An X-ray Microanalysis Study of the Salt Glands and Intracellular Calcium Crystals of Tamarix

X-ray microprobe analyses of the secreted salts from glandsof 20 Tamarix aphylla (L.) Karst trees growing in alluvial soilsin the north western district of Victoria, Australia, show thatthe glands secrete a variety of elements including calcium,magnesium and sulphur. However, sodium, potassium and chloride,common ions reported to be secreted predominantly in other studies,are present in relatively low amounts in the secreted salts.Calcium, the major cationic element in the secretory productof the gland, was not detected in the symplasm of the secretoryand collecting cells or in the vacuoles of the collecting cells.Some evidence indicates that silicon may be secreted by theglands but further corroboratory data are required. Our resultssupport previous suggestions that the glands of Tamarix havea low level of selectivity. Large, spherical aggregates of calcium crystals were observedin the vacuoles of the mesophyll cells and quantitative X-raymicroanalysis suggested that the chemical composition of thecrystalline aggregates was CaSO₄, 2H₂O. The predominant elementsin the vacuolar sap were magnesium and sulphur, probably presentas MgSO₄, whereas the major elements in the cytoplasm were potassium,sulphur and phosphorus. In Tamarix aphylla the calcium contentof the leaf appears to be regulated by both the secretion ofcalcium salts from the leaf and by intracellular calcificationin mesophyll cells.Copyright 1994, 1999 Academic Press X-ray microanalysis, Tamarix, salt gland, secretion, salts, ions, calcium crystals, compartmentation     

In vitro oxidative inactivation of glutathione S-transferase from a freeze tolerant reptile

We have previously reported that when garter snakesThamnophis sirtalis parietalis, a freeze tolerant species, were exposed to 5 h freezing at –2.5° C organs showed increases in the activities of anti-oxidant enzymes, especially catalase in skeletal muscle. This was interpreted to be an adaptation to deal with the potentially injurious postischemic situation of thawing. The present work analyzesin vitro oxidative inactivation of a possible target of postischemic-induced free radical damage, the secondary anti-oxidant defense glutathione-S transferase, and the protective role of endogenous catalase. Approximately 50% of GST activity from snake muscle homogenates was lost within 2 min after addition of H₂O₂ plus Fe(II) (0.4–2 mM) in media containing azide whereas addition of iron alone resulted in no damaging effects. The opposing effects of dimethyl sulfoxide and EDTA in modifying this process strongly suggested the involvement of ·OH radicals in the GST inactivation. A partial recovery of the activity was promoted by mercaptoethanol, indicating that sulphydryl groups oxidation participate in the mechanism of GST inactivation. Pre-incubation of the reaction media containing H₂O₂ caused protection of the GST activity only in the absence of azide, indicating that endogenous catalase modulates the extent of oxyradical damage. The protective pre-incubation effect was more efficacious when employing homogenates from lung and liver, organs that have higher catalase activities, as well as homogenates from freezing-exposed muscle (that show an 80% increase in catalase activity, compared with control). The protection against GST inactivation observed in muscle from frozen snakes demonstrates that increased anti-oxidant defenses during freezing exposure can be a key factor in controllingin vitro oxyradical damage. The implications for natural freeze tolerance are discussed.     

Suppression of reproductive development and longevity in the redshouldered leaf beetle,Monolepta australis (Col.: Chrysomelidae) by the tachinid,Monoleptophaga caldwelli (Dipt.)

This paper examines the level and seasonal incidence of parasitism of the redshouldered leaf beetle,Monolepta australis (Jacoby), by the tachinid,Monoleptophaga caldwelli Baranov, and assesses the impact of this fly on the beetle's reproductive development and longevity. Highest average numbers ofM. australis were trapped at light in north Queensland in January and February while parasitism peaked at 6.6 and 6.8% in June and July respectively, averaging 4.8% over the entire sampling period and reaching 28% for a single occasion. Overall, only 2.4% of parasitized beetles were represented in the final two stages of ovarian development compared to 29.1% of non-parasitized ones. There was a highly significant (P<0.001) difference in the distribution frequencies of parasitized and non-parasitized females in the various stages of ovarian development, and most parasitized males possessed atrophied testes. For field-collected beetles maintained in the laboratory, average longevity for those parasitized was 15.6 days compared to 72.2 days for those not parasitized.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Determination of the time course of capacitation in mouse spermatozoa using a chlortetracycline fluorescence assay

