Senescing leaves possess potential for stress adaptation: the developing leaves acclimated to high light exhibit increased tolerance to osmotic stress during senescence

Plants may experience environmental stress factors operating in nature either simultaneously or in sequence. In the study, we have acclimated the developing primary leaves of wheat seedlings to high light stress and examined their photosynthetic response to polyethylene glycol (PEG) mediated osmotic stress during different developmental phases including senescence. The high light acclimated leaves show higher level of total carotenoids as compared to their non-acclimated counterparts experiencing osmotic stress during senescence. They also exhibit greater membrane stability as indicated by the measurements of fluorescence polarisation and energy transfer efficiency in photosystem I (PSI) and Photosystem II (PSII). From the data of DCPIP photoreduction and pulse amplitude modulated (PAM) fluorimetry, a similar trend is observed for PSII photochemistry of the leaves experiencing osmotic stress during senescence. Our results may suggest that the stress adaptive potential induced by one stress during development is retained by the leaves and helps to mitigate another stress effect operating in sequence during another developmental phase, namely senescence.     

Fine needle aspiration diagnosis in HIV-related lymphadenopathy in Mangalore, India

OBJECTIVE: To evaluate the usefulness of fine needle aspiration (FNA) study of lymph nodes in HIV-positive patients. STUDY DESIGN: The study was conducted at Kasturba Medical College, Mangalore, India. Samples from lymph nodes of 48 HIV-positive patients were taken and air dried, and wet smears were made. After staining with routine cytologic stains and special stains, detailed cytomorphologic study was conducted. RESULTS: Tuberculosis accounted for nearly half (48%) the cases,followed by HIV lymphadenitis (36%), lymphoma (10%), suppurative lymphadenitis (2%), Mycobacterium avium-intracellulare lymphadenitis (2%) and metastases (2%), in descending order of their frequency. A suppurative picture, which was found in 13% of cases of tuberculous lymphadenitis in AIDS patients, occasionally was misleading without the help of acid fast bacilli stain. CONCLUSION: FNA is a useful tool in the diagnosis of lymphadenopathy in HIV-positive patients provided that proper safety measures are taken to avoid contracting the infection.     

Bath immunisation of spawn,fry and fingerlings of Indian major carps using a particulate bacterial antigen

Larval mortality in Indian major carps is one of the major problems encountered in the pond culture system. The present investigation was carried out to investigate the proper age, duration of exposure, and optimum bacterin concentration for vaccinating rohu (Labeo rohita) and catla (Catla catla) at their early stages with a formalin killed Edwardsiella tarda bacterin suspension. The development of immunological competence was recorded with spawn of rohu and catla of 3 weeks of age exposed to a bacterin at a concentration 10(9) cfu ml(-1) for 15 min, where it persisted up to 4 weeks post vaccination. They showed significant resistance against challenge with virulent E. tarda bacteria. Significant antibody titre could be recorded in advanced fries and fingerlings exposed to 10(9) cfu/ml(-1) bacterin concentration for 45 and 60 min, respectively.     

fog-2 and the Evolution of Self-Fertile Hermaphroditism in Caenorhabditis

Somatic and germline sex determination pathways have diverged significantly in animals, making comparisons between taxa difficult. To overcome this difficulty, we compared the genes in the germline sex determination pathways of Caenorhabditis elegans and C. briggsae, two Caenorhabditis species with similar reproductive systems and sequenced genomes. We demonstrate that C. briggsae has orthologs of all known C. elegans sex determination genes with one exception: fog-2. Hermaphroditic nematodes are essentially females that produce sperm early in life, which they use for self fertilization. In C. elegans, this brief period of spermatogenesis requires FOG-2 and the RNA-binding protein GLD-1, which together repress translation of the tra-2 mRNA. FOG-2 is part of a large C. elegans FOG-2-related protein family defined by the presence of an F-box and Duf38/FOG-2 homogy domain. A fog-2-related gene family is also present in C. briggsae, however, the branch containing fog-2 appears to have arisen relatively recently in C. elegans, post-speciation. The C-terminus of FOG-2 is rapidly evolving, is required for GLD-1 interaction, and is likely critical for the role of FOG-2 in sex determination. In addition, C. briggsae gld-1 appears to play the opposite role in sex determination (promoting the female fate) while maintaining conserved roles in meiotic progression during oogenesis. Our data indicate that the regulation of the hermaphrodite germline sex determination pathway at the level of FOG-2/GLD-1/tra-2 mRNA is fundamentally different between C. elegans and C. briggsae, providing functional evidence in support of the independent evolution of self-fertile hermaphroditism. We speculate on the convergent evolution of hermaphroditism in Caenorhabditis based on the plasticity of the C. elegans germline sex determination cascade, in which multiple mutant paths yield self fertility.     

Downstream processing of MDCK cell-derived equine influenza virus

A microcarrier-based process was used to produce equine influenza virus (A/Equi 2 (H3N8), Newmarket 1/93) in Madin Darby Canine kidney (MDCK) cells. The virus was purified in a sequence of downstream processing steps comprising of depth filtration, inactivation, ultrafiltration (UF) and gel filtration. In the ultrafiltration step, the hemagglutinin (HA) was recovered to 100%. A high increase of neuraminidase (NA) activity indicated the removal of some inhibitory compounds during this step. At the same time, the level of contaminating proteins and DNA was reduced by more than 88%. In the subsequent size exclusion chromatography (Sepharose CL 2B), the recovery of HA and NA in the "virus peak" was 37.8 and 59.8%, respectively compared to the concentrated feed material. Inconsistencies in the overall mass balance for HA and NA (70.0 and 69.2%) during gel filtration indicated non-specific interactions of the inactivated virus to the gel matrix which is supported by a HA recovery of about 50% in shake flask experiments performed as a control. Overall 35.8% of HA and 291.6% of NA were recovered. More than 95.7% of the host cell proteins and 98.7% of the host cell DNA were removed during downstream processing.     

