Immobilization of whole cells of Lactococcus lactis containing high levels of a hyperthermostable ß-galactosidase enzyme in chitosan beads for efficient galacto-oligosaccharide production.

Galacto-oligosaccharides (GOS) are prebiotic food ingredients that are proposed to stimulate the growth of beneficial gut microorganisms, particularly bifidobacteria. Previously, we developed a method for efficient GOS production using whole cells of Lactococcus lactis containing high levels of a hyper-thermostable ß-galactosidase enzyme from Sulfolobus solfataricus. In this study, a recombinant DNA removal and whole-cell enzyme immobilization process was developed to produce GOS from lactose before removal of the immobilized whole-cell enzyme, which could be reused for subsequent applications. Chitosan was found to be a superior immobilization material compared with alginate, as it retained its bead structure during the high temperature (90°C) used here for GOS production. Prior to immobilization, the recombinant DNA was degraded in the whole cells using UV treatment, resulting in an immobilized whole-cell enzyme that was free of recombinant DNA and with minimum effect on the efficiency of the enzyme. The optimum pH and temperature for GOS synthesis using the chitosan beads was pH = 5.5 and 90°C. The highest GOS production using the chitosan beads occurred with 40% initial lactose resulting in 150 g/L of GOS (tri-oligosaccharides and tetra-oligosaccharides) in addition to di-oligosaccharide GOS products that were not quantified. Notably, the highest lactose conversion rate was found using lower starting lactose concentrations, with more than 60% conversion into tri-oligosaccharides and tetra-oligosaccharides. The immobilized enzyme retained ∼50% activity after 2 cycles of GOS production. In conclusion, the chitosan-immobilized whole-cell enzyme can be used for efficient GOS production that is free of the whole-cell enzyme as well as detectable recombinant DNA.

Production of galactooligosaccharides using a hyperthermophilic ß-galactosidase in permeabilized whole cells of Lactococcus lactis.

Galactooligosaccharides (GOS) are novel prebiotic food ingredients that can be produced from lactose using ß-galactosidase, but the process is more efficient at higher temperatures. To efficiently express the lacS gene from the hyperthermophile Sulfolobus solfataricus, in Lactococcus lactis a synthetic gene (lacSt) with optimized codon usage for Lc. lactis was designed and synthesized. This hyperthermostable ß-galactosidase enzyme was successfully overexpressed in Lc. lactis LM0230 using a nisin-controlled gene expression system. Enzyme-containing cells were then killed and permeabilized using 50% ethanol and were used to determine both hydrolysis and transgalactosylation activity. The optimum conditions for GOS synthesis was found to be at pH 6.0 and 85 °C. A maximum production of 197 g/L of GOS tri- and tetrasaccharides was obtained from 40% initial lactose, after 55 h of incubation. The total GOS yield increased with the initial lactose concentration, whereas the highest lactose conversion rate (72%) was achieved from a low lactose solution (5%). Given that a significant proportion of the remaining lactose would be expected to be converted into disaccharide GOS, this should enable the future development of a cost-effective approach for the conversion of whey-based substrates into GOS-enriched food ingredients using this cell-based technology.

Factors influencing the stability of freeze-dried stress-resilient and stress-sensitive strains of bifidobacteria.

Freeze-drying is a common method for preservation of probiotics, including bifidobacteria, for further industrial applications. However, the stability of freeze-dried bifidobacteria varies depending on the freeze-drying method and subsequent storage conditions. The primary goals of this study were to develop an optimized freeze-drying procedure and to determine the effects of temperature, water activity, and atmosphere on survival of freeze-dried bifidobacteria. To address these goals, a commercially used bifidobacteria strain that is resilient to stress, Bifidobacterium animalis ssp. lactis Bb-12, and a characterized intestinal strain that is more sensitive to stress conditions, Bifidobacterium longum DJO10A, were used. A freeze-drying protocol was developed using trehalose as the cryoprotectant, which resulted in almost no loss of viability during freeze-drying. Resuscitation medium, temperature, and time did not significantly influence recovery rates when this cryoprotectant was used. The effects of temperature (-80 to 45°C), water activity (0.02 to 0.92), and atmosphere (air, vacuum, and nitrogen) were evaluated for the stability of the freeze-dried powders during storage. Freeze-dried B. animalis ssp. lactis Bb-12 was found to survive under all conditions tested, with optimum survival at temperatures up to 21°C, water activities up to 0.44, and all 3 atmospheres tested. The intestinal-adapted strain B. longum DJO10A was much more sensitive to the different storage conditions, but could be adequately maintained using optimum conditions. These optimum storage conditions included frozen storage, replacement of oxygen with nitrogen, and water activities between 0.11 and 0.22. These results indicated that an optimized storage environment is required to maintain viability of stress-sensitive bifidobacteria strains, whereas stress-resilient bifidobacteria strains can maintain viability over a wide range of storage conditions, which is practical in countries where controlled cold storage conditions may not be readily available.

