Physicochemical and functional characteristics of lentil starch.

The physicochemical properties of lentil starch were measured and linked up with its functional properties and compared with those of corn and potato starches. The amylose content of lentil starch was the highest among these starches. The crystallinity and gelatinization enthalpy of lentil starch were the lowest among these starches. The high amylose: amylopectin ratio in lentil starch resulted into low crystallinity and gelatinization enthalpy. Gelatinization and pasting temperatures of lentil starch were in between those of corn and potato starches. Lentil starch gels showed the highest storage modulus, gel strength and pasting viscosity than corn and potato starch gels. Peleg's model was able to predict the stress relaxation data of these starches well (R(2)>0.98). The elastic modulus of lentil starch gel was less frequency dependent and higher in magnitude at high temperature (60 °C) than at lower temperature (10 °C). Lentil starch is suitable where higher gel strengthened pasting viscosity are desired.

Interfacial and emulsifying properties of lentil protein isolate.

The dynamic interfacial tension (DIFT) at oil-water interface, diffusion coefficients, surface hydrophobicity, zeta potential and emulsifying properties, including emulsion activity index (EAI), emulsion stability index (ESI) and droplet size of lentil protein isolate (LPI), were measured at different pH and LPI concentration, in order to elucidate its emulsifying behaviour. Sodium caseinate (NaCas), whey protein isolate (WPI), bovine serum albumin (BSA) and lysozyme (Lys) were used as benchmark proteins and their emulsifying property was compared with that of LPI. The speed of diffusion-controlled migration of these proteins to the oil/water interface, was in the following order: NaCas>LPI>WPI>BSA>Lys, while their surface hydrophobicity was in the following order: BSA>LPI>NaCas>WPI>Lys. The EAI of emulsions stabilised by the above proteins ranged from 90.3 to 123.3 m(2)/g and it was 93.3 ± 0.2 m(2)/g in LPI-stabilised emulsion. However, the stability of LPI-stabilised emulsions was slightly lower compared to that of WPI and NaCas-stabilised emulsions at the same protein concentration at pH 7.0. The ESI of LPI emulsions improved substantially with decrease in droplet size when protein concentration was increased (20-30 mg/ml). Reduction of disulphide bonds enhanced both the EAI and ESI compared to untreated samples. Heat treatment of LPI dispersions resulted in poor emulsion stability due to molecular aggregation. The stability of LPI-stabilised emulsions was found to decrease in the presence of NaCl. This study showed that LPI can be as effective emulsifiers of oil-in-water emulsions as are WPI and NaCas at ≥20 mg/ml concentrations both at low and neutral pH. The emulsifying property of LPI can be improved by reducing the intra and inter-disulphide bond by using appropriate reducing agents.

Humanizing mismatch repair in yeast: towards effective identification of hereditary non-polyposis colorectal cancer alleles.

The correction of replication errors is an essential component of genetic stability. This is clearly demonstrated in humans by the observation that mutations in mismatch repair genes lead to HNPCC (hereditary non-polyposis colorectal cancer). This disease accounts for as many as 2-3% of colon cancers. Of these, most of them are in the two central components of mismatch repair, MLH1 (mutL homologue 1) and MSH2 (mutS homologue 2). MLH1 and MSH2 function as a complex with two other genes PMS2 and MSH6. Mismatch repair genes, and the mechanism that ensures that incorrectly paired bases are removed, are conserved from prokaryotes to human. Thus yeast can serve as a model organism for analysing mutations/polymorphisms found in human mismatch repair genes for their effect on post-replicative repair. To date, this has predominantly been accomplished by making the analogous mutations in yeast genes. However, this approach is only useful for the most highly conserved regions. Here, we discuss some of the benefits and technical difficulties involved in expressing human genes in yeast. Modelling human mismatch repair in yeast will allow the assessment of any functional effect of novel polymorphisms found in patients diagnosed with colon cancers.

Localization and interactions of vasoactive peptide receptors in renomedullary interstitial cells of the kidney.

Vasoactive peptides regulate renal medullary microcirculation and tubular function, but the localization of their receptors and mechanisms of actions are currently unknown. Using electron microscopic autoradiography, we have mapped the receptors for angiotensin II (Ang II [AT1 and AT2]), endothelin (ET(A) and ET(B)), and bradykinin (B2) in the rat renal medulla. Although these peptide receptors show distinct vascular and tubular distributions, they overlap strikingly in renomedullary interstitial cells (RMICs) of the inner stripe and the papilla. Using reverse transcription-polymerase chain reaction (RT-PCR) and Southern analysis, mRNAs for AT1A, ET(A), and B2 receptors were detected in cultured adult RMICs. Ang II increases intracellular inositol 1,4,5-triphosphate (IP3) and [Ca2+]i and stimulates [3H]thymidine incorporation and extracellular matrix (ECM) synthesis via AT1A receptors. Endothelin and bradykinin also stimulate cell proliferation and ECM synthesis in RMICs through ET(A) and B2 receptors, respectively, but the actions of endothelin are modulated by concurrent nitric oxide production. By contrast, AT2 receptor mRNA was detected only in embryonic RMICs, in which Ang II inhibits cell proliferation through this receptor. These results suggest that multiple vasoactive peptides may interact with RMICs to exert endocrine and/or paracrine influences on renal medullary microcirculation and tubular function.

