Fumigant toxicity of plant essential oils against Camptomyia corticalis (Diptera: Cecidomyiidae)

The toxicity of 98 plant essential oils against third instars of cecidomyiid gall midge Camptomyia corticalis (Loew) (Diptera: Cecidomyiidae) was examined using a vapor-phase mortality bioassay. Results were compared with that of a conventional insecticide dichlorvos. Based on 24-h LC50 values, all essential oils were less toxic than dichlorvos (LC50, 0.027 mg/cm3). The LC50 of caraway (Carum carvi L.) seed, armoise (Artemisia vulgaris L.), clary sage (Salvia sclarea L.), oregano (Origanum vulgare L.), lemongrass [Cymbopogon citratus (DC.) Stapf], niaouli (Melaleuca viridiflora Gaertner), spearmint (Mentha spicata L.), cassia especial (Cinnamomum cassia Nees ex Blume), Dalmatian sage (Salvia offcinalis L.), red thyme (Thymus vulgaris L.), bay [Pimenta racemosa (P. Mill.) J.W. Moore], garlic (Allium sativum L.), and pennyroyal (Mentha pulegium L.) oils is between 0.55 and 0.60 mg/cm3. The LC50 of cassia (C. cassia, pure and redistilled), white thyme (T. vulgaris), star anise (Illicium verum Hook.f.), peppermint (Mentha X piperita L.), wintergreen (Gaultheria procumbens L.), cinnamon (Cinnamomum zeylanicum Blume) bark, sweet marjoram (Origanum majorana L.), Roman chamomile [Chamaemelum nobile (L.) All.], eucalyptus (Eucalyptus globulus Labill.), rosemary (Rosmarinus officinalis L.),Virginian cedarwood (Juniperus virginiana L.), pimento berry [Pimenta dioica (L.) Merr.], summer savory (Satureja hortensis L.), lavender (Lavandula angustifolia Mill.), and coriander (Coriandrum sativum L.) oils is between 0.61 and 0.99 mg/cm3. All other essential oils tested exhibited low toxicity to the cecidomyiid larvae (LC50, >0.99 mg/cm3). Global efforts to reduce the level of highly toxic synthetic insecticides in the agricultural environment justify further studies on the active essential oils as potential larvicides for the control of C. corticalis populations as fumigants with contact action.     

CRYBA4, a novel human cataract gene, is also involved in microphthalmia

Genetic analysis of a large Indian family with an autosomal dominant cataract phenotype allowed us to identify a novel cataract gene, CRYBA4. After a genomewide screen, linkage analysis identified a maximum LOD score of 3.20 (recombination fraction [theta] 0.001) with marker D22S1167 of the beta -crystallin gene cluster on chromosome 22. To date, CRYBA4 was the only gene in this cluster not associated with either human or murine cataracts. A pathogenic mutation was identified in exon 4 that segregated with the disease status. The c.317T-->C sequence change is predicted to replace the highly conserved hydrophobic amino acid phenylalanine94 with the hydrophilic amino acid serine. Modeling suggests that this substitution would significantly reduce the intrinsic stability of the crystalline monomer, which would impair its ability to form the association modes critical for lens transparency. Considering that CRYBA4 associates with CRYBB2 and that the latter protein has been implicated in microphthalmia, mutational analysis of CRYBA4 was performed in 32 patients affected with microphthalmia (small eye). We identified a c.242T-->C (Leu69Pro) sequence change in exon 4 in one patient, which is predicted here to disrupt the beta -sheet structure in CRYBA4. Protein folding would consequently be impaired, most probably leading to a structure with reduced stability in the mutant. This is the first report linking mutations in CRYBA4 to cataractogenesis and microphthalmia.     