gamma-tubulin plays an essential role in the coordination of mitotic events

Recent data from multiple organisms indicate that gamma-tubulin has essential, but incompletely defined, functions in addition to nucleating microtubule assembly. To investigate these functions, we examined the phenotype of mipAD159, a cold-sensitive allele of the gamma-tubulin gene of Aspergillus nidulans. Immunofluorescence microscopy of synchronized material revealed that at a restrictive temperature mipAD159 does not inhibit mitotic spindle formation. Anaphase A was inhibited in many nuclei, however, and after a slight delay in mitosis (approximately 6% of the cell cycle period), most nuclei reentered interphase without dividing. In vivo observations of chromosomes at a restrictive temperature revealed that mipAD159 caused a failure of the coordination of late mitotic events (anaphase A, anaphase B, and chromosomal disjunction) and nuclei reentered interphase quickly even though mitosis was not completed successfully. Time-lapse microscopy also revealed that transient mitotic spindle abnormalities, in particular bent spindles, were more prevalent in mipAD159 strains than in controls. In experiments in which microtubules were depolymerized with benomyl, mipAD159 nuclei exited mitosis significantly more quickly (as judged by chromosomal condensation) than nuclei in a control strain. These data reveal that gamma-tubulin has an essential role in the coordination of late mitotic events, and a microtubule-independent function in mitotic checkpoint control.     

FOG-2, a novel F-box containing protein, associates with the GLD-1 RNA binding protein and directs male sex determination in the C. elegans hermaphrodite germline

Male sex determination in the Caenorhabditis elegans hermaphrodite germline requires translational repression of tra-2 mRNA by the GLD-1 RNA binding protein. We cloned fog-2 by finding that its gene product physically interacts with GLD-1, forming a FOG-2/GLD-1/tra-2 3'untranslated region ternary complex. FOG-2 has an N-terminal F-box and a novel C-terminal domain called FTH. Canonical F-box proteins act as bridging components of the SCF ubiquitin ligase complex; the N-terminal F-box binds a Skp1 homolog, recruiting ubiquination machinery, while a C-terminal protein-protein interaction domain binds a specific substrate for degradation. However, since both fog-2 and gld-1 are necessary for spermatogenesis, FOG-2 cannot target GLD-1 for ubiquitin-mediated degradation. We propose that FOG-2 also acts as a bridge, bringing GLD-1 bound to tra-2 mRNA into a multiprotein translational repression complex, thus representing a novel function for an F-box protein. fog-2 is a member of a large, apparently rapidly evolving, C. elegans gene family that has expanded, in part, by local duplications; fog-2 related genes have not been found outside nematodes. fog-2 may have arisen during evolution of self-fertile hermaphroditism from an ancestral female/male species.     

Characterization of a Gloverin-Like Antimicrobial Peptide Isolated from Muga Silkworm, <Emphasis Type="Italic">Antheraea assamensis</Emphasis>

Antimicrobial peptides (AMPs) are produced in all living organisms including insects in a non-specific manner, and act as innate immune defense arsenal against the invading pathogens. Muga silkworm (Antheraea assamensis) larvae were injected with Candida albicans and AMPs were isolated from the hemolymph after extracting with methanol, acetic acid and water mixture (90:1:9) and evaluated for antimicrobial activity against fungal and bacterial pathogens. Further purification was done through successive semipreparative and analytical reversed phase HPLC using C-18 column. The obtained fractions were collected, lyophilized and tested for antimicrobial activity. Among the HPLC fractions, one showed highest activity with MIC value of 64 µg/ml against Gram-negative bacteria, Escherichia coli and Enterobacter cloacae. Purity of this isolated peptide was confirmed by SDS-PAGE and TLC, and its molecular mass was determined as 9.052 kDa by MALDI-TOF mass spectrometry. From the mass fingerprinting analysis of this peptide after trypsin digestion a peptide fragment with molecular mass of 2622.7 Da was obtained. De novo sequencing of this peptide fragment following MS/MS analysis identified few amino acid residues as “KSGGGGWGS” with a total score of 46.9 with gloverin peptide of A. mylitta. The peptide inhibited biofilm formation of the Gram-negative bacterial pathogens. SEM study revealed that peptide disrupted bacterial cell wall to leach out intracellular materials and may be the major target for its antimicrobial activity.     

A model of lysosomal pH regulation

Lysosomes must maintain an acidic luminal pH to activate hydrolytic enzymes and degrade internalized macromolecules. Acidification requires the vacuolar-type H⁺-ATPase to pump protons into the lumen and a counterion flux to neutralize the membrane potential created by proton accumulation. Early experiments suggested that the counterion was chloride, and more recently a pathway consistent with the ClC-7 Cl^–/H⁺ antiporter was identified. However, reports that the steady-state luminal pH is unaffected in ClC-7 knockout mice raise questions regarding the identity of the carrier and the counterion. Here, we measure the current–voltage characteristics of a mammalian ClC-7 antiporter, and we use its transport properties, together with other key ion regulating elements, to construct a mathematical model of lysosomal pH regulation. We show that results of in vitro lysosome experiments can only be explained by the presence of ClC-7, and that ClC-7 promotes greater acidification than Cl^–, K⁺, or Na⁺ channels. Our models predict strikingly different lysosomal K⁺ dynamics depending on the major counterion pathways. However, given the lack of experimental data concerning acidification in vivo, the model cannot definitively rule out any given mechanism, but the model does provide concrete predictions for additional experiments that would clarify the identity of the counterion and its carrier.