Calcium ion binding to delta- and to beta-crystallins. The presence of the "EF-hand" motif in delta-crystallin that aids in calcium ion binding

Abnormal levels of endogenous calcium ions are known to induce eye lens opacity, and a variety of causative factors has been proposed, including calcium-mediated aggregation and precipitation of the lens proteins crystallins. We have specifically looked in some detail at the interaction of Ca2+ with various crystallins and its consequences. Lenses incubated in solutions containing 10 mM Ca2+ or 5 mM Tb3+ opacified. Fluorescence titration of crystallins with TbCl3 revealed that this ion binds to delta- and beta-crystallins in solution. Equilibrium dialysis showed that four Ca2+ ions bind to one delta-crystallin tetramer with an affinity of 4.3 x 10(3) M-1. Analysis of the amino acid sequence of delta-crystallin reveals the presence of a calmodulin-type "helix-loop-helix" or "EF-hand" calcium ion binding conformational motif in the region comprising residues 300-350. This is a novel feature of the molecule not reported so far. No other crystallins appear to have this motif. beta-Crystallin also binds four Ca2+ ions/aggregate unit of mass 160 kDa, with an affinity of 2.6 x 10(3) M-1, presumably in the midregion of the molecule that is rich in anionic and polar residues. Circular dichroism spectroscopy shows that the binding of calcium ion leads to subtle conformational changes in the molecules, notably in the tertiary structure.     

Poloxamer 188 decreases susceptibility of artificial lipid membranes to electroporation.

The effect of a nontoxic, nonionic block co-polymeric surface active agent, poloxamer 188, on electroporation of artificial lipid membranes made of azolectin, was investigated. Two different experimental protocols were used in our study: charge pulse and voltage clamp. For the charge pulse protocol, membranes were pulsed with a 10-micronsecond rectangular voltage waveform, after which membrane voltage decay was observed through an external 1-M omega resistance. For the voltage clamp protocol the membranes were pulsed with a waveform that consisted of an initial 10-microsecond rectangular phase, followed by a negative sloped ramp that decayed to zero in the subsequent 500 microseconds. Several parameters characterizing the electroporation process were measured and compared for the control membranes and membranes treated with 1.0 mM poloxamer 188. For both the charge pulse and voltage clamp experiments, the threshold voltage (amplitude of initial rectangular phase) and latency time (time elapsed between the end of rectangular phase and the onset of membrane electroporation) were measured. Membrane conductance (measured 200 microseconds after the initial rectangular phase) and rise time (tr; the time required for the porated membrane to reach a certain conductance value) were also determined for the voltage clamp experiments, and postelectroporation time constant (PE tau; the time constant for transmembrane voltage decay after onset of electroporation) for the charge pulse experiments. The charge pulse experiments were performed on 23 membranes with 10 control and 13 poloxamer-treated membranes, and voltage pulse experiments on 49 membranes with 26 control and 23 poloxamer-treated membranes. For both charge pulse and voltage clamp experiments, poloxamer 188-treated membranes exhibited a statistically higher threshold voltage (p = 0.1 and p = 0.06, respectively), and longer latency time (p = 0.04 and p = 0.05, respectively). Also, poloxamer 188-treated membranes were found to have a relatively lower conductance (p = 0.001), longer time required for the porated membrane to reach a certain conductance value (p = 0.05), and longer postelectroporation time constant (p = 0.005). Furthermore, addition of poloxamer 188 was found to reduce the membrane capacitance by approximately 4-8% in 5 min. These findings suggest that poloxamer 188 adsorbs into the lipid bilayers, thereby decreasing their susceptibility to electroporation.     

Production of disease-free encapsulated buds of Zingiber officinale Rosc.

Shoot buds of ginger were successfully encapsulated in 4% sodium alginate gel. Encapsulated buds were germinated in vitro to form roots and shoots. In vitro germination (emergence of sprouts) of encapsulated buds ranged from 16.7% to 81.8% on different media after 5 weeks of incubation. Normal plantlets with an average shoot length of 2.3 cm and 1.7 cm root length were successfully transplanted into unsterilized soil without any hardening process. These plantlets showed no symptoms of ginger yellows disease and the causal fungal pathogen failed to grow out on culture media (used as a diagnostic test).     