Salmonella-induced M-cell formation in germ-free mouse Peyer's patch tissue.

M cells present in Peyer's patch tissue transport enteric antigens for presentation to underlying lymphoid tissue to initiate immune responses against intestinal infection. Present work investigates how interactions taking place between bacteria, epithelial cells, and immunocytes could contribute to initial detection and later elimination of enteric antigens. Oral infection of germ-free mice with Salmonella typhimurium aroA- caused a twofold to threefold increase in M cell numbers, crypt depth, and enterocyte migration rate after 7 days. These changes were accompanied by a twofold increase in follicle-associated epithelial tissue (FAE)-associated CD4+ and a threefold decrease in FAE-associated CD8+ counts. Salmonella also increased M-cell numbers shortly after infection. Other effects on crypt size and spleen weight took longer to develop. Salmonella probably creates M cells by changing the local subepithelial immune environment in the lymphoid follicle.

Human secreted carbonic anhydrase: cDNA cloning, nucleotide sequence, and hybridization histochemistry.

Complementary DNA clones coding for the human secreted carbonic anhydrase isozyme (CA VI) have been isolated and their nucleotide sequences determined. These clones identify a 1.45-kb mRNA that is present in high levels in parotid submandibular salivary glands but absent in other tissues such as the sublingual gland, kidney, liver, and prostate gland. Hybridization histochemistry of human salivary glands shows mRNA for CA VI located in the acinar cells of these glands. The cDNA clones encode a protein of 308 amino acids that includes a 17 amino acid leader sequence typical of secreted proteins. The mature protein has 291 amino acids compared to 259 or 260 for the cytoplasmic isozymes, with most of the extra amino acids present as a carboxyl terminal extension. In comparison, sheep CA VI has a 45 amino acid extension [Fernley, R. T., Wright, R. D., & Coghlan, J. P. (1988b) Biochemistry 27, 2815]. Overall the human CA VI protein has a sequence identity of 35% with human CA II, while residues involved in the active site of the enzymes have been conserved. The human sheep secreted carbonic anhydrases have a sequence identity of 72%. This includes the two cysteine residues that are known to be involved in an intramolecular disulfide bond in the sheep CA VI. The enzyme is known to be glycosylated and three potential N-glycosylation sites (Asn-X-Thr/Ser) have been identified. Two of these are known to be glycosylated in sheep CA VI. Southern analysis of human DNA indicates that there is only one gene coding for CA VI.

Dyspnea in aging rats due to disseminated intravascular coagulation (DIC).

During an 18-month oncogenicity study using rats, approximately 10% of the animals developed a form of respiratory distress very similar to that seen in the terminal stages of chronic respiratory disease, commonly associated with Mycoplasma pulmonis infection. Investigation of the lungs of the affected rats revealed not only that they did not have the consolidation usually associated with chronic respiratory disease, but they also appeared macroscopically normal. Further investigation of a number of cases revealed systemic intravascular thrombus formation of the type usually referred to as disseminated intravascular coagulation. Using an antiserum to fibrin we have demonstrated the presence of intravascular fibrin deposits in the lungs of the affected rats and have shown them to be the same as experimentally induced intravascular fibrin deposits induced in rat lungs by the administration of thrombin after blocking the fibrinolytic system. This is the first example of such a phenomenon being recorded in aging rats.

Effect of converting enzyme inhibition on renin synthesis and secretion in mice.

We have investigated the relative importance of renal renin stores and de novo synthesis during stimulation of renin secretion and the role of transcription and posttranscriptional factors in providing increased synthesis of renin. When enalapril was administered to previously untreated mice, plasma renin concentration increased 40-fold within 1.5 hours, and remained at a high level for the 8 days of the experiment. Renal renin decreased by 82% after 24 hours and thereafter increased to levels higher than controls. Calculations of renin turnover, based on data for the rate of metabolism of renin in plasma, indicated that most of the renin released in the first 24 hours could be accounted for by the decrease in renal renin stores, indicating that de novo synthesis played only a minor role. After 24 hours, however, when both plasma renin concentration and renal renin increased, the calculated rate of renin synthesis increased to nearly 40 times the rate in controls. When enalapril was administered to mice that had been depleted of plasma and renal renin by chronic sodium loading, plasma renin concentration increased markedly within 1.5 hours, but to only half the level achieved in the previously untreated mice. No decrease in renal renin occurred, suggesting that the renal renin remaining after chronic sodium loading was not available for release. Renal renin messenger RNA increased 4.5-fold after 6 hours, and after 8 days had increased to 5.0 times the level at day 0. The increase in calculated rate of renin synthesis was maximal between 5 and 8 days, when it was 54 times greater than at day 0. During enalapril treatment, there were marked increases in the granulation of the juxtaglomerular cells and in the amount of rough endoplasmic reticulum and Golgi apparatus they contained. These results suggest that posttranscriptional factors play a major role in determining the rate of renin synthesis.

