Pressure-induced endothelial Ca(2+) oscillations in lung capillaries

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca(2+) concentration [Ca(2+)](i) by the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca(2+)](i) by Ca(2+) influx through gadolinium-inhibitable channels and amplified [Ca(2+)](i) oscillations by Ca(2+) release from intracellular stores. Endothelial [Ca(2+)](i) transients were induced by pressure elevations of as little as 5 cmH(2)O and increased linearly with higher pressures. Heptanol inhibition of [Ca(2+)](i) oscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.     

Vascular regulation of type II cell exocytosis

To determine whether lung capillary pressure regulates surfactant secretion, we viewed alveoli of the constantly inflated, isolated blood-perfused rat lung by fluorescence microscopy. By alveolar micropuncture we infused fura 2 and lamellar body (LB)-localizing dyes for fluorescence detection of, respectively, the alveolar cytosolic Ca(2+) concentration ([Ca(2+)](i)) and type II cell exocytosis. Increasing left atrial pressure (Pla) from 5 to 10 cmH(2)O increased septal capillary diameter by 26% and induced marked alveolar [Ca(2+)](i) oscillations that abated on relief of pressure elevation. The rate of loss of LB fluorescence that reflects the LB exocytosis rate increased fourfold after the pressure elevation and continued at the same rate even after pressure and [Ca(2+)](i) oscillations had returned to baseline. In alveoli pretreated with either 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, the intracellular Ca(2+) chelator, or heptanol, the gap junctional blocker, the pressure-induced exocytosis was completely inhibited. We conclude that capillary pressure and surfactant secretion are mechanically coupled. The secretion initiates in a Ca(2+)-dependent manner but is sustained by Ca(2+)-independent mechanisms.     

A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)

A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.     

Altered Ca2+ homeostasis and impaired mitochondrial function in cardiomyopathy

This work investigates whether purine metabolism and release is related to cardioprotection with hyperkalemia and hypothermia. Langendorff guinea-pig hearts were used to either monitor metabolism during ischemia or to measure functional recovery, myocardial injury and release of purine during reperfusion. Hearts underwent 30 min ischemia using one of the following protocols: control (normothermic buffer), hyperkalaemia (high-potassium buffer), hypothermia (20°C) and hyperkalemia + hypothermia. At the end of 30 min ischemia, hyperkalemia was associated with similar metabolic changes (rise in purine and lactate and fall in adenine nucleotides) to control group. Accumulation of purine was due to a rise in inosine, xanthine and hypoxanthine and was largely prevented by hypothermia and hyperkalemia + hypothermia. Upon reperfusion, there was a time-dependent release of all purine, lactate and AMP. A fast (peak in less than 20 sec) release of inosine, xanthine, hypoxanthine and lactate was highest in control followed by hyperkalemia then hypothermia and little release in hyperkalemia + hypothermia. Adenosine and AMP release was slow (peak at 3 min), only significant in control and was likely to be due to sarcolemmal disruption as the profile followed lactate dehydrogenase release. Recovery (left ventricular developed pressure) was 63% control, 82% hyperkalemia, 77% hypothermia and 98% for hyperkalemia + hypothermia. The loss of purine during reperfusion but not their production during ischemia is related to cardioprotection with hyperkalemia. The possibility that the consequences of hyperkalemia modulate a sodium-dependent purine efflux, is discussed. The reduced loss of purine in hypothermia or in hyperkalemia + hypothermia is likely to be due to a lower metabolic activity during ischemia.     