Comparative analysis of an intestinal strain of Bifidobacterium longum and a strain of Bifidobacterium animalis subspecies lactis in Cheddar cheese.

Bifidobacteria cultures were incorporated into Cheddar cheeses to conduct a comparative analysis between the commercially available strain Bifidobacterium animalis ssp. lactis Bb-12 and the wild-type intestinal isolate, Bifidobacterium longum DJO10A. They were incorporated as starter adjuncts in separate vats and as a mixed culture, and survival through manufacturing and cheese ripening was assessed. For cheese using only Bb-12, the cells may have grown during cheese manufacture as 133% of the inoculum was incorporated into the cheese, resulting in 8.00 log cfu/g. Counts remained high during ripening showing less than 1 log decrease over a 12-mo period. For cheese using a mixed culture of Bb-12 and DJO10A, both strains were incorporated at much lower levels: 3.02 and 1.11%, respectively. This resulted in cheese with 6.00 and 5.04 log cfu/g for Bb-12 and DJO10A, respectively. Bifidobacteria survival rates were low, most likely due to the moisture of the cheese being below 38%. The Bb-12 demonstrated almost 100% viability during ripening. Numbers of DJO10A started to decline after 2 mo of ripening and dropped below the level of detection (2 log cfu/g) after 4.5 mo of ripening. Neither DJO10A nor Bb-12 fortified cheeses produced detectable amounts of organic acids during ripening other than lactic acid, indicating the lack of detectable metabolic contribution from bifidobacteria during cheese production and ripening such as production of acetic acid. To determine if sublethal stresses could improve the viability of DJO10A, 2 more vats were made, 1 with DJO10A exposed to sublethal acid, cold, and centrifugation stresses, and 1 exposed to none of these stresses. Although stress-primed DJO10A survived cheese manufacture better, as 72.8% were incorporated into the cheese compared with 41.1% of the unprimed, the statistical significance of this difference is unknown. In addition, the difference in moisture levels in the cheese cannot be excluded as influencing this difference. However, the rate of decline during ripening was similar for both. After 6 mo of ripening, cell counts in cheese were 4.68 and 4.24 log cfu/g for primed and unprimed cultures, respectively. These results suggest that whereas priming bifidobacteria with sublethal stresses before incorporation in a cheese fermentation may improve the number of viable cells that get incorporated into the cheese, it does not affect viability during cheese ripening.

Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth.

BACKGROUND: Bifidobacteria are frequently proposed to be associated with good intestinal health primarily because of their overriding dominance in the feces of breast fed infants. However, clinical feeding studies with exogenous bifidobacteria show they don't remain in the intestine, suggesting they may lose competitive fitness when grown outside the gut. RESULTS: To further the understanding of genetic attenuation that may be occurring in bifidobacteria cultures, we obtained the complete genome sequence of an intestinal isolate, Bifidobacterium longum DJO10A that was minimally cultured in the laboratory, and compared it to that of a culture collection strain, B. longum NCC2705. This comparison revealed colinear genomes that exhibited high sequence identity, except for the presence of 17 unique DNA regions in strain DJO10A and six in strain NCC2705. While the majority of these unique regions encoded proteins of diverse function, eight from the DJO10A genome and one from NCC2705, encoded gene clusters predicted to be involved in diverse traits pertinent to the human intestinal environment, specifically oligosaccharide and polyol utilization, arsenic resistance and lantibiotic production. Seven of these unique regions were suggested by a base deviation index analysis to have been precisely deleted from strain NCC2705 and this is substantiated by a DNA remnant from within one of the regions still remaining in the genome of NCC2705 at the same locus. This targeted loss of genomic regions was experimentally validated when growth of the intestinal B. longum in the laboratory for 1,000 generations resulted in two large deletions, one in a lantibiotic encoding region, analogous to a predicted deletion event for NCC2705. A simulated fecal growth study showed a significant reduced competitive ability of this deletion strain against Clostridium difficile and E. coli. The deleted region was between two IS30 elements which were experimentally demonstrated to be hyperactive within the genome. The other deleted region bordered a novel class of mobile elements, termed mobile integrase cassettes (MIC) substantiating the likely role of these elements in genome deletion events. CONCLUSION: Deletion of genomic regions, often facilitated by mobile elements, allows bifidobacteria to adapt to fermentation environments in a very rapid manner (2 genome deletions per 1,000 generations) and the concomitant loss of possible competitive abilities in the gut.