Life table and predation of Neoseiulus longispinosus (Acari: Phytoseiidae) on Oligonychus coffeae (Acari: Tetranychidae) infesting tea

Life table and predation of the predatory mite Neoseiulus longispinosus (Evans) on the red spider mite (RSM), Oligonychus coffeae (Nietner), a major pest of tea in India, were studied in the laboratory. Developmental time from egg to adult varied from 4 to 14 days at 30 to 15 °C, respectively; at 35 °C no larva survived. Survival of immature stages was more than 94 % at all temperatures. Threshold temperature for development of immature stages of females and males was 10 and 9.9 °C, respectively, and thermal constant was 84.03 degree-days for females and 80 for males. Sex ratio was female biased and temperature (20–30 °C) had no clear effect on sex determination. Egg hatchability was 73 % at 35 °C and >97 % at lower temperatures. Average number of eggs laid per female/day was higher at 30 °C than at 20 or 25 °C. The highest net reproductive rate (R ₀) was 40.7, at 20 °C. Mean generation time (T) decreased from 28 to 13 days with temperature increasing from 20 to 30 °C. Weekly multiplication (6.5) and intrinsic rate of natural increase (r _m ) (0.268) were highest at 30 °C. Males lived longer than females at every temperature tested. Longevity was highest at 20 °C (50 days for females and 55 for males). Survival and longevity were adversely affected by temperature above 30 °C. Daily consumption of prey increased with the advancement of predator’s life stages; adult females consumed the highest numbers of prey items, preferably larvae and nymphs.     

Efficacy,prey stage preference and optimum predator–prey ratio of the predatory mite,Neoseiulus longispinosus Evans (Acari: Phytoseiidae) to control the red spider mite,Oligonychus coffeae Nietner (Acari: Tetranychidae) infesting tea

The predatory mite, N. longispinosus preys up on red spider mite, O. coffeae infesting tea in south India. An attempt has been made to determine the predatory potential, prey stage preference and optimum predator–prey ratio of N. longispinosus under laboratory and green house conditions. When 50 adult female O. coffeae were given, the number of adults reduced by eight days along with an increase in the number of predators. The larvae hatched from the eggs laid by O. coffeae were fed by predatory mite. N. longispinosus preyed up on all life stages with a preference to larvae and nymphs of red spider mite. Predator–prey ratios of 1:33 and 1:50 were effective in lab, and 1:25 was found to be effective in green house. These results revealed that N. longispinosus could be used as a successful biocontrol candidate of O. coffeae in tea through augmentation or mass rearing and field release.     