Investigating the association between OPA1 polymorphisms and glaucoma: comparison between normal tension and high tension primary open angle glaucoma

OPA1, the gene responsible for autosomal dominant optic atrophy, represents a good candidate gene for glaucoma, as there are similarities in the clinical phenotype and OPA1 is expressed in the optic nerve. Single nucleotide polymorphisms on intervening sequence (IVS) 8 of the OPA1gene (genotype IVS8+4 C/T;+32T/C) were recently found to be strongly associated with normal tension glaucoma (NTG). In order to investigate whether this association exists in patients with high-tension glaucoma (HTG), 90 well-characterized HTG patients were examined for the presence of these OPA1polymorphisms by PCR amplification followed by bi-directional sequencing. Five out of 90 HTG subjects (5.6%; 95% CI 1.8-12.5) were found to carry the OPA1 genotype IVS 8+4 C/T; +32 T/C, compared with 32/163 (19.6%; 95% CI 13.8-26.6) NTG subjects [chi(2)=9.2, P=0.002, OR 4.1 (95% CI 1.6-11.1)], and 7/186 (3.8%; 95% CI 1.5-7.6) control subjects [chi(2)=0.47, P=0.49, OR 1.5 (95% CI 0.5-4.9)]. These results indicate that unlike NTG, the OPA1 genotype IVS8+4 C/T,+32T/C is not significantly associated with high-tension primary open angle glaucoma, and suggest genetic heterogeneity between the conditions.     

From foe to friend: geographical and environmental factors and the control and eradication of smallpox in India

Due to the highly visible nature of the disease, smallpox received a lot of attention from the colonial and independent Indian governments. An assessment of the changing official views about the impact of geographical and environmental factors on modes of variola causation and control presents insights into themes that are generally ignored in the existing historiography. Rather than being synchronised efforts, imposed top-down, provincial level officials in charge of running vaccination programmes were able to retain a great degree of autonomy and shape the nature of local immunisation drive. As a result, vaccination work in the country was often disjointed, marked by official disagreements about the usefulness of certain strategies and technological inputs, with these trends being most noticeable in rural areas. Thus, this study seeks to highlight the importance of recognising--and studying--the fractured nature of medical and public health administration in the South Asian sub-continent.     

Evaluation of genetic diversity among Pseudomonas citronellolis strains isolated from oily sludge-contaminated sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

Unlike thrombin, protein C and activated protein C do not affect vascular tone

Because plasma levels of protein C (PC) or activated protein C (APC) are altered in certain diseases associated with vascular dysfunction, and APC has therapeutic potential in preventing microvascular coagulation in severe sepsis, potential vascular effects of PC and APC were compared to those of the vasoactive peptide, thrombin. Thrombin was a more potent relaxant agonist than contractile agonist in aorta. Unlike thrombin, cumulatively administered APC (10(-9)-10(-7) M) did not exert vascular effects in rat or rabbit aorta. Noncumulative challenge of PC (10(-7) M) and APC (8 x 10(-8) M) also did not contract rat or rabbit aortae, either with or without endothelium. Likewise, the same concentrations of PC and APC also did not relax norepinephrine-induced (10(-7) M) vascular tone in either rat or rabbit aortae. Thus, in contrast to thrombin, PC and APC failed to modulate vascular tone, suggesting that the therapeutic use of APC is unlikely to be accompanied by any direct effects on vascular motility.     

Structural and interactional homology of clinically potential trypsin inhibitors: molecular modelling of cucurbitaceae family peptides using the X-ray structure of MCTI-II

Several trypsin inhibitor peptides (with 28-32 amino acid residues) belonging to the Cucurbitaceae (LA-1, LA-2, MCTI-I, CMTI-I, CMTI-III, CMTI-IV), characterized by a distinctive tertiary fold with three conserved disulphide bonds and with mostly arginine at their active centre, were modelled using the high-resolution X-ray structure of a homologous inhibitor, MCTI-II, isolated from bitter gourd. All the inhibitors were modelled in both their native and complexed state with the trypsin molecule, keeping the active site the same as was observed in the trypsin-MCTI-II complex, by homology modelling using the InsightII program. The minimized energy profile supported the binding constants (binding behaviour) of the inhibitor-trypsin complexes in the solution state. A difference accessible surface area (DASA) study of the trypsin with and without inhibitors revealed the subsites of trypsin where the inhibitors bind. It revealed that the role of mutation of these peptides through evolution is to modulate their inhibitory function depending on the biological need rather than changing the overall structural folding characteristics which are highly conserved. The minor changes of amino acids in the non-conserved regions do not influence significantly the basic conformational and interactional sequences at the trypsin binding subsites during complex formation.