A survey of undergraduate education in dental implantology in UK dental schools.

The aim of this study was to ascertain knowledge on current teaching of implant dentistry in the undergraduate curriculum of Dental Schools in the UK. Information on the teaching modalities, including year of introduction of implant dentistry into undergraduate curriculum, departments involved in teaching, format of teaching, use of adjunctive teaching aids, and types of implant systems used in undergraduate teaching was collected by means of a questionnaire, which was sent to all undergraduate dental schools in the UK. Based on a 100% response rate, the findings indicate that all dental schools in the UK reported that they included dental implantology in their undergraduate curriculum; however there were marked variations in the content and delivery of the teaching.

The effect of a range of disinfectants on the dimensional accuracy and stability of some impression materials.

Disinfection of dental impressions should be considered as a routine procedure in dental surgeries and dental laboratories. Disinfectants can have deleterious effects on some properties of impression materials. The aim of this study was to evaluate the dimensional accuracy and dimensional stability of a model dental stone, reproduced from five commonly used impression materials (Aquasil soft putty/Aquasil Ultra LV; Aquasil Monophase; Aquasil Ultra Heavy; Impregum F and Provil putty/Provil Light CD wash) retained by their adhesives in acrylic resin trays and exposed to three disinfectant solutions (Perform ID; Haz-Tabs and MD 520). Two hundred models were used to investigate the effect of the three disinfectants on the dimensional accuracy of the five impression materials. Five impressions were taken for each impression material for each disinfection treatment group. Measurements were carried out using a High Precision Reflex Microscope. All materials demonstrated a percentage change in dimensions when subjected to no disinfection when compared to the brass master die and all materials demonstrated a percentage change in dimension when subjected to the different disinfection procedures. The results of this study have demonstrated that for all of the materials investigated, the changes in dimensional stability were small in the order of microns. These changes may however be of clinical significance for procedures requiring a high degree of accuracy, for example fixed prosthodontics. The materials respond differently depending on the disinfectant used and it may therefore be appropriate that manufacturers recommend the use of particular disinfectants for their products in order to ensure optimum dimensional accuracy and stability.

An in vitro investigation of the effect of the addition of untreated and surface treated silica on the transverse and impact strength of poly(methyl methacrylate) acrylic resin.

Silica is a commonly used filler in dental materials and as a reinforcing agent in industry. The aim of this study was to further investigate the effect of the addition of untreated and a novel surface treated silica on the transverse bend and impact strength of acrylic resin denture base material. It was hypothesized that the silica/resin composite materials would have an improved flexural and impact strength than the conventional heat-cured acrylic resin. Three types of untreated and two of treated silica powder were used in this study. The range of percentages used were 1%, 0.5%, 0.2%, 0.1%. The treated particles were coated with hexamethyldisilazane or dimethyldichloridesilazane. Conventional heat cured acrylic resin was used as a control. The modulus of rupture for all groups of acrylic resin containing silica was significantly lower than for the control. The modulus of elasticity was not significantly greater than the control group. For the impact strength statistical analysis revealed a significant difference between the groups. There was a nonsignificant increase in the impact strength for specimens compared to the control. In conclusion the addition of silica to poly(methyl methacrylate) denture base materials did not produce a significant improvement in the transverse bend or impact strength compared to conventional heat-cured acrylic resin. The incorporation of untreated and surface treated silica cannot be recommended as a method of reinforcement.

Usefulness of biochemical screening of diabetic patients for hemochromatosis.