Mcl-1 is a key regulator of the ovarian reserve

A majority of ovarian follicles are lost to natural death, but the disruption of factors involved in maintenance of the oocyte pool results in a further untimely follicular depletion known as premature ovarian failure. The anti-apoptotic B-cell lymphoma 2 (Bcl-2) family member myeloid cell leukemia-1 (MCL-1) has a pro-survival role in various cell types; however, its contribution to oocyte survival is unconfirmed. We present a phenotypic characterization of oocytes deficient in Mcl-1, and establish its role in maintenance of the primordial follicle (PMF) pool, growing oocyte survival and oocyte quality. Mcl-1 depletion resulted in the premature exhaustion of the ovarian reserve, characterized by early PMF loss because of activation of apoptosis. The increasingly diminished surviving cohort of growing oocytes displayed elevated markers of autophagy and mitochondrial dysfunction. Mcl-1-deficient ovulated oocytes demonstrated an increased susceptibility to cellular fragmentation with activation of the apoptotic cascade. Concomitant deletion of the pro-apoptotic Bcl-2 member Bcl-2-associated X protein (Bax) rescued the PMF phenotype and ovulated oocyte death, but did not prevent the mitochondrial dysfunction associated with Mcl-1 deficiency and could not rescue long-term breeding performance. We thus recognize MCL-1 as the essential survival factor required for conservation of the postnatal PMF pool, growing follicle survival and effective oocyte mitochondrial function.Estimates of the human primordial follicle (PMF) reservoir, the size of which dictates the extent of the ovarian reserve, indicates the presence of at least half a million oocytes per ovary at birth.^{1, 2} The essential decision that PMFs face is either long-term arrest with a possibility of recruitment toward the growing pool, or death. Even upon recruitment to the growing pool, intricately orchestrated crosstalk of survival signals between ovarian somatic cells and oocytes facilitate the ovulation of a single oocyte in human in each cycle. Hence, the default fate for millions of ovarian germ cells is death, as only a small fraction survive till ovulation.³ Insufficient endowment during fetal development or excessive oocyte loss during postnatal life further limits the ovarian reserve and can result in an untimely exhaustion of the follicle pool leading to premature ovarian failure (POF); a syndrome that affects around 1% of all women, with a higher prevalence (up to 30%) in families with heritable traits of this condition.^{4, 5} Mechanisms responsible for maintenance of the follicular reserve are poorly understood, however, biological assessments and mathematical modeling reveal that progressive loss of follicles with age is non-linear and accelerates, especially after 38 years.^{6, 7} With a declining ovarian reserve, poor oocyte quality is an additional factor that contributes to the reduced fertility associated with increased maternal age. Oocytes and resulting embryos of older mothers have increased rates of aneuploidies likely due to defects in chromosomal cohesion and meiotic spindle stability, decreased DNA repair capacity, altered gene expression, impaired mitochondrial function and elevated cellular redox, all contributing to increased rates of cell death.^{8, 9, 10}The marked decline of oocyte number in mammalian ovaries has been attributed to oocyte loss via stage-specific modes of death. As yet, perinatal PMF loss in mice most frequently engages apoptotic cell death,^{11, 12} whereas within the postnatal ovary, oocytes in growing follicles undergo atresia, a less ''molecularly'' defined death, carrying hallmarks of both apoptosis and autophagy.^{13, 14, 15} It is thus surprising that no member of the anti-apoptotic B-cell lymphoma 2 (Bcl-2) family has been identified with a definitive role in governing oocyte survival and the maintenance of the ovarian reserve. Bcl-2l2/Bcl-w and Bcl-2-l10/Diva deficiency had no apparent impact on the ovarian reserve, and although ablation of Bcl-2 led to a loss of one-third of the adult PMF pool, the growing follicle pool was not significantly impacted and these animals did not undergo POF.^{16, 17, 18, 19} Conditional Bcl-x (Bcl-2l1) inactivation led to increased primordial germ cell apoptosis in the embryo,²⁰ but postnatal inactivation of Bcl-x in oocytes did not compromise the ovarian reserve in young females.²¹ Bcl2a1a/Bfl-1/A1 was low to undetectable in fully grown germinal vesicle (GV) or ovulated murine oocytes,²² however, the impact of Bfl-1 deficiency on the ovarian reserve has not yet been analyzed to the best of our knowledge. Consequently, either various anti-apoptotic Bcl-2 members have overlapping roles in governing postnatal oocyte survival and maintenance of the adult ovarian reserve in mice, or the anti-apoptotic Bcl-2 member that regulates this decision has yet to be identified.     

Site-specific PEGylation of hemoglobin at Cys-93(beta): correlation between the colligative properties of the PEGylated protein and the length of the conjugated PEG chain

