Potassium Fluxes in Chlamydomonas reinhardtii (II. Compartmental Analysis)

42K+ and 86Rb+ were used to determine the subcellular distribution of potassium in Chlamydomonas reinhardtii by compartmental analysis. In both wild type and a mutant strain, three distinct compartments (referred to as I, II, and III) were apparent. Using 42K+, we found that these had half-lives for K+ exchange of 1.07 min, 12.8 min, and 2.9 h, respectively, in wild-type cells and 0.93 min, 14.7 min, and 9.8 h, respectively, for the mutants. Half-lives were not significantly different when 86Rb+ was used to trace K+. Compartments I and II probably correspond to the cell wall and cytoplasm, respectively. Based on the lack of a large central vacuole in Chlamydomonas, the effect of a dark pretreatment on the kinetic properties of compartment III and the similarity between the [K+] of compartment III and that of isolated chloroplasts, this slowly exchanging compartment was identified as the chloroplast. Growth of wild-type cells at 100 [mu]M (instead of 10 mM K+) caused no change of cytoplasmic [K+] but reduced chloroplast [K+] very substantially. The mutants failed to grow at 100 [mu]M K+.     

In vitro analysis of virus-associated RNA I (VAI RNA): inhibition of the double-stranded RNA-activated protein kinase PKR by VAI RNA mutants correlates with the in vivo phenotype and the structural integrity of the central domain.

Adenoviruses use the virus-encoded virus-associated RNA (VAI RNA) as a defense against cellular antiviral response by blocking the activation of the interferon-induced, double-stranded RNA-activated protein kinase PKR. The structure of VAI RNA consists of two long, imperfectly base-paired duplex regions connected by a complex short stem-loop at the center, referred to as the central domain. By using a series of adenovirus mutants with linker-scan mutations in the VAI RNA gene, we recently showed that the critical elements required for function in the VAI RNA molecule are in the central domain and that these same elements of the central domain are also involved in binding to PKR. In virus-infected cells, VAI RNA interacts with latent kinase, which is bound to ribosomes; this interaction takes place in a complex milieu. To more fully understand the relationship between structure and function and to determine whether the in vivo phenotype of these mutants can be reproduced in vitro, we have now analyzed these mutant VAI alleles for their ability to block the activation of a partially purified PKR from HeLa cells. We have also derived the structure of these mutants experimentally and correlated the structure with function. Without exception, when the structure of the short stem-loop of the central domain was perturbed, the mutants failed to inhibit PKR. Structural disruptions elsewhere in the central domain or in the long duplex regions of the molecule were not deleterious for in vitro function. Thus, these results support our previous findings and underscore the importance of the elements present in the central domain of the VAI RNA for its function. Our results also suggest that the interaction between PKR and VAI RNA involves a precise secondary (and tertiary) structure in the central domain. It has been suggested that VAI RNA does not activate PKR in virus-infected cells because of mismatches in the imperfectly base-paired long duplex regions. We constructed mutant VAI genes in which the imperfectly base-paired duplex regions were converted to perfectly base-paired regions and assayed in vitro for the activation of PKR. As with the wild-type VAI RNA, these mutants failed to activate PKR in vitro, while they were able to block the activation of PKR better than did the wild type. These results suggest that the failure of VAI RNA to activate PKR is not the result of mismatches in the long duplex regions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Use of photoaffinity crosslinking and molecular modeling to analyze the global architecture of ribonuclease P RNA.

Bacterial ribonuclease P (RNase P), an endonuclease involved in tRNA maturation, is a ribonucleoprotein containing a catalytic RNA. The secondary structure of this ribozyme is well established, but comparatively little is understood about its 3-D structure. In this analysis, orientation and distance constraints between elements within the Escherichia coli RNase P RNA-pre-tRNA complex were determined by intra- and intermolecular crosslinking experiments. A molecular mechanics-based RNA structure refinement protocol was used to incorporate the distance constraints indicated by crosslinking, along with the known secondary structure of RNase P RNA and the tertiary structure of tRNA, into molecular models. Seven different structures that satisfy the constraints equally well were generated and compared by superposition to estimate helix positions and orientations. Manual refinement within the range of conformations indicated by the molecular mechanics analysis was used to derive a model of RNase P RNA with bound substrate pre-tRNA that is consistent with the crosslinking results and the available phylogenetic comparisons.     

Investigations on the variability of blood group polymorphisms among sixteen tribal populations from Orissa, Madhya Pradesh and Maharashtra, India.

Sixteen tribal populations from Orissa, Madhya Pradesh and Maharashtra have been typed for the polymorphic blood group systems A1A2B0, MNSs, Rhesus, Kell, Duffy and Diego. The heterogeneity in the distribution of haplotype and allele frequencies, respectively, is partly considerable. It is supposed that this is due to the operation of several microevolutionary factors, such as genetic drift, social and geographic isolation and gene flow. This is discussed in detail.     

Isolation and Characterization of Phosphoenolpyruvate Phosphatase from Germinating Mung Beans (Vigna radiata)

A phosphoenolpyruvate (PEP) phosphatase was purified to homogeneity from germinating mung beans (Vigna radiata). It was found to be a tetrameric protein (molecular mass 240,000 daltons) made up of apparently identical subunits (subunit molecular mass 60,000 daltons). It was free from bound nucleotides. It did not show pyruvate kinase activity. The enzyme showed high specificity for PEP. Pyrophosphate and some esters (nucleoside di- and triphosphates) were hydrolyzed slowly and phosphoric acid monoesters were not hydrolyzed. The enzyme showed maximum activity at pH 8.5. At this pH, the K_m of PEP was 0.14 millimolar and the V_max was equal to 1.05 micromoles pyruvate formed per minute per milligram enzyme protein. Dialysis of the enzyme against 10 millimolar triethanolamine buffer (pH 6.5), led to loss of the catalytic activity, which was restored on addition of Mg²⁺ ions (K_m = 0.12 millimolar). Other divalent metal ions inhibited the Mg²⁺ -activated enzyme. PEP-phosphatase was inhibited by ATP and several other metabolites.     