We assessed the prevalence of previously unrecognized hemochromatosis among patients in whom diabetes mellitus was diagnosed after the age of 30 yr, and we evaluated the positive predictive value of biochemical screening tests for hemochromatosis in diabetic subjects. Thirty-eight of 572 patients screened (6.6%) had a serum ferritin level greater than 324 micrograms/L; 16 patients had normal levels on repeat testing. Four patients' serum ferritin levels fell to less than 400 micrograms/L. Seven of 18 patients with a persistently elevated serum ferritin level did not undergo a liver biopsy because of a recognized cause of hyperferritenemia (carcinoma, alcoholism, or systemic lupus erythematosus). The diagnosis of hemochromatosis seemed certain in 1 of 3 patients who were not biopsied for technical reasons. Of 8 patients biopsied, 2 had hemochromatosis, 4 had fatty liver, 1 had hemosiderosis, and 1 had a chronic inflammatory cell infiltrate with no iron deposition. Of 4 patients with a raised transferrin saturation level, 2 had raised serum ferritin levels and hemochromatosis, 1 had raised serum ferritin and hemosiderosis on liver biopsy, and 1 had a normal transferrin saturation level on repeat testing. Two of 3 cases of hemochromatosis had other clinical markers of the condition. Therefore, routine screening of diabetic patients for hemochromatosis is not necessary, because patients with hemochromatosis will often have other clinical features of the disease. When screening diabetic patients for hemochromatosis, it should be remembered that a persistently raised serum ferritin level has a low positive predictive value (16.6%) and that a normal transferrin saturation level does not exclude the diagnosis.

Exchangeable sodium and renin in hypertensive diabetic patients with and without nephropathy.

The objective of this study was to examine total exchangeable sodium, plasma-blood volume, and the status of the renin-angiotensin system in hypertensive diabetic patients with established nephropathy. We also evaluated hypertensive patients with diabetes who were free of clinically apparent nephropathy or other diabetic complications. Total exchangeable sodium (by 24Na dilution) was expressed as percentage predicted. Subjects were studied as inpatients receiving unrestricted sodium intake and in stable metabolic control. Total exchangeable sodium was 100 +/- 2% in controls (n = 42), higher (p less than 0.01) at 108 +/- 2% in normotensive patients with diabetes (n = 30), and higher still (p less than 0.005) in hypertensive patients with diabetic nephropathy (n = 16) 118 +/- 4% (p less than 0.05 vs normotensive diabetics). The value correlated with blood pressure only in diabetics with nephropathy (r = 0.61, p less than 0.01). Plasma renin activity, and blood and plasma volumes were similar in nephropathic diabetics and controls. Hypertensive patients with maturity-onset (type II) diabetes free of nephropathy (n = 18) were compared with nondiabetic controls (n = 16) and normotensive patients with type II diabetes (n = 18) of similar age. Total exchangeable sodium in the controls was 100 +/- 3%, higher (p less than 0.01) in normotensive diabetics at 109 +/- 2%, but not significantly elevated in hypertensive diabetics at 106 +/- 2%. Again, blood and plasma volumes did not differ among the groups. Plasma renin activity was suppressed (p less than 0.01) to a comparable degree in both normotensive and hypertensive patients with type II diabetes.(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetic control and the renin-angiotensin system, catecholamines, and blood pressure.

Diabetic ketoacidosis is usually associated with marked secondary hyperaldosteronism. Plasma levels of renin, angiotensin II, and aldosterone are markedly raised before treatment in most patients, with values falling rapidly toward normal as metabolic control is restored. In a few patients, mostly those with long-term complications of diabetes, plasma levels of renin, angiotensin II, and aldosterone before treatment remain within the normal range. In moderately hyperglycemic patients who have glycosuria but not ketonuria, plasma levels of all three substances are significantly higher than when control is improved. Occasionally, moderately hyperglycemic patients have mild secondary hyperaldosteronism. Improved metabolic control in such patients causes a rise in plasma volume and a rise in total exchangeable sodium, the latter to levels significantly above normal. Plasma catecholamine levels are markedly elevated in diabetic ketoacidosis, probably as a consequence of the ketoacidotic state. In nonketotic patients with moderate hyperglycemia, basal plasma norepinephrine levels are normal; catecholamine responses to exercise may be exaggerated, however. Epidemiological and animal studies suggest a relationship between blood pressure and blood glucose levels. There are few clinical studies of the effects of altering metabolic control of diabetes on blood pressure, and this is an important area for further study.

High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening.

