin or GP63, which is encoded by a family of seven genes. Targeted gene replacement was used to delete gp63 genes 1-6 encoding the highly expressed promastigote and constitutively expressed GP63. In the L. major homozygous mutants deficient in gp63 genes 1-6, there was no expression of GP63 as detected by reverse transcription-polymerase chain reaction (RT-PCR) or fluorescent staining in promastigotes from the procyclic stage (logarithmic growth phase). The remaining L. major gP63 gene 7 was shown to be developmentally regulated, as it was expressed exclusively in infectious metacyclic stage (late stationary growth phase) promastigotes and in lesion amastigotes. The gp63 genes 1-6-deficient mutants showed increased sensitivity to complement-mediated lysis. The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equivalent wild-type stages. Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was restored to the same levels as wild-type promastigotes by transfection with gp63 gene 1. Thus, expression of GP63 is clearly involved in conferring resistance to complement-mediated lysis. The L. major GP63 1-6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. Similar levels of infection and subsequent intracellular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1-6 mutants and mutants transfected with gp63 gene 1. The GP63 1-6 mutants were capable of lesion formation in BALB/c mice and, thus, gp63 genes 1-6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1-6 in parasite development within the sandfly vector was studied. GP63 1-6 mutants grew normally in the blood-engorged midgut of both Phlebotomus argentipes and P. papatasi However, both wild-type and mutant promastigotes were lost after 2 days' growth in P. papatasi. The complete developmental pathway in P. argentipes was observed for wild-type promastigotes, GP63 1-6 mutants and mutants transfected with gp63 gene 1. Normal stage differentiation from amastigotes to procyclics, to nectomonads, to haptomonads and to infectious metacyclics was observed. Thus, the highly expressed promastigote forms of GP63, encoded by gp63 genes 1-6, do not appear to be required for nutrient utilization in the bloodmeal during the early stages of development in the sandfly or for midgut attachment and further development. gp63 1-6 genes do, however, play a major protective role against complement-mediated lysis when promastigotes are introduced into the mammalian host.

Transfection of cultured myoblasts in high serum concentration with DODAC:DOPE liposomes.

The inhibitory effect of serum is one of the main obstacles to the in vivo use of cationic liposomes as a DNA delivery system. We have found that a novel liposome formulation, DODAC:DOPE (1:1) is totally resistant to the inhibitory effects of serum for transfection of cultured myoblasts and myotubes. Transfection with a lacZ reporter gene in the presence of 95% fetal bovine serum gave up to 25% beta-gal-positive cells in C2C12 myoblasts and about six-fold less in primary human myoblasts. The lower transgene expression in primary cells does not appear to be a result of less DNA uptake but might result from differences in intracellular trafficking of the complexes. DODAC-based liposomes are unique in their resistance to serum inhibition and may therefore be valuable for the systemic delivery of genetic information to muscle and other tissues.

Leishmania major: molecular cloning, sequencing, and expression of the heat shock protein 60 gene reveals unique carboxy terminal peptide sequences.

Heat shock proteins (HSP) in the size range of M(r) 60,000 are major targets of the immune response in vivo. The leishmania heat-inducible proteins of M(r) 65-67,000 are expressed at relatively high levels in infected macrophages (Infection and Immunity 1993, 61, 3265-3272) and may be important targets of the host response. To facilitate further studies concerned with these proteins, the HSP60 gene of Leishmania major was cloned, sequenced, and expressed. A lambdaEMBL-3 L. major genomic library was screened with a PCR-generated DNA probe derived from a highly conserved region of the leishmania HSP60 gene. A single clone that hybridized strongly was characterized. Sequence analysis revealed an open reading frame of 1770 bp encoding a putative polypeptide of 589 amino acids with a predicted size of M(r) 64,790 and with the highest degree of amino acid sequence similarity (56%) to HSP60 from Trypanosoma cruzi. Less extensive amino acid sequence similarity (48%) was observed between that leishmania HSP60 and the corresponding human protein. Notably, significant regions of sequence dissimilarity between the leishmania and human proteins were identified principally within the carboxy-terminal regions of the proteins. The entire coding region of the leishmania HSP60 gene was subcloned into the pET-3a vector and expressed in Escherichia coli. Purified recombinant protein was used to examine sera from patients with tegumentary leishmaniasis from Colombia for the presence of antibodies to HSP60. Unlike sera from healthy, uninfected controls, sera from patients reacted strongly with recombinant leishmania HSP60. This recognition had specificity in that these same sera showed little or no reactivity with either recombinant mycobacterial HSP65 or recombinant human HSP60. These findings indicate that patients with tegumentary forms of leishmaniasis have humoral responses to leishmania HSP60. Further studies of this protein will clarify its importance as a target of the immune response and as a potential antigen for serodiagnosis.

The gene encoding streptothricin acetyltransferase (sat) as a selectable marker for Leishmania expression vectors.