Increasing the molecular size of acellular hemoglobin (Hb) has been proposed as an approach to reduce its undesirable vasoactive properties. The finding that bovine Hb surface decorated with about 10 copies of PEG5K per tetramer is vasoactive provides support for this concept. The PEGylated bovine Hb has a strikingly larger molecular radius than HbA (1). The colligative properties of the PEGylated bovine Hb are distinct from those of HbA and even polymerized Hb, suggesting a role for the colligative properties of PEGylated Hb in neutralizing the vasoactivity of acellular Hb. To correlate the colligative properties of surface-decorated Hb with the mass of the PEG attached and also its vasoactivity, we have developed a new maleimide-based protocol for the site-specific conjugation of PEG to Hb, taking advantage of the unusually high reactivity of Cys-93(beta) of oxy HbA and the high reactivity of the maleimide to protein thiols. PEG chains of 5, 10, and 20 kDa have been functionalized at one of their hydroxyl groups with a maleidophenyl moiety through a carbamate linkage and used to conjugate the PEG chains at the beta-93 Cys of HbA to generate PEGylated Hbs carrying two copies of PEG (of varying chain length) per tetramer. Homogeneous preparations of (SP-PEG5K)(2)-HbA, (SP-PEG10K)(2)-HbA, and (SP-PEG20K)(2)-HbA have been isolated by ion exchange chromatography. The oxygen affinity of Hb is increased slightly on PEGylation, but the length of the PEG-chain had very little additional influence on the O(2) affinity. Both the hydrodynamic volume and the molecular radius of the Hb increased on surface decoration with PEG and exhibited a linear correlation with the mass of the PEG chain attached. On the other hand, both the viscosity and the colloidal osmotic pressure (COP) of the PEGylated Hbs exhibited an exponential increase with the increase in PEG chain length. In contrast to the molecular volume, viscosity, and COP, the vasoactivity of the PEGylated Hbs was not a direct correlate of the PEG chain length. There appeared to be a threshold for the PEG chain length beyond which the protection against vasoactivity is decreased. These results suggest that the modulation of the vasoactivity of Hb by PEG could be a function of the surface shielding afforded by the PEG, the latter being a function of the disposition of the PEG chain on the protein surface, which in turn is a function of the length of the PEG chain. Thus, the biochemically homogeneous PEGylated Hbs described in the present study, surface-decorated with PEG chains of appropriate size, could serve as potential candidates for Hb-based oxygen carriers.     

Developmental heterogeneity in DNA packaging patterns influences T-cell activation and transmigration

Cellular differentiation programs are accompanied by large-scale changes in nuclear organization and gene expression. In this context, accompanying transitions in chromatin assembly that facilitates changes in gene expression and cell behavior in a developmental system are poorly understood. Here, we address this gap and map structural changes in chromatin organization during murine T-cell development, to describe an unusual heterogeneity in chromatin organization and associated functional correlates in T-cell lineage. Confocal imaging of DNA assembly in cells isolated from bone marrow, thymus and spleen reveal the emergence of heterogeneous patterns in DNA organization in mature T-cells following their exit from the thymus. The central DNA pattern dominated in immature precursor cells in the thymus whereas both central and peripheral DNA patterns were observed in naïve and memory cells in circulation. Naïve T-cells with central DNA patterns exhibited higher mechanical pliability in response to compressive loads in vitro and transmigration assays in vivo, and demonstrated accelerated expression of activation-induced marker CD69. T-cell activation was characterized by marked redistribution of DNA assembly to a central DNA pattern and increased nuclear size. Notably, heterogeneity in DNA patterns recovered in cells induced into quiescence in culture, suggesting an internal regulatory mechanism for chromatin reorganization. Taken together, our results uncover an important component of plasticity in nuclear organization, reflected in chromatin assembly, during T-cell development, differentiation and transmigration.     

Pair-wise interactions of polymerization inhibitory contact site mutations of hemoglobin-S

The linkage of pair-wise interactions of contact site mutations of HbS has been studied using Le Lamentin [His-20 (α)→Gln], Hoshida [Glu-43 (β)→Gln] and α₂β₂^T87Q mutations as the prototype of three distinct classes of contact sites of deoxy HbS fiber. Binary mixture experiments established that β^A-chain with the Thr-87 (β)→Gln mutation is as potent as the γ-chain of HbF (α₂γ₂) in inhibiting polymerization. On combining the influence of Le Lamentin mutation with that of β₂^T87Q mutations; the net influence is only partial additivity. On the other hand, in binary mixture studies, combined influence of Hoshida mutation with that of β₂^T87Q mutations is synergistic. Besides, a significant level of synergistic complementation is also seen when the Le Lamentin and Hoshida mutations are combined in HbS (symmetrical tetramers). Le Lamentin and Hoshida mutation introduced into the cis-dimer of the asymmetric hybrid tetramer completely neutralizes the Val-6 (β) dependent polymerization. Accordingly, we propose that combining the perturbation of intra-double strand contact site with that of an inter-double strand contact site exhibit synergy when they are present in two different chains of the αβ dimer. A comparison of the present results with that of the earlier studies suggest that when the two contact site perturbations are from the same sub-unit of the αβ dimer only partial additivity is observed. The map of interaction linkage of the contact site mutations exposes new strategies in the design of novel anti-sickling Hbs for the gene therapy of sickle cell disease.     