High- and low-copy-number shuttle cloning vectors were constructed by incorporating the Escherichia coli P15A plasmid origin of replication into the pAM beta 1-derived vectors, pIL252 and pIL253. The resulting vectors were structurally stable in Lactococcus, which is a common feature of theta-replicating plasmids, and also displayed good structural stability in E. coli, possibly due to lack of a resolvase-encoding gene. All the vectors expressed erythromycin resistance (ErR) in both; brain heart infusion medium allowed clear selection of ErR in E. coli. Some of the vectors provided insertional inactivation of a cat (pTRKH1; pTRKL1) or tet (pTRKH1; pTRKH3; pTRKH5) gene to facilitate screening for clones. Multiple cloning sites in a lacZ gene, which expresses beta-galactosidase in lacZ alpha-complementing E. coli strains, were included in some vectors (pTRKH2/H5 and pTRKL2) to enable blue/white screening of clones on XGal plates. The 'H' and 'L' prefixes signify if the vector exists at high (H) or low (L) copy number in Lactococcus. Successful introduction of these vectors into Lactococcus, Enterococcus, Streptococcus and Lactobacillus highlights their utility for expanding the possibilities for genetic manipulation of these industrially significant bacteria.

A leucine repeat motif in AbiA is required for resistance of Lactococcus lactis to phages representing three species.

The abiA gene encodes an abortive bacteriophage infection mechanism that can protect Lactococcus species from infection by a variety of bacteriophages including three unrelated phage species. Five heptad leucine repeats suggestive of a leucine zipper motif were identified between residues 232 and 266 in the predicted amino acid sequence of the AbiA protein. The biological role of residues in the repeats was investigated by incorporating amino acid substitutions via site-directed mutagenesis. Each mutant was tested for phage resistance against three phages, phi 31, sk1, and c2, belonging to species P335, 936, and c2, respectively. The five residues that comprise the heptad repeats were designated L234, L242, A249, L256, and L263. Three single conservative mutations of leucine to valine in positions L235, L242, and L263 and a double mutation of two leucines (L235 and L242) to valines did not affect AbiA activity on any phages tested. Non-conservative single substitutions of charged amino acids for three of the leucines (L235, L242, and L256) virtually eliminated AbiA activity on all phages tested. Substitution of the alanine residue in the third repeat (A249) with a charged residue did not affect AbiA activity. Replacement of L242 with an alanine elimination phage resistance against phi 31, but partial resistance to sk1 and c2 remained. Two single proline substitutions for leucines L242 and L263 virtually eliminated AbiA activity against all phages, indicating that the predicted alpha-helical structure of this region is important. Mutations in an adjacent region of basic amino acids had various effects on phage resistance, suggesting that these basic residues are also important for AbiA activity. This directed mutagenesis analysis of AbiA indicated that the leucine repeat structure is essential for conferring phage resistance against three species of lactococcal bacteriophages.

Lesion of thalamic centromedian--parafascicular complex after chronic deep brain stimulation.

A patient with PD who exhibited disabling tremor and prominent dyskinesia underwent deep brain stimulation (DBS) of the left thalamic ventral intermediate nucleus. The electrode migrated and was replaced but with suboptimal clinical response. Two years later, postmortem analysis found the second electrode tip had entered the thalamic centromedian-parafascicular complex. There was a small thalamotomy and cell loss exceeding that found in PD. Thalamic damage may occur in association with DBS for PD.

Nisin independent induction of the nisA promoter in Lactococcus lactis during growth in lactose or galactose.

Nisin biosynthesis is autoregulated extracellularly by the mature and modified peptide. To investigate other regulatory effects on nisin biosynthesis, a transcription fusion of the nisA promoter from Lactococcus lactis ATCC 11454 to the promoterless lacZ gene from Streptococcus thermophilus was constructed. This fusion construct, pDOC99, expressed beta-galactosidase in L. lactis ATCC 11454 growing in M17 medium containing glucose (M17G). Consistent with the known model for transcription of nisA, pDOC99 did not express beta-galactosidase in the non-nisin producer, L. lactis LM0230 grown in M17G, unless the nisRK genes (cloned in pDOC23) were included in trans and nisin was added to the medium. Growth of this strain in M17 containing lactose or galactose, resulted in nisA transcription, even in the absence of exogenous nisin. This expression was independent of pDOC23. Furthermore, nisA transcription in L. lactis LM0230(pDOC99) grown in M17G could be induced by the addition of exogenous galactose, with maximum induction occurring at concentrations > 5 mM.

Evaluation of using a short region of the recA gene for rapid and sensitive speciation of dominant bifidobacteria in the human large intestine.