The pLEX series of vectors was developed for the stable expression of exogenous genes in the protozoan parasite Leishmania. These pUC-based constructs contain one of three independent selectable markers and a multiple cloning site inserted between the upstream and downstream untranslated regions of the previously cloned Leishmania major HEXBP gene. Selection was based on resistance to the aminoglycosides, hygromycin B and neomycin, and to nourseothricin, a novel independent selectable marker for transfection of Leishmania. The vectors were introduced into Leishmania promastigotes by electroporation and were maintained as extrachromosomal circular concatemers containing between four and eight repeat units of the pLEX monomer. To demonstrate the efficient expression of cloned exogenous genes using the pLEX system, promastigotes were transfected with a pLEX construct that contained a second drug-resistant selectable marker gene cloned into the expression site, and clones were obtained that grew on media containing two antibiotics. These vectors, together with the novel selectable marker, will further facilitate the molecular analysis of gene expression in Leishmania.

Study of exfoliative dermatitis.

A study of 46 cases of exfoliative dermatitis revealed peak distribution in sixth decade and very high preponderance in males (M:F = 6.67 : 1). The highest single cause was found to be psoriasis (41.30%). Injudicious use of topical medicaments including herbal medicines and haphazard treatment were found to be the precipitating factors in 34% cases of pre-existing dermatoses. Laboratory investigations contributed little towards diagnosis except in lymphoma group; but were helpful in knowing systemic implications of the disease. Skin biopsy was found to be helpful in diagnosing underlying skin disorders in 18 cases (40.91%). Two cases (43.5%) had malignancy as the cause of exfoliative dermatitis.

Mutational and functional analysis of the Leishmania surface metalloproteinase GP63: similarities to matrix metalloproteinases.

The major surface glycoprotein of Leishmania, referred to as GP63, is a zinc metalloproteinase of 63,000 M(r) present on promastigotes and amastigotes from diverse species of Leishmania. GP63 shares several characteristics with the members of the matrix metalloproteinase family including degradation of at least one component of the extracellular matrix, location at the cell surface, requirement for Zn2+ for proteinase activity and inhibition of the proteinase activity by chelating agents and alpha 2-macroglobulin. Site-directed mutagenesis of the cloned L. major GP63 genes was carried out to determine whether the proposed active site of Leishmania GP63 was homologous to those of other zinc metalloproteinases. The codon encoding the catalytic glutamic acid was modified to encode an aspartic acid and when expressed in COS-7 cells the resulting mutant GP63 had no demonstrable proteinase activity compared to wild type GP63. GP63 was predicted to be synthesized as a precursor protein containing a pro region at the NH2-terminus of GP63 implicated to be involved with the regulation of proteinase activity. As with many other proteinases, including matrix metalloproteinases, these enzymes are synthesized as latent proteinases that require activation for full proteinase activity. L. major recombinant GP63 (rGP63) has been produced in the baculovirus expression system where rGP63 was secreted as a latent proteinase. To study the activation of baculovirus rGP63, purified rGP63 was incubated with the mercurial compound, HgCl2, at concentrations previously shown to result in activation of other latent matrix degrading metalloproteinases and resulted in a significant enhancement of GP63 proteinase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibrinolytic phenomenon in leprosy.

Fibrinolytic activity in eighty-one patients with different types of leprosy and thirty-two normal healthy controls was studied by Euglobulin Lysis Time Method, Fibrinolytic activity was markedly decreased in patients with lepromatous leprosy and those with ENL reaction. Decline in fibrinolytic activity during ENL was independent of frequency of attacks. Fibrinolytic activity was partly restored after subsidence of ENL reaction, though it failed to attain normal levels. Cutaneous vasculitis seems to be most probable cause of fall in fibrinolytic activity in lepromatous leprosy and ENL reaction.

Incontinentia Pigmenti Stage - II.

Eight weeks old female child with linear verruc,ous lesions on e)dtemities, eosinohilia and characteristic calcified dyskeratotic cells on histopathological examination confirming the diagnosis of second stage of incontinentia pigmenti is reported. Brief account of genetic counselling is given.'

Pilocarpine test in assessment of therapeutic efficacy in maculoanaesthetic leprosy.

Pilocarpine test has been used since long to study the functional status of sweat glands. This article deals with its use in the assessment of Maculoanaesthetic patches of leprosy before and after therapy. This being an objective method it eliminates the possible pitfalls in subjective testing of sensations over the affected areas. Therefore, it is recommended that this test should be routinely employed in field work as its technique is simple. The test should be repeated at an interval of six months.

Ocular manifestations of leprosy

This study covers 654 patients of leprosy who were predominatly of non-lepromatous type. Detailed examination of eyes has been done and ocular manifestations noted. Ocular manifestations with a bearing on status of treatment, extent of disease and duration of disease have been studied. The salient changes include sluggish corneal reflex, loss of eyebrows, madarosis and nodules on eyebrows. Patients suffering for 4 years and more manifested damage to iris, cornea and lens. the article stresses the importance of routine examinations of eyes (including slit lamp examination) at the beginning of therapy and periodical check-up thereafter. It also emphasizes that the non lepromatous patients also exhibit eye changes to a sizeable degree, through not to the same extent as lepromatous variety.