Effect of mineralocorticoids and salt loading on renin release, renal renin content and renal renin mRNA in mice.

1. DOCA and 9 alpha-fludrocortisone were given to mice on a high-sodium diet for periods of up to 20 weeks, resulting in decreases in plasma renin concentration, renal renin concentration and renal renin mRNA with both treatments. 2. Plasma renin concentration was suppressed prior to suppression of renin mRNA and renal renin levels, indicating that suppression of synthesis and secretion of renin occur separately. 3. The decrease in renal renin concentration that occurred with DOCA was greater and more rapid than the decrease that occurred with 9 alpha-fludrocortisone, suggesting that DOCA caused intra-renal breakdown of renin. 4. When DOCA was given to mice on a low-sodium diet, plasma renin concentration and renal renin concentration increased, indicating that the effects of DOCA on renin levels were dependent on dietary sodium. 5. Renin secretion and synthesis appeared to be controlled by different mechanisms and sodium balance has an important effect on both processes.

Tissue and species distribution of the secreted carbonic anhydrase isoenzyme.

The secreted carbonic anhydrases, CA VI, are high molecular mass, oligomeric enzymes originally found in the sheep parotid gland and saliva. The enzymes have been purified from the saliva or parotid glands of several different species. All the CA VI enzymes studied have an apparent subunit Mr of about 45,000 as previously reported for the sheep enzyme. By Western analysis, CA VI from human, cow and dog cross-reacted with antibody raised against the purified sheep enzyme whereas that of the mouse did not. The N-terminal sequences of the sheep, human, cow and mouse enzymes are reported. The sheep, cow and human N-terminal sequences are similar to one another while the mouse sequence is substantially different. Nevertheless, the amino acids in the aromatic cluster I (Trp-5, Tyr-7, Trp-16 and Tyr/Phe-20) have all been conserved, as is the case with the cytoplasmic carbonic anhydrases. Eighteen tissues from the sheep have been examined for the presence of CA VI by Western analysis but it has been found only in the salivary glands. Northern analysis and hybridization histochemistry show that the mRNA for CA VI in sheep is expressed specifically in the acinar cells of the parotid and submandibular glands.

The gene for human carbonic anhydrase VI(CA6) is on the tip of the short arm of chromosome 1.

The gene encoding the human secreted carbonic anhydrase isozyme CAVI(CA6) maps to chromosome 1 by Southern analysis of a somatic cell hybrid panel and to 1p36.22----p36.33 by in situ hybridization. CA6 is therefore not linked to the cytoplasmic carbonic anhydrase genes on chromosome 8 or to CA7 on chromosome 16.

Use of a DNA probe in the diagnosis of adult polycystic kidney disease.

Adult polycystic kidney disease (APKD) is one of the most common inherited diseases in man. A diagnosis based on the demonstration of renal cysts with ultrasonography or computerised tomography may be inconclusive in early adulthood, the crucial years before child-bearing is complete. Here we describe the improved diagnostic probability that is possible using genetic linkage studies. A 24-year-old woman, whose father and younger sister were affected by APKD, was demonstrated to have a single cyst in each kidney. These findings were insufficient for a diagnosis of APKD and for this reason genetic linkage studies were undertaken. DNA was extracted from peripheral blood leukocytes from the presenting individual, and her immediate and extended family; the DNA was cut with the restriction enzyme PvuII, electrophoresed in a 0.7% agarose gel and blotted onto nitrocellulose before probing with a 32P-labelled 4 kb fragment. This contained DNA from the hypervariable region (3' hypervariable region, 3'HVR) that is linked to the gene for APKD on the short arm of chromosome 16 and has been used in other family studies by Reeders et al. We correlated the findings on Southern blotting with ultrasound evidence of APKD and found that the disease segregated with a 7.0 kb fragment in the presenting individual's father and sister. She was shown to have inherited this allele also; the use of this technique thus increased the probability of her having APKD from 50% to 96.5%.

Embryonic death in pregnant rats owing to intercurrent infection with Sendai virus and Pasteurella pneumotropica.