The heads of mouse spermatozoa obtained 5 min after release from the excised caudae epididymides showed a characteristic fluorescence pattern in the presence of the fluorophore chlortetracycline (CTC). There was uniform fluorescence over the entire head with about half the sperm population showing a brighter line of fluorescence across the equatorial segment; this fluorescence pattern was designated “F.” After 90-min incubation in culture medium (CM) containing 2% (w/v) bovine serum albumin, most of the sperm heads showed a dark band of nonfluorescence over the equatorial and postequatorial segment, while the anterior portion of the head showed bright fluorescence. This fluorescence pattern was designated “B.” The time course for the disappearance of pattern F matched the time course of the appearance of pattern B, with a half-time of 30 min. The transformation was complete in 90 min. At longer times of incubation in CM, the percentage of spermatozoa showing pattern B declined; fluorescence over the entire head was lost, characteristic of the pattern for acrosome-reacted sperm (P. M. Saling and B. T. Storey (1979). J. Cell Biol.83, 544–555). Mouse sperm showing pattern B were able to undergo the acrosome reaction, either spontaneously or by induction with acid-solubilized zonae pellucidae from mouse eggs (H. M. Florman and B. T. Storey (1982). Dev. Biol.91, 121–130). The latter reaction was blocked by its specific inhibitor 3-quinuclidinyl benzilate (QNB). Mouse sperm showing pattern F could not be induced to undergo the acrosome reaction by exposure to solubilized zonae. This implies that the change from fluorescence pattern F to fluorescence pattern B corresponds with changes in the sperm which make them susceptible to undergo the acrosome reaction. This change occurs during the time interval previously determined to be needed for capacitation of mouse sperm in vitro in CM (M. Inoue and D. P. Wolf (1975). Biol. Reprod.13, 340–346). These results imply that spermatozoa showing CTC fluorescence pattern B can be considered to be capacitated and that a functional definition for capacitation is the acquired ability to undergo the acrosome reaction rapidly when treated with acid-solubilized zonae pellucidae. The CTC fluorescence assay provides for the first time a means to monitor the time course of epididymal mouse sperm capacitation in vitro.     

Mouse gamete interactions during fertilization in vitro. Chlortetracycline as a fluorescent probe for the mouse sperm acrosome reaction

We have developed an assay for detecting the acrosome reaction in mouse sperm using chlortetracycline (CTC) as a fluorescent probe. Sperm known to be intact with nonreacted acrosomes show CTC fluorescence in the presence of Ca2+ over the anterior portion of the sperm head on the plasma membrane covering the acrosome. Sperm which have undergone the acrosome reaction do not show fluorescence on the sperm head. Mouse sperm bind to zonae pellucidae of cumulus-free eggs in vitro in a Ca2+-dependent reaction; these sperm are intact by the CTC assay. Intact sperm bind to mechanically isolated zonae under the same conditions: the egg is apparently unnecessary for this inital reaction. Sperm suspensions, in which greater than 50% of the motile population had completed the acrosome reaction, were prepared by incubation in hyperosmolal medium followed by treatment with the divalent cation ionophore, A23187. Cumulus-free eggs challenged with such sperm suspensions preferentially bind intact sperm; acrosome-reacted sperm do not bind. We conclude that the plasma membrane of the mouse sperm is responsible for recognition of the egg's zona pellucida and that the obligatory sequence of reactions leading to fusion of mouse gametes is binding of the intact sperm to the zona pellucida, followed by the acrosome reaction at the zona surface, followed in turn by sperm penetration of the zona.     

High TSH concentrations in "euthyroidism": explanation based on control-loop theory.

High concentrations of thyroid-stimulating hormone (TSH) in the serum have often been reported in apparently euthyroid patients with damaged thyroids. We have confirmed this finding in 14 patients 18 months after subtotal thyroidectomy for Graves''s disease (group 1) and in 14 patients with manic-depressive psychosis (group 2) receiving lithium carbonate, which reduces thyroid reserve. One factor common to groups 1 and 2 but not to the controls was reduced thyroid reserve or functioning capacity, and, using established physical principles of servo-control, we have tried to define the mechanism. A series of curves were projected to indicate how TSH might be expected to vary with functioning thyroid capacity.     

Gas-liquid chromatography and enzymatic determination of alanopine and strombine in tissues of marine invertebrates

Gas-liquid chromatography (GLC) and enzymatic assays were developed for quantitating the imino acids, alanopine and strombine, alternate products of anaerobic glycolysis (replacing lactate) in the tissues of many marine invertebrates. For GLC analysis, d-strombine (2-methyliminodiacetic acid) and meso-alanopine (2,2′-iminodipropionic acid) were chromatographeo as N-trifluoroacetyl isobutyl esters. Modifications of techniques used for GLC analysis of amino acids were required to overcome steric hindrance in the acylation reaction caused by the presence of imino, rather than amino, groups. Both imino acids were separated from each other and from all amino acids by GLC. Detection limit of the technique was 0.05 μg imino acid. Enzymatic determination of imino acids made use of the alanopine-specific alanopine dehydrogenase (ADH) purified from the periwinkle, Littorina littorea, and the strombine/alanopine utilizing strombine dehydrogenase (SDH) from the clam, Mercenaria mercenaria, with assay conditions: 300 mm hydrazine buffer, pH 9.0, 5 mm NAD, and 0.3 unit ADH or 1.0 unit SDH. Enzymatic determinations of mixtures of alanopine and strombine in tissue samples required a dual analysis using both enzymes. Production of alanopine and strombine during anoxic stress in two species of marine molluscs was quantitated.