Thyroid function during intermittent exposure to hypobaric hypoxia

Circulatory levels of triiodothyronine (T₃) and thyroxine (T₄) and their kinetics were studied in rabbits exposed to intermittent hypobaric hypoxia (5200 m, 395 mm Hg,PO₂ 83 mm Hg) 6 h daily for 5 weeks in a decompression chamber maintained at room temperature of 22°–24° C. Kinetics of T₃ and T₄ were studied on days 21 and 28 of hypoxic exposure. The T₃ and T₄ values were found to be significantly lower on day 8 of exposure to hypoxia compared to the pre-exposure values. The decreased levels were maintained throughout the entire period of hypoxic stress. The metabolic clearance rate, production rate, distribution space and extrathyroidal T₃ and T₄ pools were significantly decreased in animals under hypoxic stress compared to the control animals. The decline in thyroid hormone levels and their production in rabbits under hypoxic stress indicate an adaptive phenomenon under conditions of low oxygen availability.     

Interodontoblastic collagen (von Korff fibers) and circumpulpal dentin formation: an ultrathin serial section study in the cat

The collagenous fibers of von Korff pass from the dentin matrix between the odontoblasts into the dental pulp. Although collagen fibrils are known to be present between odontoblasts, the existence of von Korff fibers has remained controversial. This may be because their continuity between the dentin matrix and the pulp has not been demonstrated ultrastructurally. In this study we have examined the odontoblast layer in the middle to apical regions of perfusion-fixed permanent canine teeth of cats by using transmission electron microscopy. Ultrathin sections of demineralized specimens revealed frequent bundles of collagen fibrils 1) entering the odontoblast layer from the predentin, 2) present between odontoblast cell bodies, and 3) passing from between the odontoblasts into the pulp. The question of continuity of these bundles from the predentin, across the odontoblast layer into the pulp was examined in ultrathin serial sections. Unbroken continuity of a collagen bundle from the predentin between the odontoblasts into the pulp was established in a reconstruction of one series of 22 serial sections and was very strongly suggested by a number of other series in which the numbers of available sections restricted their full visibility. This investigation has shown, therefore, that classical von Korff fibers are present and that these fibers are present in fully erupted teeth with closed apices, i.e., at a time when secondary circumpulpal dentinogenesis is in progress. The findings call for a reexamination of the question of von Korff fibers during mantle dentinogenesis and primary circumpulpal dentinogenesis. Resolution of their existence at the earlier stages of dentinogenesis should be possible by using the ultrathin serial-sectioning technique.     

Glucoregulatory hormones in man at high altitude

Concentrations of glucose, lactic acid, free fatty acid (FFA), insulin, cortisol and growth hormone (GH) in the blood were monitored in 15 euglycaemic men (sojourners, SJ) at sea level (SL) and while at altitudes of 3500 m and 5080 m, in acclimatised low landers (ALL) and in high altitude natives (HAN). In SJ, blood glucose and insulin concentrations showed a significant increase on the 3rd and 7th day after arrival at high altitude (HA), thereafter returning to sea level values and remaining the same during the entire period of their stay at 3500 m. Subsequently, on arrival at higher altitude (5080 m) the glucose concentrations again showed an increase over the preceding values and returned to SL values on day 41 while at 5080 m. A significant increase in cortisol concentrations was seen on day 3 after arrival at HA and the increased levels were maintained until day 21 at 3500 m. The cortisol concentrations on day 30 after arrival at 5080 m came down to SL values and remained unchanged thereafter. No appreciable change in GH and FFA was seen during the sojourn at HA. On the other hand, blood lactic acid concentration decreased significantly. There was no difference between the fasting glucose concentrations in ALL at 3500 m and in HAN at 3500 m and 4200 m compared to values of SJ at SL, whereas ALL at 4200 m had higher glucose values. Concentrations of plasma insulin and GH in ALL and HAN were higher than the values of SJ at SL, whereas cortisol values did not show any difference. These observations indicated that at HA the glucose values were high for the insulin concentration observed and might have been due to increased secretion of GH by the pituitary gland.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

Dissection of a single round of vesicular transport: sequential intermediates for intercisternal movement in the Golgi stack

We take advantage of a cell-free system that reconstitutes essentially a single round of transport of the VSV-encoded G protein between Golgi cisternae to identify discrete stages in the maturation of carrier vesicles. Using GTP gamma S and N-ethylmaleimide (NEM) as selective inhibitors to accumulate coated and uncoated vesicles, respectively, we find these to be successive and obligatory transport intermediates. We find that the coated and uncoated vesicles that accumulate when transport is blocked have already transferred from donor to acceptor stacks but not yet fused. Similar coated and uncoated vesicles accumulate in appropriately treated whole cells. Our studies imply that a coated bud (pit)-coated vesicle-uncoated vesicle system analogous to that responsible for receptor-mediated endocytosis carries biosynthetic protein transport across the Golgi stack. However, "Golgi"-coated buds do not contain clathrin and seem to act as bulk carriers, whereas endocytic clathrin-coated pits carry a highly selective cargo.