During an outbreak of Sendai virus infection in a colony of rats used for embryo production, severe lung lesions due to secondary colonization of the rat lungs with Pasteurella pneumotropica were noted. The effect on pregnancy was to cause embryonic death and resorption, leaving a deciduoma with trophoblastic giant cells as the only embryonic remnants. Up to 30% of fetuses in each pregnant animal were affected at the time of severe maternal lung lesions. Heavy growths of Pasteurella pneumotropica were obtained from the lungs of all affected dams. The formation of deciduomas as a result of embryonic death was due to the indirect effect of damage to the lungs during pregnancy rather than the direct pathogenic effect on the developing embryo of the microbial organisms isolated.

Location of gene expression in CNS using hybridization histochemistry.

In this chapter we have placed heavy emphasis on our own recent work to lay out a workable recipe for hybridization histochemistry. Only a trickle of papers followed the initial benchmark excursions into in situ labeling of tissue sections. Our own entry into this field was as late starters in 1978, but since then a confluence of important questions and technical advances has served to make hybridization histochemistry much more attractive and universally applicable as a research tool. Hybridization histochemistry allows the location of anatomical sites of gene expression and viral replication with unique specificity and is able to solve some problems for which there is no other suitable technique available in the central nervous system. For example, allowing that peptides may enter neurons by a variety of mechanisms and then be christened neuroendocrine peptides, it has become a compelling issue to know which cells are manufacturing the peptide. Thus, much can be learned by the approach elegantly demonstrated by Gee et al., of locating mRNA and its peptide product within the same neuron. The intracellular location of specific mRNA for a neuropeptide in a cell body indicates a very high probability that the peptide is secreted as a neurotransmitter or a neuromodulator from sites associated with the cell body. Our introduction of the use of whole mouse sections and large sections of brain of large animals in hybridization histochemistry has great potential in locating hormonal, enzymatic, and growth factor gene expression. The technique has been applied most elegantly by others to developmental studies and for the examination of viral infection. Resolution down to a single cell in heterogeneous tissue was beyond the original expectation of the capability of 32P-labeled probes and single cells in sections shown in Fig. 2 is probably the limit of resolution with this isotope. There is no reason why other isotopes, fluorescent labels, or labels suitable for EM should not take the resolution of the technique to intracellular. The horizon of application is widened enormously by the successful application of synthetic oligonucleotide probes, and at the same time unshackles the procedure from dependence upon a fully functional molecular biology laboratory. Although hybridization is a valuable research tool which we have applied to location of neuropeptides in the brain, it should soon find a niche in many fields and in a short time should become a key diagnostic tool.(ABSTRACT TRUNCATED AT 400 WORDS)

Hybridization histochemistry.

In this review we have used our own recent work as a flagship to illustrate the recent renaissance of interest in hybridization histochemistry. A trickle of papers followed the initial key excursion into the in situ labeling of tissue sections (48-50). Our own entry into this field started in 1978 and since then a confluence of important questions and technical advances has served to make hybridization histochemistry much more attractive as a research tool. Hybridization histochemistry is able to solve some problems for which there is no other suitable technique at this time. Hybridization histochemistry provides the location of anatomical sites of gene expression, and viral replication, with uniquely high specificity. We have taken 32P-labeled probes to what appears to be their limit of resolution, which is single cells in thin sections. While 32P has clear disadvantages, exposure time is relatively short and the use of fast-X-ray film to preview the results and estimate exposure time for emulsion has been turned to advantage. Our introduction (27) of the use of whole-mouse sections in hybridization histochemistry has great potential in hormonal, enzymatic, and growth factor gene expression and will no doubt prove of great use in developmental studies and examination of viral infection. The use of synthetic DNA (synthetic oligonucleotides) unshackles the technique from the need for an associated molecular biology laboratory and at once widens the horizon of application of the technique. Although hybridization histochemistry is a valuable research tool which will soon find a niche in many fields, in a short time it should become a key diagnostic aid. It may well become the method of preference for detection of the expression of oncogenes and other cancer-related genes and for viruses which for other reasons are difficult to detect.

Renin gene expression in vessels of the ovine renal cortex.

Using hybridization histochemistry, a technique which localizes specific mRNA populations in tissue sections with a 700 base pair recombinant DNA probe which codes for ovine renin, we have localized renin gene expression in the afferent arteriole of the juxtaglomerular apparatus (JGA) in the sheep renal cortex. Specific labelling representing renin gene expression was also found at a distance from the glomerular tuft in the walls of the afferent arteriole and also in cells in the medial layer of larger vessels of the renal cortex, specifically the interlobular arteries. These observations provide morphological evidence of renin gene expression at these sites and, combined with ultrastructural and immunocytochemical evidence suggest that renin is synthesized and stored in the afferent arteriole either within the JGA or at a distance from the glomerulus, and in the smooth muscle coat of the interlobular arteries in the sheep kidney.