Life table and predation of Oligota pygmaea (Coleoptera: Staphylinidae) a major predator of the red spider mite, Oligonychus coffeae (Acarina: Tetranychidae) infesting tea

The staphylinid beetle, Oligota pygmaea (Solier) is an important predator of the red spider mite, Oligonychus coffeae (Nietner) infesting tea. Biology, life table and predatory efficiency of O. pygmaea were studied under laboratory conditions. Duration of developmental stages of O. pygmaea was 3.2, 5.7 and 12.5 d for eggs, larvae and pupae, respectively with an average of 23.0 d from egg to adult emergence. After a mean preovipostion period of 2.9 d, each female laid an average of 400.5 eggs in its life span. Adult O. pygmaea lived for an average of 54.1 d. Adult females lived for a longer period of 58.8 d compared to the longevity of 49.4 d of adult male. Studies revealed that its life table characterized by an intrinsic rate of natural population increase (r) of 0.118 d, net reproductive rates (R_o) of 243.693 eggs/female, gross reproduction rate (Σm_x) of 245.313 eggs/female, generation time (T) of 46.575 d, doubling time (DT) of 5.874 d and finite rate of increase (λ) of 1.125 d. Seasonal abundance of O. pygmaea and its prey, O. coffeae was monitored by sampling 25 tea leaves randomly from each experimental block grown under the prevailing field conditions. O. pygmaea showed a typical pattern of population dynamics with a peak during January to March and low incidence during June to September. Peak in the population of O. pygmaea coincided with the abundance of O. coffeae in the tea fields. Weather factors such as high temperature, low relative humidity and low sunshine hours adversely affected the populations of O. pygmaea. The first to third instar larvae of O. pygmaea consumed 31.0–133.2 eggs of mites per day. Third instar larva of O. pygmaea consumed an average of 133.2 eggs, 46.4 hexapod larvae, 39.6 nymphs and 11.4 adults per day. Adult females consumed more number of red spider mites compared to the males.     

Life table and predatory efficiency of Stethorus gilvifrons (Coleoptera: Coccinellidae), an important predator of the red spider mite, Oligonychus coffeae (Acari: Tetranychidae), infesting tea

The ladybird beetle, Stethorus gilvifrons, is a major predator of the red spider mite, Oligonychus coffeae, infesting tea. Biology, life table and predatory efficiency of S. gilvifrons were studied under laboratory conditions. Its average developmental period from egg to adult emergence was 19.2 days. After a mean pre-oviposition period of 5.3 days, each female laid an average of 149.3 eggs. Adult females lived for 117.3 days and males for 41.5 days. The life table of the beetle was characterized by an intrinsic rate of increase (r) of 0.066 day⁻¹, net reproductive rate (R ₀) of 72.2 eggs/female, gross reproduction rate (Σm _x ) of 82.3 eggs/female, generation time (T) of 64.9 days, doubling time of 10.5 days and finite rate of increase (λ) of 1.07 day⁻¹. Population dynamics of S. gilvifrons and its prey, O. coffeae, was monitored by sampling 25 tea leaves from each experimental block grown under the prevailing field conditions. Populations of S. gilvifrons reached a peak during January to March and had low incidence during June to November. Peaks in the populations of S. gilvifrons coincided with the abundance of O. coffeae in tea fields. Weather factors such as low temperature, high humidity and heavy rainfall adversely affected the populations of S. gilvifrons. The predatory efficiency of S. gilvifrons increased during the growth of larval instars. An adult female consumed 205.0 eggs, 92.2 larvae, 81.8 nymphs and 52.4 adult mites per day.