Identification of photo-inactive phytochrome A in etiolated seedlings and photo-active phytochrome B in green leaves of the aurea mutant of tomato

The contents of spectrophotometrically measurable phytochrome A (PhyA) and phytochrome B (PhyB) and the corresponding immunochemically detectable apoproteins (PHYA and PHYB) were examined in dark- and light-grown tissues of the aurea mutant of tomato and its wild-type (WT). The amount of PHYA in etiolated aurea seedlings was found to be about 20% of that in the WT; this PHYA showed no photoreversible changes in absorbance, no downregulation of the level of PHYA in light-grown seedlings, and no differential proteolysis of Pr and Pfr species in vitro which was seen in the case of the WT. By contrast, the amount of PHYB in aurea seedlings was not significantly different from that in WT seedlings. Phytochrome isolated from green leaves of the aurea mutant and purified by ion-exchange chromatography showed a red/far-red reversible spectral change, and its elution profile during chromatography was essentially similar to that of PHYB. The results indicate that aurea is a mutant that is deficient in photoactive PhyA at the etiolated stage, when it contains a spectrally inactive PHYA. However, the mutant contains spectrally active PhyB in its green tissue as does the WT.     

Citric acid fermentation by the mutant strain of theAspergillus niger resistant to manganese ions inhibition

Summary A new mutant strain,Aspergillus niger GS-III, showing resistance to manganese ions inhibition of citric acid fermentation on a sugarcane molasses containing medium was induced fromAspergillus niger KCU 520, a high citric acid-yielding strain. In submerged, surface or continuous cultures in the presence of manganese ions concentration upto 1.5 ppm the mutant strain yielded citric acid about 90 KgM^–3 . The citric acid yield was comparable to that obtained with the parental strain KCU 520 in the absence of manganese ions, but it was atleast 3-fold higher than that obtained by the latter in the presence of manganese ions. The mutant strain immobilized in calcium alginate beads was used in combination with surface-stabilized cultures for about 36-days in a continuous flow horizontal fermenter without any apparent loss in citric acid productivity. These results indicate that the manganese-resistant mutant is stable and may be used in the presence of sufficient manganese ions concentration (1.5 ppm) in the fermentation medium. This capability of the mutant strainA. niger GS-III has been correlated with greatly reduced levels (about one-thirds) of the NADP⁺ -isocitric dehydrogenase, one of the control points for citric acid accumulation.     

Target antigens in malaria transmission blocking immunity

Malaria transmission blocking immunity has been found to operate against two distinct phases of development of malaria parasites in the mosquito midgut: (i) against the extracellular gametes and newly fertilized zygotes shortly after ingestion by a mosquito of parasitized blood and (ii) against the zygotes during their subsequent development into ookinetes. Immunity is antibody-mediated and stage-specific. A set of three proteins, synthesized in the gametocytes, expressed on the surface of the gametes and newly fertilized zygotes and subsequently shed during later transformation of the zygotes, has been identified as the target antigens of anti-gamete fertilization blocking antibodies. A single protein, synthesized and expressed on the zygote surface during its development to ookinetes, has been identified as the target of antibodies which block the development of the fertilized parasites in the mosquito. Immunization of human populations against gamete or zygote antigens, while not directly protecting an immunized individual from inflection, would reduce the transfer of malaria within the population. Such immunity, in addition to reducing the overall rate of malaria transmission, would, if combined with a vaccine against the asexual (disease-causing) stages, reduce the chance of selection of parasites that are resistant to the asexual vaccine by preventing their entry into the mosquito population.     

Water relations in three species of theConvolvulaceae in the Indian arid zone

The relationship of water deficit, actual water content, area, water loss and stomatal behaviour in leaves of different insertion levels of three members of Indian arid zoneConvolvulaceae—Merremia aegyptia, M. dissecta andIpomoea pes-caprae were measured simultaneously in the month of May, 1974, when field conditions were extremely adverse for growth. The two former species loose far more water than the latter. The values measured varied considerably with the leaf insertion levels and they reversed inI. pes-caprae when compared withMerremia species.