The feasibility of intragenerically characterizing bifidobacteria by a comparison of a short region within the recA gene was tested. An approximately 300 bp fragment of the recA gene was PCR-amplified from six species from the genus Bifidobacterium using primers directed to two universally conserved regions of the recA gene. A phylogenetic analysis of the sequenced recA products compared favorably to classification based on the 16S rRNA sequences of the species tested. To apply this rapid methodology to unknown human intestinal bifidobacteria, 46 isolates were randomly chosen from the feces of four subjects and initially characterized by RFLP analysis of a PCR-amplified region of their 16S RNA genes. From a representative of the dominant RFLP family in each of the subjects, the recA segment was PCR-amplified, sequenced and phylogenetically analyzed. All four isolates were found to be related to one another and to B. longum and B. infantis. These results illustrate that the recA gene may be useful for intrageneric phylogenetic analysis as well as for the identification of unknown fecal bifidobacteria.

Escherichia coli ferric uptake regulator (Fur) can mediate regulation of a pseudomonad iron-regulated promoter.

Introduction of a Pseudomonas iron-regulated promoter lacZ fusion (SP1) and a Pseudomonas transcriptional factor into Escherichia coli allowed expression of the promoter in this heterologous host. Evaluation of this promoter in wild-type and fur mutants of E. coli, by measuring beta-galactosidase activity, indicated that E. coli Fur can regulate the Pseudomonas promoter in response to iron starvation. Gel retardation assays suggested that purified Fur protein could interact with the SP1 promoter upstream of the transcriptional start. DNase I footprinting analysis established that Fur protected a primary 58-bp region (-50 to -106 bp). These protein/DNA interactions correlate with the observed in vivo regulation of the SP1 promoter in E. coli and indicate that Fur can functionally regulate a Pseudomonas iron-regulated promoter.

Procedure for quantifiable assessment of nutritional parameters influencing nisin production by Lactococcus lactis subsp. lactis.

A modified rapid plate assay procedure was developed, that allowed quantifiable measurement of nisin production by Lactococcus lactis growing directly on agar media. Using this direct plate assay, several nutritional parameters were assessed for their influence on nisin production (as distinct from their influence on growth) by L. lactis subsp. lactis ATCC 11454 growing on standard M17 based media over 3 and 6 h incubation periods. Glucose was found to be the optimal carbon source tested, with glycerol having the greatest suppressive effect. The addition of salts suppressed nisin production on a per cell basis, except MnCl2. This direct plate method proved to be a good pilot assay for rapidly and quantifiably investigating the initial effects of different parameters on nisin production by L. lactis, prior to conducting more intensive broth batch culture assays. The data obtained in this study indicate that certain nutritional parameters can impose a repressive effect on nisin production. Elucidation of how these parameters control the amount of nisin produced will provide further insight into the regulation of nisin biosynthesis in L. lactis.

The pressor response to exercise and stress in uncomplicated insulin-dependent diabetes.

We investigated whether or not an increased pressor response to exercise or stress is a feature of the diabetic state per se or a feature of its complications was investigated. Twelve insulin-dependent diabetic patients without clinical evidence of complications and with normal albumin excretion rates (less than 20 micrograms/min) were studied together with 12 matched control subjects. Each underwent a study protocol of isometric handgrip exercise at 30% of maximum capacity for four minutes, a cold pressor test with immersion of one hand in ice-cold water for two minutes, and bicycle ergometry at a resistance of 105 watts per minute for six minutes. The tests were undertaken in the same order in all subjects. There was, in both groups, a similar and significant rise in systolic blood pressure and pulse rate in response to each stimulus. Diastolic pressure also rose significantly in response to handgrip exercise and to cold pressor stimulation, but fell slightly during bicycle ergometry in both groups. Mean plasma noradrenaline concentration rose in response to each stimulus but the changes reached conventional significance in both groups only in response to handgrip exercise. Pressor responses to exercise and stress, as tested here, are concluded to be normal in insulin-dependent diabetic patients without complications due to their disease.

Enalapril and microalbuminuria in diabetic and non-diabetic hypertension.

Nine patients with non-insulin-dependent diabetes mellitus (NIDDM) and ten non-diabetic patients with mild hypertension were treated with enalapril 20 mg daily. None had overt nephropathy, though 4 diabetic subjects had microalbuminuria. Subjects with the highest baseline albumin excretion rates (AER) showed the greatest fall on therapy. Metabolic control of diabetes did not deteriorate. Enalapril had no significant effect on AER in NIDDM patients with AER below 20 micrograms/minute or in the non-diabetic group.